Early host responses to avian influenza A virus are prolonged and enhanced at transcriptional level...

Transcriptomics of host-virus interactions:

Immune responses to avian influenza virus in chicken

The research described in this thesis was conducted at the Division of Immunology,

Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine,

Utrecht University, The Netherlands.

COLOFON

© S. Reemers, 2010. No part of this publication may be reproduced or transmitted without

prior permission of the author.

Published papers were reprinted with permission from the publisher.

ISBN: 978-90-393-5331-8

Cover: Erwin van Hoof - SiteOn - www.siteon.nl

Printed by: Wöhrmann Print Service, Zutphen, The Netherlands.

The research described in this thesis was financed by a BSIK VIRGO consortium grant

(Grant no. 03012), The Netherlands.

The printing of this thesis was financially supported by: Intervet Schering-Plough Animal

Health, Shimadzu and the Infection & Immunity Center Utrecht.

Transcriptomics of host-virus interactions: Immune responses to avian influenza virus in chicken

Transcriptomics van virus-gastheer interacties: Immuunresponsen tegen aviair influenza virus in de kip

(met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. J C Stoof, ingevolge het besluit van het college voor promoties

in het openbaar te verdedigen op maandag 10 mei 2010 des middags te 12.45 uur

door

Sylvia Samantha Natascha Reemers

geboren op 22 augustus 1978 te Eindhoven

Promotor: Prof. dr. W. van Eden Co-promotor: Dr. ir. L. Vervelde

Contents

Chapter 1 General introduction 7

Chapter 2 Cellular host transcriptional responses to Influenza A virus 23

in chicken tracheal organ cultures differ from responses in

in vivo infected trachea

Chapter 3 Chicken Lung Lectin is a functional C-type lectin and 45

inhibits haemagglutination by Influenza A Virus

Chapter 4 Transcriptional expression levels of chicken collectins 63

are affected by avian influenza A virus inoculation

Chapter 5 Differential gene expression and host response profiles 75

against avian influenza virus within the chicken lung

due to anatomy and airflow

Chapter 6 Lack of immune activation as a correlate of protection 113

after challenge with avian influenza virus:

a transcriptomics analysis of adjuvanted vaccines

Chapter 7 Early host responses to avian influenza A virus are 135

prolonged and enhanced at transcriptional level depending

on maturation of the immune system

Chapter 8 General discussion 167

Summary 181

Nederlandse samenvatting 187

Dankwoord 193

List of abbreviations 198

Curriculum vitae 200

General introduction

Chapter 1


9

“Fowl plague” was first described in 1878 as a disease affecting chickens in Italy. In 1902

the causative agent was isolated, though it was not until 1955 that it was identified as a

member of the genus Influenzavirus A belonging to the Orthomyxoviridae family

(Horimoto et al., 2001; Tollis et al., 2002). Today influenza viruses are a major cause of

respiratory infections in both humans and animals and outbreaks are carefully monitored.

Over a time span of 45 years, from 1959-2004, there has been a sharp increase in the

number of outbreaks in poultry worldwide. These range from minor outbreaks to outbreaks

having devastating effects worldwide on human health issues with fear for generation of

new pandemics and on the poultry industry like the Italian 1999-2000, Dutch 2003,

Canadian 2004 and ongoing Asian outbreaks (Capua et al., 2006).

Influenza A virus

Influenza A virus is an enveloped, single stranded RNA virus and the genome consists of 8

negative sensed RNA segments variable in size coding for 10 proteins (Fig. 1). The three

surface proteins are haemagglutin in (HA), neuraminidase (NA) and matrix protein 2 (M2).

The seven internal proteins are matrix protein 1 (M1), nucleoprotein (NP), the polymerase

complex (PB1, PB2 and PA) and non-structural proteins NS1 and NS2. A relatively new

protein was encoded named PB1-F2, which is expressed from a second open reading frame

from PB1 (Chen et al., 2001).

Of the surface proteins, HA and NA are the most prominent features of the viral envelope

and currently 16 HA and 9 NA subtypes are identified which are used to subtype the virus

strains (Fouchier et al., 2005). HA and NA are spike-like proteins embedded in the viral

membrane and play a role in virus attachment, while M2 acts as an ion channel and is

important in the uncoating of the virus in the endosome. Of the internal proteins NP

encapsidates each genomic RNA segment, which associates with the viral polymerase

composed of PB1, PB2 and PA to form the coiled ribonucleoprotein (RNP). The RNP is

linked to the viral envelope via M1, which is associated to both. NS2 is present in small

quantities in the virions where it is associated with the RNP, making NS1, which plays a

critical role in inhibiting the host antiviral defenses, the only true non-structural protein that

is not packed into the virion (Brown, 2000; Horimoto et al., 2001; Tollis et al., 2002; Lee et

al., 2009). PB1-F2 is suggested to be involved in cell death of immune cells which react to

influenza virus infection (Chen et al., 2001).

Chapter 1

10

A. B.

Figure 1. A) Electron microscopy image of Influenza A virus (www.esrf.eu/files/press/ Influenza-Virus.jpg). B)

Schematic overview of an influenza A virion (Horimoto, et al., 2005).

Life cycle

Influenza virus binds to a target cell via an interaction of the HA protein with a sialic acid

linked to galactose by an α-2,3 or α-2,6 linkage on the host cell. Once bound, virions are

internalised in the target cell mainly via clathrin-dependent endocytosis, but entry via

clathrin- and caveolin-independent endocytosis has also been reported and is equally

efficient for viral fusion once the virus is internalized (Lakadamyali et al., 2004). Once

vesicles fuse with endosomes the low pH within the endosome causes a conformational

change of the HA protein exposing the fusion protein leading to fusion of the viral and

endosomal membranes. Through opening of the M2 ion channel protein, the M1 protein is

also exposed to an increased proton concentration. This causes dissociation of M1, which is

associated with the RNPs and the viral envelope, releasing the RNPs into the cytoplasm.

The RNPs are imported into the nucleus where transcription and replication of viral RNA

(vRNA) occurs via the vRNA polymerase consisting of PB1, PB2 and PA (Baigent et al.,

2003). During transcription the vRNA polymerase catalyzes RNA polymerization,

catalyzes polyadenylation of mRNA and cleaves capped host cell RNAs in the nucleus to

generate capped RNA fragments serving as primers for viral mRNA synthesis, which is

called cap-snatching (Shih et al., 1996; Honda et al., 2002). The viral mRNA is translated

in the cytoplasm into viral proteins that are transported into the nucleus. In the nucleus

replicated vRNA binds to newly synthesised polymerase complex, NP and M1 proteins to

form M1-vRNP complexes which are exported to the cytoplasm. Envelop proteins migrate

via the Golgi apparatus to the plasma membrane where they envelop the M1-vRNP

complex to form sialylated progeny virions. During budding of the new virions the NA

protein cleaves the sialic acids leading to the release of mature virions (Baigent et al.,

2003).


11

Host range

Members of the influenzavirus A, of which influenza A is the only serotype (Brown, 2000),

infect a wide variety of species like, pigs, birds, horses, minks, seals, whales and humans

often causing serious disease. However, the virus is considered avirulent in the natural hosts

for avian influenza virus, wild waterfowl, gulls and shorebirds, which maintain all subtypes

of HA and NA proteins (Suarez, 2000; Webby et al., 2001; Suzuki, 2005).

Host range restrictions of influenza virus are based on effective entry and replication of the

virus in a specific host. This requires the balanced functions of HA and NA, but also of the

internal proteins. Influenza virus infectivity is mainly influenced by the type of galactose

linkage of the sialic receptor on the host cell to which HA binds and thus also depends on

the distribution of these receptors in the host. Avian influenza viruses prefer α-2,3 linkage,

while human influenza viruses prefer a α-2,6 linkage (Van Riel et al., 2007; Lee et al.,

2009). It was generally believed that avian influenza viruses are limited in their host range

and thus were restricted in their ability to infect humans, although rare interspecies

transmissions have been detected. However, in 1997 an outbreak of 18 human infections in

Hong Kong was caused by avian influenza virus that was directly transmitted to humans

(Ito et al., 2000; Subbarao et al., 2000). Single amino acid substitutions at the receptor

binding site of HA converted avian H5N1 viruses to recognize the human receptor

(Yamada et al., 2006). Through the antigenic variability of influenza A the virus is able to

subvert the immune response. This antigenic variability is obtained due to mutation

(antigenic drift) and reassortment (antigenic shift). The RNA polymerase of influenza has a

low fidelity and also lacks proofreading capacity, which results in a rapid rate of mutation

in the viral genome. Antigenic drift occurs when genes coding for HA and NA undergo

point mutations at the antigenic site of the proteins. This change in antigenicity causes the

annual outbreaks of influenza epidemics and necessity to annually adjust the virus strains in

the influenza vaccine. Antigenic shift occurs when 2 different virus particles, possibly from

different species, co-infect a single target cell. By reassortment of genome segments the

progeny viruses can have RNA segments from both the original viruses. Such a new virus

has the potential to cause a pandemic (Parvin et al., 1986; Zambon, 1999; Horimoto et al,

2001).

Pathogenicity in chickens

The disease caused by avian influenza viruses infecting poultry ranges from asymptomatic

infection to acute, fatal disease. According to their ability to cause disease in chickens avian

influenza viruses can be divided into two groups, highly pathogenic avian influenza (HPAI)

and low pathogenic avian influenza (LPAI). HA protein has a key role in influenza virus

pathogenicity since proteolytic cleavage of HA precursor by host proteases is essential for

Chapter 1

12

viral infectivity. HA of HPAI viruses are cleaved by ubiquitous proteases which are present

in a wide range of host organs thus supporting systemic infection, while HA of LPAI

viruses are thought to be cleaved by proteases localized mostly in respiratory and intestinal

organs resulting in mild or asymptomatic infection.

HPAI viruses are very virulent viruses in which mortality rates may approach 100% and

appear to be pantropic with respect to systemic virus replication and ability to produce

gross lesions. The most severe lesions result in inflammation, haemorrhage with necrosis

and cellular death of skin, brain, heart, adrenal gland, pancreas and other visceral organs

(Tollis et al., 2002). Post-mortem examination revealed oedema of the head and upper part

of the neck, presence of bile-green mucous liquid in the oral cavity and oesophagus,

pancreatitis, congestion of internal organs, occasionally necrotic foci in the spleen and

hemorrhagic cecal tonsils and in some cases urate deposits in the kidney could be seen

(Mutinelli et al., 2003). These viruses have been restricted to subtypes H5 and H7 and have

a preference for infecting the lower respiratory tract (Alexander, 2000; Horimoto et al.,

2005).

LPAI viruses cause milder infections and appear to be restricted in their capability for

replication and production of lesions in individual organs and tissues (Alexander, 2000;

Tollis et al., 2002; Horimoto et al., 2005). Lesions were detected in the ovary and oviduct

with colliquation of ovarian follicles and congestion of the lung and trachea (Mutinelli et

al., 2003). LPAI viruses seem more transmissible from infected to susceptible birds than

HPAI viruses. This is probably due to the rapid death of birds infected with HPAI virus,

which reduces the amount of virus excreted, and the preference of LPAI virus for infecting

the upper part of the respiratory tract, increasing the transmission via aerosols breath or

coughing (Tollis et al., 2002).

Avian respiratory tract

The structure and airflow of the avian respiratory system is unique among vertebrates.

Birds have rather small, rigid lungs compared to the large, flexible lungs of mammals. They

lack a diaphragm and instead use airsacs that act like bellows to ventilate the lungs. The

trachea bifurcates at the syrinx into the primary bronchus, like in mammals, and runs

throughout the entire length of the lungs, exiting into the ostium of the abdominal airsacs.

Four groups of secondary bronchi (medioventral, lateroventral, mediodorsal and

laterodorsal) originate from the primary bronchus. Branching off from the secondary

bronchi are the parabronchi that connect the dorsal and ventral secondary bronchi (Fig. 2A).

The parabronchi, like the alveoli in mammals, are surrounded by a network of blood

capillaries and are the functional units of gas exchange in the lung (Duncker, 1974; Reese

et al., 2006).


13

Air enters the respiratory tract via the trachea and flows into the lung via the primary

bronchus. While the airflow in mammals is two directional, birds have a unidirectional

airflow which is driven by difference in pressure throughout the respiratory tract. During

inspiration the posterior and anterior airsacs expand lowering the pressure. This causes the

air to flow from the trachea and bronchi into the posterior airsacs, simultaneously, flowing

into the lung through the parabronchi into the anterior airsacs (Fig. 2B). During expiration

the volume of the posterior and anterior airsacs is reduced. This causes the air to flow out of

the posterior airsacs into the lung through the parabronchi, simultaneously air from the

anterior airsacs flows into the trachea out of the body (Fig. 2C). The advantage of this

unidirectional airflow is that birds constantly have oxygen rich air in their lungs (Duncker,

1974; Reese et al., 2006).

Besides its primary function for gas exchange, the respiratory tract is a barrier between the

external and internal environment. In the lung highly organised lymphoid structures and

diffusely distributed lymphoid cells are present. Lymphoid nodules, named bronchus

associated lymphoid tissue (BALT), in the lung have much similarity with Peyer’s patches

in the gut (Kothlow et al., 2008). BALT is located at the bifurcations of primary bronchus

into secondary bronchi (Fig. 2A) and is influenced by age and environmental stimuli like

infection. BALT is not observed before 2-3 weeks after hatching and develops into mature

structures found in 6-8 week old birds. Distinct T cell and B cell areas and frequently

germinal centres are displayed covered by characteristic follicle-associated epithelium

(FAE). The FAE consist of ciliated and non-ciliated cells of which the numbers differ

according to the age of the bird in that the FAE of older birds consists of more ciliated cells.

Cells displaying some features similar to M-cells in the gut have been described, but

particle uptake by these cells has not been seen. The germinal centre is mainly made up by

IgM+ B cells, while smaller numbers of IgY+ and IgA+ B cells are present. CD4+ T cells

cover the germinal centre, while CD8+ T cells are diffusely distributed between the

lymphoid nodules (Jeurissen et al., 1994; Reese et al., 2006; Kothlow et al., 2008).

A. B. Inspiration C. Expiration

Figure 2. A) Schematic representation of the right avian lung showing the main bronchi. Red dots mark the

localization of the BALT nodules in the primary bronchus. Legend: PB primary bronchus; 1: medioventral

secondary bronchi; 2: mediodorsal secondary bronchi; 3: lateroventral secondary bronchi; A: ostium to the

clavicular air sac; B: ostium to the cranial thoracis air sac; C: ostium to the caudal thoracis air sac; D: ostium to the

6

1

2

3

54

1

2

3

5

6

4

Chapter 1

14

abdominal air sac. (Reprinted from “The avian lung-associated immune system: a review”, Vol. 37, S. Reese et al.,

p. 313, 2006, with permission from EDP Sciences). Schematic representation of airflow through the avian lung

during B) inspiration and C) expiration. Legend: 1. Primary bronchus, 2. lung, 3. clavicular air sac, 4. cranial

thoracic air sac, 5. caudal thoracic air sac, 6. abdominal air sac. (Adapted and reprinted from “Avian

Immunology”, first edition 2008, Kothlow S. et al., chapter 14, The Avian Respiratory immune system, 2008, with

permission from Elsevier).

Early immune response in the respiratory tract

Although adaptive responses are important for the control and clearance of AIV infection,

innate responses are critical for blocking of virus entry and virus replication, and for the

induction of virus specific adaptive responses (Fig. 3). Virus entry in the respiratory tract is

blocked by several mechanisms. Mucus on top of the epithelial cells is the first barrier that

blocks virus entry by trapping the virus, which together with the mucus is transported out of

the respiratory tract by the constant movement of cilia. Neutralizing components located in

the mucus like secretory IgA (sIgA), complement molecules, collectins or antimicrobial

peptides such as β-defensins, also block virus entry. sIgA has been shown to neutralize

influenza virus infectivity in both mammals and chicken (Taylor et al., 1985; Tamura et al.,

2004). Both mammalian collectins and defensins have neutralizing activity against

influenza (Hartshorn et al., 1997; Klotman et al., 2006), but for chicken collectins

(Hogenkamp et al., 2006) and β-defensins, which are named gallinacins (Xiao et al., 2004)

this has not been clarified yet. Complement molecules become activated by binding directly

to the virus or to an antibody-virus complex or by hydrolysis of C3 (Favoreel et al., 2003).

This leads to the activation of the complement cascade inducing inflammatory responses,

enhancing phagocytosis of the antigens and enhancing cytolysis of target cells.

When virus entry is not successfully blocked, AIV will infect the epithelial cells resulting in

the production of pro-inflammatory cytokines such as IL-1β and IL-6, chemokines such as

CXCLi2 (IL-8) and RANTES, and interferons IFN-α and -β (Julkunen et al., 2000). This

attracts macrophages and DC to the place of infection, which upon activation or AIV

infection also start producing cytokines and chemokines attracting more antigen presenting

cells (APC) but also heterophils, T cells, B cells and natural killer (NK) cells. IL-18

produced by APC can prime T cells and NK cells for IFN-γ production (Julkunen et al.,

2001).

Influenza virus that passes the epithelial cells is recognized by Toll-like receptors (TLRs)

which are pattern recognition receptors that recognize pathogen-associated molecular

patterns (PAMP). Upon recognition of PAMP, the TLR signalling pathway is activated

leading to production of pro-inflammatory cytokines, chemokines, IFN-α and -β. TLR-

activated DC mature and can activate naïve T cells via antigen presentation on MHC in

combination with co-stimulatory molecules such as CD80 and CD86 thereby inducing an


15

adaptive response (Lee et al., 2007). In mammals TLR3, TLR7, TLR8 and TLR9 recognize

viral nucleic acids of which dsRNA is recognized by TLR3, ssRNA by TLR7 and TLR8,

while TLR9 recognizes viral DNA (Kawai et al., 2008). TLR3, TLR7 and TLR8 have been

directly implicated in influenza infection in mice (Sen et al., 2005). TLR9 has not, however

TLR9-agonists are assumed to play an potential role in protection against influenza virus

infection (Wong et al., 2009). In chickens, TLR3, TLR7 and TLR8 are found of which

TLR8 is disrupted and non-functional. No TLR9 orthologue is found although chickens

respond to TLR9-agonists (Juul-Madsen et al., 2008; Jenkins et al., 2009). Two TLR7

splice variants, TLR15 and TLR21, are speculated to elicit the response to TLR9 ligands

(Philbin et al., 2005; Jenkins et al., 2009). Several studies report that TLR expression in

chicken is affected by influenza infection and imply induction of anti-viral responses via

TLR signalling triggered by influenza virus infection (Degen et al., 2006; Karpala et al.,

2008; Xing et al., 2008).

Figure 3. Early immune responses to AIV infection in the respiratory tract.

Influenza viruses have evolved strategies to ensure their survival in the host. Influenza virus

can directly interfere with IFN-γ-stimulated signal transduction via the Jak/Stat pathway

and enables the virus to inhibit IFN-γ-inducible gene expression of MHC class II (Uetani et

al., 2008). Viral NS1 protein is a tri-functional viral immunosuppressor. Innate immunity is

Interferons IFN-α IFN-β

Antiviral effect

Influenza A virus

Mucus

Epithelial cells

IgA Collectins

Chemokines RANTES IL-

8

Chemokines RANTES MIP-3α MIP-1β

Cytokines IL-1β IL-6

IL-18

Macrophage Dendritic cell

NK cell

T cell

B cell

Chemotatic effect

Interferons IFN-α IFN-β

Proinflammatory effect

IFN-γ production

Chapter 1

16

inhibited due to binding of NS1to RIG-I which inhibits activation of IRF-3 blocking the

transcription of IFN-β (Mibayashi et al., 2007), while inhibition of NF-κB by NS1 blocks

induction of both IFN-α and -β (Wang et al., 2000). Furthermore, NS1 inhibits adaptive

immunity by attenuating human DC maturation and the capacity of DC to induce T-cell

responses (Fernandez-Sesma et al., 2006) and NS1 is able to block apoptosis via inhibition

of caspase 9 (Ehrhardt et al., 2007). Besides blocking host responses influenza viruses also

use host proteins for virus replication and transcription. Viral factors such as the

polymerase complex, RNP and NP are known to interact with cellular factors such as

histones, the cytoskeleton and importins. These cellular factors are not only used for viral

RNA synthesis, but are also implicated in processes needed for virus replication such as

import and export of viral factors from the nucleus and transport to the cell membrane

(Engelhardt et al., 2006; Naffakh et al., 2008; Nagata et al., 2008). Efficient infection by

influenza viruses thus depends on the ability of the virus to interact with host factors needed

for virus replication and counteracting host defense responses.

Control of avian influenza virus outbreaks

Influenza outbreaks in humans and animals have been reported with increased frequency in

recent years and LPAI H5 and H7 viruses have the property to become HPAI viruses during

circulation in poultry. Therefore much research is conducted on improving surveillance and

diagnostic methods and development of antiviral drugs and effective vaccines. Poultry

vaccines preferably provide a broad protection against different influenza virus strains,

especially HPAI strains, provide clinical protection and prevent transmission to achieve

efficient control of the disease. In order to induce protection, a vaccine has to give a desired

type of adaptive response which is mostly a combination between humoral and cell-

mediated immunity. Neutralizing antibodies against HA or NA protein provide immediate

protection against influenza virus infection by reducing infectivity or restricting replication

, while viral clearance mainly depends on cell-mediated immunity. Due to the mutation rate

of influenza virus strains humoral immunity is poorly protective against antigenic drift

variants of strains to which immunity was originally obtained. Cytotoxic T lymphocytes

recognize epitopes of NP, PB2 and PA proteins which are highly conserved among

influenza strains. Therefore cell-mediated immunity against these epitopes could protect

against infection of various influenza strains (Subbarao et al., 2007; Van den Berg et al.,

2008).

The type of response induced by a vaccine largely depends on the composition of the

antigen and the type of adjuvant present in the vaccine. There are several vaccines licensed

for poultry; inactivated vaccines, reverse genetics inactivated vaccines and vector based

vaccines (Swayne, 2008; Van den Berg et al., 2008). Inactivated vaccines contain whole

influenza virus which is chemically or physically inactivated. For poultry homologous (HA


17

and NA subtype similar to the field strain) or heterologous (same HA but different NA

subtype) inactivated vaccines are used. Inactivated vaccines induce large cross-protection

against diverse field strains in poultry in contrast to humans. Reverse genetics inactivated

vaccines are created in cell lines using plasmid based techniques. The backbone of the virus

is made with 6 plasmids containing the sequence of the internal proteins. Together with two

plasmids containing the sequence of the HA and NA protein of the circulating strain, all

plasmids are co-transfected in appropriate cell lines. Recombinant virus is isolated and

inactivated. For this type of vaccine a homologous H5N1 and heterologous H5N3 are

licensed and used in traditional oil emulsified inactivated vaccines in poultry (Swayne,

2008; Van den Berg et al., 2008). Vector based vaccines use a virus, bacterial or plasmid

DNA as a vector to deliver protective proteins. For chickens, vectors based on fowlpox

virus and Newcastle Disease Virus (NDV) containing influenza virus H5 protein sequences

are licensed (Swayne, 2008; Van den Berg et al., 2008).

The inactivated virus is associated with an adjuvant to increase the immunogenicity of the

vaccine. In poultry, adjuvants like oil emulsions, for example water-in-oil (w/o),

aluminium-based formulations, such as aluminium hydroxide, and nonmethylated CpG

oligonucleotides (CpG) are used of which CpG is not commercially available but used for

experimental purposes (Hilgers et al., 1998; Spickler et al., 2003; Wang, et al., 2009).

Oil emulsions and aluminium-based adjuvants are known to generally induce a Th2

response (Hogenesch, 2002; Hilgers et al., 1998), but oil emulsions may also be capable of

activating CTL under certain conditions (Spickler et al., 2003). In mammals it is assumed

that oil emulsions and aluminium-based formulations act as a depot slowly releasing the

antigen at the site of injection, although actual mechanisms are still under debate

(Aucouturier et al., 2001; Marrack et al., 2009). Both types of adjuvants are able to induce

inflammation stimulating recruitment of eosinophils, neutrophils, APC and lymphocytes

and enhance antigen uptake and activation of APC (Aucouturier et al., 2001; Marrack et al.,

2009). Aluminium salts and oil emulsions do not act directly on dendritic cells (DC), but

they promote a pro-inflammatory environment which may indirectly activate DC in a TLR

independent manner (Tritto et al., 2009). In chickens immunisation of inactivated NDV

with an aluminium-based adjuvants caused proliferation and accumulation of macrophages

(Yamanaka et al., 2003). Vaccination of birds against O. rhinotracheale using w/o or

aluminium salts inhibits pathology of the airways and results in an increased HI titre

(Murthy et al., 2007), and birds vaccinated with a w/o adjuvanted inactivated H5N1 vaccine

had an increased HI titer, a strong secondary antibody response as shown by a high HI titer

after challenge and were protected from clinical signs and mortality (Sasaki et al., 2009).

CpG induces a Th1 like response inducing APC function and the production of cytokines

and chemokines supportive for antigen specific immunity in mammals (Dalpke et al., 2001;

Klinman, et al., 2004). CpG acts directly on DC in a TLR dependent manner (Ishii et al.,

Chapter 1

18

2007). Addition of CpG to H5N1 AIV vaccines induces a protective response in chickens

with enhanced antigen specific antibody production, increased HI titre and IFN-γ and a

decreased IL-10 protein concentration in serum (Wang, et al., 2009). Birds immunised with

NDV vaccine adjuvanted with CpG, showed virus-specific antibodies, stronger PBMC

proliferation and protection against clinical signs and mortality to NDV challenge

(Linghua, et al., 2007).

Scope of the thesis

Influenza virus infection is one of the major causes of respiratory disease in both humans

and animals. Although much research is performed to elucidate the course of events that

follow AIV infection, the interaction between the virus and the host at molecular and

cellular level at an early stage of infection is unclear. The aim of this thesis is to gain

insight in early host responses after primary avian influenza virus infection and viral

challenge in immunised chickens at host transcriptional level. Elucidating mechanisms

involved in innate and specific immune responses may contribute to a better understanding

of influenza virus induced pathology and to new concepts for vaccine development.

To define early immune responses to avian influenza virus infection, gene expression of

immune-related genes in epithelial cells after exposure to avian influenza virus was studied

in vitro. Since no chicken epithelial cell line was available we used tracheal organ cultures

(TOC) as an in vitro infection model. In chapter 2 we determined early immune responses

to infection with avian influenza virus H9N2 in TOC. We also assessed whether TOC was a

suitable model to study early immune responses to avian influenza infection in vitro at host

transcriptional level by comparing early immune responses in TOC with early immune

response in in vivo infected trachea.

Part of the early innate response are collectins, which for mammalians have been described

to have strong neutralizing activity against influenza A virus in vitro. In chapter 3 we

investigated whether recombinant chicken collectin cLL has a similar protective capacity

and was able to inhibit in vitro H9N2 infection of TOC. Since mammalian collectins are

implicated to play an important role in the innate defense to influenza A virus infection in

vivo, we also studied in vivo whether chicken collectins could play a similar role during

H9N2 infection in chickens in chapter 4. Furthermore, the possible effect of age on chicken

collectin expression was determined.

Gene expression patterns after H9N2 infection in primary infected chickens and in

immunised chickens after viral challenge were studied in vivo to define early host responses

throughout the respiratory tract and to define correlates of protection. To be able to study

immune responses in the respiratory tract we first had to know where the virus was located.

It was unknown if the virus is distributed equally within the respiratory tract and whether


19

possible differences affect local immune responses. Therefore, the effect of differences in

anatomy and airflow in the lung and the trachea on gene expression, virus distribution and

subsequently host responses was studied in uninfected and H9N2 infected chickens in

chapter 5. Once the distribution of the virus in the respiratory tract was determined we

focused on early host responses throughout the respiratory tract. Avian influenza virus

infection triggers innate responses, which the virus tries to inhibit. However, the virus also

uses certain host factors for replication and transcription as described in mammals. In

chapter 6 we determined early host responses to H9N2 infection and analysed the effect of

host-factor hijacking by the virus on host gene expression. Since differences in maturity of

the immune system may affect innate responses, 1-week and 4-week-old birds were used.

After defining early host responses in primary infected birds we aimed at defining

correlates of protection after challenge of birds vaccinated with various adjuvants.

Vaccination can provide protection to avian influenza virus infections. However, adjuvants

stimulate the immune response via different mechanisms and may provide protection via

different response patterns. In chapter 7 we aimed at getting a better understanding of the

characteristics of these response patterns and determining correlates of protection.

References

1. Alexander, D.J., 2000. A review of avian influenza in different bird species. Vet. Microbiol. 74, 3-13.

2. Aucouturier, J., Dupuis, L., Ganne, V., 2001. Adjuvants designed for veterinary and human vaccines.

Vaccine 19, 2666-2672.

3. Baigent, S.J., McCauley, J.W., 2003. Influenza type A in humans, mammals and birds: determinants of

virus virulence, host-range and interspecies transmission. Bioessays 25, 657-671.

4. Brown, E.G., 2000. Influenza virus genetics. Biomed. Pharmacother. 54, 196-209.

5. Capua, I., Alexander, D.J., 2006. The challenge of avian influenza to the veterinary community. Avian

Pathol. 35, 189-205.

6. Chen, W., Calvo, P.A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., Basta, S., O'Neill, R., Schickli, J.,

Palese, P., Henklein, P., Bennink, J.R., Yewdell, J.W., 2001. A novel influenza A virus mitochondrial

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Suppl B, 3-9.

Cellular host transcriptional responses to Influenza A

virus in chicken tracheal organ cultures differ from

responses in in vivo infected trachea

Sylvia S. Reemers1, Marian J. Groot Koerkamp2, Frank C. Holstege2, Willem van Eden1,

Lonneke Vervelde1

1Department of Infectious Diseases and Immunology, Faculty Veterinary Medicine, Utrecht University, Yalelaan

1, 3584 CL Utrecht, The Netherlands 2Genomics Laboratory, Department of Physiological Chemistry, Utrecht Medical Centre, Universiteitsweg 100,

3584 CG Utrecht, The Netherlands

Veterinary Immunology and Immunopathology, 2009; 132: 91-100

Chapter 2

Transcriptional host responses to AIV infection in TOC

25

Abstract

In this study a viral infection of a tissue culture model system was compared to an in vivo

infection, which is of importance to gauge the utility of the model system. The aim was to

characterize early immune responses induced by avian influenza virus using tracheal organ

cultures (TOC) as a model system. First, the in vitro system was optimized to ensure that

host transcription responses were only influenced by virus infection and not by differences

in viral load. Upper and lower trachea could both be used in the cultures because the virus

load was the same. Cilia motility was not affected in non-infected TOC and only slightly in

infected TOC at 24 h post inoculation. Gene expression profiles of early immune responses

were analyzed in in vitro infected TOC, and were compared to responses found in in vivo

infected trachea. The gene expression profile in infected TOC suggested up regulation of

innate anti-viral responses that were triggered by attachment, entry and uptake of virus

leading to several signalling cascades including NF-κB regulation. Genes associated with

IFN mediated responses were mainly type I IFN related. Overlapping gene expression

profiles between non-infected and infected TOC suggested that tissue damage during

excision induced wound healing responses that masked early host responses to the virus.

These responses were confirmed by real-time quantitative RT-PCR showing up regulation

of IL-1β and IL-6. Microarray analysis showed that gene expression profiles of infected and

non-infected TOC had a large overlap. This overlap contained many immune-related genes

associated with inflammatory responses, apoptosis and immune system process and

development. Infected TOC and in vivo infected trachea shared few significantly

differentially expressed genes. The gene expression profile of infected TOC contained

fewer genes which were expressed at reduced amplitude of change. Genes that were

common between TOC and trachea were associated with early immune responses likely

triggered by virus attachment and entry. Most of the genes were associated with IFN-

mediated responses, mainly type I IFN related. Our study implicates that although the TOC

model is suitable for culturing of virus and lectin or virus binding studies, it is not suitable

for measuring early immune responses upon viral infection at host transcriptional level.

Introduction

Avian influenza virus (AIV) causes infection of the respiratory tract, triggering a cascade

of both innate and specific immune responses. In the last decade our knowledge on these

host defenses against and pathogenic mechanisms of influenza virus infections has

tremendously increased. It has been demonstrated that early immune responses are critical

in defining the severity and pathological outcome of AIV infection in mice and macaques

(Kash et al., 2006; Kobasa et al., 2007). Yet little is known about the early immune

response and host factors that play a role in the induction phase of the responses to AIV in

Chapter 2

26

the chicken. By using in vitro models, experimental conditions can be highly controlled and

both mechanisms of viral infection and host defenses can be studied.

Numerous respiratory epithelial cell lines have been frequently used to study influenza

virus infection (Chan et al., 2005; Veckman et al., 2006). An alternative for cell lines is the

use of primary cell cultures. Primary respiratory epithelial cell cultures have been

developed for various species including human (Matrosovich et al., 2004), hamster (Newby

et al., 2006), mouse (You et al., 2002), pig (Steimer et al., 2006) and since recently chicken

(Zaffuto et al., 2008). Another alternative has been the use of organ cultures as infection

model, such as lung slice cultures (Booth et al., 2004) and tracheal organ cultures (TOC),

which mimic natural infection events more closely and can also be used under controlled

conditions. To date early immune responses to AIV in the chicken have not been

systematically characterized at global gene transcriptional levels. This report is the first one

exploring the use of organ cultures for illustrating these responses at host transcriptional

level.

TOC are susceptible to respiratory virus infections in vitro and are commonly used to study

host-pathogen interaction (Schmidt et al., 1974), to grow virus, and as diagnostic tool

(Cook et al., 1976). In this study an organ culture model system was compared to an in vivo

infection which is of importance to gauge the utility of the model system. We investigated

early immune responses to AIV infection and explored the value of using the in vitro TOC

model for influenza studies by comparing responses in infected TOC with in vivo AIV

infected trachea using a high-throughput genomics approach.

Materials and methods

Virus

Avian influenza A virus, subtype H9N2, isolate A/Chicken/United Arab Emirates/99 was

produced in eggs according to routine procedures. Virus was kindly provided by Intervet

Schering-Plough Animal Health.

In vitro infection model

Tracheas of 18 day-old Cobb broiler embryos (Lagerwey) were aseptically removed and

transferred to warm culture medium containing DMEM, 20 mM HEPES and penicillin/

streptomycin. Tracheas were cut into 3 mm rings resulting in 6 rings per trachea. For

optimization of the in vitro infection model, rings from 1 trachea were divided into two

groups. Each group consisted of 3 rings derived from either the lower or the upper part of

the trachea. These were transferred to a 12-well plate (3 rings per well; TOC) containing

culture medium. TOC were inoculated with increasing concentrations of virus; 101-105

EID50 H9N2/ml in a total volume of 0.5 ml culture medium per well and incubated at 37°C,


27

5 % CO2. After 1 h 1.5 ml culture medium was added and incubation continued. TOC were

harvested at 24 h post inoculation (p.i.) and stored in RNAlater (Ambion) at -80°C.

For the experimental set up, infected and non-infected TOC were incubated in either 0.5 ml

culture medium or 0.5 ml culture medium containing 105 EID50 H9N2/ml. After 1 h 1.5 ml

culture medium was added and incubation continued. TOC were harvested at 0, 3, 6, 15 and

24 h.p.i. and stored in RNAlater at -80°C.

TOC harvested at 0 h were non-infected controls harvested directly after preparation and

were not cultured or inoculated. Infected and non-infected TOC originated from the same

trachea and were time matched during harvesting, which enabled us to control for

variations in physiology and bird to bird variation thus only detect changes due to virus

infection.

In vivo infection model

One-day-old White Leghorns were housed under SPF conditions and all experiments were

carried out according to protocols approved by the Intervet Animal Welfare Committee.

Chickens were divided into 2 groups over 2 isolators, infected and non-infected, containing

20 animals per group. Fourteen-day-old chickens were inoculated via aerosol spray with 20

ml 107.7 EID50 H9N2 AIV. The control group was inoculated via aerosol spray with 20 ml

saline. Aerosol spray was made with an aerographer and compressor at a pressure of 1.5

Atm, resulting in droplets with an average diameter of 50 μm. Chickens remained in the

aerosol spray in a closed isolator (0.79 m3) for 10 min, after which the isolator was

ventilated as before. At day 1 p.i. chickens were killed (n=5 per time point per group) and

the upper part of the tracheas were isolated and stored in RNAlater at -80°C.

RNA isolation

TOC or trachea, stored in RNAlater, were homogenized (Retsch Mixer Mill 301, Fisher

Scientific) and total RNA was isolated using the RNeasy Mini Kit and DNase treated using

the RNase-free DNase Set following manufacturer’s instructions (Qiagen Benelux B.V).

Purified RNA was eluted in 30 μl RNase-free water and stored at -80°C. All RNA samples

were checked for quantity using a spectrophotometer (Shimadzu) and quality using a 2100

Bioanalyzer (Agilent Technologies). Isolated RNA was used for both real-time quantitative

RT-PCR and microarray analysis.

Real-time quantitative reverse transcription-PCR (qRT-PCR)

cDNA was generated with reverse transcription using iScript cDNA Synthesis Kit (Biorad

Laboratories B.V.).

Real-time qRT-PCR was performed with a MyiQ Single-Color real-time PCR Detection

System (Biorad) using iQ SYBR green supermix (Biorad) and the TaqMan Universal PCR

Chapter 2

28

Master Mix (Applied Biosystems). Primers (Invitrogen-Life Technologies) and probes

(Applied Biosystems) were designed according to previously published sequences (Sijben

et al., 2003; Rothwell et al., 2004; Degen et al., 2006; Eldaghayes et al., 2006).

Amplification and detection of specific GAPDH and H9 haemagglutinin (HA) products

was achieved with SYBR green, 400 nM primers and using the following cycle profile: one

cycle of 95°C for 5 min, 40 cycles of 92°C for 10 s, 55°C for 10 s and 72°C 30 s. For

detection of the cytokines and 28S, product specific probes were used according to the

cycle profile described in Ariaans et al. (2008). Primers were used at 600 nM and probes at

100 nM concentration.

To generate standard curves GAPDH, H9 HA and IL-4 PCR-fragments were cloned and

used to generate log10 dilution series regression lines. RNA from LPS stimulated HD11

cells was used to generate standard curves for IL-10 and RNA from ConA stimulated

splenocytes was used for standard curves of the remaining cytokines and 28S. GAPDH was

used as a reference gene for correction of H9 HA RNA expression and 28S as reference

gene for the cytokines. Corrections for variation in RNA preparation and sampling were

performed according to Eldaghayes et al. (2006). Results are expressed in terms of the

threshold cycle value (Ct) and given as corrected 40-Ct values.

A paired t-test was used to determine the statistical significance between infected and non-

infected TOC samples from the same bird. To determine the statistical significance between

infected and non-infected trachea and between TOC and trachea at 24 h an ANOVA with a

Tukey post-hoc test was used. For both tests a p-value < 0.05 was considered significant.

Microarray analysis

For microarray analysis the Gallus gallus Roslin/ARK CoRe Array Ready Oligo Set V1.0

(Operon Biotechnologies) was used. The array was spotted onto Codelink activated slides

(GE Healthcare) and contains 20,460 oligo probes representing chicken genes, and 3828

control spots, used for QC and normalisation purposes (Van de Peppel et al., 2003). RNA

amplification and labelling was performed according to Roepman et al. (2005). All

hybridizations contained 2.5 µg cRNA per channel on a HS4800Pro hybstation (Tecan

Benelux BVBA).

For determining early responses in TOC to H9N2 infection, infected samples were co-

hybridised with non-infected samples of the same trachea. In order to investigate the gene

expression profile over time in infected TOC, a 0 h non-infected control reference sample

was co-hybridized with infected and non-infected TOC samples at 24 h.p.i. The 0 h non-

infected control reference sample was created by pooling equal amounts of RNA extracted

from 0 h non-infected control TOC samples.


29

For the in vivo experiment, infected and non-infected trachea samples were co-hybridised

with a trachea reference sample. This reference sample consisted of pooled RNA extracted

from tracheas of 4 chickens that were not included in the infection experiment.

Slides were scanned with an G2565AA scanner (Agilent Technologies) at 100% laser

power, 30% PMT. Resulting image files were analyzed using Imagene 8.0 (BioDiscovery,

Inc.). Within slide normalisation was performed with Printtip Loess on mean data without

background subtraction. Groups of replicates were analysed using ANOVA (R version

2.2.1/MAANOVA version 0.98-7, http://www.r-project.org/). In a fixed effect analysis,

sample, array and dye effects were modelled. P-values were determined by a permutation

F2-test, in which residuals were shuffled 5000 times globally. Genes with p < 0.05 after

family wise error correction were considered significantly differentially expressed and were

selected to be included for further analysis. Visualisation and cluster-analysis was

performed using GeneSpring 7.2 (Agilent Technologies). Ensembl Gallus gallus (assembly:

WASHUC2, May 2006, genebuild: Ensembl, Aug 2006, database version: 47.2e) was used

for gene names, description and GO-annotations.

In accordance with proposed MIAME standards primary data are available in the public

domain through Expression Array Manager at

http://www.ebi.ac.uk/arrayexpress/?#ae=main[0] under accession number E-MTAB-12.

Results

In vitro TOC infection model

The TOC infection model had to be optimised to ensure that host transcription responses

were only influenced by virus infection and not by differences in viral load.

To ensure that the inoculation dose would result in consistent viral load of the TOC, H9 HA

RNA levels were measured with real-time qRT-PCR. TOC infected with 103 EID50/ml had

significant differences in H9 mRNA levels between the replicates and between TOC of the

upper and lower part of trachea (data not shown). There was no significant difference in H9

HA RNA levels between TOC of the upper and lower part of trachea for TOC infected with

a dose of 104 and 105 EID50/ml. However, based on standard deviations TOC infected with

105 EID50/ml had a 0.4 Ct smaller difference between the replicates than TOC infected with

104 EID50/ml (data not shown). Based on these results an infection dose of 105 EID50/ml

was used for further experiments and TOC of upper and lower part of trachea were divided

randomly.

In order to determine the kinetics of H9 HA RNA expression in TOC, infected and non-

infected TOC were harvested at 0, 3, 6, 15 and 24 h.p.i. H9 HA RNA was detected from 3

h.p.i. and levels increased over time (Fig. 1A). The differences between replicates were

very small at every time point. Therefore, differences in responses caused by a difference in

viral load will not affect the gene expression within a treatment group.

Chapter 2

30

To ensure that the tracheal rings were viable in non-infected TOC and to investigate if

influenza virus infection affected cilia motility like avian pneumoviruses and influenza

viruses in respiratory epithelium do (Diaz-Rodriguez and Boudreault, 1982; Naylor and

Jones, 1994) we microscopically examined the motility of cilia in both infected and non-

infected TOC at 0, 3, 6, 15 and 24 h.p.i. No difference in cilia motility was detected in non-

infected TOC. In infected TOC at 24 h.p.i., 10-20% of the epithelial cell layer showed no

cilia motility, whereas in non-infected TOC at 24 h of culturing the whole epithelial cell

layer showed cilia activity (data not shown).

Expression of genes relevant for immune responses to H9N2 infection in TOC

In order to elucidate the expression of genes involved in host defenses in H9N2 infected

TOC, infected and non-infected TOC within the same time point were compared using

microarray analysis. The data were consistent and even small differences were measured as

significantly differentially expressed. At 3, 6 and 15 h.p.i. no significantly differentially

expressed genes were found, but at 24 h.p.i. 108 genes were significantly differentially

expressed. Of these genes, 106 genes were up regulated and 2 genes were down regulated

(supplemental Table S1). Based on the GO terms host-pathogen interaction, external

stimulus and immune response, we created an immune-related term that was used for

selection of immune-related genes. Of the 108 genes, 22 were immune-related genes that

were divided into GO terms using GSEA to obtain more details on the biological processes

they were involved in (Table 1). All 22 immune-related genes were up regulated and 8 of

these genes are associated with IFN mediated responses like ISG12-2. STAT4, OASL,

MDA5, LY6E are induced by type I IFNs, while CYSLTR2 is induced by IFN-γ. On the

other hand SOCS1 is able to inhibit IFN-γ expression and USP18 can inhibit signal

transduction pathways triggered by type I IFNs. When looking at the cellular position based

on GO terms of the immune-related genes LY6E, ISG12-2, CYSLTR2, DGKE, CD8A,

STAB1, TIMD4 and TLR5 reside in a membrane, PTK2B, OASL, MDA5 and SOCS1

reside in the cytoplasm and STAT4, USP18 and ZEB1 reside in the nucleus. IL19 and

MASP2 are secreted molecules. There was no significant difference in gene expression

between infected TOC and non-infected TOC at 3, 6 and 15 h.p.i.. This suggested that

tissue excision and culturing caused host responses in non-infected TOC similar to host

responses caused by H9N2 infection in infected TOC. Therefore the gene expression

profiles of infected and non-infected TOC within a 24 h period were determined by

comparing infected or non-infected TOC at 24 h to non-infected control TOC at 0 h. The

comparison of non-infected TOC at 24 h of culturing with non-infected control TOC at 0 h

represented wound healing responses. Responses in infected TOC at 24 h.p.i. compared to


31

Fig

ure

1. A

) R

eal-

tim

e qR

T-P

CR

of H

9 H

A R

NA

leve

ls n

orm

alis

ed to

GA

PDH

ove

r ti

me

in T

OC

infe

cted

in v

itro

wit

h 10

5 EID

50/m

l H9N

2 an

d at

24

h.p.

i. in

trac

hea

of b

irds

spra

y in

ocul

ated

wit

h 10

7.7 E

ID50

/ml H

9N2.

B)

Rea

l-ti

me

qRT

-PC

R o

f m

RN

A le

vels

nor

mal

ised

to 2

8S o

f IL

-1β,

IL

-6 a

nd I

FN-β

ove

r ti

me

in in

fect

ed a

nd n

on-i

nfec

ted

TO

C

and

at 2

4 h

p.i i

n tr

ache

a of

infe

cted

and

non

-inf

ecte

d bi

rds

(* p

< 0

.05)

. Res

ults

are

pre

sent

ed a

s 40

-Ct a

nd e

rror

s ba

rs s

how

SE

M f

or p

oole

d ex

pres

sion

leve

l in

four

TO

C

(fro

m f

our

bird

s) a

nd f

our

trac

heas

per

trea

tmen

t.

Tab

le 1

. Im

mun

e-re

late

d ge

nes

sign

ific

antl

y di

ffer

entia

lly

expr

esse

d (p

< 0

.05)

in H

9N2

infe

cted

com

pare

d to

non

-inf

ecte

d T

OC

at 2

4 h.

p.i.

wit

h as

soci

ated

GO

term

s. A

ll

gene

s w

ere

up r

egul

ated

.

Gen

e n

ame

GO

an

nota

tion

STAT4*

PTK2B

OASL*

MDA5*

LY6E*

ISG12-2*

CYSLTR2*

CCDC18

DGKE

GENE A*1

GENE B2

CD8A

IL19

MASP2

SOCS1*

STAB1

TIAL1

TIMD4

TLR5

TRAF3IP2

USP18*

ZEB1

Mem

bran

e

x

x x

x

x

x

x x

Cyt

opla

sm

x

x x

x

Nuc

leus

x

x

x

Ext

race

llul

ar r

egio

n

x

x

Sign

al tr

ansd

uctio

n x

x

x

x

x

x

x

x

Infe

cted

TO

C

Infe

cted

trac

hea

Infe

cted

TO

C

Infe

cted

trac

hea

0481216

03

615

2424

0481216

03

615

2424

0481216

03

615

2424

6 01216 4

Corrected 40-Ct + SEM

Hou

rs p

.i.

IL-1β

03

61

524

24*

Hou

rs p

.i.

03

61

52

424

Hou

rs p

.i.

03

61

524

24

6 0

12

16 4


6 0

1216 4


IL-6

*

*

IFN

-β

**

0481216

03

615

2424

0481216

03

615

2424

0481216

03

615

2424

6 01216 4


Hou

rs p

.i.

IL-1β

03

61

524

24*

Hou

rs p

.i.

03

61

52

424

Hou

rs p

.i.

03

61

524

24

6 0

12

16 4


6 0

1216 4


IL-6

*

*

IFN

-β

**

No

n-in

fect

ed T

OC

Infe

cted

TO

C

No

n-in

fect

ed tr

ach

ea

Infe

cted

trac

hea

No

n-in

fect

ed T

OC

Infe

cted

TO

C

No

n-in

fect

ed tr

ach

ea

Infe

cted

trac

hea

0510152025

36

1524

24

36

1524

24

Hou

rs p

.i.

25 20 15 10 5 0


0510152025

36

1524

24

36

1524

24

Hou

rs p

.i.

0510152025

36

1524

24

36

1524

24

Hou

rs p

.i.

25 20 15 10 5 0


25 20 15 10 5 0


A.

B.

Chapter 2

32

Prot

ein

bind

ing

x

x

x

x

x

x

Imm

une

resp

onse

x

x

x x

x

Apo

ptos

is

x

x

x

x

Res

pons

e to

vir

us

x x

Def

ense

res

pons

e

x x

T c

ell a

ctiv

atio

n

x

x

Infl

amm

ator

y re

spon

se

x

x

JAK

-ST

AT

cas

cade

x

x

Reg

ulat

ion

of N

F-k

B c

asca

de

x x

Cel

l adh

esio

n

x

Cel

l pro

life

rati

on

x

Lys

osom

e

x

Res

pons

e to

str

ess

x

Ant

igen

pre

sent

atio

n/pr

oces

sing

x

Com

plem

ent a

ctiv

atio

n

x

Def

ense

res

pons

e to

bac

teri

um

x

Cel

l adh

esio

n

x

Cel

l-ce

ll s

igna

llin

g

x

Ubi

quiti

n-de

p. p

rote

in c

atab

olic

pr

oces

s

x

Neg

. reg

. epi

thel

ial

cell

diff

eren

tiat

ion

x

Tra

nspo

rt

x

* G

enes

invo

lved

in in

terf

eron

med

iate

d re

spon

ses;

1 G

EN

E A

: EN

SGA

LG

0000

0006

384;

2 G

EN

E B

: EN

SGA

LG

0000

0007

208


33

non-infected control TOC at 0 h represented host responses to H9N2 infection and wound

healing responses. The overlap between significantly differentially expressed genes in the

gene expression profile of infected TOC in one set and in the gene expression profile of

wound healing in non-infected TOC in the other set represents the overlap in responses

caused by wound healing and virus infection and was depicted in a Venn diagram (Fig.

2A). An overlap of 1268 genes between the profiles was found, containing 84 genes

involved in immune responses, of which 41 genes were up regulated and 43 genes were

down regulated (Fig. 2B). Immune-related genes were divided into GO terms to obtain

more details on the biological processes they were involved in. GO terms that mostly

contained down regulated genes could be divided into three categories containing relating

GO terms. Most genes are associated with response to stress, chemical and biotic stimuli,

defense to pathogens and regulation of NF-κB. The other two categories consisted of GO

terms negative regulation of apoptosis and negative regulation of programmed cell death,

and protein folding and protein binding. Together these genes suggest that cells are not

responding to stimuli, protein production has come to a hold and apoptosis and

programmed cell death are no longer inhibited. Death of the cells can either be induced via

virus infection or tissue damage due to preparation and culturing of TOC. GO terms that

contained more up regulated than down regulated genes could also be divided into

categories containing relating GO terms. Response to wounding and to external stimuli

indicated wound healing responses of the TOC due to tissue damage caused by excision

and culturing of TOC and to virus infection. This resulted in cell death as suggested by up

regulation of genes involved in regulation of apoptosis and programmed cell death, anti-

apoptosis and negative regulation of cellular process. Genes associated with immune

system processes and development were also up regulated suggesting the initiation of an

immune response. This response involved genes that are related to inflammatory responses,

leukocyte migration and differentiation, defense responses, hemapoiesis, hemapoietic or

lymphoid organ development and carbohydrate binding. These suggested cell attraction,

migration and adhesion and phagocytosis of virus or cell debris by MΦs inducing an

inflammatory response. Virus and cell debris were likely degraded in lysosomes or lytic

vacuoles suggested by the up regulation of genes that were part of the GO terms lysosome,

lytic vacuole and vacuole. Besides cell death, the TOC structure was possibly also renewed

as suggested by up regulation of genes associated with cell development, positive

regulation of cell differentiation, organelle part and regulation of developmental processes.

Up regulation of genes involved in protein metabolic process, cellular protein process and

lipid raft was in accordance with this and suggested an active communication in and

between cells. These data showed a large overlap of host responses, particularly early

immune responses, between H9N2 infection and wound healing.

Chapter 2

34

A.

B.

Figure 2. A) Venn diagram of significantly differentially expressed genes (p < 0.05) involved in host responses to

H9N2 infection and in wound healing in TOC on the left and host responses involved in wound healing on the

right (n = 4). The grey area represents the overlapping genes between the expression profiles and indicates the

overlap between responses to H9N2 infection and wound healing responses. The numbers in the circles indicate

the altered genes for each condition. B) Overlap of 93 immune-related genes between responses to H9N2 infection

in infected TOC and wound healing responses in non-infected TOC. Red represents up regulation and green down

regulation in infected (T24-i) and non-infected (T24-ni) TOC at 24 h.p.i. compared to non-infected control TOC at

0 h.

Cytokine mRNA expression in infected and non-infected TOC

In order to confirm the microarray data on the early responses induced by viral entry and

replication, we measured mRNA expression of different cytokines using real-time qRT-

PCR. mRNA levels of infected TOC were compared to non-infected TOC within the same

0 hr non-infected TOC vs

24 hr non-infected TOC

906 2771268


24 hr p.i. infected TOC


24 hr non-infected TOC

906 2771268


24 hr p.i. infected TOC

ni

nini

ni

T24

-ni

T24

-i

T24

-i

T24

-ni


35

time point. In infected TOC IL-6, IL-8, IFN-α and IFN-β were significantly up regulated at

24 h.p.i., but no significant changes were detected in IL-1β, IL-4, IL-10, IL-18 and IFN-γ

levels compared to non-infected TOC (Fig. 1B and data not shown). IL-6, IL-8 and IL-10

mRNA levels were also up regulated from 3 h of culturing in non-infected TOC compared

to non-infected control TOC at 0 h and remained elevated up to 24 h of culturing (Fig. 1B

and data not shown). With microarray analysis IL-6, IL-8, IFN-α and IFN-β were not

significantly differentially expressed in infected TOC compared to non-infected TOC. Real-

time qRT-PCR is more sensitive than microarray analysis and differences in IL-6, IL-8,

IFN-α and IFN-β levels between infected and non-infected TOC measured with real-time

qRT-PCR were small, which explain why these cytokines were not found with microarray

analysis. Moreover, for real-time qRT-PCR a paired T-test was used to determine

significant differences, which rules out biological differences, because infected and non-

infected TOC from the same animal were compared. For microarray analysis, stringent

statistics with multiple testing was used, which does not take into account that infected and

non-infected TOC hybridized together on a slide were from the same animal. Resulting

from this is that small differences were significant for real-time qRT-PCR, but not for

microarray analysis, and are not shown in the gene lists.

Comparison of in vitro and in vivo gene expression after H9N2 infection

To compare responses in in vitro infected TOC with in vivo infected trachea, results should

not be affected by differences in viral load between TOC and trachea. H9 HA RNA levels

of infected trachea at 24 h.p.i. were compared with TOC at 24 h.p.i. (Fig. 1A). There was

no significant difference in H9 HA RNA level between infected TOC and trachea at 24

h.p.i.

Scatterplots were made of the expression ratio of infected samples compared to a reference

sample on one axis and of non-infected samples to a reference sample on the other axis for

both TOC and trachea (Fig. 3A to C). For trachea a broad scatterplot was seen (Fig. 3A),

suggesting a high amplitude of change in infected trachea compared to non-infected

trachea. Whereas for TOC the response is more moderate with a scatterplot close to the

diagonal 1-fold indication line (Fig. 3B and C), suggesting a low amplitude of change

between infected and non-infected TOC. Moreover, the expression profile of infected

trachea involved more significantly differentially expressed genes than that of infected

TOC (Fig. 3A-C white dots), and few genes were shared between both expression profiles.

Responses in infected samples were compared to non-infected samples at 24 h for both

TOC and trachea representing responses to H9N2 infection. A Venn diagram was generated

of significantly differentially expressed genes to elucidate the overlap between the

expression profile of H9N2 infection in TOC in one set and in trachea in the other set (Fig.

3D). A small overlap in response was seen between TOC and trachea consisting of 34

Chapter 2

36

A. B. C.

D.

Figure 3. A to C) Scatterplots of host responses to H9N2 infection in TOC and trachea at 24 h.p.i. (n = 4). The

intensities for gene expression depicted in fold change in infected samples compared to a reference sample on the

y-axis and in non-infected samples compared to a reference sample on the x-axis. The middle diagonal blue line

represents a 1-fold change and the outer two diagonal lines a 2-fold change in infected compared to non-infected

samples. A) A scatterplot of trachea with the significantly differentially expressed genes (p < 0.05) in the trachea

given in white. B) A scatterplot of TOC with the significantly differentially expressed genes (p < 0.05) in the TOC

given in white. C) A scatterplot of TOC with the significantly differentially expressed genes (p < 0.05) in the

trachea given in white. D) Venn diagram of significantly differentially expressed genes (p < 0.05) involved in host

responses to H9N2 infection in TOC and trachea at 24 h.p.i. (n = 4). The grey area represents the overlap between

the expression profile of H9N2 infection of TOC and trachea. The numbers in the circles indicate the altered genes

for each condition.

genes that were all up regulated in both TOC and trachea. Of these 34 genes, 10 were

immune-related which were divided into GO terms to obtain more details of the biological

processes they were involved in (Table 2). Of the 10 immune-related genes, which were all

up regulated in both TOC and trachea, 8 are associated with IFN mediated responses. All

genes are related to type I IFN mediated responses, except for SOCS1 which is able to

inhibit IFN-γ. Genes involved in IFN mediated responses were expressed at a > 6-fold

change in the trachea whereas responses of these genes in TOC were 3 to 6-fold lower

compared to trachea. These 10 immune-related genes were related to small parts of several

core immune responses induced by virus infection. Together they suggested that several

signal transduction cascades were triggered by attachment and entry of virus in the

epithelial cells which likely lead to defense responses via activation of the NF-κB cascade.

TOC Trachea

74 74139

TOC Trachea

74 74139

Expression in infected samples

0.2 0 5

Non-infected

Infe

cte

d

Trachea genes in trachea scatterplot

1010.1

10

1

0.1

Non-infected

Infe

cted

TOC genes in TOC scatterplot

1010.1

10

1

0.1

Non-infected

Infe

cte

d

Trachea genes in TOC scatterplot

1010.1

10

1

0.1


0.2 0 5


0.2 0 5

Non-infected

Infe

cte

d


1010.1

10

1

0.1

Non-infected

Infe

cte

d


1010.1

10

1

0.1

Non-infected

Infe

cted


1010.1

10

1

0.1

Non-infected

Infe

cted


1010.1

10

1

0.1

Non-infected

Infe

cte

d


1010.1

10

1

0.1

Non-infected

Infe

cte

d


1010.1

10

1

0.1


37

Possibly, viral Ags were processed in APC or epithelial cells for Ag presentation on the cell

surface. In TOC, fewer genes were differentially expressed compared with trachea and at

lower amplitude of change.

We also compared mRNA levels of IL-1β, IL-6 and IFN-β in TOC with trachea (Fig. 1B).

These cytokines were significantly up regulated in trachea, whereas in TOC only IL-6 and

IFN-β were significantly up regulated. The mRNA levels of IL-1β and IL-6 were higher in

infected trachea compared to infected TOC, whereas these levels were lower in non-

infected trachea compared to non-infected TOC. Thus differences between IL-1β and IL-6

mRNA levels in non-infected compared to infected samples were larger in trachea than in

TOC, suggesting that wound healing responses in the TOC masked early immune responses

which was not found in trachea.

Table 2. Immune-related genes significantly differentially expressed (p < 0.05) in H9N2 infected compared to

non-infected samples at 24 h.p.i. in TOC and trachea with associated GO terms. All genes were up regulated.

Gene name

TR

AF

3IP

2

TIM

D4

LY

6E*

ST

AT

4*

USP

18*

MD

A5*

SO

CS1

*

IS

G12

-2*

OA

SL

*

GE

NE

C*1

TOC 2.2 1.3 2.0 1.7 2.0 1.8 1.7 4.4 4.6 7.6

Fol

d

chan

gea

Trachea 1.6 1.6 6.1 7.4 7.7 8.5 11.6 29.2 29.3 50.7

Membrane x x x

Cytoplasm x x x

Nucleus x x

Signal transduction x x x x

Protein binding x x x

Immune response x x

Response to virus x x

JAK-STAT cascade x x

Apoptosis x

Ubiquitin-dep. protein catabolic process x

T cell activation x

GO

an

nota

tion

Regulation of NF-κB cascade x

a Fold changes represent the difference in expression levels in infected TOC and trachea compared to non-infected

samples; * Genes involved in interferon mediated responses; 1 GENE C: ENSGALG00000006384

Discussion

Early immune responses are important in defining the pathological outcome of AIV

infection and have been demonstrated to differentiate the highly pathogenic and low

pathogenic AIV H1N1 infection in mice (Perrone et al., 2008). Yet in chicken, little is

known about these responses against AIV. In this study we investigated early immune

Chapter 2

38

responses upon viral infection at host transcriptional level in the chicken and explored the

use of TOC as an in vitro model system. The virus strain H9N2 is important as it has

regularly been detected in land-based poultry and it is able to make cross species infection

(Butt et al., 2005). TOC were susceptible to H9N2 infection in vitro and H9 HA RNA

levels increased over time, indicating not only viral entry but also replication. Several

respiratory viruses such as influenza, infectious bronchitis virus and turkey rhinotracheitis

virus (or avian pneumovirus) are known to cause ciliostasis in respiratory epithelium (Diaz-

Rodriguez and Boudreault, 1982; Naylor and Jones, 1994; Cavanagh et al., 2007).

Macroscopical examination revealed that ciliostasis was detected in 10-20% of the

epithelium of infected TOC at 24 h.p.i. Pathogenesis of influenza virus infection has been

investigated in equine and duck TOC (Kocan et al., 1977; O'Niell et al., 1984), where

scattered loss of cilia and disruption of the epithelial cell layer was reported. Our results in

chickens are consistent with these studies.

Immune responses to influenza infection in the respiratory tract have been studied

extensively in vitro in human and mouse. Elevated expression levels of inflammatory

cytokines and chemokines such as IL-1β, IL-6, IL-8, IL-18, IFN-α and -β have been

reported as early as 6 h.p.i. depending on the strain of influenza used (Julkunen et al., 2001;

Chan et al., 2005). In our in vitro study also elevated expression levels of IL-6, IL-8, IFN-α

and IFN-β were found in infected TOC at 24 h.p.i., suggesting the initiation of an acute host

response by induction of an increased MHC class I expression and anti-viral activity by

IFNs and inflammatory effects of IL-6 and IL-8 mobilizing and activating lymphocytes.

Genomics tools are more and more used to study host-pathogen interactions (Kash et al.,

2006). Our study was focused on gene expression profiles of early responses to low

pathogenic AIV using the TOC model system. Responses in infected and non-infected TOC

at 24 h showed very similar gene expression profiles. In both cultures, genes associated

with responses to external stimuli and stress, regulation of NF-κB, protein folding and

processing, apoptosis, and immune system process and development were significantly

differentially expressed, suggesting the initiation of an immune response. Genes related to

inflammatory responses found in the gene profile of non-infected TOC corresponded to

those found using real-time qRT-PCR in non-infected TOC from 3 h of culturing. IL-6, IL-

8 and IL-10 expression levels were up regulated in non-infected TOC. In mammals, IL-6,

IL-8 and IL-10 are known to be expressed in damaged tissue (Yamamura et al., 1992). As

expected, excision of the trachea into rings caused damage and resulted in up regulation of

IL-6, IL-8 and IL-10. The preparation of TOC likely induced strong inflammatory

responses, largely masking an early immune response triggered by AIV infection. Culturing

of TOC several days before infection will most likely not improve the inflammatory

responses induced by preparation of the TOC. Cilia activity slowly changed over a period

of 5 days with a maximum loss of 20%, but histopathological changes in the TOC were


39

found (data not shown). Still several genes associated with innate anti-viral responses were

significantly differentially up regulated in the gene expression profile of infected compared

to non-infected TOC at 24 h. Together these genes related to a virus induced immune

response in which attachment and entry of virus in the epithelial cells triggered several

signal transduction cascades leading to defense responses via activation of the NF-κB

cascade, inflammatory responses and complement activation via the lectin pathway. Via

these responses chemokines and IFNs can be secreted, likely triggering expression of IFN-

induced genes and attracting APC and T cells, residing in the lamina propria of the TOC, to

the place of infection. Virus might have been taken up by APC and transported to

lysosomes where the virus is degraded or virus is processed in the endosomes for Ag

presentation on the cell surface.

To validate whether early immune responses to AIV in in vitro infected TOC are similar to

in vivo responses at host transcriptional level, a comparison was made with in vivo H9N2

infected trachea. Higher levels of IL-1β and IL-6 were found in trachea, which could be

caused by an influx of MΦs, that cannot occur in TOC. Differences in fold change and the

small overlap in genes found between TOC and trachea are most likely caused by masking

of the immune responses in TOC due to wound healing responses, although some genes are

involved in both wound healing and immune responses to pathogens. The number of genes

that were in common in host response to H9N2 between TOC and trachea was very low.

These genes up regulated and related to innate anti-viral responses, likely triggered by

attachment and entry of virus leading to several signal transduction cascades. Most of the

common genes are associated with IFN mediated responses. These were mainly type I IFN

related corresponding with the up regulation in both TOC and trachea of IFN-α and IFN-β.

These findings suggest that these common immune responses are more involved in host

responses to AIV infection than in wound healing responses and likely play a dominant

role.

There is a clear need for detailed knowledge of the mechanisms of early immune responses

in the induction phase of AIV infection to enable modulating of the outcome. Studies

conducted in in vitro respiratory models can play an important role in elucidating host-

pathogen interactions. However, tissue culture model systems have to be compared to in

vivo infections to gauge the utility of the model system. This study showed that host

responses in TOC are masked by wound healing responses and thus influence any study

into host transcriptional responses. Although TOC and trachea shared similar immune-

related genes, which may play a dominant role in immune responses to influenza virus

infection, the number of shared genes significantly differentially expressed in both TOC

and trachea was very low. Therefore the results obtained from organ cultures or primary

cells for investigating any host responses at transcriptional level should be interpreted with

Chapter 2

40

caution. This makes TOC a good in vitro model for culturing of virus, and lectin or virus

binding studies, but not for studying gene expression profiles of host responses.

Acknowledgements

We thank Daphne van Haarlem from the Faculty of Veterinary Medicine for technical

assistance and Winfried Degen and Virgil Schijns from Intervet Schering-Plough Animal

Health for their collaboration on the in vivo experiment. From the Genomics Laboratory we

would like to thank Diane Bouwmeester, Dik van Leenen and Tony Miles for technical

assistance and Erik Sluiters and Patrick Kemmeren for bioinformatical assistance. This

work was supported by a BSIK VIRGO consortium grant (Grant no. 03012), The

Netherlands.

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L., 2008. The role of phagocytic cells in enhanced susceptibility of broilers to colibacillosis after

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Supplementary data

Table S1. Genes significantly differentially expressed (p < 0.05) in H9N2 infected compared to non-infected TOC

at 24 h.p.i. with associated Ensembl ID.

Description Ensembl ID Fold

changea

Fibromodulin precursor ENSGALG00000003546 1.26

Stabilin-1 precursor ENSGALG00000001535 1.27

TIA1 cytotoxic granule-associated RNA binding protein-like 1 ENSGALG00000009427 1.27

Matrilin-3 precursor ENSGALG00000016478 1.27

T-cell immunoglobulin and mucin domain containing 4 ENSGALG00000003876 1.28

Hypothetical protein (Uniprot/SPTREMBL;Acc:Q5ZJE8) ENSGALG00000008104 1.28

Protein DmX-like 2 ENSGALG00000004805 1.28

Laforin ENSGALG00000012275 1.29

Formimidoyltransferase-cyclodeaminase ENSGALG00000006131 1.29

Hypothetical protein (Uniprot/SPTREMBL;Acc:Q5ZLQ2) ENSGALG00000006378 1.30

Collagen alpha-1(VI) chain precursor ENSGALG00000005974 1.30

Hypothetical protein LOC419466 ENSGALG00000003774 1.30

Sodium channel protein type 3 subunit alpha ENSGALG00000011040 1.31

Probable urocanate hydratase ENSGALG00000006306 1.33

Teneurin-1 ENSGALG00000008442 1.33

Histone-lysine N-methyltransferase, H3 lysine-36 and H4 lysine-20 specific ENSGALG00000002971 1.33

Interleukin-19 precursor ENSGALG00000000911 1.33

Rho-associated coiled coil forming kinase 2 ENSGALG00000016451 1.35

Hyaluronan synthase 3 ENSGALG00000000630 1.38

Protein NOV precursor ENSGALG00000016112 1.39

Hypothetical protein (Uniprot/SPTREMBL;Acc:Q5ZI42) ENSGALG00000008517 1.44

CD8 alpha chain ENSGALG00000015816 1.47

Signal transducer and activator of transcription 4 ENSGALG00000007651 1.72

Cartilage matrix protein precursor ENSGALG00000000548 1.48

Protein PTHB1 ENSGALG00000012188 1.50

High-affinity cAMP-specific and IBMX-insensitive 3',5'-cyclic phosphodiesterase 8B ENSGALG00000004339 1.52

Septin 5 ENSGALG00000001688 1.53

Toll-like receptor 5 ENSGALG00000009392 1.53

Serine/threonine kinase NLK ENSGALG00000005699 1.57

Sn1-specific diacylglycerol lipase alpha ENSGALG00000013306 1.58


43

Similar to egg envelope component ZPAX ENSGALG00000016465 1.58

Diacylglycerol kinase epsilon ENSGALG00000003131 1.60

Leucine-rich repeat and fibronectin type-III domain-containing protein 5 precursor ENSGALG00000012505 1.60

Mannan-binding lectin serine protease 2 ENSGALG00000003016 1.61

Suppressor of cytokine signaling 1 ENSGALG00000007158 1.66

Glycerophosphodiester phosphodiesterase domain containing 5 ENSGALG00000017331 1.66

Ceramide glucosyltransferase ENSGALG00000015682 1.68

Vacuolar protein sorting-associated protein 13B ENSGALG00000016029 1.68

WD repeat protein 85 ENSGALG00000008750 1.70

Coiled-coil domain-containing protein 18 ENSGALG00000005889 1.71

Interferon-induced helicase C domain-containing protein 1 ENSGALG00000011089 1.80

Beta-1,3-galactosyltransferase 2 ENSGALG00000002477 1.83

Uncharacterized protein C3orf17 ENSGALG00000015182 1.86

Gallus gallus similar to AT rich interactive domain 4B (LOC425840) ENSGALG00000014327 1.91

Voltage-dependent N-type calcium channel subunit alpha-1B ENSGALG00000008456 1.91

Hypothetical protein (Uniprot/SPTREMBL;Acc:Q5F463) ENSGALG00000003477 1.94

Epithelial stromal interaction 1 isoform 2 ENSGALG00000016964 1.94

Regulator of G-protein signaling 22 ENSGALG00000016030 1.97

Lymphocyte antigen 6E precursor ENSGALG00000016152 2.03

Radical S-adenosyl methionine domain containing 2 ENSGALG00000016400 2.17

Adapter protein CIKS ENSGALG00000015026 2.17

Uncharacterized protein C12orf34 ENSGALG00000005149 2.17

Probable G-protein coupled receptor 20 ENSGALG00000016163 2.19

Cysteinyl leukotriene receptor 2 ENSGALG00000017001 2.34

Protein tyrosine kinase 2 beta ENSGALG00000016564 2.37

Hypothetical protein (Uniprot/SPTREMBL;Acc:Q5ZKQ9) ENSGALG00000006457 2.38

Zinc finger E-box-binding homeobox 1 ENSGALG00000007260 2.43

Cytoplasmic polyadenylation element-binding protein 2 ENSGALG00000014516 2.49

Thymidylate kinase family LPS-inducible member ENSGALG00000016398 2.66

Choline/ethanolaminephosphotransferase 1 ENSGALG00000000142 3.46

Phosphorylated CTD-interacting factor 1 ENSGALG00000023547 3.70

Zinc finger and BTB domain-containing protein 24 ENSGALG00000015231 3.96

Putative ISG12-2 protein ENSGALG00000013575 4.36

Titin sequence3 ENSGALG00000009089 4.54

Gallus gallus 2'-5'-oligoadenylate synthetase-like ENSGALG00000013723 4.63

ENSGALESTG00000007682 0.78

ENSGALG00000001074 1.27

ENSGALG00000004617 1.29

ENSGALG00000002574 1.30

ENSGALG00000004028 1.35

ENSGALESTG00000026335 1.35

ENSGALG00000009479 1.37

ENSGALG00000021226 1.49

ENSGALG00000006018 1.50

Chapter 2

44

ENSGALG00000020220 1.50

ENSGALG00000023709 1.54

ENSGALG00000011784 1.56

ENSGALG00000006396 1.60

ENSGALG00000023644 1.63

ENSGALG00000001744 1.71

ENSGALG00000005567 1.91

ENSGALG00000013057 1.97

ENSGALG00000021863 2.04

ENSGALG00000020219 2.33

ENSGALG00000002902 2.43

ENSGALG00000017206 2.43

ENSGALG00000007208 3.11

ENSGALG00000002806 4.42

ENSGALESTG00000009290 5.27

ENSGALG00000006384 7.59

ChEST832f13 0.71

ChEST583m7 1.26

ChEST300f17 1.36

ChEST834i23 1.41

ENSGALT00000008995.1 1.42

ENSGALT00000009361.1 1.44

ChEST267l24 1.55

ChEST721o9 1.68

Contig_28_forward 1.71

ChEST432a1 1.71

ChEST1017a23 1.73

ChEST622g7 1.75

ENSGALT00000019233.1 1.95

ENSGALT00000022021.1 2.19

ENSGALT00000025401.1 2.54

ChEST822p4 2.72

Contig_12_reverse 4.43

ENSGALT00000023527.1 5.52

a Fold changes represent the difference in expression levels in infected TOC and trachea.

Chicken Lung Lectin is a functional C-type lectin and

inhibits haemagglutination by Influenza A Virus

Astrid Hogenkamp1, Sylvia S.N. Reemers1, Najiha Isohadouten1, Roland A.P. Romijn2,

Wieger Hemrika2, Mitchell R. White3, Boris Tefsen4, Lonneke Vervelde1, Martin van Eijk1,

Edwin J.A. Veldhuizen1, Henk P. Haagsman1

1 Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, P.O.

Box 80.175, 3508 TD Utrecht, The Netherlands 2ABC Expression Center, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands 3Boston University School of Medicine, Department of Medicine, Boston MA, USA 4 Department of Molecular Cell Biology and Immunology, Vrije Universiteit Medical Center, 1081 BT

Amsterdam, The Netherlands

Adapted from Veterinary Microbiology, 2009; 130: 37-46

Chapter 3

Structure and functionality of cLL

47

Abstract

Many proteins of the calcium-dependent (C-type) lectin family have been shown to play an

important role in innate immunity. They can bind to a broad range of carbohydrates, which

enables them to interact with ligands present on the surface of micro-organisms. We

previously reported the finding of a new putative chicken lectin, which was predominantly

localized to the respiratory tract, and thus termed chicken lung lectin (cLL). In order to

investigate the biochemical and biophysical properties of cLL, the recombinant protein was

expressed, affinity purified and characterized. Recombinant cLL was expressed as four

differently sized peptides, which is most likely due to post-translational modification.

Crosslinking of the protein led to the formation of two high-molecular weight products,

indicating that cLL forms trimeric and possibly even multimeric subunits. cLL was shown

to have lectin activity, preferentially binding to a-mannose in a calcium-dependent manner.

Furthermore, cLL was shown to inhibit the haemagglutination-activity of human isolates of

Influenza A Virus (IAV), subtype H3N2 and H1N1 and potentially has an anti-viral activity

against avian IAV. These result show that cLL is a true C-type lectin with a very distinct

sugar specificity, and that this chicken lectin could play an important role in innate

immunity.

Introduction

The innate immune system provides a first line defense against potential pathogens,

bridging the interval between exposure to the pathogen and the specific response of the

adaptive immune system. The effectiveness of the innate immune system highly depends on

the recognition of pathogens. For this purpose, the innate immune system relies on pattern

recognition molecules which are capable of binding to regular patterns of carbohydrates

present on the surface of pathogens. Within this group of pattern recognition molecules, the

calcium dependent (C-type) lectins represent a family of proteins which are found

throughout the animal kingdom (Day, 1994). These proteins share a structural homology in

their carbohydrate recognition domains (CRD) but differ with respect to their carbohydrate

specificity. We recently reported the discovery of a chicken lectin which was designated

chicken Lung Lectin (cLL) due to its predominant expression in the chicken respiratory

system (Hogenkamp et al., 2006). Based on sequence homology, cLL was identified as a C-

type lectin, and although it could not be classified as a collectin due to its lack of a collagen

domain, the sequence of its CRD was found to be most homologous to that of the collectin

Surfactant Protein A (SP-A) (39% - 45% similarity, depending on the species, (Hogenkamp

et al., 2006)). To our knowledge, no other C-type lectins with more structural similarities to

cLL have been identified to date. Collectins and several other C-type lectins have been

identified as important molecules in innate immunity. For example, SP-A plays an

Chapter 3

48

important role in innate defense against invading bacterial pathogens in the lung

(Hogenkamp et al., 2007; Wright et al., 2001), as well as viruses, including Influenza A

Virus (IAV). Other lectins such as RegIIIγ (Narushima et al., 1997), and its human

homologue HIP/PAP (Lasserre et al., 1992) that directly affect the gut flora composition,

and have been shown to have direct antimicrobial properties (Cash et al., 2006). Tetranectin

is a secreted C-type lectin (Berglund et al., 1992; Sorensen et al., 1995) which has also been

found in chicken (unpublished data, GenBank accession no. AJ277116). Its function is not

quite clear yet, but it has been suggested to play a role in cellular immunity (Stoevring et

al., 2005). In this study, we expressed recombinant cLL to investigate the structural and

functional characteristics of this protein. The carbohydrate-specificity of cLL was tested,

and the putative protective role of cLL was studied by investigating haemagglutination-

inhibition and neutralization of viral infectivity of various IAV strains.

Materials and Methods

RNA extraction and cDNA synthesis

Total cellular RNA from lung tissue from healthy female Ross 308 broiler chicken was

extracted using TRIzol (Invitrogen) and Magnalyser Green Beads (Roche Diagnostics

GmbH). The cDNAs used as templates in PCR were synthesized using 1 µg DNase I

treated RNA with M-MLV-RT and 500 µg/ml oligo dT12-18 primers (Invitrogen) in a 20

µl reaction volume with incubation at 37°C for 50 min.

Polymerase chain reaction and amplified DNA fragment isolation

To amplify cDNA of the mature peptide sequence of cLL, a PCR reaction was performed

using FastStart DNA Taq-polymerase (Roche Diagnostics Gmbh), cLL-specific forward

primer 5'- GGATCCAAACCAACACAGATTTTTCC-3', and cLL-specific reverse primer

5'- GCGGCCGCAAACTGGCAGACAACAAG-3' (Hogenkamp et al., 2006). The forward

primer contained the BamHI restriction site sequence, and cLL reverse primer contained the

NotI restriction site sequence. Amplification comprised of initial denaturation at 95°C for 2

min, followed by 40 cycles consisting of 95°C for 30s, 49°C for 30s, 72°C for 1 min and a

final extension at 72°C for 7 min. PCR-products were analyzed by agarose gel

electrophoresis and purified with a QIAEX agarose gel extraction kit (Qiagen).

Cloning and sequencing of the PCR products

The purified PCR fragments were ligated into a pCR 4-TOPO plasmid vector (Invitrogen).

Ligated plasmids were transfected into TOP10 Escherichia coli cells by heatshock. Clones

were selected by growth on Luria-Bertani broth (LB)-plates containing 100 µg/ml

kanamycin. Positive clones were screened with PCR for correct product size and

sequenced. Sequence reactions were performed using an ABI PRISM BigDye Terminator


49

v3.0 Ready Reaction Cycle Sequencing kit (Applied Biosystems). All reactions were

carried out in both directions using the T7 and T3 primer sites and separated on an ABI

PRISM 3100 fluorescent DNA sequencer (Applied Biosystems). cLL-inserts were isolated

from positive clones using BamHI and NotI restriction sites and ligated into a pABC-

cystatin-hisN vector with a cystatin signal peptide and an in-frame N-terminal His-tag.

Transfection

HEK293-EBNA cells (ATCC CRL10852) were grown in 90% Freestyle (Gibco) and 10%

Ca2+-free Dulbecco's modified Eagle's medium (DMEM) (Gibco), containing 5% FCS

(Invitrogen), 1% pluronic (Sigma-Aldrich), 10 mM HEPES, 4 mM L-glutamine, 200 U/l

penicillin G, 0.1 mg/l streptomycin and 50 μg/ml geneticin. Cells were maintained in

exponential growth using Erlenmeyer flasks at 120 rpm on an orbital shaker mounted in a

Reach-In CO2 incubator (Clean Air Techniek). HEK293-EBNA cells were transfected

using DNA-PEI (Polysciences) according to Durocher et al. (2002). Briefly, 24 hrs before

transfection, cells were seeded at 2.5 x 105/ml in medium without FCS. The next day DNA-

PEI complexes were formed by a 10 min incubation of plasmid DNA at 20 μg/ml with PEI

at 40 μg/ml in Optimem (Gibco), 25 μl of this mixture was used for each ml of cell culture

to be transfected. Small scale transfections (4 ml) were performed in 6 well plates, large

scale transfections were performed in a Bioreactor (New Brunswich Scientific).

Purification of cLL

The supernatant of transfected HEK293-EBNA cells was collected after 6 days and

concentrated to a final volume of approximately 250 ml using a hollow fiber column

(molecular-mass cut-off 10 kDa, Amersham Biosciences). Purification of cLL was

performed by affinity chromatography (adapted from Van Eijk et al., 2002). Briefly, 1 ml

(bed-volume) mannan-sepharose (Sigma), equilibrated in 50 mM Tris-HCl, 5 mM CaCl2,

and 0.05% (vol/vol) Tween-80, pH 7.4 was added to the supernatant and CaCl2 was added

to a final concentration of 5 mM. The mixture was stirred overnight at 4°C. Sepharose

beads were washed with 25 ml washing buffer (50 mM Tris-HCl, 5 mM CaCl2, 500 mM

NaCl, 0.05% Tween-80, pH 7.4). This washing procedure was repeated with 25 ml washing

buffer without Tween-80. cLL was eluted with 50 mM Tris-HCl, 5 mM EDTA, pH 7.4.

The eluted protein was concentrated using Amicon Ultra centrifugal filter units with a

molecular weight cutoff of 10 kDa (Millipore), after which the buffer was changed to 5 mM

Tris-HCl, 150 mM NaCl, pH 7.4 by repetitive washing. Purified cLL was stored in aliquots

at -20°C.

Chapter 3

50

Electrophoresis and Western Blot Analysis

Proteins (0.1-1 µg/lane) were analyzed by SDS-PAGE as described by Laemmli (Laemmli,

1970) using 10% polyacrylamide gels. Protein bands were visualized by Coomassie

staining. For immunoblot analysis, proteins (0.1-1 µg/lane) were transferred

electrophoretically from the gels onto nitrocellulose membrane. Immunostaining was

performed using mouse anti-His6 monoclonal antibody (Roche Diagnostics Gmbh).

Primary antibodies were detected by peroxidase-conjugated goat anti-rabbit

immunoglobulin G (IgG) (Sigma).

Enzymatic deglycosylation of cLL

N-deglycosylation treatment of cLL was performed using N-glycanase PNGase F

(ProZyme, Inc.) according to the manufacturer's instructions. Briefly, 45 µl cLL (230

µg/ml) was mixed with a 10 µl incubation buffer (0.25 M sodium phosphate, pH 7.0) 2.5 µl

denaturation solution (2% SDS and 1 M β-mercaptoethanol). The sample was heated to

100°C for 5 min, after which the sample was cooled and 2.5 µl detergent solution (15%

NP-40) was added. 2 µl of N-glycanase (5 U/ml) was added, and after 16 hr of incubation

at 37°C, samples were concentrated using Microcon centrifugal filter units (MWCO 10

kDa) (Millipore). Samples were immediately processed for further analysis. O-

deglycosylation treatment was performed using 1 µl 0.5 mU/µl O-Glycosidase (Roche

Diagnostics Gmbh) according to the protocol used for N-glycanase-PNG-ase. However, for

O-glycosidase treatment the cLL storage buffer was changed to phosphate buffer (pH 7.0)

using Microcon centrifugal filter units (MWCO 10 kDa) (Millipore). Deglycosylation by

both N-glycanase-PNG-ase and O-glycosidase was carried out similarly to O-

deglycosylation, but in this case both enzymes were added simultaneously.

DIG-Glycan detection

To assess possible glycosylation of cLL and enzymatically treated cLL, the DIG-Glycan

detection kit (Roche Diagnostics Gmbh) was used according to the manufacturer's

instructions. Briefly, samples containing cLL were loaded onto gel and subsequently

transferred from the gel onto nitrocellulose membrane.

The presence of glycoconjugates was assessed by labeling oxidized sugars with

DIG-0-3-succinyl-ε-aminocaproic acid hydrazide, which was subsequently detected using

anti-digoxigenin-AP and staining with NBT/X-phosphate.

Bis (Sulfosuccinimidyl) suberate (BS)-crosslinking of cLL

Crosslinking of cLL using Bis (Sulfosuccinimidyl) suberate (BS3) (Pierce) was performed

according to the manufacturer's instructions. Briefly, the cLL storage buffer was changed to

10 mM HEPES buffer, pH 7.5 containing 3 mM EDTA using Microcon centrifugal filter


51

units (MWCO 10 kDa) (Millipore). Final concentration of cLL was approximately 170

µg/ml. BS3 (50 mM) was added to the protein solution and incubated at room temperature

for 30 min. The reaction was stopped by adding SDS-PAGE sample buffer to the mixture,

which was subsequently heated to 100°C and loaded onto the gel for further analysis.

MALDI TOF-TOF

After protein separation on 12% SDS-PAGE and fixation in 50% methanol and 7% acetic

acid, cLL was visualized using GelCode Blue Stain reagent (Pierce). The four visible bands

were cut from the gel individually and subjected to in-gel tryptic digestion as previously

described (Van Balkom et al., 2005). Subsequently, these bands were identified by matrix-

assisted laser desorbtion/ionization (MALDI) TOF-TOF analysis. The samples, dissolved

in 0.1% acetic acid, were concentrated using μC18-ZipTips (Millipore) and eluted directly

on the target plates in 1 μl of a saturated solution of R-cyano-4-hydroxycinnamic acid in

50% (v/v) acetonitrile. Data were acquired on a MALDI TOF-TOF instrument (Applied

Biosystems 4700 Proteomics Analyzer) in positive reflectron mode at a laser intensity of

3800 and a bin time of 0.5 ns.

Carbohydrate specificity

Polyacrylamide (PAA)-coupled glycoconjugates (~20% substitution; Lectinity) were coated

(5 µg/ml) in 0.2 M sodium cacodylate buffer (pH 9.2) on maxisorb plates (NUNC)

overnight at 4°C. Plates were blocked with 1% ELISA-grade BSA (Fraction V, fatty acid

free; Calbiochem) in TSM (20 mM Tris-HCL pH 7.4, 150 mM NaCl, 2 mM CaCl2, 2 mM

MgCl2) and cLL was added (10 µg/ml) for 2-3 hrs at 37°C in the presence or absence of 10

mM EDTA. After washing with TSM containing 0.1% Tween-20, 1 μg/ml anti-His6

antibody (Roche Diagnostics Gmbh) was added for 1 hr at room temperature. Binding was

detected using a peroxidase-labeled anti-mouse IgG antibody (Jackson) and measured in a

Benchmark Microplate Reader using the Microplate Manager software (both from Biorad).

Virus inhibition assays

A. Testing the activity of cLL against avian IAV

Tracheal organ cultures (TOC)

Tracheas of 18 day-old Cobb broiler embryos were aseptically removed and placed

immediately in warm culture medium containing DMEM (Gibco) containing 2.38 g HEPES

(Sigma) and 50,000 units penicillin/50,000 μg streptomycin (Gibco) per 500 ml. They were

cut into 3 mm rings resulting in 6 TOC per trachea. TOC were transferred to a 24-well plate

containing culture medium.

Chapter 3

52

Virus and immune serum

Avian IAV, subtype H9N2, isolate A/Chicken/United Arab Emirates/99 was produced in

eggs according to routine procedures. Chicken immune serum was obtained from SPF

chickens 5 weeks after vaccination with H9N2. Virus and immune serum was kindly

provided by Intervet Schering-Plough Animal Health.

Experimental design

TOC were divided randomly and transferred to a 24-well plate (2 TOC per well) containing

culture medium. Culture medium was modified to infection medium by adding 3.5 mM

CaCl2 (Merck). Pre-mixes of virus and lectin were made by combining virus (2*105 EID50

H9N2 / ml infection medium) with chicken lung lectin (cLL; 25 μg/ml), virus with chicken

immune serum, and virus with infection medium as negative control. All tubes were

vortexed briefly and incubated for 1 hour at 37°C, 5 % CO2. Subsequently, culture medium

was removed and 0.25 ml per well premix was added to TOC. Plates were incubated for 1

hour at 37°C, 5 % CO2 after which 0.5 ml infection medium was added. TOC were

harvested at 4 hours post inoculation and stored in RNAlater (Ambion) at -80°C. Test were

run in quintuplicate.

In the first experiment cLL batch 1 (1.1) was used, in the second experiment cLL batch 2

(2.2), in the third experiment cLL batch 3 (3.3) and batch 1 (3.1) and 2 (3.2) were retested.

Real-Time quantitative reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated using the RNeasy Mini Kit (Qiagen) following manufacturer’s

instructions. Purified RNA was eluted in 30 μl RNase-free water and stored at -80°C.

Reverse transcription was performed using iScript cDNA Synthesis Kit (Biorad

Laboratories B.V.).

Quantitative RT-PCR was performed with a MyiQ Single-Color Real-Time PCR Detection

System (Biorad) using iQ SYBR green supermix (Biorad). Primers (Invitrogen) for

detection of specific GAPDH and H9 haemagglutinin (HA) products were designed

according to previously published sequences (Degen et al., 2006) and used at 400 nM

concentration. Amplification and detection of specific products was achieved with the

following cycle profile: one cycle of 95°C for 5 min, 40 cycles of 92°C for 10 sec, 55°C for

10 sec and 72°C 30 sec. To generate standard curves GAPDH and H9 PCR-fragments were

cloned and used to generate log10 dilution series regression lines. GAPDH was used as a

reference gene for correction of H9 HA RNA expression and 28S as reference gene for the

cytokines. Corrections for variation in RNA preparation and sampling were performed

according to Eldaghayes et al. (2006). Results are expressed in terms of the threshold cycle

value (Ct) and given as corrected 40-Ct values.


53

B. Testing the activity of cLL against human isolates of IAV

Virus preparations

IAV was grown in the chorioallantoic fluid of 10-day-old chicken eggs and purified on a

discontinuous sucrose gradient as described previously (Hartshorn et al., 1988). Virus

stocks were dialyzed against PBS, aliquoted, and stored at -70°C. A/Phillipines/82 (H3N2)

(Phil), and its bovine serum β inhibitor-resistant variant Phil/BS, were provided by Dr. E.

M. Anders (Department of Microbiology, University of Melbourne, Melbourne, Australia).

A/Puerto Rico/8/34 (H1N1) (PR-8), which lacks the high-mannose glycans on the

haemagglutinin molecule, was provided by Dr. J. Abramson (Department of Pediatrics,

Bowman Gray School of Medicine, Wake Forest University, Winston Salem, NC).

Neutralization of infectivity

Madin-Darby canine kidney (MDCK) cell monolayers were prepared in 96-well plates and

grown to confluence. These layers were then infected with a PBS++ (PBS with 1 mM

calcium and 0.5 mM magnesium; Gibco) -diluted IAV preparation (Phil strain) which was

preincubated for 30 min at 37°C in the presence or absence (control) of increasing amounts

of cLL. After exposure of the MDCK cells to the IAV or IAV/cLL mixture for 30 min at

37°C, the cells were washed three times in serum-free DMEM (Gibco) containing 1% (w/v)

penicillin-streptomycin and subsequently incubated for 7 hrs at 37°C. Next, the monolayers

were washed, fixed, and FITC-labeled for IAV nucleoprotein as described previously

(Hartshorn et al., 2002), after which fluorescent foci were counted.

Haemagglutination-inhibition assay

Haemagglutination (HAA)-inhibition was measured by serially diluting collectin

preparations in round-bottom 96-well plates (Serocluster U-Vinyl plates; Costar) using

PBS++ as diluent (25 µl per well). After adding 25 µl of IAV solution, giving a final

concentration of 40 HAA U/ml or 4 HAA U/well, the IAV/cLL mixture was preincubated

for 15 min, followed by the addition of 50 µl of human erythrocyte suspension in PBS++.

The entire procedure was performed at room temperature. The minimal concentration of

cLL, required to fully inhibit the HAA caused by the virus, was determined by reading the

plates after 2 hrs. HAA was detected as the formation of a pellet of erythrocytes.

Statistical analysis

Statistical analysis was performed using SPSS version 12.0.1 for Windows. Analysis of

mean values between groups was carried out using Levene's Test for Equality of Variances.

When equality of variance was assumed, mean values between groups were compared with

an independent t-test, in which p<0.05 was considered to indicate statistical significance.

Chapter 3

54

<---------------------- Signal peptide ---------------------><-1-><-- HIS-tag ---><-2-><----------------------- M-A-R-P-L-C-T-L-L-L-L-M-A-T-L-A-G-A-L-A-G-S-H-H-H-H-H-H-G-S-K-P-T-Q-I-F-P- ---------- N-terminal region -------------------------->< ---------------------------- Neck Domain -------------- V-P-G-F-K-A-E-R-G-I-S-Q-A-Y-L-P-G-F-P-S-V-A-G-S-E-M-D-D-A-V-L-Q-L-K-D-R-I-S- ------------------------------><------------------------------------------------------------------------------------------------------K-L-E-G-V-L-Y-L-Q-G-K-I-T-K-S-G-G-K-I-F-A-T-S-G-K-T-A-D-F-H-A-T-V-K-M-C-Q-E- ------------------------------------------ Carbohydrate Recognition Domain ---------------------------------- A-G-G-C-I-A-S-P-R-N-A-D-E-N-A-A-I-L-H-F-V-K-Q-F-N-R-Y-A-Y-L-G-I-K-E-S-L-I-P- ----------------------------------------------------------------------------------------------------------------------------------------G-T-F-Q-F-L-N-G-G-E-L-S-Y-T-N-W-Y-S-H-E-P-S-G-K-G-E-E-E-C-V-E-M-Y-T-D-G-T- ---------------------------------------------------------->< --3-- > W-N-D-R-R-C-N-Q-N-R-L-V-V-C-Q-F-A-A-A

Figure 1. Predicted amino acid sequence of recombinant cLL. 1: extra Gly-Ser-residues added after signal peptide;

2: extra Gly-Ser-residues added as a result of inserting the BamHI restriction site into the sequence; 3: extra

Alanine-residues added as a result of inserting the NotI restriction site into the sequence

Results

Production and characterization of recombinant cLL

Preliminary results from the small scale transfections (not shown) showed that cLL was

most effectively secreted using the mature peptide sequence ligated into the pABC-cystatin-

hisN vector with a cystatin signal peptide and an in-frame N-terminal His-tag. Therefore,

this vector was selected for large-scale (1 liter) transfections. The predicted amino-acid

sequence of cLL expressed in this vector is shown in Figure 1. Affinity purification of the

recombinant protein using mannan-sepharose yielded approximately 500-800 µg of protein

per 1L batch (Fig. 2A). Analysis of the purified product on SDS PAGE showed that four

different bands varying in size between approximately 22 kDa and 27 kDa were present.

Western Blot analysis using anti-His6 antibody showed a positive signal for all four bands

(Fig. 2B) in the untreated cLL-sample (Lane 1) indicating that all four bands were

recombinant products. To identify whether different glycosylation patterns of the protein

could account for the observed multiplet, a DIG-glycan staining was performed. Results of

this DIG-glycan detection are shown in Figure 2C and indicate that all of the 4 recombinant

protein bands contained sugar moieties. However, enzymatic treatment with N-glycanase /

PNGase or O-glycanase of cLL did not result in a size shift (lane 2-6), that would indicate

loss of sugar moieties. The multimeric state of cLL was investigated by crosslinking

experiments using Bis (Sulfosuccinimidyl) suberate. SDS PAGE analysis of the crosslinked

cLL showed that monomeric cLL bands were not present anymore while two high-

molecular weight bands of approximately 85 kDa and 160 kDa were present (Fig. 3).


55

Figure 2. Production and structural characterization of

cLL. (A) Coomassie staining of cLL eluted from mannan-

sepharose beads. (B) Western Blot-analysis of the eluted

cLL and enzymatically treated cLL using anti-His6. (C)

DIG-glycan detection for both Sham-treated and

enzymatically treated cLL. (B&C): 1: untreated cLL, 2:

Sham-treated N-Glycanase-PNGase, 3: N-Glycanase-

PNGase-treated cLL, 4: N-Glycanase-PNGase and O-

glycosidase-treated cLL, Lane 5: O-glycosidase-treated

cLL, Lane 6: Sham-treatment O-glycosidase.

Analysis by MALDI-TOF-TOF

MALDI-TOF-TOF analysis was performed in order to investigate the cause of appearance

of four bands. However, the spectra of the trypsin-digested protein of all four bands were

similar, and could not explain the size difference observed on SDS-PAGE. Several peaks in

the spectra could be assigned to sequences in the recombinant protein, including the N- and

C-terminus which were retrieved for all four bands (data not shown).

Figure 3. BS-crosslinking of cLL. Left lane: untreated cLL; Right lane:

crosslinked cLL.

Carbohydrate specificity

Carbohydrate specificity of cLL was tested by use of PAA-coupled glycoconjugates. cLL

was observed to preferentially bind to α-mannose-PAA and trimannose-PAA (Man3), but

not to any of the other glycoconjugates (Fig. 4). Binding to α-mannose-PAA and Man3-Paa

was also tested in the presence of EDTA, which significantly reduced the binding,

indicating that cLL binding to α-mannose and Man3 is calcium-dependent.

Chapter 3

56

Figure 4. Carbohydrate specificity of cLL. cLL binds to α-mannose and Man3 in a calcium-dependent manner.

Man3: trimannose; LeX: Lewis X; LeY: Lewis Y; LeA: Lewis A; H-type 2: Lewis H-type 2; H-type 3: Lewis H-

type 3; LN: Gal-β-GlcNAc; LDN: GalNAc-beta-GlcNAc; chi-3: GlcNAc-GlcNAc-GlcNAc

Viral inhibition assays

A. Testing the activity of cLL against avian IAV

In order to assess whether cLL is capable of protecting TOC from infection by avian IAV

H9N2, viral inhibitions assays were carried out. Results of these tests are depicted in Figure

5. In experiment 1, inoculation of TOC with a premix of H9N2 and cLL from the first batch

(1.1) led to a significant decline of viral RNA expression compared to the negative control

(H9N2-medium premix), suggesting a cLL-mediated inhibition of H9N2 entry in TOC.

Chicken immune serum was used as a positive control and also caused a significant decline

in viral RNA expression. In experiment 2, the second batch of cLL (2.2.) failed to induce

this inhibition in viral RNA expression. The positive control, chicken immune serum, also

failed to cause a significant decline in viral RNA expression in this experiment. Therefore,

both cLL batches from the first (3.1) and second (3.2) experiment were retested in

experiment 3 together with a third cLL batch (3.3). Due to the low amount of recombinant

protein in batch 1 and 2 the tests were run in duplicate and triplicate respectively. The

positive control, chicken immune serum, caused a significant decline in viral RNA

expression. A decline in viral RNA expression was not found in TOC treated with either

cLL batch 2 (3.2) and 3 (3.3). However, for cLL batch 1 (3.1) a decline in viral RNA

expression was found in one of the replicates.


57

Figure 5. Limited virus entry inhibition by cLL. Viral RNA expression in TOC after inoculation with H9N2 +

medium (negative control), H9N2 + chicken immune serum (positive control) and H9N2 + cLL for 4 h. In the first

experiment cLL batch 1 (1.1.) was used, in the second experiment cLL batch 2 (2.1) and in the third experiment

cLL batch 3 (3.3). In the third experiment batch 1 (3.1) and batch 2 (3.2) were retested. * = significantly different

from the negative control within an experiment (p<0.05).

B. Testing the activity of cLL against human isolates of IAV

Neutralization of infectivity

In order to investigate whether cLL was capable of protecting MDCK cell monolayers from

infection by Phil-strain, Phil/BS-strain or PR-8 strain IAV, virus preparations were

preincubated with increasing concentrations of cLL prior to adding them to the cells. No

differences were observed in the number of fluorescent foci, indicating that cLL does not

interfere with infection of these cells (data not shown).

cLL mediated HAA-inhibition

In the HAA-inhibition-assay, cLL was capable of inhibiting HA-activity of all three human

isolates of IAV tested. The mean concentrations at which erythrocytes were no longer

agglutinated by the virus are depicted in Figure 6. Phil/BS was inhibited at a much lower

concentration of cLL than its parent strain Phil. Haemagglutination-activity of PR-8 was

inhibited at a concentration comparable to that of Phil.

Discussion

We previously reported the finding of cLL which, based on sequence homology, was

predicted to be a C-type lectin (Hogenkamp et al., 2006). The sequence of its CRD is most

similar to SP-A, but cLL does not contain a collagenous domain and therefore could not be

classified as a collectin. To our knowledge, this protein lacks further similarity to other C-

type lectins and should therefore be regarded as a novel type of lectin (Hogenkamp et al.,

2006).

1.1

2.2

3.3

3.1

3.2

Mean value0

2

4

6

8

10

12

14

H9N2 + medium

H9N2 + immune serum

H9N2 + cLL

Cor

rect

ed 4

0-C

t

* *

*

1.1

2.2

3.3

3.1

3.2

Mean value0

2

4

6

8

10

12

14

H9N2 + medium

H9N2 + immune serum

H9N2 + cLL

Cor

rect

ed 4

0-C

t

* *

*

Chapter 3

58

Figure 6. Haemagglutination activity-inhibition by cLL. Minimal concentration of cLL (μg/ml) necessary to

inhibit HA-activity of human isolates of IAV. Values are mean concentration ± s.d. of three separate experiments.

In this study, recombinant cLL was used to further characterize structural and functional

properties of this lectin. It was found that cLL appeared as four differently sized products

on SDS-PAGE gel (Fig. 2A and B). No potential GPI-modification sites were found in the

sequence, and since the sequence of cLL does not contain any potential N- glycosylation

sites, it is highly unlikely that differential N-glycosylation could account for the appearance

of four products instead of one. However, a positive signal was observed for all bands using

the DIG-glycan detection kit (Fig. 2C). Aspecific DIG-labeling or staining may explain this

result. Accordingly, enzymatic treatment with N-Glycanase and/or O-glycosidase did not

result in a size shift (Fig. 2B and C), and no difference in intensity of DIG-glycan staining

was observed between enzymatically treated samples. However, it cannot be excluded that

cLL contains sugar moieties that are insufficiently removed by enzymatic treatment. As for

O-linked glycans, it is known that these glycans are highly heterogeneous while in general

O-glycosidases show a restricted specificity for subclasses of O-linked sugars. Therefore,

we can not exclude the possibility that O-linked glycans are indeed present and result in

heterogeneity of the cLL polypeptide chains as illustrated by results presented in Figure 2.

The four bands obtained by SDS-PAGE analysis on purified cLL were subjected to

MALDI-TOF-TOF and resulted in identical spectra in which the C- and N-terminal

fragments could be retrieved for all four bands, identifying all bands as (isoforms of) cLL.

This method is rather stringent, therefore it is possible that posttranslational modifications

are removed during the procedure. Further analysis using 2D gel electrophoresis combined

with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) may

reveal what causes the differences in mass. As this concerns a recombinant protein, it

would be very interesting to find out whether the same size differences are present in native

cLL.

In mammalian species, collectin monomers are known to organize into higher order

multimers (King et al., 1989; Crouch et al., 1994). Crosslinking cLL led to the formation of

two high molecular weight products (Fig. 3), suggesting that this protein is capable of

forming trimeric subunits and higher order subunits. As the largest product observed in the


59

crosslinking was ~160 kDa, corresponding to six monomeric subunits of cLL, it is possible

that cLL is capable of forming hexameric multimers. In support of the assumption that cLL

can form trimers at the least, the presence of heptad repeats in the cLL-amino acid sequence

could allow for the formation of trimeric subunits (Hogenkamp et al., 2006). Furthermore,

it is known that the carbohydrate-binding activity of C-type lectins is quite weak when they

are in their monomeric form (Kishore et al., 1996). During the procedure of affinity-

purifying cLL by use of mannan-sepharose beads, the beads were washed twice with a

relatively large volume of washing buffer. These conditions would most likely be too

stringent if carbohydrate-binding by cLL were weak, making it more likely that this protein

is present as a trimer or a higher multimeric form.

Analysis of the carbohydrate-specificity of cLL revealed that the protein binds to α-

mannose and Man3 in a calcium-dependent manner, but other carbohydrates tested

(including galactose) were not bound by cLL (Fig. 4). It is possible that the presence of a

Glu-Pro-Ser motif in the CRD of cLL accounts for the relatively high specificity of cLL.

Most other mannose type-collectins contain a Glu-Pro-Asn motif (Drickamer, 1992) and

most SP-As contain a Glu-Pro-Arg motif, whilst retaining their preference for mannose

over galactose (McCormack et al., 1994). However, it was shown that substitution of one

amino acid is enough to change the sugar binding specificity of C-type lectins (Drickamer,

1992). It would be interesting to determine if construction of a similar Glu-Pro-Ser motif in

other C-type lectins would result in a comparable specificity for mannose.

The activity of cLL against avian and human isolates of IAV was tested in viral inhibition

assays. Incubating TOC with a premix of avian IAV H9N2 and cLL led to a decline in viral

RNA expression in TOC when the first batch of cLL was used. This result was not

reproducible using other batches of cLL. These results indicate that cLL may have

neutralising activity against avian IAV H9N2, but the activity is not strong. Differences in

posttranslational modifications have been shown to affect collectin functionality while the

binding activity is not affected (Mikerov et al., 2007). Differences in posttranslational

modifications between the chicken and human HEK cells used for protein expression might

have an influence on the cLL neutralisation activity.

In HAA-inhibition assays cLL showed strong activity against the Phil/BS strain (Fig. 6).

Two other strains tested, Phil strain and PR-8, were less susceptible to cLL inhibition. The

infection of MDCK cells by IAV could not be inhibited by cLL which may be explained by

the, compared to collectins, relative weak interaction of cLL with these IAV strains. The

change in equilibrium between cLL-bound and free virus particles could result in a constant

supply of free IAV as MDCK cell infection draws free virus particles from the medium.

This may explain the discrepancy between the results from the MDCK cell infections and

the HAA-inhibition assays. It is not yet clear what mechanism underlies the susceptibility

of the different IAV strains with respect to the inhibition of haemagglutination by cLL. The

Chapter 3

60

Phil/BS strain (subtype H3N2) differs from the parent Phil strain, in that the high-mannose

oligosaccharide overlying the sialic acid receptor-binding site of the HA molecule is absent

(Hartley et al., 1992). It is possible that this reveals targets for cLL binding that are not

available in the parent strain, resulting in increased binding. The PR-8 strain also lacks high

mannose glycans on the HA molecule (Schwarz et al., 1981), but since this concerns a

H1N1 subtype of IAV it is possible that differences in targets available on the HA molecule

or the neuraminidase may result in decreased binding. It will be interesting to see what

mediates cLL binding to IAV, since binding of SP-A (to which cLL is most similar) is

thought to occur via binding of the sialic acid receptor of the virus to sialylated N-linked

oligosaccharide present in the CRD of SP-A (Benne et al., 1995). It is possible that cLL,

similar to SP-D (Hartshorn et al., 2000), binds to HA and neuraminidase in a C-type lectin-

like manner, but the exact mechanism remains to be elucidated.

In summary, the chicken lung lectin was successfully expressed in HEK293-EBNA cells.

The purified protein proved to be a C-type lectin, as predicted from its sequence. Analysis

of its carbohydrate specificity revealed that this protein has an unusual high preference for

binding a-mannose and trimannose. The results of the viral inhibition assays showed that

cLL might have an anti-viral activity against avian IAV. Furthermore, cLL has moderate

HAA-inhibition activity against the human IAV strains Phil and PR-8 and a strong

inhibitory activity against Phil/BS. These results indicate that cLL could play an important

part in the innate immune system of chickens.

Acknowledgements

The authors would like to thank Kevan Hartshorn, Irma van Die, and Albert van Dijk for

support and helpful discussions. Laurie Bruinsma is thanked for her practical assistance.

This work was supported by a research grant (Adaptation and Resistance Program) from the

Animal Sciences Group (Wageningen University and Research Center) and the Faculty of

Veterinary Medicine (Utrecht University), The Netherlands. The authors acknowledge the

support by the Commission of the European Community (Contract Number 512093).

Sylvia Reemers was financially supported by a BSIK VIRGO consortium grant (Grant no.

03012), The Netherlands.

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Transcriptional expression levels of chicken collectins

are affected by avian influenza A virus inoculation

Sylvia S. Reemers, Edwin J.A. Veldhuizen, Cherina Fleming, Daphne A. van Haarlem,

Henk Haagsman, Lonneke Vervelde

Department of Infectious Diseases and Immunology, Faculty Veterinary Medicine, Utrecht University, Yalelaan 1,

3584 CL Utrecht, The Netherlands

Veterinary Microbiology, 2010; 141: 379-384

Chapter 4

Effects of AIV inoculation on expression of chicken collectins

65

Abstract

Mammalian collectins have been found to play an important role in the defense against

influenza A virus H9N2 inoculation, but for chicken collectins this has not yet been

clarified. The aim of this study was to determine the effect of avian influenza A virus (AIV)

inoculation on collectin gene expression in the respiratory tract of chickens and whether

this was affected by age. For this purpose 1- and 4-week-old chickens were inoculated

intratracheally with PBS or H9N2 AIV. Chickens were killed at 0, 8, 16 and 24 h post

inoculation and trachea and lung were harvested for analysis. Viral RNA expression and

mRNA expression of chicken collectins 1 and 2 (cCL-1 and cCL-2), chicken lung lectin

(cLL) and chicken surfactant protein A (cSP-A) were determined using real-time

quantitative RT-PCR. In lung, a decrease in mRNA expression of cCL-2, cLL and cSP-A

after inoculation with H9N2 was seen in both 1- and 4-week-old birds, although at different

time points, while in trachea changes were only seen in 4-week-old birds and expression

was increased. Moreover, collectin expression correlated with viral RNA expression in lung

of 1-week-old birds. These results suggest that both age and location in the respiratory tract

affect changes in collectin mRNA expression after inoculation with H9N2 and indicate a

possible role for collectins in the host response to AIV in the respiratory tract of chickens.

Introduction

The first line of defense against invading pathogens in the respiratory tract is provided by

the innate immune system. This phylogenetically ancient system does not require previous

exposure to a pathogen and acts within hours after the first encounter. Especially in the

early stages of life, the host is highly dependent on innate immunity for protection because

the adaptive immune system is not fully developed yet. An important group of effector

molecules of the innate immune system comprises collectins, proteins characterized by a

collagen- and a C-type lectin domain. These proteins are pattern recognition molecules that

can bind and neutralize a wide array of pathogens including bacteria and viruses

(Hogenkamp et al., 2007).

For mammalian collectins, several in vitro studies have described a strong neutralizing

activity against influenza A virus (Hartshorn et al., 1997; Van Eijk et al., 2003; White et al.,

2008). Collectins are thought to bind carbohydrate residues present on influenza

haemagglutinin and neuraminidase, thereby aggregating viral particles and preventing

invasion of host cells (Reading et al., 2007). In addition, a highly reduced clearance of

influenza virus was observed in mice lacking either surfactant protein A or surfactant

protein D, implicating the importance of these mammalian collectins in defense against

influenza virus inoculations in vivo (Sano et al., 2005). In chicken, five collectins have been

described thus far, which are mannan binding lectin (Laursen et al., 1998), surfactant

Chapter 4

66

protein A (cSP-A) and chicken collectins 1, 2 and 3 (cCL-1-3) (Hogenkamp et al., 2006). In

addition, a chicken lung lectin (cLL) with high sequence homology to cSP-A was reported.

Whether chicken collectins can protect against influenza A virus inoculation has not been

clarified, but cLL has been shown to possess in vitro haemagglutination inhibiting activity

(Hogenkamp et al., 2008).

Avian influenza virus (AIV) enters the respiratory epithelium using sialic acid receptors

linked to galactose by an α-2,3 linkage, although the pattern of receptor distribution in

chickens is less defined than in mammals (Wan et al., 2006). H9N2 AIV has been shown to

have a preference for infecting the upper part of the respiratory tract (Nili et al., 2002).

After spray inoculation, viral replication was detected at 24 hours post inoculation in both

trachea and lung of chickens. Virus was first detected in the epithelium of the trachea,

intrapulmonary bronchus and in adjacent parabronchi, and thereafter spread to other

parabronchi deeper in the lung (Reemers et al., 2009).

The aim of the current work was to determine changes in gene expression of chicken

collectins upon AIV inoculation in the respiratory tract of 1- and 4-week-old chickens and

the effect of age on collectin gene expression after AIV inoculation.


Inoculation model

AIV, subtype H9N2, isolate A/Chicken/United Arab Emirates/99 was kindly provided by

Intervet Schering-Plough Animal Health. The experiment was carried out according to

protocols approved by the Animal Experiment Committee of Utrecht University. One-week

and four-week-old Lohmann Brown layer chickens were inoculated intratracheally with

either 0.1 ml PBS or 107.7 EID50 H9N2. Before inoculation 5 control chickens per age were

killed and at 8, 16 and 24 h post inoculation (p.i.) 5 control and 5 H9N2 inoculated

chickens per age were killed. The cranial part of the lung containing the intrapulmonary

bronchus and the upper trachea were used for analysis.

Real-time quantitative RT-PCR (qRT-PCR)

Lung and trachea were homogenized and total RNA was isolated using the RNeasy Mini

Kit and DNase treated (Qiagen) as described previously (Reemers et al., 2009). cDNA was

generated with reverse transcription using iScript cDNA Synthesis Kit (Biorad Laboratories

B.V.).

Detection of GAPDH, H9 haemagglutinin (HA) and 28S products was performed as

described by Reemers et al. (2009). Real-time qRT-PCR was performed for cCL-1, cCL-2,

cLL and cSP-A using primers and probes depicted in Table 1 according to the following

cycle protocol: 2 min at 50°C, 10 min at 95°C (denaturation); 40 cycles: 15 s at 95°C and

60 s at 60°C. GAPDH was used as a reference gene for correction of viral HA RNA


67

expression and 28S as reference gene for collectin mRNA expression. Corrections for

variation in RNA preparation and sampling were performed as previously described

(Reemers et al., 2009). Results are expressed in terms of the threshold cycle value (Ct) and

given as corrected 40-Ct values.

Table 1. Real-time qRT-PCR primers and probes

Target Probe or primera

sequence (5’-3’) Accession No.b

cCL-1 F R Probe

5'- ATTGTCAAAGAAGAGAAGAATTACAGAG -3' 5'- GAGGAGATGTAATCAGCAAGCAG -3' 5'- (FAM) – CGTTGGTCACCTCATCTTTAGGCATGGC – (BHQ) - 3'

DQ129668

cCL-2 F R Probe

5'- GGGAGCCCAACAATGCCTATG -3' 5'- GTAATATGACATGCAACATCATTCCAC -3' 5'- (FAM) – TGCCACCATCTCCACACAGTCCTCCTC – (BHQ) -3'

DQ129669

cLL F R Probe

5'- CTTACAAGGGAAGATAACAAAGTCTGG -3' 5'- CATTCCTTGGAGATGCAATACACC -3' 5'- (FAM) - CTCCTGGCACATTTTCACCGTAGCATGG – (BHQ) - 3'

DQ129667

cSP-A F R Probe

5'- GGAATGACAGAAGGTGCAATCAG -3' 5'- GCAATGTTGAGTTTATTAGCTACAAATG -3' 5'- (FAM) – CCGCCTTGTTGTCTGCCAGTTTTAGTGC – (BHQ) - 3'

AF411083

a F, forward; R, reverse; b Genomic DNA sequence

Statistical analysis

Significance between H9N2 inoculated and control samples and between age groups within

a time point was determined with an ANOVA. Significance between time points within age

groups and treatment groups was determined with an ANOVA and Tukey post-hoc test.

Correlation between viral RNA and collectin mRNA expression was based on the Pearson

correlation coefficient (r) and determined using SPSS 15.0 software. Data were expressed

as means with standard error of the mean (SEM). A p-value < 0.05 was considered

significant.

Results

Viral RNA expression in lung and trachea

Viral RNA expression was detected in lung and trachea of all H9N2 inoculated birds (Fig.

1). There was no significant difference in viral RNA levels in lung between 1- and 4-week-

old birds within a time point or between time points within an age group. In trachea there

was no significant difference between 1-week and 4-week-old birds within a time point.

Only the decline of viral RNA expression in trachea of 1-week-old birds at 24 h.p.i. was

significantly different from the expression at 16 h.p.i.

Chapter 4

68

Figure 1. Viral RNA expression in 1-week and 4-week-old H9N2 inoculated birds at 8, 16 and 24 h.p.i. in lung

and trachea determined with real-time qRT-PCR. * Indicates significant difference in viral RNA expression

between 16 and 24 h.p.i. in 1-week-old birds (p < 0.05).

Collectin mRNA expression in lung and trachea

Expression of cCL-1, cCL-2, cLL and cSP-A mRNA was determined in individual birds at

all time points in both lung and trachea (Fig. 2 and 3). cCL-2, cLL and cSP-A mRNA levels

in lung of H9N2 inoculated birds were generally lower compared to control birds. In 1-

week-old birds, cCL-2, cLL and cSP-A mRNA levels were significantly decreased after

H9N2 inoculation at 16 h.p.i. In 4-week-old birds, there was a significant decrease in

mRNA levels of cCL-2 at 8 h, cSP-A at 16 h and cLL at 24 h.p.i. between lung of H9N2

inoculated and control birds.

Although expression in lung was mostly decreased after H9N2 inoculation, collectin

mRNA expression in trachea was mostly increased in H9N2 inoculated birds. The cCL-1

mRNA level was significantly higher at 16 h.p.i. in H9N2 inoculated birds compared to

control birds in trachea of both 1- and 4-week-old birds. In 4-week-old birds, cCL-2, cLL

and cSP-A mRNA levels were significantly increased at 8 h.p.i. after H9N2 inoculation. All

observed changes were between 1 and 2 Ct values corresponding to a 2-4 fold change in

mRNA levels.

Correlations between viral RNA and collectin mRNA expression in lung and trachea

First the correlation between viral RNA and collectin mRNA expression was determined

per age group for all collectins in lung and trachea over time. If a correlation is found this

means that viral RNA expression and collectin mRNA expression correlate at every time

point. There was no significant correlation between viral RNA expression and mRNA

expression of any of the collectins in lung and trachea of 4-week-old birds over time. No

significant correlation was seen in trachea of 1-week-old birds for any of the collectins,

however in lung there was a significant strong negative correlation over time between viral

0

5

10

15

20

8 hr 16 hr 24 hr 8 hr 16 hr 24 hr

1 wk 4 wk

16hpi16hpi 24hpi 8hpi 24hpi

1 wk old 4 wk old

0

Viral RNA in lung

15

20

8hpi

5

10C

orre

cted

40-

Ct

+ S

EM

0

5

10

15

20

8 hr 16 hr 24 hr 8 hr 16 hr 24 hr

1 wk 4 wk

16hpi16hpi 24hpi 8hpi 24hpi

1 wk old 4 wk old

0

Viral RNA in lung

15

20

8hpi

5

10C

orre

cted

40-

Ct

+ S

EM

0

5

10

15

20

8 hr 16 hr 24 hr 8 hr 16 hr 24 hr

1wk 4w k

8hpi 16hpi 24hpi 8hpi 16hpi 24hpi

1 wk old 4 wk old

0

Viral RNA in trachea

5

10

15

20

Cor

rect

ed4

0-C

t +

SE

M

*

0

5

10

15

20

8 hr 16 hr 24 hr 8 hr 16 hr 24 hr

1wk 4w k

8hpi 16hpi 24hpi 8hpi 16hpi 24hpi

1 wk old 4 wk old

0

Viral RNA in trachea

5

10

15

20

Cor

rect

ed4

0-C

t +

SE

M

*


69

Figure 2. cCL-1, cCL-2, cLL and cSP-A mRNA expression in lung of 1- and 4-week-old birds at 0, 8, 16 and 24

h.p.i. determined with real-time qRT-PCR. * Indicates significant difference in collectin mRNA expression

between control and H9N2 inoculated birds (p < 0.05).

RNA expression and mRNA expression of cCL-2 (r=-0.668, p=0.007), cLL (r=-0.655,

p=0.008) and cSP-A (r=-0.657, p=0.008).

Although no correlation was found between viral RNA and collectin mRNA expression in

trachea of 4-week-old birds over time, there was a significant difference in collectin mRNA

expression after H9N2 inoculation at certain time points. Only cCL-2 mRNA expression

correlated with viral RNA expression at 8 h.p.i.

Discussion

The aim of this study was to determine changes in chicken collectin gene expression due to

AIV inoculation in the chicken respiratory tract and whether this was affected by age.

Collectins have only relatively recently been described in the chicken and their role in the

innate immune response is largely unknown. Based on sequence homology and gene

expression profiles, two chicken homologues for SP-A were found: cSP-A and cLL.

However, the chicken genome did not seem to contain a homologue for the second

pulmonary collectin SP-D. In addition, chicken collectins 1, 2 and 3 were found which

resemble the mammalian collectin liver 1 (CL-L1) (Ohtani et al., 1999), collectin kidney 1

Control H9N2 inoculatedControl H9N2 inoculated

6

9

12

0 hr 8 hr 16 hr 24 hr 0 hr 8 hr 16 hr 24 hr

1 wk 4 w k

12

9

Cor

rect

ed4

0-C

t +

SE

M

cCL-1

60hpi 8hpi 16hpi 24hpi

1 wk old


4 wk old

6

9

12


1 wk 4 w k

12

9

Cor

rect

ed4

0-C

t +

SE

M

cCL-1


1 wk old


4 wk old


1 wk old


4 wk old

6

9

12


1 w k 4 wk

12

9

6

Cor

rect

ed4

0-C

t +

SE

M

cCL-2

**


1 wk old


4 wk old

6

9

12


1 w k 4 wk

12

9

6

Cor

rect

ed4

0-C

t +

SE

M

cCL-2

**


1 wk old


4 wk old


1 wk old


4 wk old

12

15

18


1 w k 4 wk

18

15

12

Cor

rect

ed4

0-C

t +

SE

M

cLL

**


1 wk old


4 wk old

12

15

18


1 w k 4 wk

18

15

12

Cor

rect

ed4

0-C

t +

SE

M

cLL

**


1 wk old


4 wk old


1 wk old


4 wk old

12

15

18


1 wk 4 w k

18

15

12

Cor

rect

ed4

0-C

t +

SE

M

cSP-A

**


1 wk old


4 wk old

12

15

18


1 wk 4 w k

18

15

12

Cor

rect

ed4

0-C

t +

SE

M

cSP-A

**


1 wk old


4 wk old


1 wk old


4 wk old

Chapter 4

70

Figure 3. cCL-1, cCL-2, cLL and cSP-A mRNA expression in trachea of 1- and 4-week-old birds at 0, 8, 16 and

24 h.p.i. determined with real-time qRT-PCR. * Indicates significant difference in collectin mRNA expression

between control and H9N2 inoculated birds (p < 0.05).

(CL-K1) (Keshi et al., 2006) and collectin placenta 1 (CL-P1) (Ohtani et al., 2001). The

biological roles of CL-L1 and CL-K1 have not been determined yet, leaving no indication

for a possible role of chicken collectins cCL-1 and cCL-2. However, both cCL-1 and cCL-2

are expressed in the chicken respiratory tract (Hogenkamp et al., 2007). The cCL-3

homologue CL-P1 is different from other collectins in the way that it is a type II membrane

protein. It acts as a scavenger receptor on endothelial cells, while all other collectins are

soluble effector molecules involved in direct neutralization of pathogens. A role for cCL-3

in direct neutralization of viruses in the lung is therefore unlikely.

Based on this similarity to mammalian collectins and the location of expression in the

chicken we investigated the changes in mRNA expression of cSP-A, cLL, cCL-1 and cCL-

2 after an H9N2 AIV inoculation in the chicken respiratory tract. In the lung of 1 week-old

birds, a down regulation was observed for cLL, cSP-A and cCL-2 mRNA, at 16 h.p.i. This

time point significantly correlated to the observed peak in viral load in the lung. In addition

the expression of cCL-2 and cSP-A mRNA was down regulated in 4-week-old birds at 8

and 16 h.p.i., but no correlation to viral RNA expression was found. This indicates that

expression of cSP-A, cLL and cCL-2 mRNA expression is affected by H9N2 inoculation

3

6

9

0hr 8hr 16hr 24hr 0hr 8hr 16hr 24hr

1 wk 4wk

*

*

9

6

3

Cor

rect

ed4

0-C

t +

SE

M

cCL-1


1 wk old


4 wk old

3

6

9


1 wk 4wk

*

*

9

6

3

Cor

rect

ed4

0-C

t +

SE

M

cCL-1


1 wk old


4 wk old


1 wk old


4 wk old

3

6

9


1 wk 4wk

9

6

3

Cor

rect

ed4

0-C

t +

SE

M

cCL-2

*


1 wk old


4 wk old

3

6

9


1 wk 4wk

9

6

3

Cor

rect

ed4

0-C

t +

SE

M

cCL-2

*


1 wk old


4 wk old


1 wk old


4 wk old

12

15

18


1 wk 4wk

*18

15

cLL

12

Cor

rect

ed4

0-C

t +

SE

M


1 wk old


4 wk old

12

15

18


1 wk 4wk

*18

15

cLL

12

Cor

rect

ed4

0-C

t +

SE

M


1 wk old


4 wk old


1 wk old


4 wk old

9

12

15


1 wk 4wk

15

12

9

Cor

rect

ed4

0-C

t +

SE

M

cSP-A

*


1 wk old


4 wk old

9

12

15


1 wk 4wk

15

12

9

Cor

rect

ed4

0-C

t +

SE

M

cSP-A

*


1 wk old


4 wk old


1 wk old


4 wk old

Control H9N2 inoculatedControl H9N2 inoculated


71

early after inoculation and in 1-week-old birds is directly related to viral RNA expression in

lung. Interestingly, in the trachea, all significant changes due to H9N2 inoculation were

related to up regulation of collectin mRNA expression, suggesting host collectin responses

after H9N2 inoculation are site specific. cCL-1 was up regulated at 16 h.p.i. in trachea of

both 1- and 4-week-old birds, while cCL-1 mRNA expression was not affected by H9N2

inoculation in lung. This suggests that changes of cCL-1 mRNA expression after H9N2

inoculation are dependent on the location in the respiratory tract. cCL-2 expression was

affected by H9N2 inoculation in 4-week-old birds at 8 h.p.i. in both lung and trachea,

although down regulated in lung and up regulated in trachea, indicating that cCL-2 mRNA

expression changes time specifically at 8 h.p.i. after H9N2 inoculation. In lung, changes in

mRNA expression of cCL-2, cLL and cSP-A after H9N2 inoculation were seen in both 1-

and 4-week-old birds, although at different time points, while in trachea changes were only

seen in 4-week-old birds. This suggests that both age and location in the respiratory tract

affect changes in collectin mRNA expression after H9N2 inoculation, though one has to

keep in mind that changes found at early time points are caused by the virus inoculum

whereas at 24 h.p.i. viral replication might affect the outcome. In mammals site specific

expression and secretion of collectins in the airways has been reported (Coalson et al.,

1998; Rooney, 2001). Furthermore, down regulation of SP-A gene expression through p38

MAPK and PI-3 kinase pathways have been described in lung epithelial cells (Miakotina et

al., 2002a; Miakotina et al., 2002b). Epithelial cells in the lung differ from tracheal ciliated

epithelial cells supporting the possibility of site specific collectin expression after H9N2

inoculation in the chicken. Whether similar signalling pathways are involved in regulation

of chicken collectins needs to be addressed to clarify our observed tissue specific changes

of these collectins.

Without knowing the exact function of chicken (col)lectins in innate defense , it is difficult

to relate the observed changes in gene expression to a biological effect. In mammals, an up

regulation of collectins has frequently been observed in both bacterial and viral infections

(Murray et al., 2002; Grubor et al., 2004), which is usually interpreted as a direct response

of the host to increase its defense against the incoming pathogens. A down regulation of

collectins upon infection is less frequently reported, but could reflect a survival strategy of

the pathogen to overcome the host’s hostile environment, as has been described for other

innate immune proteins such as Toll like receptors and defensins (Wang et al., 2000; Shin

et al., 2007). However, whether this applies to AIV in the chicken lung and why the altered

expression of chicken (col)lectins is tissue specific is not clear at this moment and requires

further investigation.

In conclusion, the results obtained in this study show that mRNA expression of chicken

collectins cCL-1, cCL-2, and cSP-A and cLL are affected by H9N2 AIV inoculation. The

effect is tissue specific, showing up regulated mRNA expression in the trachea and down

Chapter 4

72

regulation in the lung. Furthermore, changes in collectin mRNA expression are age

specific, showing differential gene expression in 1- and 4-week old birds. These observed

changes in collectin mRNA expression implicate that chicken collectins can play an

important role in innate defense against viral infection, especially in neonates. More

research is needed to relate the changes in collectin mRNA expression after H9N2 AIV

inoculation to a biological function and clarify the function of collectins within the innate

response to infection.

Acknowledgements

We thank Astrid Hogenkamp from the Department of Pharmaceutical Sciences for

stimulating discussions. This study is financially supported by a BSIK VIRGO consortium

grant (Grant no. 03012), the EU sixth framework program Flupath (Grant no. 044220) and

the Dutch consortium Veterinary Network Avian Influenza (FES).

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Differential gene expression and host response profiles

against avian influenza virus within the chicken lung due

to anatomy and airflow

Sylvia S. Reemers1, Daphne A. van Haarlem1, Marian J. Groot Koerkamp2, Lonneke

Vervelde1

1Department of Infectious Diseases and Immunology, Faculty Veterinary Medicine, Utrecht University, Yalelaan

1, 3584 CL Utrecht, The Netherlands 2Genomics Laboratory, Department of Physiological Chemistry, Utrecht Medical Centre, Universiteitsweg 100,


Journal of General Virology, 2009; 90: 2134-2146

Chapter 5

Airflow and anatomy affect host responses to AIV infection in the avian lung

77

Abstract

Sampling the complete organ instead of defined parts might affect the analysis at both

cellular and transcriptional level. We defined host responses to H9N2 avian influenza virus

(AIV) in trachea and different parts of the lung. Chickens were spray inoculated with either

saline or H9N2 AIV. Trachea and lung were sampled at 1 and 3 days post inoculation

(d.p.i.) for immunocytochemistry, real-time qRT-PCR and gene expression profiling. The

trachea was divided into upper and lower trachea and lung into 4 segments according to

anatomy and airflow. Two segments contained the primary and secondary bronchi cranial

vs caudal (part L1 and L3) and two segments contained the tertiary bronchi cranial vs

caudal (part L2 and L4). Between upper and lower trachea in both control and infected

birds minor differences in gene expression and host responses were found. In the lung of

control birds differences in anatomy were reflected in gene expression, and in the lung of

infected birds virus deposition enhanced the differences in gene expression. Differential

gene expression in trachea and lung suggested common responses to a wide range of agents

and site specific responses. In trachea site specific response were related to heat shock and

lysozyme activity. In lung L1, containing most virus, site specific responses related to

genes involved in innate responses, interleukin activity and endocytosis. Our study

implicates that anatomy of the chicken lung has to be taken into account when investigating

in vivo responses to respiratory virus infections.

Introduction

Avian influenza virus (AIV) infection is a continuing threat to both humans and birds

worldwide. In order to control outbreaks, research into new intervention strategies and

vaccines is ongoing. Therefore studies into pathogenesis and host-virus interactions are

being performed (Kash et al., 2004; Baskin et al., 2005; Degen et al., 2006). Host-virus

interactions depend on the ability of the virus to enter to host cells. The molecules used by

avian and human influenza viruses to enter the respiratory tract are sialic acid linked to

galactose by respectively an α-2,3 or α-2,6 linkage and are not equally distributed.

Differences in virus entry generally correspond with variation in the type of sialic acid

expressed in the respective host species (Shinya et al., 2006; Van Riel et al., 2007). The

pattern of receptor distribution in chickens was shown to be less defined than in mammals

(Wan et al., 2005). In chickens uneven spread of low pathogenic AIV (LPAI) has been

shown for H9N2 with a preference for infecting the upper part of the respiratory tract (Nili

et al., 2002). Difference in distribution of virus within the respiratory tract has an impact on

the host responses, as shown for infected macaques (Baas et al., 2006). Host responses to

respiratory viral infections have only been investigated in the whole lung, one part of the

Chapter 5

78

lung or a pool of samples without taking the anatomy and airflow, bidirectional in

mammals and unidirectional in birds, through the respiratory tract into account.

Herein we describe that gene expression within the lung of control chickens was affected

by anatomy, and airflow affected virus deposition and thereby the localised host responses.

The trachea and lung of saline and H9N2 AIV inoculated birds were sampled 1 and 3 days

post inoculation (d.p.i.). The trachea was divided in upper and lower trachea and the lung

into 4 parts based on airflow (Fig. 1). The airflow is unidirectional and ventilation is

performed by the air sacs (Fedde et al., 1998). Between upper and lower trachea in both

control and H9N2 infected birds minor differences in gene expression were seen. In the

different parts of the lung in control birds gene expression differed significantly in that

expression was similar in L1 and L3 and in L2 and L4. In infected birds L1 and L3

contained larger and more virus infected areas than L2 and L4, which enhanced differences

in gene expression already seen in control birds. Our data showed that anatomy and airflow

in lung of both control and infected birds affect gene expression, virus deposition and

subsequently host responses. The trachea and lung shared expression of genes involved in

common host responses, but several genes were expressed site specifically. Our study

suggests that anatomy and airflow especially of the avian lung have to be taken into account

when investigating in vivo host responses to respiratory virus infections.

Methods

Infection model


provided by Intervet Schering-Plough Animal Health.

One-day-old White Leghorn chickens were housed under SPF conditions and all

experiments were carried out according to protocols approved by the Intervet Animal

Welfare Committee.

Chickens were divided into 2 groups over 2 isolators, infected and non-infected, containing

20 birds per group. Fourteen-day-old chickens were inoculated via aerosol spray with either

20 ml 107.7 EID50 H9N2 AIV or with 20 ml saline per isolator. Each isolator contained 20

birds, and of these birds 8 were used for the experiment described here. Chickens remained

in the closed isolator for 10 min, after which the isolator was ventilated as before. At 1 and

3 d.p.i. chickens were killed (n = 4 per time point per group) and trachea and left lung were

isolated and stored in RNAlater (Ambion) at -80°C for RNA isolation and liquid nitrogen

for immunocytochemistry. Trachea was divided into upper and lower trachea and lung was

divided into 4 pieces (Fig. 1), lung L1 to L4.


79

A. B. C.

Figure 1. A) Right chicken lung with a blunt probe indicating the localization of the primary bronchus. The lung

was divided in 4 parts, L1 to L4 according to airflow and lung anatomy, with the primary bronchus entering the

lung in L1. The syrinx with the primary bronchus is shown on the left side. The white blocks indicate the part of

the lung segments used for RNA isolation. The remainder of the segments was used for immunocytochemistry.

Illustration of the air flow pattern during B) inspiration and C) expiration in the avian lung. During inhalation, air

flows through the primary bronchus into the caudal air sacs or through the mediodorsal and lateroventral

secondary bronchi. This caudal to cranial flow pattern is also evident during expiration. 1. Primary bronchus, 2.

lung, 3. clavicular air sac, 4. cranial thoracic air sac, 5. caudal thoracic air sac, 6. abdominal air sac. Adapted and

reprinted with permission (Kothlow et al., 2008), Copyright 2008.

Immunocytochemistry

Detection of viral NP and cellular influxes in cryosections was described previously by

Vervelde et al. (1996). Viral NP was detected with a mouse monoclonal antibody to NP of

H9N2 (provided by Intervet Int.). KUL-01+ cells (macrophages; Mast et al., 1998) and

CD4+ cells were detected with monoclonal antibodies KUL-01 and CT-4 (Southern

Biotech). For the detection of CD8α+ cells a mixture of monoclonal antibodies EP72

(Southern Biotech) and AV14 (kind gift of Dr T.F. Davison, Institute for Animal Health,

Compton UK; Withers et al., 2005) was used to avoid differences in staining due to

polymorphism in the chicken CD8α molecule (Breed et al., 1996; Luhtala et al., 1997).

RNA isolation

Total RNA was isolated from upper and lower trachea (5 mm per part of the trachea) and

all 4 segments of the lung (an 1x5 mm part per segment of the lung; Fig. 1A) using the

RNeasy Mini Kit and DNase treated using the RNase-free DNase Set following

manufacturer’s instructions (Qiagen). All RNA samples were checked for quantity using a

spectrophotometer (Shimadzu) and quality using a 2100 Bioanalyzer (Agilent

Technologies).

Real-time quantitative RT-PCR (qRT-PCR)

cDNA was generated from 500 ng RNA with reverse transcription using iScript cDNA

Synthesis Kit (Biorad Laboratories B.V.). Real-time qRT-PCR was performed using iQ

SYBR green supermix (Biorad) and the TaqMan Universal PCR Master Mix (Applied

Biosystems, AB). Primers (Invitrogen) and probes (AB) were described by Degen et al.

L2

L1 L3

L4

Primary bronchus

6

1 2

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Inspiration

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Chapter 5

80

(2006) and Eldaghayes et al. (2006). Detection of GAPDH and H9 haemagglutinin (HA)

was described by Degen et al. (2006). Primers were used at 400 nM concentration.

Detection of interleukins (IL), interferon beta (IFN-β) and 28S was described by Ariaans et

al. (2008). Primers were used at 600 nM and probes at 100 nM concentration. Corrections

for variation in RNA preparation and sampling were performed according to Eldaghayes et

al. (2006).

A paired t-test was used to determine the statistical significance between upper and lower

trachea samples from the same bird. To determine the statistical significance between parts

of the lung an ANOVA with a Tukey post-hoc test was used. A p-value < 0.05 was

considered significant.

Oligonucleotide microarray analysis

For microarray analysis the Gallus gallus Roslin/ARK CoRe Array Ready Oligo Set V1.0

(Operon Biotechnologies) was used. The array was spotted onto Codelink activated slides

(GE Healthcare) and contains 20,460 oligo probes representing chicken genes, and 3828

control spots, used for QC and normalisation purposes (Van de Peppel et al., 2003). RNA

amplification and labelling were performed according to Roepman et al. (2005). All

hybridizations contained 2.5 µg cRNA per channel on a HS4800Pro hybstation (Tecan

Benelux BVBA). All trachea and lung samples were co-hybridised with respectively a

trachea or lung reference sample. These reference samples consisted of RNA extracted

from tracheas or lungs of 4 chickens that were not included in the infection experiment.

Slides were scanned with an G2565AA scanner (Agilent Technologies) at 100% laser

power, 30% PMT. Resulting image files were analyzed using Imagene 8.0 (BioDiscovery,

Inc.). Within slide normalisation was performed with Printtip Loess (Yang et al., 2002) on

mean data without background subtraction. Groups of replicates were analysed using

ANOVA (Wu et al., 2003). In a fixed effect analysis, sample, array and dye effects were

modelled. P-values were determined by a permutation F2-test, in which residuals were

shuffled 5000 times globally. Genes with p < 0.05 after family wise error correction were

considered significantly differentially expressed and were selected to be included for

further analysis. Visualisation and cluster-analysis were performed using GeneSpring 7.2

(Agilent Technologies). Ensembl Gallus gallus (assembly: WASHUC2, May 2006,

genebuild: Ensembl, Aug 2006, database version: 47.2e) was used for gene names,

description and Gene Ontology (GO) annotations. Primary data are available in the public

domain through Expression Array Manager (www.ebi.ac.uk/arrayexpress/?#ae=main[0])

under accession numbers E-TABM-637 for trachea and E-TABM-636 for lung.


81

Results

Responses in upper and lower trachea are similar

Trachea of control birds at 3 d.p.i. was used to analyse global and immune related gene

expression. The immune related category was based on the Gene Ontology (GO) terms

host-pathogen interaction, external stimulus and immune response. Gene expressions were

depicted in a scatterplot in which upper and lower trachea were plotted against each other

(Fig. 2A). Only few genes were significantly (p < 0.05) and with ≥ 2-fold difference

expressed (Table 1). These genes are involved in clathrin-independent vesicular transport,

nuclear organization, energy metabolism and calcium binding.

IL-1β, IL-6 and IFN-β mRNA expression was measured in trachea of control birds at 3

d.p.i. (Fig. 2B) and no significant differences in expression were found between upper and

lower trachea.

In the trachea of control birds the lamina propria consisted of one to three cell layers with

no lymphoid infiltrates. Few CD4+, CD8α+ and mainly KUL-01+ cells were found in the

lamina propria of both upper and lower trachea (Fig. 2E).

Upon infection we found that the viral RNA level in upper trachea was significantly higher

than in lower trachea at both 1 and 3 d.p.i. (Fig. 2C). Samples of 3 d.p.i. were used to

analyse cytokine expression and global gene expression. The difference in viral RNA level

did not result in significant differences in IL-1β, IL-6 and IFN-β mRNA expression

between upper and lower trachea (Fig. 2B), but in infected birds the mRNA levels of IL-1β

and IL-6 mRNA increased ~6 Ct values compared to control birds. IFN-β mRNA

expression also increased, but to a lesser extend, ~2 Ct values. Gene expression in upper

and lower trachea of infected birds was plotted against each other in a scatterplot (Fig. 2D).

Inoculation with H9N2 resulted in differential gene expression of ~1700 genes compared to

control birds, but only few genes were significantly and with ≥ 2-fold difference expressed

(Table 1). Three genes were down regulated, cysteine, glycine-rich protein 3 and a

chemokine gene. The up regulated genes were involved in metabolic processes,

metallopeptidase activity, regulation of cytokinesis, and endothelial cell activation.

At 1 and 3 d.p.i. virus was located in the epithelial cells and the mucoid glands. The lamina

propria was slightly swollen at 1 d.p.i., with influxes of CD4+ and KUL-01+ cells, whereas

at 3 d.p.i. the lamina propria consisted of multiple cell layers due to cellular influxes. KUL-

01+ cells were located in the epithelial cell layer and in the lamina propria, these cells had a

round appearance, indicative for activation, whereas in the submucosa these cells were

more dendritic shaped. Influxes of CD8α+ cells were found in the lamina propria from 3

d.p.i. and were smaller than the CD4+ cell influxes. All cellular influxes were localised and

not found throughout the whole trachea with no differences between upper and lower

trachea (Fig. 2E).

Chapter 5

82

A.

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E.


83

Figure 2. A) Scatterplot of global and immune related genes in upper versus lower trachea of control birds at 3

d.p.i. Genes in red were up regulated and in green down regulated. The middle diagonal line represents a 1-fold

change and the two outer diagonal lines a 2-fold change difference between upper and lower trachea. B) IL-1β, IL-

6 and IFN-β mRNA expression in trachea at 3 d.p.i. C) Viral RNA levels in trachea of infected birds at 1 and 3

d.p.i. Bars within a time point with different letters were significantly different (p < 0.05). D) Scatterplot of global

and immune related genes in upper versus lower trachea of infected birds at 3 d.p.i. E) Cryosections of trachea at 1

and 3 d.p.i. stained for viral NP, KUL-01+, CD4+ and CD8α+ cells. Upon infection, KUL-01+ cells (macrophages)

change from dendritic (arrows) to round morphology (arrowheads). Bar, 50 μm.

Table 1. Genes expressed significantly at ≥ 2-fold difference in upper trachea versus lower trachea at 3 d.p.i. in

control birds and infected birds.

Gene description Ensembl ID Fold

change†

Control Syntaxin 8 ENSGALT00000001879 0.50

birds Nesprin-1 ENSGALT00000021996 2.04

ENSGALG00000018428 2.08

genomic:Un_random+27728607-27728677

2.09

ENSGALESTG00000000019 * 2.11

Hypothetical protein LOC418424 ENSGALT00000024836 2.14

ENSGALT00000035596 2.21

AMP deaminase 1 ENSGALT00000003247 2.22

ENSGALT00000003550 2.26

ENSGALG00000023715 2.58

Parvalbumin, thymic ENSGALT00000005489 * 3.93

Infected Cysteine and glycine-rich protein 3 ENSGALT00000006436 0.30

birds Cardiac phospholamban ENSGALT00000024024 0.32

Chemokine ENSGALT00000041379 * 0.44

Potassium channel subfamily K member 2 ENSGALG00000009687 2.09

Aminoacylase 1-like protein 2 ENSGALT00000025446 2.09

ENSGALT00000025298 2.18

Proline-rich protein 6 ENSGALT00000007174 2.26

ENSGALT00000007194 2.60

ENSGALT00000015324 2.60

Coiled-coil domain containing 78 isoform 1 ENSGALT00000003811 2.63

GTP-binding protein RAD ENSGALT00000008251 2.84

Ankyrin repeat domain-containing protein 1 ENSGALT00000010491 3.81

* Immune related gene; † Expression ratio in lower trachea dived by expression ratio in upper trachea

Differential responses in lung segments L1, L2, L3 and L4

Lung was divided into 4 pieces according to unidirectional airflow and lung anatomy. In

this study the airflow through the air sacs is being ignored for simplicity, because it does

not affect the airflow through the lung.

Chapter 5

84

Global and immune related gene expression in lung L1 to L4 of control birds at 3 d.p.i.

were compared by hierarchical clustering. L1 clustered with L3 and L2 clustered with L4

for both global and immune related genes (Fig. 3A), coinciding with airflow and lung

anatomy.

No significant difference in IL-1β and IFN-β mRNA expression between L1 to L4 was

found (Fig. 3B). However, IL-6 mRNA expression in L1 and L3 was significantly higher

than in L2 and L4.

In a non-infected lung, the KUL-01+, CD4+ and CD8α+ cells were spread throughout the

lung in the parabronchi, interparabronchial septa and lamina propria of larger airways.

KUL-01+ and CD8α+ cells were found closer to the lumen compared to CD4+ cells, and the

number of KUL-01+ cells exceeded that of CD4+ and CD8α+ cells (Fig. 3F).

After inoculation (Fig. 3C) at both 1 and 3 d.p.i. no significant differences in viral RNA

levels were found between L1 and L3 and between L2 and L4. Though, at both time points

the viral RNA level was higher in L1 and L3 than in L2 and L4, being significantly

different at 1 d.p.i. for L2 and at both time points for L4. The effect of uneven virus

distribution on IL-1β, IL-6 and IFN-β mRNA expression was shown at 3 d.p.i. (Fig. 3B).

IL-1β and IL-6 mRNA levels were similar in L1 and L3, but significantly higher than in L2

and L4. In all 4 parts of the lung IL-1β and IL-6 mRNA expression was significantly higher

in infected birds than in control birds. The IFN-β mRNA levels in infected birds were low

and not significantly different between L1 to L4, nor different from control birds.

A hierarchical clustering on both global and immune related gene expression was

performed on infected birds at 3 d.p.i. (Fig. 3D). In infected birds the same clustering was

observed as in control birds, but the clustering of L1-L3 and of L2-L4 in infected birds was

even stronger for both global and immune related genes, since the branches leading to

cluster L1-L3 and L2-L4 were longer and the branches leading to the individual lung parts

were shorter compared to the cluster tree in control birds.

Using immunocytochemistry virus deposition and cellular influxes in the lung parts were

studied (Fig. 3E). In both L1 and L3 virus infected areas were found in the primary and

secondary bronchi and adjacent parabronchi. At 3 d.p.i. more virus was located in the

parabronchi compared to 1 d.p.i. In L2 and L4 infected areas were located in the

parabronchi and an increase over time was seen. In L1 the largest infected areas were found

followed by L3, L4 and L2 were similar and contained fewer and smaller infected areas

(Fig. 3E). At 1 d.p.i. influxes of KUL-01+, CD4+ and CD8α+ cells were seen in both L1 and

L4 in the bronchi and parabronchi (Fig. 3F). Cellular influxes co-localised with virus


85

LL

LL

LL

LL

L1 L2 L3 L4

LL

LL

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LL

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NP KUL 01 ll CD4 ll CD8 ll

L1 L2L3 L4

Globalgenes

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Immune relatedgenes

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Chapter 5

86

Figure 3. A) Hierarchical clustering on expression of global and immune related genes in lung L1 to L4 in control

birds at 3 d.p.i. Genes in red were up regulated and in green down regulated. B) IL-1β, IL-6 and IFN-β mRNA

expression in lung L1 to L4 at 3 d.p.i. Bars with different letters were significantly different (p < 0.05). C) Viral

RNA levels in infected birds at 1 and 3 d.p.i. Bars within a time point with different letters were significantly

different (p < 0.05). D) Hierarchical clustering on expression of global and immune related genes in lung L1 to L4

in infected birds at 3 d.p.i. Genes in red were up regulated and in green down regulated. E) Cryosections of lung

L1 to L4 stained for viral NP at 3 d.p.i. The parabronchi of the different parts of the lung are affected to a different

extend. L indicates luminal side of a parabronchus. Bar, 75 μm. F) Cryosections of lung L1 stained for viral NP

and for KUL-01+, CD4+ and CD8α+ cells at 3 d.p.i. Upon infection, KUL-01+ cells (macrophages) change from

dendritic (arrows) to round morphology (arrowheads). Bar, 75 μm.

infected areas, and the number of cells positively correlated with the size of the virus

infected areas. At 1 d.p.i. KUL-01+ cells in infected areas were mainly round, whereas in

non-infected areas they were dendritic shaped. The number of KUL-01+, CD4+ and CD8α+

cell influxes increased over time.

Differences between lung L1 and L4

In both control and infected birds gene expression differed most between L1 and L4. These

significantly differentially expressed genes were depicted in a graph showing three major

GO categories and an immune related category (Fig. 4A). In control birds more genes were

expressed at a higher rate in L1 compared to L4 for all four categories. Immune related

genes that significantly differed between L1 and L4 were depicted in a heatmap (Fig. 4B).

Genes expressed in a higher rate in L1 were mainly involved in cell adhesion and motility

like VTN and TSPAN1, T cell and B cell related like ITFG1 and PIGR and involved in

chemotactic activities like CCL20, CX3CR1 and CHIA. In infected birds more genes were

expressed at a higher rate in L4 compared to L1 for the categories molecular function,

cellular component and biological processes, but not for immune related genes. Immune

related genes that significantly differed between L1 and L4 were depicted in a heatmap

(Fig. 4C). These genes were mainly involved in lymphocyte activation, apoptosis,

chemokine and interleukin activity. Genes expressed in a higher rate in L1 were mainly

involved in lymphocyte activation, mainly T cell related, such as CD3E, CD28 and ICOS,

and chemokine and interleukin activity like CCL19, SOCS1, CXCLi1, IL-1β, IL-7Rα and

IL-16. In L4 mainly genes involved in apoptosis, stress responses and MHC pathways were

more expressed compared to L1 like BCLX, BNIP3L, HSP70, HSPB2, MHC class II.

Induction of host responses against H9N2 in upper trachea and lung L1 and L4

To determine chicken host responses against H9N2 in the respiratory tract at transcriptional

level, immune related genes that were significantly differentially expressed between control

and infected birds were analysed. Most genes were significantly differentially expressed in

trachea and lung L1, respectively 244 and 272 immune related genes, whereas lung L4


87

Figure 4. Differences in responses between lung L1 and L4. A) Genes that significantly (p < 0.05) differed in

expression between L1 and L4 in control and infected birds at 3 d.p.i. are categorized into three major GO terms

and an immune related category. Heatmap of immune related genes significantly (p < 0.05) differentially

expressed in L1 compared to L4, B) in control birds and C) in infected birds. Genes expressed at a higher rate in

L1 are shown in red and at a higher rate in L4 in green.

significantly differentially expressed 86 immune related genes. Genes were divided into

functional groups based on GO interpretations of which the groups containing most genes

were depicted in a graph (Fig. 5). The complete gene sets were listed in the supplemented

data (Table S1-S3).

A shortened gene list, indicating the location in the respiratory tract at which genes were

significantly induced due to H9N2 infection, was depicted in Table 2. This list was based

on the functional groups shown in Figure 5 and depicted a global overview of the location

of expression and expression rate of genes (up or down) within a functional group. Most

(b) (c)

L1 L4L1 L4L1 L4

0 50 100 150

Immune related

GO biologicalprocess

GO cellularcomponent

GO molecularfunction

150# genes sign. diff. expressed

in L1 and L4

0 50 100 150

Immune related



GO molecularfunctionGO molecular function

GO Cellular component

GO Biological process

50 100 150# genes sign. diff. expressed

in L1 and L4

0

Control birds

GO molecular function



Immune related

50 100

Infected birds

0

Lung L1

Lung L4

Immune related

0 50 100 150

Immune related



GO molecularfunction

150# genes sign. diff. expressed

in L1 and L4

0 50 100 150

Immune related



GO molecularfunctionGO molecular function



50 100 150# genes sign. diff. expressed

in L1 and L4

0

Control birds

GO molecular function



Immune related

50 100

Infected birds

0

Lung L1

Lung L4

Lung L1

Lung L4

Immune related

A.

B. C.

Chapter 5

88

striking facts were that much fewer genes were differentially expressed in lung L4, and

most genes were up regulated independent of the location and functional group.

In trachea and both lung L1 and L4 genes involved in chemokine and cytokine signalling

were most common, sharing many similar genes. In contrast to the up regulation of

chemokine related genes, several genes related to growth factors were down regulated.

Of the genes involved in innate responses and interferon signalling, only the genes

negatively involved in interferon signalling (FLN29, USP18) and related to IFN-γ

signalling (IRF10) were expressed in trachea, lung L1 and L4. Other genes involved in

IFN-γ signalling (IFI35, MIME, IFI30), and type I interferon signalling (IRFs) were

expressed at certain locations rather than in trachea and both lung segments. Genes

involved in NF-κB signalling were expressed in trachea and L1, but in L4 no genes related

to NF-κB signalling were found.

Genes involved in antigen presentation were all up regulated. MHC class IV or B-G, which

is only expressed in lung L1 and ASB2, which was only expressed in trachea were down

regulated.

Up regulation of genes that supported an increased T cell response were mostly expressed

in lung L1 and trachea. Several genes were exclusively expressed in L1 and were involved

in co-stimulation (CD86, CTLA4, PDL2), T cell activation (ZAP70, TXK), T cell

proliferation IGSF2, (TNFRSF4) and CD3 signal transduction (TRAT1). Many genes

involved in B cell activity were expressed especially in lung L1 and trachea. All B cell

related genes were up regulated except for BRAG, which is expressed only in trachea, and

PIGR which were both down regulated. In lung L4, genes related to T and B cell responses

were rarely expressed.

Several genes involved in cell migration and adhesion were exclusively expressed in

trachea (CD9, THBS4) or in lung L1 (MAEA, VCAM1). Genes involved in monocyte

activity were equally expressed in all three parts of the respiratory tract, sharing similar

genes.

Infection with H9N2 caused mostly up regulation of genes involved in the regulation of

apoptosis mainly via caspase activity. Most genes involved in apoptosis were expressed in

trachea and lung L1, whereas in lung L4 few apoptosis related genes were expressed.

Several genes involving oxidative stress were up regulated, but mainly in trachea and lung

L1. Lung L4 only expressed p40-phox. Gene expression related to complement activity

mainly seemed to occur via the classical route. In L4 only CD93 and ITGB2 were

expressed.


89

Discussion To be able to study host responses to respiratory viruses, we first wanted to know whether

sampling certain locations would affect the outcome of the study. The aim of this study was

to determine if gene expression differed between upper and lower trachea and between

different parts of the lung in control birds, and whether this was affected by H9N2 AIV

infection, and secondly to study the early host responses against LPAI H9N2.

Besides the trachea and lung, other structures such as the paranasal organs, paraocular

Harderian gland (HG) and the conjunctiva associated lymphoid tissue come in contact with

aerosolized virus or particles (Corbanie et al., 2006). Although cells capable of rapid

responses against pathogens are present in these tissues (Kothlow and Kaspers, 2008), only

the humoral responses to pathogens have been described for the chicken. Upon infection

with infectious bronchitis virus or Newcastle disease virus an increase in virus-specific

IgA-antibody forming cells (AFC) in the HG is found (Van Ginkel et al., 2008; Russell et

al., 1993), whereas most IgG-AFC reside in the spleen (Russell et al., 1993). In mice

intranasal immunisation with influenza virus results in local long-term specific antibody

production. These AFC reside in larger numbers in the diffuse region rather than in the

organised region of the NALT (Liang et al., 2001; Etchart et al., 2006) showing that also

within the nasal compartment of mice sampling of specific parts of the tissue will results in

a different outcome as shown for antibody production, but which most likely will also be

found at host transcriptional level.

In our study, trachea was divided into upper and lower trachea as H9N2 LPAI has a

preference for infecting the upper part of the respiratory tract (Nili et al., 2002), which

coincided with our data at both 1 and 3 d.p.i.. However, only minor differences in gene

expression were seen between upper and lower trachea in both control and infected birds,

suggesting that gene expression within the trachea was not affected by a difference in virus

deposition.

Lung was divided into 4 pieces according to unidirectional airflow and lung anatomy. Lung

L1 was most similar to L3 and L2 resembled L4. In this paper we described that the

differences in anatomy and airflow were reflected upon gene expression in control birds

and upon infection with H9N2, host responses in the different parts of the lung were even

more diverse. Furthermore, airflow and anatomy affected virus deposition within the lung.

During inhalation, air flows through the primary bronchus, bypassing the cranially-located

openings of the medio-ventral secondary bronchi and flows into the caudal air sacs or

through the mediodorsal and lateroventral secondary bronchi (Fig. 1). This caudal to cranial

flow pattern is also evident during expiration. In the caudal part of L1 and L3, which

contained the bifurcations to the mediodorsal and lateroventral secondary bronchi, viral

Chapter 5

90

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Chapter 5

92

RNA levels were significantly higher and virus infected areas were larger than in L2 and

L4. This correlated with results of studies on deposition of airborne microspheres in the

avian respiratory tract, which indicate that airborne particles are not distributed uniformly

in the lung and deposition is influenced by particle size, size of the airway and that particles

are intercepted at bifurcations within the respiratory tract (Hayter et al., 1974; Mensah et

al., 1982; Corbanie et al., 2006). Differences in virus deposition enhanced in infected birds

the differences in gene expression already seen in control birds. This coincided with the

finding in the lung of macaques that differences in H1N1 influenza virus mRNA result in

differences in gene expression (Baas et al., 2006). A functional consequence of the flow

pattern in the avian lung resulting in an uneven spread of virus may explain the absence of

bronchus associated lymphoid tissue in the cranial part of the lung. Although organized

BALT nodules are not observed before the third week after hatching, a similar time course

of BALT development in SPF and conventional chickens was found (Fagerland et al.,

1993) and infections with pathogenic micro-organisms increase the number of BALT

nodules significantly (Van Alstine et al., 1988). This most likely resulted in the higher

expression rate of genes involved in cell adhesion and motility, chemotactic and interleukin

activities and T and B cell related genes in L1 compared to L4, in both control and infected

birds.

By analyzing host responses in lung L1 and L4 separately, gene expression patterns were

not diluted and could be used to define overall and site specific responses to H9N2

infection. After comparing gene expression in trachea, lung L1 and L4 we found that most

changes occurred in trachea and L1. Most genes expressed in L4 were also expressed in

trachea and L1 indicating these were part of a common host response, independent of the

amount viral RNA. Most of these shared responses are described previously as being part of

a common response to pathogens (Jenner et al., 2005; Pennings et al., 2008) and involve up

regulation of genes related to chemokine activity and inflammatory responses as described

in primate and rodent influenza models (Pennings et al., 2008). These corresponded with

increased IL-1β and IL-6 mRNA expression and the massive influx of KUL-01+ cells in

trachea and lung of infected birds. Trachea and L1 had overlap in gene expression that was

not shared by L4 indicating that these responses were affected by viral load.

Several site specific responses were measured upon infection. TLRs contribute to innate

responses of which TLRs 3, 7-9 recognise nucleosides that are important in viral

recognition. In chicken TLR9 has not been identified, but it is speculated that the two TLR7

splice variants (Philbin et al., 2005; Jenkins et al., 2009), TLR15 or TLR21, may elicit

responses to the mammalian TLR9 ligands. After infection with H9N2, up regulation of

TLR3 was found in trachea correlating to an increase in TLR3 mRNA in lung and brain of

chickens upon infection with high pathogenic AIV (Karpala et al., 2008). TLR1 and 7 were

up regulated in trachea, lung L1 and L4. Both TLR1 and TLR7 are up regulated in human


93

primary macrophages infected with influenza or Sendai virus (Miettinen et al., 2001)

indicating that up regulation of these genes is most likely part of a common host response

to viruses. TLR15 expression remains unchanged after in vitro H9N2 infection (Xing et al.,

2008) in contrast to our data showing that upon H9N2 infection in vivo TLR15 was up

regulated in L1.

Several genes were especially expressed in L1 and were involved in innate responses,

interleukin activity and vesicle trafficking. Molecules involved in innate immunity to

pathogens such as CLL1, PTX3 and GAL4 were only up regulated in L1. C-type lectins and

PTX3 are known to have antiviral activity against influenza (Hogenkamp et al., 2008;

Reading et al., 2008). In mice beta-defensins, like GAL4, are up regulated in the airway

epithelial cells due to influenza virus infection (Chong et al., 2008).

ANXA6, DDEF2, PICALM and ZFYVE20 are involved in vesicle trafficking like

endocytosis and phagocytosis and were only expressed in L1, and in contrast to most genes

they were down regulated. Changes in expression of these genes have not been described in

a virus infection model yet, although the endocytosis pathway is known to play an

important role in the entry of influenza virus into host cells (Lakadamyali et al., 2004).

Whether down regulation of these genes was induced by host cells to inhibit viral entry or

by antigen presenting cells to control dissemination of virus remains to be investigated.

Although not described in this paper, the avian respiratory tract also contains multiple air

sacs which work as bellows to enable ventilation of the lungs. Upon inoculation with IBV

or influenza virus decreased lucency of the air sacs and cellular influxes containing CD4+,

CD8+, γδ-TCR+ and KUL-01+ cells and heterophilic infiltrates are found (Perkins et al.,

2002; Matthijs et al., 2009). It is highly likely that the air flow also results in different

responses in the air sacs, because only certain air sacs will be exposed to the inhaled air

containing the virus.

To our knowledge we are the first to give an overview of host responses to AIV at

transcriptional level in the trachea and in the lung taking into account the effect of anatomy

and airflow through the respiratory tract. Gene expression within the lungs of control birds

already differed significantly. Moreover, airflow affected virus deposition and subsequently

the gene expression indicating that sampling at specific sites within the lung affects the

outcome of the study into respiratory infections in chickens. Changes in gene expression

and cellular influxes were significantly more pronounced in the parts of the respiratory tract

where virus deposition was highest. These findings suggest that not only responses against

avian influenza virus are affected locally, but most pathogens that are not evenly spread

throughout an organ will induce localised responses and careful sampling of the organ will

be essential.

Chapter 5

94

Acknowledgements

We thank Peter van de Haar, Dik van Leenen, Cheuk Ko and Linda Bakker for their

technical assistance and Dr Winfried Degen for the collaboration on the in vivo experiment.

This work was supported by a BSIK VIRGO consortium grant (Grant no. 03012), The

Netherlands.

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Supplementary data

Table S1. Expression of immune related genes induce by H9N2 infection in upper trachea 3 d.p.i.

Decription Ensembl ID Fold

change†

Cytokine/chemokine activity

Eosinophil chemotactic cytokine ENSGALG00000023760 0.02

Cytokine-like protein 1 ENSGALG00000015015 0.33

TNF receptor-associated factor 3 interacting protein 1 ENSGALG00000004236 0.44

Cytokine ENSGALG00000024470 0.46

Transforming growth factor beta-3 precursor ENSGALG00000010346 0.48


97

Growth hormone receptor precursor ENSGALG00000014855 0.54


Chemokine (C-C motif) receptor 2 ENSGALG00000011733 1.62


CX3C chemokine receptor 1 ENSGALG00000011955 1.74

CX3C chemokine ENSGALG00000021467 1.77

Lymphocyte antigen 75 ENSGALG00000011153 1.89

Chemokine (C-C motif) ligand 20 (MIP-3a) ENSGALG00000003003 1.99


Tumor necrosis factor (ligand) superfamily, member 13b (BAFF) ENSGALG00000016852 2.03

Granulocyte colony stimulating factor 3 receptor ENSGALG00000002112 2.20

Decoy receptor 3 ENSGALG00000006106 2.22

Small inducible cytokine B13 precursor ENSGALG00000010336 2.38

Platelet-activating factor receptor ENSGALESTG00000001511 2.83

N-myc-interactor ENSGALG00000012480 3.17


Chemokine ENSGALG00000024466 4.05




Interferon-induced GTP-binding protein Mx ENSGALG00000016142 6.16

Macrophage inflammatory protein 1-beta homolog (CCL4) ENSGALG00000000951 6.96

Lymphocyte antigen 96 precursor ENSGALG00000015648 7.19

K60 protein ENSGALG00000011668 8.93

2'-5'-oligoadenylate synthetase-like (OASL) ENSGALG00000013723 14.22

Chemokine ah221 ENSGALG00000014585 14.26

Apoptosis

Cell death activator CIDE-A (Cell death-inducing DFFA-like effector A) ENSGALG00000000018 0.23

Death-associated protein ENSGALG00000013002 0.50

Sprouty homolog 2 ENSGALG00000016906 0.58

Uveal autoantigen with coiled-coil domains and ankyrin repeats ENSGALG00000008143 0.59

RING finger protein 122 ENSGALG00000001640 0.63

Myeloid cell leukemia protein MCL-1 ENSGALG00000014628 1.41

Receptor (TNFRSF)-interacting serine-threonine kinase 1 ENSGALG00000012827 1.64

Apoptosis facilitator Bcl-2-like 14 protein ENSGALG00000011544 1.66

Tumor necrosis factor, alpha-induced protein 8-like protein 1 ENSGALG00000004266 1.78

TNF, alpha-induced protein 8 ENSGALG00000002196 1.85

Tumor necrosis factor receptor superfamily member 21 precursor ENSGALG00000016719 1.89

Fas ligand receptor soluble form ENSGALG00000006351 1.89

Initiator caspase ENSGALG00000008346 1.92

Caspase-7 precursor ENSGALG00000008933 1.93

Caspase 1 ENSGALG00000001049 2.09

BCL2-antagonist/killer 1 ENSGALG00000003182 2.26


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98

Granzyme A ENSGALG00000013548 2.61

Ras association domain family 2 ENSGALG00000000206 3.41

Pannexin-1 ENSGALG00000017213 8.28

T cell

Gallus gallus Thy-1 cell surface antigen (THY1) ENSGALG00000006751 0.47

CD82 antigen ENSGALG00000008044 0.53

Tumor necrosis factor receptor superfamily member 11A precursor ENSGALG00000012891 1.43

DNA-binding protein Ikaros ENSGALG00000013086 1.44

CD2-associated protein ENSGALG00000016720 1.57

Psychosine receptor ENSGALG00000010595 1.68

FYN binding protein ENSGALG00000003792 1.81


TNF receptor superfamily, member 18 ENSGALG00000001882 2.01

T-cell-specific surface glycoprotein CD28 homolog precursor ENSGALG00000008669 2.07

Inducible T-cell co-stimulator precursor ENSGALG00000008656 2.08

TCR gamma alternate reading frame protein isoform 2 ENSGALG00000019602 2.46

GTPase, IMAP family member 7 ENSGALG00000005136 2.59

Lymphocyte cytosolic protein 2 ENSGALG00000002113 2.63

T-cell surface glycoprotein CD3 epsilon chain ENSGALG00000007416 2.65

CD3D antigen, delta polypeptide ENSGALG00000007418 3.15

GTPase,IMAP family member 4 ENSGALG00000005650 3.44

Src-like-adaptor ENSGALG00000016234 3.59

Lymphocyte cytosolic protein 1 (L-plastin) ENSGALG00000016986 4.68

Interferon induced

Mimecan/Osteoglycin. ENSGALG00000004732 0.44

Interferon regulatory factor 3 (IRF-3) ENSGALG00000014297 1.48

Interferon-induced 35 kDa protein (IFP 35) ENSGALG00000002832 1.85


TRAF-type zinc finger domain containing 1 (FLN29) (TRAFD1) ENSGALG00000004802 2.06

Interferon regulatory factor 10 ENSGALG00000006448 3.71



Ubiquitin specific peptidase 18 ENSGALG00000013057 4.57

Interferon induced transmembrane protein 5 ENSGALG00000004243 7.19


Interleukin

Oncostatin-M specific receptor subunit beta precursor. ENSGALG00000003747 0.51

Interleukin 7 ENSGALG00000022798 1.36

Interleukin 22 receptor, alpha 1 ENSGALG00000004221 1.57

Interleukin-7 receptor alpha chain ENSGALG00000013372 2.04

Interleukin-18 receptor 1 precursor ENSGALG00000016786 2.30

Interleukin 10 receptor, beta ENSGALG00000015941 2.67


Interleukin 2 receptor, gamma ENSGALG00000005638 3.66


99


Interleukin 1 beta ENSGALG00000000534 12.89


B cell

Polymeric-immunoglobulin receptor precursor (PIGR) ENSGALG00000000919 0.22

N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase ENSGALG00000009704 0.59

B-cell CLL/lymphoma 6 (zinc finger protein 51) ENSGALG00000007357 1.39

BCR-associated protein Bcap29 ENSGALG00000008009 1.68

Pre-B-cell colony enhancing factor 1 ENSGALG00000008098 1.87

Tyrosine-protein kinase BTK ENSGALG00000004958 1.94

B-cell linker protein ENSGALG00000006973 2.02

Syndecan-4 precursor ENSGALG00000003932 2.17


Lysosomal-associated transmembrane protein 5 ENSGALG00000000562 3.50

Cell adhesion

Thrombospondin-4 ENSGALG00000014804 0.32

Hematopoietic progenitor cell antigen CD34 precursor ENSGALG00000001177 0.35


Cadherin-11 ENSGALG00000005278 0.39


Cell surface A33 antigen precursor ENSGALG00000015455 1.46

Zyxin ENSGALG00000014688 1.97

CD44-like protein ENSGALG00000007849 2.84

Heat shock

Heat shock 70kDa protein 4-like ENSGALG00000010185 1.46

HSP 105 ENSGALG00000017077 1.62

Heat shock 10kDa protein 1 (chaperonin 10) ENSGALG00000009070 1.63

78 kDa glucose-regulated protein precursor (Heat shock 70 kDa protein 5) ENSGALG00000001000 1.72

Heat shock cognate 70 ENSGALG00000006512 1.94

60 kDa heat shock protein, mitochondrial precursor ENSGALG00000008094 2.09

DnaJ (Hsp40) homolog, subfamily A, member 1 ENSGALG00000023066 2.14

Heat shock protein 25 ENSGALG00000023818 3.21

Antigen presentation/processing

Ankyrin repeat and SOCS box protein 2 ENSGALG00000010881 0.55

MHC class II M alpha chain (DMA) ENSGALG00000000158 1.87

Cathepsin B precursor ENSGALG00000016666 1.88

Tetraspanin-13 (Tspan-13) ENSGALG00000010819 1.94


MHC class I antigen ENSGALG00000000178 3.37

Tetraspanin-8 ENSGALG00000010152 3.68

Cathepsin C precursor ENSGALG00000017239 4.09

Complement

Complement C1q tumor necrosis factor-related protein 7 precursor ENSGALG00000014515 0.52

Complement C1q TNF-related protein 2 ENSGALG00000001471 0.54

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100

Complement component C7 precursor ENSGALG00000014835 2.09

Integrin, beta 2 (CD18 antigen) ENSGALG00000007511 2.21

Complement C1q subcomponent subunit C precursor ENSGALG00000023605 3.05

Complement C1q subcomponent subunit B precursor ENSGALG00000021569 3.13

Complement component 3a receptor 1 ENSGALG00000013218 3.71

Monocyte

CD200 receptor precursor ENSGALG00000019231 1.40

DAZ associated protein 1 ENSGALG00000015200 1.63

Regulator of G-protein signaling 18 (RGS18) ENSGALG00000021143 2.35

Immunoglobulin superfamily, member 6 ENSGALG00000007059 2.69

Cystatin-A ENSGALG00000014412 2.93

TNFAIP3-interacting protein 3 (ABIN-3) ENSGALG00000011961 3.71

TLR

Mitogen-activated protein kinase kinase kinase 8 ENSGALG00000007356 1.47

Myeloid differentiation primary response gene (MyD88) ENSGALG00000005947 1.52


Interferon (alpha, beta and omega) receptor 1 ENSGALG00000015942 1.72



NFkB pathway

TNF receptor-associated factor 1 ENSGALG00000001583 1.41

NF-kappa-B inhibitor epsilon ENSGALG00000010171 1.51

TRAF interacting protein ENSGALG00000002908 1.62

NF-kappa-B inhibitor alpha ENSGALG00000010063 1.83

Caspase recruitment domain-containing protein 9 ENSGALG00000001889 2.06

TNFAIP3 interacting protein 2 ENSGALG00000015666 2.68

Innate defense

Bactericidal permeability-increasing protein ENSGALG00000006756 3.72

Macrophage receptor with collagenous structure ENSGALG00000012119 4.23

Matrix metalloproteinase 7 ENSGALG00000017184 20.75

Avidin-related protein 2 precursor ENSGALG00000002441 22.07

Avidin precusor ENSGALG00000023622 68.36

JNK pathway

SPARC precursor ENSGALG00000004184 0.31

TRAF3-interacting JNK-activating modulator ENSGALG00000001373 1.40

Jun dimerization protein p21SNFT ENSGALG00000009816 4.11

Superoxide stress

Neutrophil cytosolic factor 1 ENSGALG00000001189 1.52

ERO1-like protein alpha precursor ENSGALG00000012404 1.60


NADPH oxidase organizer 1 (Nox organizer 1) ENSGALG00000005572 1.94

Migration

Lysosome-associated membrane glycoprotein 2 ENSGALG00000008572 0.56



101


Lysozym activity

Lysozyme C precursor ENSGALG00000009963 0.18

Lysozyme g precursor ENSGALG00000016761 2.13

Vesicule trafficking

Microtubule-associated proteins 1A/1B light chain 3C precursor ENSGALG00000010741 0.61

C-type lectin domain family 3, member B ENSGALG00000011883 0.20

NK cell

Peroxiredoxin-1(Natural killer cell-enhancing factor A) ENSGALG00000010243 4.57

Ig-like receptor

Similar to immunoglobulin-like receptor CHIR-B4 ENSGALG00000022376 2.08

Differentiation

CCAAT/enhancer-binding protein beta ENSGALG00000008014 2.17

Hemapoetic cell linage

Myeloid leukemia factor 1 ENSGALG00000009654 0.39

Respiratory gas exchange

Surfactant, pulmonary-associated protein A2 (SP-A1) ENSGALG00000002503 0.02

Miscellaneous

Surfactant, pulmonary-associated protein A2 (SP-A2 ENSGALG00000002496 0.07

Lactoperoxidase precursor ENSGALG00000000979 0.11

Uncharacterized protein CXorf41 (Sarcoma antigen NY-SAR-97) ENSGALG00000004870 0.19

CDNA FLJ44366 fis, clone TRACH3008629, weakly similar to Cadherin- related tumor suppressor (Hypothetical protein FLJ23834)

ENSGALG00000008109 0.20

Prostate stem cell antigen precursor ENSGALG00000016153 0.22

LIM and senescent cell antigen-like-containing domain protein 1 ENSGALG00000014148 0.35

Serine/threonine/tyrosine-interacting-like protein 1 ENSGALG00000001963 0.36

Podoplanin precursor ENSGALG00000004095 0.37

72 kDa type IV collagenase precursor (Matrix metalloproteinase-2) ENSGALG00000003580 0.38


Regulator of G-protein signalling 3 RGS3L isoform ENSGALG00000008881 0.47


Dual specificity phosphatase 26 ENSGALG00000001633 0.50

Rhombotin-1 ENSGALG00000005967 0.51

Beta-galactoside-binding lectin (C-16) ENSGALG00000011480 0.51

GDNF family receptor alpha-1 precursor ENSGALG00000009173 0.51

Ly-6 antigen/uPA receptor-like domain-containing protein ENSGALG00000012467 0.51

Sperm-associated antigen 16 protein ENSGALG00000003148 0.54

Heat shock factor protein 3 ENSGALG00000004644 0.57

Thymic dendritic cell-derived factor 1 ENSGALG00000010746 0.59

Metalloproteinase inhibitor 3 precursor ENSGALG00000012568 0.63

NADH dehydrogenase [ubiquinone] flavoprotein 3, mitochondrial precursor

ENSGALG00000022813 0.64

Platelet-derived growth factor D precursor ENSGALG00000017179 0.64

RAS-like family 11 member B ENSGALG00000013948 0.66

Calcium binding atopy-related autoantigen 1 ENSGALG00000004372 0.70

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102

Dolichyl-diphosphooligosaccharide--protein glycosyltransferase subunit DAD1

ENSGALG00000000196 0.75

Sarcoma antigen NY-SAR-48 isoform a ENSGALG00000003727 1.30

52 kDa repressor of the inhibitor of the protein kinase ENSGALG00000000838 1.33

NMDA receptor-regulated protein 1 ENSGALG00000009782 1.40

Sarcoma antigen NY-SAR-79 ENSGALG00000012103 1.42

RRP5 protein homolog (Programmed cell death protein 11) ENSGALG00000008265 1.48

Leptin receptor (CD295 antigen) ENSGALG00000011058 1.51

Hypothetical protein LOC422107 ENSGALG00000007713 1.53

Condensin complex subunit 3 ENSGALG00000014425 1.54

Putative RNA methyltransferase NOL1 ENSGALG00000014445 1.64

Small nuclear ribonucleoprotein Sm D1 ENSGALG00000011842 1.64

Sjogren syndrome antigen B ENSGALG00000009677 1.65

Fanconi anemia, complementation group L ENSGALG00000007879 1.69

Tissue factor precursor (CD142 antigen) ENSGALG00000005619 1.71

Ribosomal protein S6 kinase 2 alpha ENSGALG00000000366 1.75

SEC6-like protein C14orf73 ENSGALG00000011443 1.86

Proliferating cell nuclear antigen (PCNA) ENSGALG00000000169 1.97

Shugoshin-like 1 ENSGALG00000011281 2.05


Gallus gallus hypothetical LOC426928 (LOC426928), mRNA ENSGALG00000002869 2.21

Programmed cell death 1 ligand 1 precursor (CD274) ENSGALG00000015031 2.41

Targeting protein for Xklp2 ENSGALG00000006267 2.77

PHD finger protein 11 ENSGALG00000019094 2.96

Ankyrin repeat domain-containing protein 22 ENSGALG00000006315 3.07

Undescribed

genomic:1+100209867-100209940 genomic:1+100209867-100209940

0.12

ENSGALG00000001004 ENSGALG00000001004 0.32

ENSGALG00000024386 ENSGALG00000024386 0.34

ENSGALG00000007735 ENSGALG00000007735 0.48

ENSGALG00000003338 ENSGALG00000003338 0.51

ENSGALG00000002431 ENSGALG00000002431 0.54

genomic:Un_random-9337222-9337263 genomic:Un_random-9337222-9337263

0.68

genomic:14-4187818-4187887 genomic:14-4187818-4187887

1.92

genomic:2+863896-863967 genomic:2+863896-863967 1.95

ENSGALG00000000974 ENSGALG00000000974 2.19

genomic:2+61818365-61818434 genomic:2+61818365-61818434

2.54

ENSGALG00000010338 ENSGALG00000010338 2.60

ENSGALESTG00000000019 ENSGALESTG00000000019 2.64

ENSGALG00000001478 ENSGALG00000001478 2.80

ENSGALG00000011806 ENSGALG00000011806 3.21

ENSGALG00000006384 ENSGALG00000006384 17.21

† Expression ratio in upper trachea of infected birds compared to expression ratio in upper trachea of naïve birds


103

Table S2. Expression of immune related genes induce by H9N2 infection in lung L1 at 3 d.p.i..


change†




Tumor necrosis factor (ligand) superfamily, member 10 (TRAIL) ENSGALG00000009179 0.57


Attractin precursor ENSGALG00000016034 0.67

Protein FAM3B precursor (Cytokine-like protein 2-21) ENSGALG00000016140 0.68

Lymphocyte antigen 75 ENSGALG00000011153 1.45


Chemokine (C-X-C motif) receptor 4 ENSGALG00000012357 1.53


Decoy receptor 3 ENSGALG00000006106 1.68

C-C chemokine receptor type 11 ENSGALG00000011706 1.68

Chemokine K203 ENSGALG00000000956 1.76


LPS-induced TNF-alpha factor homolog ENSGALG00000003217 2.02

Lymphotactin ENSGALG00000015235 2.12


K60 protein ENSGALG00000011668 2.30

Lymphocyte antigen 86 precursor (MD-1 protein) ENSGALG00000012801 2.44

Tumor necrosis factor (ligand) superfamily, member 13b (BAFF) ENSGALG00000016852 2.54


Chemokine ENSGALG00000024466 2.77


Platelet-activating factor receptor ENSGALESTG00000001511 3.21


Chemokine (C-C motif) ligand 19 (MIP-3-beta) ENSGALG00000005851 4.47



Macrophage inflammatory protein 1-beta homolog (CCL4) ENSGALG00000000951 6.97






T cell

T-cell immunomodulatory protein ENSGALG00000004049 0.63

Immunoglobulin superfamily member 2 precursor ENSGALG00000015467 0.68

E3 ubiquitin-protein ligase CBL-B ENSGALG00000015351 1.45

Tumor necrosis factor receptor superfamily member 11A precursor ENSGALG00000012891 1.45


Chapter 5

104

Tyrosine-protein kinase ZAP70 ENSGALG00000001486 1.71

CD8 alpha chain ENSGALG00000015816 1.80

Psychosine receptor ENSGALG00000010595 1.81

T lymphocyte activation antigen CD86 precursor ENSGALG00000014362 1.82

Tumor necrosis factor receptor superfamily member 4 precursor ENSGALG00000001875 1.82

CD8b antigen ENSGALG00000015902 1.87

DNA-binding protein Ikaros ENSGALG00000013086 1.89

T-cell receptor-associated transmembrane adapter 1 ENSGALG00000015362 2.01

Tyrosine-protein kinase TXK ENSGALG00000014127 2.02



GTPase,IMAP family member 4 ENSGALG00000005650 2.31

Cytotoxic T-lymphocyte-associated protein 4 ENSGALG00000008666 2.44

Inducible T-cell co-stimulator precursor ENSGALG00000008656 2.47

TNF receptor superfamily, member 18 ENSGALG00000001882 2.73


GTPase, IMAP family member 7 ENSGALG00000005136 2.88

T-cell surface glycoprotein CD3 epsilon chain ENSGALG00000007416 2.89


CD3D antigen, delta polypeptide ENSGALG00000007418 3.19


T-cell-specific surface glycoprotein CD28 homolog precursor ENSGALG00000008669 3.79


Apoptosis


Death-associated protein ENSGALG00000013002 0.60

TNF-related apoptosis inducing ligand-like protein ENSGALG00000007240 0.61

Cell death activator CIDE-A ENSGALG00000000018 0.70

RING finger protein 122 ENSGALG00000001640 0.73

Receptor (TNFRSF)-interacting serine-threonine kinase 1 ENSGALG00000012827 1.45

BCL2-antagonist/killer 1 ENSGALG00000003182 1.52


Tumor necrosis factor, alpha-induced protein 8-like protein 1 ENSGALG00000004266 1.70





Serine/threonine kinase 17b ENSGALG00000007924 2.27

Inhibitor of apoptosis protein ENSGALG00000017186 2.59

Apoptosis facilitator Bcl-2-like 14 protein ENSGALG00000011544 2.77

BCL2-related protein A1 ENSGALG00000006511 4.09

Granzyme A ENSGALG00000013548 4.57



105

Interleukin

Interleukin 1 receptor-like 1 isoform LV ENSGALG00000016785 1.45

Interleukin-22 precursor ENSGALG00000009904 1.47

Interleukin 21 receptor ENSGALG00000006318 1.59



Interleukin 15 precursor ENSGALG00000009870 1.76

Sushi domain-containing protein 3. ENSGALG00000005231 1.97

Interleukin 10 receptor, beta ENSGALG00000015941 2.03



Interleukin-18 receptor 1 precursor ENSGALG00000016786 3.08


Interleukin-18 ENSGALG00000007874 3.62





Interferon induced

Interferon-related developmental regulator 1 ENSGALG00000009448 0.56

Mimecan/Osteoglycin. ENSGALG00000004732 0.67


Interferon (alpha, beta and omega) receptor 1 ENSGALG00000015942 1.64


Interferon-induced 35 kDa protein (IFP 35) ENSGALG00000002832 1.89





Gamma- interferon-inducible protein IP-30 ENSGALG00000003389 3.75



Antigen presentation

MHC B-G antigen isoform 1 precursor ENSGALG00000024357 0.49

MHC class IV antigen ENSGALG00000024365 0.57

L-amino-acid oxidase ENSGALG00000000081 1.75


MHC class II beta chain ENSGALG00000000141 2.06




Bone marrow stromal cell antigen 1 ENSGALG00000014509 2.94

Cathepsin B precursor ENSGALG00000016666 3.81


Chapter 5

106

B cell

Polymeric-immunoglobulin receptor precursor (PIGR) ENSGALG00000000919 0.39


Kelch-like 6 ENSGALG00000002263 1.45

BCR-associated protein Bcap29 ENSGALG00000008009 1.60

Syndecan-4 precursor ENSGALG00000003932 1.69

Raftlin ENSGALG00000011241 1.72


B6.1 ENSGALG00000015461 2.64

B-cell CLL/lymphoma 6 ENSGALG00000007357 2.79



Innate defence

Collectin sub-family member 10 ENSGALG00000016113 0.50

Beta-galactoside-binding lectin (14 kDa lectin) ENSGALG00000012420 0.62

Gallinacin-4 precursor ENSGALG00000019843 1.53

Macrophage receptor with collagenous structure ENSGALG00000012119 3.09


Pentraxin-related PTX3 ENSGALG00000009694 4.86




Adhesion



cAMP-dependent protein kinase type II-beta regulatory subunit ENSGALG00000008060 0.65

Catenin (cadherin-associated protein), beta 1, 88kDa ENSGALG00000011905 0.71

Lectin, galactoside-binding, soluble, 3 (galectin 3) ENSGALG00000012173 1.43

Alpha 4 integrin (CD49D) ENSGALG00000008978 1.66

Zyxin ENSGALG00000014688 2.29

Gallus gallus vascular cell adhesion molecule 1 (VCAM1) ENSGALG00000005257 2.75


NF-kB pathway

Nuclear receptor coactivator 6 ENSGALG00000001182 0.46

Receptor-interacting serine/threonine-protein kinase 4 ENSGALG00000016145 0.62


NF-kappa-B inhibitor epsilon ENSGALG00000010171 1.67

TNFAIP3-interacting protein 3 (ABIN-3) ENSGALG00000011961 1.71


TNFAIP3 interacting protein 2 ENSGALG00000015666 1.78


Caspase recruitment domain-containing protein 9 ENSGALG00000001889 2.52

Complement



107

CD59 glycoprotein precursor ENSGALG00000024468 0.58


Complement component 1, s subcomponent ENSGALG00000014603 1.53


Complement C1q subcomponent subunit C precursor ENSGALG00000023605 3.98

Complement C1q subcomponent subunit B precursor ENSGALG00000021569 4.44

Complement component 3a receptor 1 ENSGALG00000013218 5.02

JNK pathway

TRAF3-interacting JNK-activating modulator ENSGALG00000001373 0.28


Dual specificity protein phosphatase 22 ENSGALG00000012829 0.61

Dual specificity protein phosphatase 14 ENSGALG00000005455 0.68

C-jun-amino-terminal kinase-interacting protein 1 ENSGALG00000008430 1.47


Monocyte

Macrophage erythroblast attacher. ENSGALG00000013310 0.69

Triggering receptor expressed on myeloid cells 2 ENSGALG00000023781 2.04


DAZ associated protein 1 ENSGALG00000015200 2.40



Hemapoetic lineage

Myeloid leukemia factor 1 ENSGALG00000009654 0.49

ADP-ribosyl cyclase 1 (Lymphocyte differentiation antigen CD38) ENSGALG00000014508 1.86


SH2 domain-containing protein 1A (SAP) ENSGALG00000008447 2.25

Transcription factor MAFB ENSGALG00000003670 2.25

Vesicule trafficking

C-type lectin domain family 3, member B ENSGALG00000011883 0.42

Phosphatidylinositol-binding clathrin assembly protein ENSGALG00000013256 0.58

Annexin A6 ENSGALG00000004357 0.60

Rabenosyn-5 ENSGALG00000008500 0.66

Development and differentiation-enhancing factor 2 ENSGALG00000016419 0.71

Heat shock

DnaJ (Hsp40) homolog, subfamily B, member 9 ENSGALG00000009492 0.61

DnaJ (Hsp40) homolog, subfamily C, member 7 ENSGALG00000003449 1.37

Heat shock cognate 70 ENSGALG00000006512 1.48


Oxidative stress

ERO1-like protein alpha precursor ENSGALG00000012404 1.37

Transcription factor MafF ENSGALG00000012277 1.92


Extracellular superoxide dismutase [Cu-Zn] precursor ENSGALG00000018557 2.43

Chapter 5

108

TLR





Ig-like receptor

Similar to immunoglobulin-like receptor CHIR-AB3 ENSGALG00000017760 1.58

Similar to immunoglobulin-like receptor CHIR-B2 precursor ENSGALG00000000134 1.76

Similar to immunoglobulin-like receptor CHIR-AB1 ENSGALG00000018703 2.12

Differentiation

Early growth response protein 4 ENSGALG00000016086 1.36

Dual specificity mitogen-activated protein kinase kinase 2 ENSGALG00000001267 1.91


NK cell

Class-I MHC-restricted T cell associated molecule ENSGALG00000006527 1.47

Peroxiredoxin-1 (natural killer-enhancing factor A) ENSGALG00000010243 2.19

Migration


LIM and senescent cell antigen-like domains 2 ENSGALG00000001995 0.55

Miscellaneous


CDNA FLJ44366 fis, clone TRACH3008629, weakly similar to Cadherin- related tumor suppressor

ENSGALG00000008109 0.35


Gallus gallus similar to RDC1 like protein (LOC428997), mRNA ENSGALG00000021031 0.47

Integrin alpha-1 ENSGALG00000014891 0.49

72 kDa type IV collagenase precursor (Matrix metalloproteinase-2) ENSGALG00000003580 0.49

Similar to G protein-coupled receptor, family C, group 5, member A [Gallus gallus]

ENSGALG00000011796 0.50

Ras association (RalGDS/AF-6) domain family 3 ENSGALG00000009842 0.50

Prostate stem cell antigen precursor ENSGALG00000016153 0.50

SH3 domain containing, Ysc84-like 1 ENSGALG00000016362 0.51

Beta-galactoside-binding lectin (C-16) ENSGALG00000011480 0.58


Similar to annexin VIII; VAC beta [Gallus gallus] ENSGALG00000005956 0.60

T-cell lymphoma breakpoint associated target protein 1 ENSGALG00000014844 0.61

Kit ligand precursor ENSGALG00000011206 0.62

Collectin sub-family member 11 ENSGALG00000016396 0.63

Serine/threonine/tyrosine-interacting-like protein 1 ENSGALG00000001963 0.66

Troy-long ENSGALG00000017119 0.66


Sprouty homolog 2 ENSGALG00000016906 0.68

Grave disease carrier protein ENSGALG00000004065 0.68

Calpain 8 (NCL-2) ENSGALG00000009382 0.68

Gallus gallus acireductone dioxygenase 1 (ADI1), Mrna ENSGALG00000019885 0.72


109

ATP-binding cassette sub-family G member 2 ENSGALG00000005792 0.73

SH3 domain-binding protein 1 3BP-1 ENSGALG00000012422 1.40

Platelet receptor Gi24 precursor ENSGALG00000004673 1.41

hypothetical protein LOC422107 ENSGALG00000007713 1.41

Putative RNA methyltransferase NOL1 ENSGALG00000014445 1.46

Ribosomal protein S6 kinase 2 alpha ENSGALG00000000366 1.51

B-cell CLL/lymphoma 11A ENSGALG00000007866 1.90


Transmembrane 7 superfamily member 4 ENSGALG00000016075 2.32


Undecribed

ENSGALG00000006699 ENSGALG00000006699 0.26

genomic:1+100209867-100209940 genomic:1+100209867-100209940

0.32

ENSGALG00000007735 ENSGALG00000007735 0.42

ENSGALG00000002431 ENSGALG00000002431 0.53

ENSGALG00000024386 ENSGALG00000024386 0.54

ENSGALG00000011796 ENSGALG00000011796 0.59

ENSGALG00000008759 ENSGALG00000008759 0.61

ENSGALG00000014349 ENSGALG00000014349 0.62

ENSGALG00000006229 ENSGALG00000006229 1.38

ENSGALG00000003338 ENSGALG00000003338 1.48

ENSGALG00000023017 ENSGALG00000023017 1.52

ENSGALG00000013546 ENSGALG00000013546 1.70

ENSGALG00000014659 ENSGALG00000014659 1.85



genomic:2+863896-863967 genomic:2+863896-863967 2.31

ENSGALG00000023540 ENSGALG00000023540 2.49



ENSGALG00000001478 ENSGALG00000001478 2.81

ENSGALG00000000974 ENSGALG00000000974 3.01

genomic:2+61818365-61818434 genomic:2+61818365-61818434

3.15

ENSGALG00000010338 ENSGALG00000010338 5.25

ENSGALG00000009103 ENSGALG00000009103 5.76

ENSGALG00000006384 ENSGALG00000006384 12.82

† Expression ratio in lung L1 of infected birds compared to expression ratio in lung L1 of naïve birds

Chapter 5

110

Table S3. Expression of immune related genes induce by H9N2 infection in lung L4 3 d.p.i.


change†



Chemokine K203 ENSGALG00000000956 1.70



Platelet-activating factor receptor (PAF-R) ENSGALESTG00000001511 2.47





Lymphocyte antigen 86 precursor (MD-1 protein) ENSGALG00000012801 4.37






Interferon induced






Gamma- interferon-inducible protein IP-30 ENSGALG00000003389 2.96


Antigen presentation


L-amino-acid oxidase ENSGALG00000000081 2.08




Bone marrow stromal cell antigen 1 ENSGALG00000014509 3.37

Monocyte

Triggering receptor expressed on myeloid cells 2 ENSGALG00000023781 1.92

Protein PTDSR ENSGALG00000001802 1.91

CD200 receptor precursor ENSGALG00000019231 1.87




Apoptosis





111

BCL2-related protein A1 ENSGALG00000006511 3.24


B cell

B-cell CLL/lymphoma 6 ENSGALG00000007357 1.86




T cell





Adhesion



Annexin A1 ENSGALG00000015148 3.76

Differentiation

Early growth response protein 4 ENSGALG00000016086 1.46

Dual specificity mitogen-activated protein kinase kinase 2 ENSGALG00000001267 1.92


Heat shock

HSP 105 ENSGALG00000017077 1.75

Heat shock 70 kDa protein ENSGALG00000011715 4.49


Innate defense




TLR




Complement

Complement component C1q receptor precursor (CD93 antigen) ENSGALG00000008359 0.53


Hemapoetic cell linage

Cystatin-F precursor ENSGALG00000008660 1.51


Interleukin



JNK pathway


Chapter 5

112

Migration


Oxidative burst


Miscellaneous



T-cell lymphoma breakpoint associated target protein 1 ENSGALG00000014844 0.59

Targeting protein for Xklp2 ENSGALG00000006267 0.61

Fanconi anemia, complementation group L ENSGALG00000007879 0.63

ENSGALG00000006152 ENSGALG00000006152 1.67


Glioma pathogenesis-related protein ENSGALG00000010224 1.94

Transmembrane 7 superfamily member 4 ENSGALG00000016075 2.94

Undescribed

ENSGALG00000023540 ENSGALG00000023540 1.90

ENSGALG00000000974 ENSGALG00000000974 2.30

ENSGALG00000001478 ENSGALG00000001478 2.33


ENSGALG00000009103 ENSGALG00000009103 6.51

ENSGALG00000006384 ENSGALG00000006384 8.73

† Expression ratio in lung L4 of infected birds compared to expression ratio in lung L4 of naïve birds

Lack of immune activation as a correlate of protection

after challenge with avian influenza virus: a

transcriptomics analysis of adjuvanted vaccines

Sylvia S. Reemers1, Christine Jansen1, Marian J. Groot Koerkamp2, Daphne van Haarlem1,

Peter van de Haar1, Winfried Degen3, Willem van Eden1, Lonneke Vervelde1

1 Department of Infectious Diseases and Immunology, Faculty Veterinary Medicine, Utrecht University, Yalelaan

1, 3584 CL Utrecht, The Netherlands 2 Genomics Laboratory, Department of Physiological Chemistry, Utrecht Medical Centre, Universiteitsweg 100,

3584 CG Utrecht, The Netherlands 3 Intervet Schering-Plough Animal Health, Wim de Körverstraat 35, 5831 AN Boxmeer, The Netherlands

Submitted for publication

Chapter 6

Lack of immune activation relates to protection after AIV challenge

115

Abstract

To gain more insight in underlying mechanisms correlating to protection against avian

influenza virus (AIV) infection, we investigated correlates of protection after AIV H9N2

infection and studied the contribution of different adjuvants to a protective response at host

transcriptional level. One-day-old chickens were immunised with inactivated H9N2

supplemented with w/o, Al(OH)3, CpG or without adjuvant. Two weeks later, birds were

homologously challenged and at 1 to 4 days post challenge (d.p.c.) trachea and lung were

collected. Birds immunised with H9N2+w/o or H9N2+Al(OH)3 were protected against

challenge infection based on low viral RNA expression. Microarray analysis showed that in

protected birds the number of immune related genes induced after challenge and the

amplitude of change of gene expression were lower compared to unprotected birds. The

gene expression profile of protected birds showed that expression was especially up

regulated at 1 d.p.c., while in unprotected birds higher and prolonged gene expression was

found. This suggests that lack of immune activation correlates to protection.

Immunocytochemical examination of the lung confirmed the microarray data; smaller

cellular influxes were found in protected birds which correlated to the number and location

of virus infected cells. In conclusion, we show that in addition to a limited number of

differentially expressed genes correlating with protection, such as CCL20, IAP and CD59,

lack of immune activation is the most important correlate of protection after challenge with

AIV.

Introduction

Avian influenza virus (AIV) infection causes problems world wide and affects both humans

and animals. Therefore, much effort is invested in development of new vaccines and

improvement of vaccine efficacy of which vaccine adjuvants are one aspect. Vaccine

adjuvants are molecules with immune stimulating properties and enhance the

immunogenicity of a co-administered antigen. There is a large variety of adjuvants such as

inorganic salts like aluminium hydroxide (Al(OH)3), emulsions like water-in-oil (w/o) and

synthetic products like non-methylated CpG oligonucleotides (CpG), which enhance

immunogenicity via diverse and often not fully understood mechanisms. Recent findings

show that adjuvants act either Toll-like receptor (TLR) independent like Al(OH)3 and w/o

or TLR dependent like CpG. This difference in mode of action consequently results in a

difference in immune activation and induction of a protective host response (Ishii et al.,

2007).

The use of aluminium salts and oil emulsions as adjuvants in human and veterinary

vaccines is generally associated with induction of Th2 responses (Cox et al., 1997, Hilgers

et al., 1998, Hogenesch, 2002, Marrack et al., 2009). Some studies have suggested that

Chapter 6

116

aluminium salts and oil emulsions act as a depot slowly releasing the antigen at the site of

injection (Aucouturier et al., 2001, Tritto et al., 2009). Both types of adjuvants have the

ability to enhance antigen uptake and activation of mammalian APC, and induce

inflammation which stimulates recruitment of eosinophils, neutrophils, APC and

lymphocytes (Aucouturier et al., 2001, Marrack et al., 2009). Although aluminium salts and

oil emulsions do not act directly on dendritic cells (DC), they promote a pro-inflammatory

environment which may indirectly activate DC in a TLR independent manner (Tritto et al.,

2009). In birds vaccinated against O. rhinotracheale, addition of w/o or aluminium salts

inhibits pathology of the airways and results in an increased haemagglutination inhibition

(HI) titre especially in w/o adjuvanted birds (Murthy et al., 2007). Vaccination with a w/o

adjuvanted inactivated H5N1 vaccine results in an increased HI titer, a strong secondary

antibody response indicated by a high HI titer after challenge and protection from clinical

signs and mortality (Sasaki et al., 2009).

CpG has been known to induce Th1 responses leading to a protective cytotoxic response in

a TLR dependent manner in mammals acting directly on DC (Ishii et al., 2007). CpG

induces the enhancement of virus-specific antibodies, APC function and the production of

cytokines and chemokines supportive for cellular antigen specific immunity in mice

(Dalpke et al., 2001, Klinman et al., 2004). In chicken addition of CpG to recombinant

plasmids DNA vaccine encoding the VP2 gene of infectious bursal disease virus (IBDV)

induces an increased antigen specific serum antibody titer, increased lymphoproliferation

and provides protection against gross lesions, clinical signs and mortality (Mahmood et al.,

2006). Addition of CpG to E. coli bacterin also induces an increased antigen specific serum

antibody titer, reduces the number of bacteria from the airsacs, pericardium and cellulites

and provides protection against mortality (Gomis et al., 2007).

In this study we set out to investigate correlates of protection after challenge with the AIV

H9N2 and study the contribution of different adjuvants to this protective immune response.

Therefore, chickens were immunised with H9N2 vaccine in presence of w/o, Al(OH)3, CpG

or without adjuvant, and these birds together with non-immunised birds were challenged

with live H9N2 14 days post immunisation. At 1 to 4 days post challenge (d.p.c.) viral

RNA expression, gene expression profiles and cellular influxes were determined in trachea

and lung. Together with HI and AIV nucleoprotein (NP)-specific antibody titers in serum

we defined characteristics of protective responses and differences in the mode of action

between the different adjuvants. Birds immunised in presence of w/o and Al(OH)3 were

protected and had similar correlates of protection that related to a limited number of

differentially expressed genes and to lack of immune activation and high HI and AIV NP-

specific antibody titers


117


Infection model


kindly provided by Intervet/Schering-Plough Animal Health.

One-day-old White Leghorn chickens were housed under SPF conditions and all

experiments were carried out according to protocols approved by the Animal Welfare

Committee.

Chickens were divided into 6 groups over 6 isolators, containing 20 animals per group.

One-day-old chickens were immunised s.c. with 0.25 ml inactivated AIV H9N2

(Intervet/Schering-Plough Animal Health) supplemented with w/o, Al(OH)3, CpG or

without adjuvant. The non-immunised-non-challenged (NINC) and non-immunised-

challenged (NIC) groups were immunised s.c. with saline. The w/o adjuvant was kindly

provided by Intervet/Schering-Plough Animal Health. Al(OH)3 consisted of an Al(OH)3-gel

(Brenntag) with a final concentration in the vaccine of 0.5%. CpG was custom made and

consisted of the whole phosphorothioate backbone purified via ethanol precipitation

(TibMolbiol). Two weeks after the immunization blood samples were taken from each bird

for measuring HI titers and AIV NP-specific antibody titers after which they were

inoculated via aerosol spray with 20 ml 107.7 EID50 H9N2 AIV per isolator. The NINC

group was inoculated via aerosol spray with 20 ml saline. Chickens remained in the aerosol

spray in a closed isolator for 10 min, after which the isolator was ventilated as before. This

led to the following 6 experimental groups: non-immunised-non-challenged (NINC), non-

immunised and H9N2 challenged (NIC), H9N2 immunized and challenged (IC), H9N2 +

adjuvant w/o immunized and challenged (IC+w/o), H9N2 + adjuvant Al(OH)3 immunized

and challenged (IC+Al(OH) 3), H9N2 + adjuvant CpG immunized and challenged

(IC+CpG).

At 1 to 4 days post challenge (d.p.c.) chickens were killed (n = 4 per time point per group;

n=2 for NIC at 4 d.p.c. due to death caused by infection of 2 birds) and trachea and left lung

were isolated and stored in RNAlater (Ambion) at -80°C for RNA isolation or fixed in

liquid nitrogen for immunocytochemistry. The trachea was divided into upper and lower

trachea of which upper trachea was used for analysis. The lung was divided into 4 parts L1

to L4 according to anatomy and airflow (Fig. 1). Segment L1, containing the primary

bronchus and secondary bronchi, and L4, containing the paleopulmonic parabronchi, were

used for analysis. Selection of organ parts used for analysis was based on viral load and

virus induced gene expression as previously described (Reemers et al., 2009a).

Chapter 6

118

A. B. C.

Figure 1. A) “Right chicken lung with a blunt probe indicating the localization of the primary bronchus.” The lung

was divided in 4 parts, L1 to L4 according to airflow and lung anatomy (27), with the primary bronchus entering

the lung in L1 shown on the left side. Segment L1, containing the primary bronchus and secondary bronchi, and

L4, containing the paleopulmonic parabronchi, were used for analysis. The white blocks indicate the part of the

lung segments used for RNA isolation, while the adjacent part within the segment was used for

immunocytochemistry. “Illustration of the unidirectional air flow pattern, which flows from caudal to cranial

during B) inspiration and C) expiration in the avian lung. 1. Primary bronchus, 2. lung, 3. clavicular air sac, 4.

cranial thoracic air sac, 5. caudal thoracic air sac, 6. abdominal air sac.” Adapted and reprinted with permission

(Kothlow et al., 2008), Copyright 2008.

H9-specific HI and AIV NP-specific antibody titers

H9-specific haemagglutination inhibition (HI) titers in serum were determined by a HI

assay as previously described (Degen et al., 2006).

AIV NP-specific antibody titers in serum were determined by enzyme-linked

immunosorbent assay (ELISA). Briefly, Nunc Maxisorp flatbottom plates were coated with

mouse monoclonal antibody (mAb) to nucleoprotein (NP) of H9N2 at 37ºC overnight.

Antibody coated plates were washed three times with PBS/Tween and blocked with 10 mM

PBS + 1% BSA for 1h at room temperature. Formalin inactivated AIV H9N2 antigen was

added and incubated for 1h at 37ºC after which plates were washed three times with

PBS/Tween. Chicken sera were 2-fold serially diluted in PBS/Tween and incubated for 1h

at 37ºC. Plates were washed with PBS/Tween and incubated for 30 minutes at 37ºC with

HRP-conjugated rabbit anti-chicken IgG (Nordic Immunological Laboratories). After

washing with PBS/Tween plates were developed with TMB substrate for 10 minutes at

room temperature in the dark. The reaction was stopped by adding 4N H2SO4 and the

absorbance was measured at 450 nm.

Immunocytochemistry

Virus and cellular influxes were detected in lung by immunocytochemistry as previously

described (Vervelde et al., 1996). Viral NP was detected with mAb to NP of H9N2

(provided by Intervet/Schering-Plough Animal Health). Macrophages and CD4+ cells were

detected with mAb KUL-01 (Mast et al., 1998) and CT-4 (Southern Biotech). For detection

of CD8α+ cells a mix of mAb EP72 (Southern Biotech) and AV14 (kind gift of Dr T.F.

Davison, Institute for Animal Health, Compton UK (Withers et al., 2005)) was used to

L2

L1 L3

L4

Primary bronchus

6

1 2

3

5

Inspiration

1

2

3

5

6

Expiration

4 4


119

avoid differences in staining due to polymorphism in the chicken CD8α molecule (Breed et

al., 1996, Luhtala et al., 1997).

RNA isolation

Total RNA was isolated from trachea (5 mm part) and lung L1 and L4 (1x5 mm part) using

the RNeasy Mini Kit (Qiagen) as previously described (Reemers et al., 2009b). All RNA

samples were checked for quantity using a spectrophotometer (Shimadzu) and quality using

a 2100 Bioanalyzer (Agilent Technologies).


cDNA was generated from 500 ng RNA with reverse transcription using iScript cDNA

Synthesis Kit (Biorad Laboratories B.V).

Real-time qRT-PCR was used for detection of GAPDH and H9 haemagglutinin (HA)

products as previously described (Reemers et al., 2009b). Expression of GAPDH mRNA,

which was used as a reference gene for correction of viral RNA expression, was not

affected by H9N2 AIV (data not shown). Corrections for variation in RNA preparation and

sampling were performed as previously described (Eldaghayes et al., 2006). Results are

expressed in terms of the threshold cycle value (Ct) and given as corrected 40-Ct values.

To determine the statistical significance between groups and time points of trachea and lung

an ANOVA with a Tukey post-hoc test was used. A p-value < 0.05 was considered

significant.


Microarray analysis was performed as previously described (Reemers et al., 2009a) using

the Gallus gallus Roslin/ARK CoRe Array Ready Oligo Set V1.0 (Operon

Biotechnologies). All trachea and lung samples were co-hybridised with respectively a

trachea or lung reference sample. These reference samples consisted of pooled RNA

extracted from tracheas or lungs of 4 chickens that were not included in the inoculation

experiment.

Microarray arrays were analysed as previously described (Reemers et al., 2009a). Ensembl

Gallus gallus, (assembly: WASHUC2, May 2006, genebuild: Ensembl, Aug 2006, database

version: 47.2e) was used for gene names, description and Gene Ontology (GO) annotations.

For pathway analysis Database for Annotation, Visualization and Integrated Discovery

(DAVID) 2008 was used.

Chapter 6

120

Microarray data accession numbers

Primary data are available in the public domain through ArrayExpress at

http://www.ebi.ac.uk/microarray-as/ae/ under accession numbers E-MTAB-136 for L1, E-

MTAB-137 for L4 and E-MTAB-138 for upper trachea.

Results

Effect of adjuvants on protection against challenge with avian influenza virus

To determine the protective effect of the adjuvants, viral RNA expression was measured

using real-time qRT-PCR (Fig. 2). IC+w/o and IC+Al(OH)3 birds had significantly lower

(p<0.05) viral RNA expression in upper trachea, L1 and L4 compared to unprotected NIC

and IC birds. There were no significant differences in the viral RNA levels in IC+CpG

compared to unprotected NIC and IC birds at any time point. The viral RNA levels were

significantly higher (p<0.05) in all unprotected groups in lung L1, which contained the

larger airways and the bifurcations to the secondary bronchi, compared to lung L4, which

contained the paleopulmonic parabronchi. This suggests, based on viral RNA expression,

that immunisation in the presence of w/o and Al(OH)3 results in protection against

challenge with AIV.

Figure 2. Viral RNA levels in trachea and lung of birds immunised with H9N2 adjuvanted with w/o or Al(OH)3

lower compared to the other challenged groups. The groups non-immunised-challenged (NIC), immunised-

challenged (IC) and immunised with H9N2 adjuvanted with CpG were not protected against challenge with H9N2. Data were expressed as means (n = 4, † n =2) with standard error of the mean (SEM). * Indicates significant

differences (p<0.05) in viral RNA expression compared to unprotected (NIC and IC) birds.

Global gene expression profiles

In order to determine whether differences in protection to AIV challenge based on viral

RNA level were also reflected in gene expression profiles, microarray analysis was

performed on upper trachea and lung L1 and L4 at 1 to 4 d.p.c. By connecting the gene

expression level per gene over time, a global gene expression pattern was created reflecting

the global host response. Based on the expression patterns, the different treatment groups

fell into two groups: one group with a low amplitude of change and one group with a high

NIC IC+CpG IC+Al(OH)3

IC IC+w/o

0

5

10

15

20

25

1 2 3 4

Days p.c.

1 2 3 4

25

20

15

10

5

0Co

rrec

ted

40

-Ct

+ S

EM Upper trachea

0

5

10

15

20

25

1 2 3 4

Lung L1

1 2 3 4

25

20

15

10

5

0Co

rrec

ted

40

-Ct

+ S

EM

Days p.c.

0

5

10

15

20

25

1 2 3 4

Lung L4

1 2 3 4

25

20

15

10

5

0Co

rre

cte

d 4

0-C

t +

SE

M

Days p.c.


IC IC+w/o


IC IC+w/o

0

5

10

15

20

25

1 2 3 4

Days p.c.

1 2 3 4

25

20

15

10

5

0Co

rrec

ted

40

-Ct

+ S

EM Upper trachea

0

5

10

15

20

25

1 2 3 4

Lung L1

1 2 3 4

25

20

15

10

5

0Co

rrec

ted

40

-Ct

+ S

EM

Days p.c.

0

5

10

15

20

25

1 2 3 4

Lung L4

1 2 3 4

25

20

15

10

5

0Co

rre

cte

d 4

0-C

t +

SE

M

Days p.c.


121

Figure. 3. A) A lower amplitude of change in global gene expression patterns over time was found in upper

trachea, lung L1 and L4 of protected (IC+w/o and IC+Al(OH)3) versus unprotected (NIC, IC and IC+CpG) birds

(n = 4, except for NIC 4 d.p.c. n = 2). Red indicates up regulation and green down regulation. Gene expression

rates of individual birds are compared to expression rates in a reference sample consisting of pooled lungs or

0.01

100

10

1

0.1

NINC IC+ CpG

IC+ w/o

IC+ Al(OH)3

ICNIC0.01

100

10

1

0.1

NINC IC+ CpG

IC+ w/o

IC+ Al(OH)3

ICNIC

100

10

1

0.1

0.01NINC IC+

CpGIC+ w/o

IC+ Al(OH)3

ICNIC

100

10

1

0.1

0.01NINC IC+

CpGIC+ w/o

IC+ Al(OH)3

ICNIC

NINC IC+ CpG

IC+ w/o

IC+ Al(OH)3

IC

100

10

1

0.1

0.01NICNINC IC+

CpGIC+ w/o

IC+ Al(OH)3

IC

100

10

1

0.1

0.01NIC

A. B. Genes significantly differentially expressed between IC and adjuvanted birds

261

150

138

267

5041

143

IC+ w/o

IC+ Al(OH)3

IC+ CpG

261

150

138

267

5041

143

261

150

138

267

5041

143

IC+ w/o

IC+ Al(OH)3

IC+ CpG

1256549 449

176

16547

40

IC+ w/o

IC+ Al(OH)3

IC+ CpG

1256549 449

176

16547

40

1256549 449

176

16547

40

IC+ w/o

IC+ Al(OH)3

IC+ CpG

127

31

728

1358

525

122111

IC+ w/o

IC+ Al(OH)3

IC+ CpG

127

31

728

1358

525

122111

127

31

728

1358

525

122111

IC+ w/o

IC+ Al(OH)3

IC+ CpG

Upper trachea

Lung L1

Lung L4

Upper trachea

Lung L1

Lung L4

A. B.

Chapter 6

122

pooled trachea of uninfected birds. B) Venn diagrams showing genes significantly differentially expressed at any

day post challenge between IC birds and IC+CpG, IC+w/o and IC+Al(OH)3 birds in upper trachea, lung L1 and L4

(n = 4). The numbers within the segments indicate the number of genes that were significantly differentially

expressed between the IC group and an adjuvanted group. Most overlap in gene expression was found between the

protected groups IC+w/o and IC+Al(OH)3.

amplitude of change (Fig. 3A). The gene expression patterns of the unprotected NIC, IC

and IC+CpG group showed that many genes were significantly differentially expressed

with a high amplitude of change. The protected groups, IC+w/o and IC+Al(OH)3, and the

non-challenged NINC group had a gene expression pattern with a low amplitude of change.

In the trachea more genes were expressed at a higher amplitude of change compared to lung

L1 and L4, but the global gene expression patterns of trachea, lung L1 and to a lesser extent

in L4 showed a similar trend. Thus, gene expression patterns can act as correlate of

protection in which a signature with a low amplitude of change was related to the protected

and non-challenged birds and a signature with a high amplitude of change to the

unprotected birds.

We determined which genes were significantly differentially expressed in IC+w/o,

IC+Al(OH)3 and IC+CpG compared to IC birds to determine adjuvant induced genes. Venn

diagrams were made to show number of genes that were overlapping or unique to the

adjuvant (Fig. 3B). Most overlap in genes was seen between the protected IC+w/o and

IC+Al(OH)3 birds. Innate defense and interferon related genes were differentially expressed

in all adjuvanted groups (in white), but are not correlates of protection. The number and the

genes expressed differed between locations within the respiratory tract, with lung L1 and

L4 being most similar.

Immune related gene expression profiles

To determine immune related correlates of protection after challenge, gene expression

profiles of unprotected IC and IC+CpG birds and of protected IC+w/o and IC+Al(OH)3

birds were compared to the non-immunised-non-challenged NINC birds. An immune

related gene category was created based on the GO terms host-pathogen interaction,

external stimulus and immune response. Immune related genes that were significantly

differentially expressed in the protected groups compared to the unprotected IC group were

selected, divided into functional groups according to the GO annotations, and depicted in

heatmaps (Fig. 4). In general these immune related genes expressed in protected birds were

also significantly differentially expressed in unprotected birds, but at a much lower

expression level in protected birds. Of these genes, 44 genes were significantly

differentially expressed throughout the respiratory tract, trachea, lung L1 and L4, in

protected compared to unprotected IC birds (Supplemented data Fig. S1) and related mainly

to defense and inflammatory responses and cell differentiation. Again these genes were


123

Cyt

okin

e / c

hem

okin

eT

cel

lB

cel

lIn

terl

euki

nIn

terf

eron

Com

plem

ent

TLR

rel

ated

Lung L1 Lung L4Upper trachea

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3

1-4 d.p.c

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3

1-4 d.p.cIC+ w/o

IC IC+ CpG

IC+ Al(OH)3

1-4 d.p.c

Cyt

okin

e / c

hem

okin

eT

cel

lB

cel

lIn

terl

euki

nIn

terf

eron

Com

plem

ent

TLR

rel

ated

Lung L1 Lung L4Upper trachea

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3

1-4 d.p.c

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3

1-4 d.p.c1-4 d.p.c

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3

1-4 d.p.c

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3

1-4 d.p.c1-4 d.p.cIC+ w/o

IC IC+ CpG

IC+ Al(OH)3

1-4 d.p.c

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3

1-4 d.p.c1-4 d.p.c

1

5

0.2

Fold ch

ang

e

1

5

0.2

Fold ch

ang

e

Chapter 6

124

Figure. 4. Lower expression rates of immune related genes were found in protected (IC+w/o and IC+Al(OH)3)

compared to unprotected (IC and IC+CpG) birds. Heatmaps over time showing genes significantly differentially

expressed at any day post challenge between IC birds and IC+w/o and/or IC+Al(OH)3 birds in upper trachea, lung

L1 and L4 (n = 4). Gene expression rates in the heatmaps are the result of comparing expression in challenged

birds to expression in non-challenged birds (NINC). Red indicates up regulation and green down regulation. Genes

were divided into functional groups based on GO annotations.

induced at a lower expression level in protected IC+w/o and IC+Al(OH)3 birds in all three

parts of the respiratory tract, with two exceptions; CCL20 and TGFB2. CCL20 was up

regulated in protected birds and a correlate of protection, whereas TGFB2 was down

regulated in unprotected birds and a correlate of disease. In lung L4 differences in gene

expression level between unprotected and protected birds were less profound than in L1 and

trachea, which correlated to the lower viral RNA expression in L4.

Pathway analysis of immune related genes

Next, we studied expression of genes within immune related pathways that were

significantly differentially expressed in protected compared to unprotected birds to obtain a

general signature of protection induced after challenge.

Genes involved in leukocyte transendothelial migration were only expressed on 1 d.p.c in

protected birds, but continually in unprotected birds, suggesting that in protected birds only

a short period of cellular recruitment occurred. This expression pattern was also seen in the

other pathways that were part of the general signature of protection (Fig. 5). Based on

differential gene expression the signature of protection after challenge mainly consisted of

increased gene expression at 1 d.p.c in protected birds after which gene expression

declined. In contrast, in unprotected birds stronger and prolonged gene expression over

time was found.

Genes differentially expressed in the complement and coagulation cascade were mainly

involved in the classical pathway and followed the expression pattern of the general

signature of protection, except for CD59. This gene, which prevents lysis of cells by

complement, was down regulated in protected birds especially at 1 and 2 d.p.c., and a

correlate of protection.

In the cytokine-cytokine receptor interaction pathway several genes of many subfamilies

were induced. In contrast to the other cytokine and chemokine gene subsets, growth factors

were increasingly down regulated in unprotected birds, whereas in protected birds

expression was less down regulated.

The apoptosis pathway was the only pathway in the general signature of protection in

which most genes differed in expression pattern from the other pathways. In contrast to

other genes IAP and IL1R1 were oppositely expressed between protected and unprotected

birds. IAP was up regulated over time in unprotected birds, while down regulated from 2 to


125

ThThTcTc

IC IC+

w/o

IC+

Al(O

H)3

1-4 d.p.c

Heatmap legend

IC IC+

w/o

IC+

Al(O

H)3

1-4 d.p.c

Heatmap legend

Leukocyte transendothelial migration

ITGB2 VCAM1MMP9 THY1ITGB2 VCAM1MMP9 THY1

APCAPC

Antigen presentation and processing

MHCI

CIITA DMA CTSB IFI30CD74

MHC I pathway

MHC II pathway


MHCI

CIITA DMA CTSB IFI30CD74

MHC I pathway

MHC II pathway


MHCIMHCI

CIITA DMA CTSB IFI30CD74CIITA DMA CTSB IFI30CD74

MHC I pathway

MHC II pathway

Toll-like receptor signaling

IRF3 LY96 TLR15 IFNAR1TLR7

Toll-like receptor signaling

IRF3 LY96 TLR15 IFNAR1TLR7 TLR4

Cytokine-cytokine receptor interaction

CCL20CCLi7 CCL4 CCLi3 CCL19 CCR8

LEPR LIFRCSF3R

TNFRSF18 TNFRSF13B GHR TGFB2 TGFB3

CXCL13 CXCLi1XCL1 CXCLi2

Cytokine-cytokine receptor interaction

CCL20CCLi7 CCL4 CCLi3 CCL19 CCR8

LEPR LIFRCSF3R

TNFRSF18 TNFRSF13B GHR TGFB2 TGFB3

CXCL13 CXCLi1XCL1 CXCLi2

Transcription factors

NMI SOCS2STAT4

Transcription factors

NMI SOCS2STAT4

Complement cascade

CD59C1QC C3AR1 CS1C1QB

Complement cascade

CD59C1QC C3AR1 CS1C1QB

CD3Z CD3D LCP2CD3E ZAP70

Cell adhesion molecules and T cell receptor signaling

CD8B CD4 ICOSCD8A CD86 CCR5CTL4A

CD28CBLB CCR2CD3Z CD3D LCP2CD3E ZAP70

Cell adhesion molecules and T cell receptor signaling

CD8B CD4 ICOSCD8A CD86 CCR5CTL4A

CD28CBLB CCR2

IL16 IL7RAIL15

Interleukin- receptor interaction

IL6 IL1B IL13RA IL10RBIL22

IL2RB IL2RG IL20RAIL21 IL18

IL16 IL7RAIL15

Interleukin- receptor interaction

IL6 IL1B IL13RA IL10RBIL22

IL2RB IL2RG IL20RAIL21 IL18

BCL2A1 CASP8 IAPBCL2L14

Apoptosis

CASP9IL1R1BCL2A1 CASP8 IAPBCL2L14

Apoptosis

CASP9IL1R1BCL2A1 CASP8 IAPBCL2L14

Apoptosis

CASP9IL1R1

15 0.2

Fold change

15 0.2

Fold change

Chapter 6

126

Figure 5. Brief and low expression of significantly differentially expressed pathways in protected birds as

correlate of protection. Pathways showing genes significantly differentially expressed at any day post challenge

between IC birds and both IC+w/o and IC+Al(OH)3 birds in lung L1 (n = 4). Gene expression rates in the gene

individual heatmaps are the result of comparing expression in IC+w/o, IC+Al(OH)3 and IC birds to expression in

NINC birds. Red indicates up regulation and green down regulation. Pathways analysis was performed using

DAVID and based on KEGG.

4 d.p.c. in protected birds. IL1R1 was down regulated in unprotected birds and up regulated

in protected birds. CASP9 was strongly down regulated in unprotected birds over time, but

in protected birds was less down regulated and only from 1 to 3 d.p.c.

Subsequently we looked for pathways that were significantly differentially expressed in

either IC+w/o or IC+Al(OH)3 birds compared to unprotected IC birds. Not one immune

related pathway was uniquely significantly induced in either w/o or Al(OH)3 immunised

birds. However, within the pathways described above, unique genes were found within an

adjuvant group (Supplemented data Fig. S2). For w/o 41 adjuvant unique genes were found

and for Al(OH)3 35 genes. Again expression rates of the adjuvant specific genes were lower

in protected IC+w/o or IC+Al(OH)3 birds compared to unprotected IC birds. In conclusion,

although IC+w/o or IC+Al(OH)3 birds expressed some adjuvant specific genes, the

expression pattern of these genes remained similar to the general gene expression signature

of protection after challenge emphasising the relation of this pattern with a protective

response.

Effect of adjuvants on recruitment of leukocytes in the lung

Gene profiling resulted in a signature of protection after AIV challenge: a low viral RNA

expression associated with a low amplitude of change in gene expression. Next, we

analysed cell influxes in the respiratory tract using immunocytochemical staining for viral

NP, KUL-01+ (macrophages), CD4+ and CD8α+ cells.

In general, independent of the treatment, virus infected cells were detected at 1 d.p.c. in the

larger airways and in adjacent parabronchi in L1. Thereafter more virus infected cells were

found in the adjacent parabronchi. In L4 less and smaller virus infected areas were found

than in L1, corresponding to the viral RNA levels. In protected IC+w/o and IC+Al(OH)3

birds, less and smaller virus infected areas in L1 were found compared to the unprotected

(NIC, IC and IC+CpG) birds, but the location of infected areas was similar. No differences

were found between unprotected groups, the number and size of virus infected areas was

similar in IC and IC+CpG birds to those seen in NIC birds for both L1 and L4.

Influxes of KUL-01+, CD4+ and CD8α+ cells were seen in lung L1 and L4 in all birds from

1 d.p.c. Independent of the adjuvant, larger influxes of KUL-01+ macrophages and CD4+

cells were seen in L1 than in L4 correlating to the number of virus infected cells. The cell


127

influxes co-localised with virus infected areas. The size of the CD4+ and KUL-01+ cell

influxes correlated to the number of virus infected cells, resulting in fewer influxes in

protected birds. Influxes of CD8α+ cells did not differ between protected and unprotected

birds. Influxes of CD8α+ cells were less dense than KUL-01+ and CD4+ cell influxes (Fig.

6), but also co-localised with virus infected areas.

The recruitment of leukocytes in the lung corresponded to the gene profiles of protected

and unprotected birds. Less virus entry into the lung resulted in smaller influxes of

macrophages and CD4+ cells, linked to absence of immune activation after challenge

therefore having fewer and lower gene expression in protected birds.

Figure 6. Less influx of CD4+ cells and KUL-01+ macrophages in lung of protected (IC+w/o and IC+Al(OH)3)

compared to unprotected (NIC, IC and IC+CpG) birds. Cryosections of lung L1 of non-immunised-non-challenged

(NINC) birds, unprotected non-immunised-challenged (NIC) birds, unprotected immunised-challenged (IC+CpG)

birds and protected immunised-challenged (IC+w/o) birds stained for viral NP and KUL-01+ macrophages at 2

d.p.c. and for CD4+ and CD8α+ cells at 4 d.p.c. L indicates luminal side of a parabronchus. Bar is 100 μm.

Effect of adjuvants on humoral responses

Since the cause of the lower viral load in the respiratory tract might be prevention of viral

entry by virus-specific antibodies, H9-specific HI titres were measured in blood samples

taken two weeks after immunisation prior to challenge (Fig. 7A). A significant increase in

HI titer was only found in IC+w/o and IC+Al(OH)3 birds. IC+Al(OH)3 birds had the

NIC

NINC

IC+ CpG

Viral NP KUL-01+ CD4+ CD8a+

IC+ w/o

L

LL

L

L

L

L

L

NIC

NINC

IC+ CpG

Viral NP KUL-01+ CD4+ CD8a+

IC+ w/o

L

LL

L

L

L

L

L

Chapter 6

128

highest HI titre, which was significantly higher compared to IC+w/o birds. However, this

was to be expected because the maximum antibody titer post-vaccination is generally

delayed in w/o vaccinated birds when compared to Al(OH)3 vaccinated birds. AIV NP-

specific antibody titers were also measured (Fig. 7B) and a significant increase was found

in protected IC+w/o and IC+Al(OH)3 birds.

A. B.

Figure 7. Protected IC+w/o and IC+Al(OH)3 birds had a significant increase in A) HI titer and B) AIV NP-

specific antibody titer in contrast to unprotected NIC, IC and IC+CpG birds. Serum was collected at 14 days after

immunisation prior to challenge of all birds (n = 16 per group). Data are expressed as means with standard error of

the mean (SEM). * Indicates a significant difference (p<0.05).

Discussion

Avian influenza virus (AIV) infection has severe consequences, not only for the worldwide

economy but also for human health. As no vaccines have been found yet that protect

against a broad range of influenza virus strains much effort is invested in development of

new vaccines and improvement of vaccine efficacy. To this end, knowledge on correlates of

protection against AIV infection is warranted.

In this study we set out to investigate possible correlates of protection after challenge with a

low pathogenic AIV and studied the role of the different immunepotentiators w/o, Al(OH)3

and CpG. Correlates of protection were investigated using microarrays, and gene

expression profiles were studied in more detail using qRT-PCR and immunocytochemical

analysis of the lung. Immunisation of day-old birds in with H9N2+w/o or HN2+Al(OH)3

provided protection based on low viral RNA expression against homologues H9N2

challenge 2 weeks later, while immunisation with H9N2+CpG did not. When differences in

gene expression patterns of protected and unprotected birds were compared, no specific sets

of genes were associated with protection. Previous studies report subsets of genes defining

disease pathology of influenza virus infection in mammals (Baskin et al., 2002, Pennings et

al., 2008, Cillóniz et al., 2009), but no gene subsets have been described relating to

protection. Differences in the patterns of gene expression were observed. In protected birds

gene expression rates in lung and trachea were lower and diminished after 1 d.p.c., while

gene expression patterns and pathway analysis showed stronger and prolonged gene

0

5

10

15

10

5

10

15

Mea

n 2l

og s

erum

tite

r

0

5

10

15

10

5

10

15

Mea

n 2l

og s

erum

tite

r

NINC

NIC

IC

IC+CpG

IC+w/o

IC+Al(OH)3

NINC

NIC

IC

IC+CpG

IC+w/o

IC+Al(OH)3

NINC

NIC

IC

IC+CpG

IC+w/o

IC+Al(OH)30

1

2

3

4

5

10

1

2

3

4

5

Mea

n 2l

og s

erum

tite

r

0

1

2

3

4

5

10

1

2

3

4

5

Mea

n 2l

og s

erum

tite

r

*

*

*

0

5

10

15

10

5

10

15

Mea

n 2l

og s

erum

tite

r

0

5

10

15

10

5

10

15

Mea

n 2l

og s

erum

tite

r

NINC

NIC

IC

IC+CpG

IC+w/o

IC+Al(OH)3

NINC

NIC

IC

IC+CpG

IC+w/o

IC+Al(OH)3

NINC

NIC

IC

IC+CpG

IC+w/o

IC+Al(OH)3

NINC

NIC

IC

IC+CpG

IC+w/o

IC+Al(OH)3

NINC

NIC

IC

IC+CpG

IC+w/o

IC+Al(OH)3

NINC

NIC

IC

IC+CpG

IC+w/o

IC+Al(OH)30

1

2

3

4

5

10

1

2

3

4

5

Mea

n 2l

og s

erum

tite

r

0

1

2

3

4

5

10

1

2

3

4

5

Mea

n 2l

og s

erum

tite

r

*

*

*

*

*


129

expression in unprotected birds. These data suggest that short term activation of the

immune system is associated with protection after challenge, while prolonged activation of

the immune system leads to progressive AIV infection. Indeed immunocytochemistry

showed smaller influxes of KUL-01+ macrophages and CD4+ cells in the lung of protected

birds compared to unprotected birds which parallels the microarray data. Thus, rather than

finding certain genes or pathways that correlate with protection, we conclude that lack of

immune activation is the strongest predictor and correlate of progression after challenge.

That lack of immune activation is associated with protection against progressive infection

has been reported previously for other viral infections. For example, persistent immune

activation after HIV-1 infection has been shown to correlate with progressive disease

(Hazenberg et al., 2003) and non-pathogenic SIV infection in sooty mangabeys was

associated with attenuated immune activation despite the presence of high levels of viral

replication (Silvestri et al., 2003). Apparently uncontrolled immune activation is also

reported for high pathogenic influenza virus. The broad tissue specificity and systemic

replication seem to determine the pathogenicity of H5N1 viruses in animals. Virus induced

cytokine dysregulation contributes to disease severity, in that high viral load results in

intense inflammatory responses and fatal outcome (Baskin et al., 2002, De Jong et al.,

2006, Kash et al., 2006, Perrone et al., 2008). Thus, although activation of the immune

system is necessary in order to get an anti-viral immune response, too much activation may

lead to immune pathology and progressive disease.

To our knowledge, this is the first time that a correlation between lack of immune

activation and absence of pathogenesis after challenge with AIV is described for the

chicken. This lack of immune activation in protected birds is likely caused by presence of

virus-specific antibodies that reduce viral entry and subsequently attenuate immune

activation. Protected birds had a high HI titre while unprotected birds had no HI titre

correlating to the difference in viral RNA expression.

These results have severe implications for vaccine design. Vaccines that are merely directed

to induce virus-specific CTL do not necessarily prevent immune pathology (Moskophidis et

al., 1998), suggesting that antibody based vaccines are better candidates to induce

protection. However, vaccines that only induce antibodies may be very effective for a

certain strain of AIV, but cross protection against other strains is limited (Rimmelzwaan et

al., 2008). It may also induce escape mutants due to antigenic shift under the influence of

immune pressure (Rimmelzwaan et al., 2008). In contrast, CTL have been shown to be

largely directed against epitopes within conserved viral proteins and contribute to

protection against AIV infection (Jameson et al., 1999). Furthermore, H9N2 viruses have

been demonstrated to induce cross-protection against highly pathogenic H5N1 based on

cellular immunity (Seo et al., 2001). This suggests that a vaccine inducing both humoral

and cellular immunity may prevent immune pathology and induce strong CTL responses.

Chapter 6

130

Furthermore, the type of adjuvant used is also important. We showed that protection to low

AIV was only obtained after immunisation in the presence of immunopotentiators w/o and

Al(OH)3. These adjuvants are known to induce a humoral response (Hogenesch, 2002,

Jansen et al., 2007) providing high virus-specific antibody responses. Furthermore, w/o

emulsions have been demonstrated to also enhance cellular immune responses (Aucouturier

et al., 2001, Tritto et al., 2009). Immunisation in the presence of CpG, which induces

activation of macrophages, NK cells and antigen specific CTL did not result in protection

(Klinman et al., 2004) .

In our study the differences between the adjuvants is most likely explained by the

difference in HI titre; adjuvants that induce neutralizing antibodies may prevent viral entry

and are associated with protection. This observation is in contrast with other studies which

show that addition of CpG to E. coli bacterin or recombinant plasmids DNA vaccine

encoding the VP2 gene of IBDV provide protection from mortality and induced increased

antigen specific antibody titers (Mahmood et al., 2006, Gomis et al., 2007). This may be

due to the age of the birds that were used in the different studies. We immunised day-old

birds which were challenged 14 days later, while the other studies immunised birds from 7

or 10 days of age and challenged birds at 21 or 30 days of age. Previous data showed that

immunization of 1-day-old broilers with BSA resulted in a much lower and slower antibody

production compared to immunization at 1 or 2 weeks of age (Mast et al., 1999). This

suggests that age of the vaccinated birds may be an important factor in the choice of

adjuvant, which is highly relevant for the poultry industry.

In summary, we show that in addition to high HI and AIV NP-specific antibody titers and a

limited number of differentially expressed genes correlating with protection, such as

CCL20, IAP and CD59, lack of immune activation is the most important correlate of

protection after challenge with AIV.

Acknowledgements

We thank Marijn Driessen, Nicolette Scholtes and Virgil Schijns from Intervet/Schering-

Plough Animal Health for their collaboration on the in vivo experiment. From the Animal

Breeding and Genomics Centre, Wageningen UR, we would like to thank Dirkjan Schokker

and Ina Hulsegge for pathway analysis assistance. From the Genomics Laboratory we

would like to thank Dik van Leenen and Diane Bouwmeester for technical assistance and

Erik Sluiters and Patrick Kemmeren for bioinformatical assistance. This work was

supported by a BSIK VIRGO consortium grant (Grant no. 03012), The Netherlands.


131

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133

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recovering bursa. Viral Immunol. 18, 127-137.

Supplementary data

Figure S1. Lower expression rates of immune related genes were found in protected (IC+w/o and IC+Al(OH)3)

compared to unprotected (IC and IC+CpG) birds of genes expressed in trachea, lung L1 and L4. Heatmaps over

time showing genes significantly differentially expressed at any day post challenge between IC and IC+w/o birds

and/or IC+Al(OH)3 birds that were shared between upper trachea, lung L1 and L4 (n = 4). Gene expression rates in

the heatmaps are the result of comparing expression in challenged birds to expression in NINC birds. Red

indicates up regulation and green down regulation.

Upper trachea Lung L1 Lung L4

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3

unprotected protected

1-4 d.p.c

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3


1-4 d.p.c

IC+ w/o

IC IC+ CpG

IC+ Al(OH)3


1-4 d.p.c

15 0.2

Fold

Chapter 6

134

Figure S2. Significant differentially expressed genes that are uniquely expressed within an adjuvant group. These

genes were mainly expressed at a higher rate and for a longer period of time in unprotected birds. Heatmaps over

time showing adjuvant unique genes expressed at any day post challenge between IC birds and IC+w/o and

IC+Al(OH)3 birds in lung L1 (n = 4). Gene expression rates in the heatmaps are the result of comparing expression

in challenged birds to expression in NINC birds. Red indicates up regulation and green down regulation.

Al(OH)3 specific w/o specific

IC IC+ w/o

1-4 d.p.c

IC IC+ Al(OH)3

1-4 d.p.c

15 0.2

Fold change

Early host responses to avian influenza A virus are

prolonged and enhanced at transcriptional level

depending on maturation of the immune system

Sylvia S. Reemers1, Dik van Leenen2, Marian J. Groot Koerkamp2, Daphne van Haarlem1,

Peter van de Haar1, Willem van Eden1, Lonneke Vervelde1

1 Department of Infectious Diseases and Immunology, Faculty Veterinary Medicine, Utrecht University, Yalelaan

1, 3584 CL Utrecht, The Netherlands 2 Genomics Laboratory, Department of Physiological Chemistry, Utrecht Medical Centre, Universiteitsweg 100,


Molecular Immunology, 2010; in press

Chapter 7

Immunological maturation affects the strength and duration of transcriptional host responses to AIV infection

137

Abstract

Newly hatched chickens are more susceptible to infectious diseases than older birds

because of an immature immune system. The aim of this study was to determine to what

extent host responses to avian influenza virus (AIV) inoculation are affected by age in 1-

and 4-week (wk) old birds. Birds were inoculated with H9N2 AIV or saline control. The

trachea and lung were sampled at 0, 8, 16 and 24 hours post inoculation (h.p.i.) and gene

expression profiles determined using microarray analysis. Firstly, saline controls of both

groups were compared to analyse the changes in gene profiles related to development.

Genes related to tissue development and immunological maturation were significantly

differentially expressed. In 1-wk-old birds, higher expression of genes related to

development of the respiratory immune system and innate responses were found, whereas

in 4-wk-old birds genes were up regulated that relate to the presence of higher number of

leukocytes in the respiratory tract. After inoculation with H9N2, gene expression was most

affected at 16 h.p.i. in 1-wk-old birds and at 16 and 24 h.p.i. in 4-wk-old birds in the

trachea and especially in the lung. In 1-wk-old birds less immune related genes including

innate related genes were induced and which might be due to age-dependent reduced

functionality of APC, T cells and NK cells. In contrast cytokine and chemokines gene

expression was related to viral load in 1-wk-old birds and less in 4-wk-old birds.

Interestingly, expression of cellular host factors that block virus replication by interacting

with viral factors was independent of age or tissue for most host factors. These data show

that differences in development are reflected in gene expression and suggest that the

strength of host responses at transcriptional level may be a key factor in age-dependent

susceptibility to infection, and the cellular host factors involved in virus replication are not.

Introduction

Young animal are highly susceptible to opportunistic pathogens that are common in their

environment. Susceptibility to disease decreases as the bird matures, suggesting that this

phenomenon is due to immaturity of the immune system (Raj et al., 1997, Hume et al.,

1998, Beal et al., 2005). Neonatal immune dysfunction has also been reported for mammals

(Gasparoni et al., 2003, Chelvarajan et al., 2004, Velilla et al., 2006). At present,

vaccination of young chicks is commonly practised to establish immunity in a flock. In

newly hatched birds, the activation, phagocytosis and bactericidal activities of heterophils

and macrophages were shown to be age-dependent in that they increase with age (Kodama

et al., 1976, Wells et al., 1998, Kogut et al., 2002). T cells from 1-day-old chicks hardly

proliferate in response to mitogens and produce less IFN and IL-2 (Lowenthal et al., 1994).

Immunization of 1-day-old broilers with BSA resulted in a much lower and slower antibody

production compared to immunization at 1 or 2 weeks of age (Mast et al., 1999).

Chapter 7

138

The respiratory tract is constantly exposed to pathogens like influenza virus and to provide

adequate protection, inhaled pathogens are removed by mucus, neutralizing molecules like

IgA, complement and antimicrobial peptides. When virus entry is not successfully blocked,

influenza virus will infect the epithelial cells resulting in the production of pro-

inflammatory cytokines, chemokines and interferons (Julkunen et al., 2001). This attracts

macrophages and DC, which upon activation or influenza virus infection also start

producing cytokines and chemokines attracting more APC and lymphocytes to the place of

infection. The trachea, lung and air sacs contribute to the respiratory immune system, but

the lung plays a special role because it contains secondary lymphoid structures (Kothlow et

al., 2008). Newly hatched birds have dispersed T cells, B cells, leukocytes and monocytes

present throughout the lung. At 2 weeks of age areas with lymphocyte infiltrates are found

around the bifurcations of the caudal secondary bronchi. These organized structures show

similarity to Peyer’s patches and are called bronchus associated lymphoid tissue (BALT;

Jeurissen et al., 1989, Fagerland et al., 1993). Avian BALT is not comparable to inducible

BALT in mice and human (Moyron-Quiroz et al., 2004, Rangel-Moreno et al., 2006),

because its presence is independent of antigenic stimulation (Reese et al., 2006). In the

trachea no organized lymphoid structures have been reported, but infection with various

pathogens results in lymphoid infiltration and in formation of lymphoid follicles (Gaunson

et al., 2006, Matthijs et al., 2009, Reemers et al., 2009b).

For replication and transcription of the influenza virus genome, the virus uses both viral and

cellular host factors. Viral factors like the polymerase complex or nucleoprotein have been

known to interact with mammalian host factors like importins or histones during influenza

virus infection (Engelhardt et al., 2006, Naffakh et al., 2008, Nagata et al., 2008). The virus

must stimulate expression of host factors that are needed for replication ongoing during

infection to ensure virus multiplication. However, some host factors that interact with viral

factors are used in host defense responses and can limit viral replication.

To investigate the effect of age on the early host response and on host factors affecting viral

replication in the respiratory tract, we inoculated 1- and 4-week (wk) old birds with H9N2

avian influenza virus (AIV) or saline. The trachea and lung were sampled at 0, 8, 16 and 24

hours post inoculation (h.p.i.) and gene expression was studied using microarray analysis.

Differences between 1- and 4-wk-old saline inoculated control birds were mainly related to

tissue development and immunological maturation. Differences between 1- and 4-wk-old

H9N2 inoculated birds were related to strength and the timing of host responses at

transcriptional level. Expression of cellular host factors that block viral replication by

interacting with viral factors was independent of age and tissue.


139


Experimental design

Lohmann Brown chickens of 1 and 4 weeks of age were divided into 2 groups per age, a

saline and AIV inoculated group. The birds were inoculated intratracheally (i.t.) with either

0.1 ml PBS or with 0.1 ml 107.7 EID50 H9N2 AIV, isolate A/Chicken/United Arab

Emirates/99 (kindly provided by Intervet Schering-Plough Animal Health). At 0, 8, 16 and

24 hours post inoculation (h.p.i.) 5 birds per time point per group and per age were killed.

The upper trachea and the left lung were isolated and stored in RNAlater (Ambion) at -

80°C for RNA isolation. The segment of the lung containing the primary and secondary

bronchi was used for analysis. Selection of the upper trachea and the lung segment used for

analysis was based on high viral load and high virus-induced gene expression as described

previously (Reemers et al., 2009b). All experiments were carried out according to protocols

approved by the Animal Experiment Committee of Utrecht University (Utrecht, The

Netherlands).

RNA isolation

The trachea (5 mm part) and lung (1x5 mm part) were homogenized (Mixer Mill 301,

Retsch) and total RNA was isolated using the RNeasy Mini Kit and DNase treated using the

RNase-free DNase Set following manufacturer’s instructions (Qiagen). All RNA samples

were checked for quantity using a spectrophotometer (Shimadzu) and quality using a 2100

Bioanalyzer (Agilent Technologies).


cDNA was generated from 500 ng RNA using reverse transcription using iScript cDNA

Synthesis Kit (Biorad Laboratories B.V.). Real-time qRT-PCR was used for detection of

GAPDH, viral H9 haemagglutinin (HA), interleukins (IL-1β, IL-8, IL-18), interferon alpha

(IFN-α) and 28S as previously described (Reemers et al., 2009a). Amplification and

detection of specific GAPDH and viral H9 HA products was achieved using iQ SYBR

green supermix (Biorad). For amplification and detection of IL-1β, IL-8, IL-18, IFN-α and

28S TaqMan Universal PCR Master Mix (Applied Biosystems) was used.

Statistical analysis qRT-PCR data

To determine the statistical significance in viral RNA expression between time points

within an age group and between age groups within a time point in the trachea and lung an

ANOVA with a Tukey post-hoc test was used. To determine the statistical significance in

cytokine mRNA expression between control birds and H9N2 inoculated birds within a time

point and age group in the trachea and lung an ANOVA was used. Correlations between

viral RNA and cytokine mRNA expression were based on the Pearson correlation

Chapter 7

140

coefficient (r) and determined using SPSS 15.0 software. A p-value < 0.05 was considered

significant.


Microarray analysis was performed as described previously (Reemers et al., 2009b) using

the Gallus gallus Roslin/ARK CoRe Array Ready Oligo Set V1.0 (Operon

Biotechnologies). All the trachea and lung samples were co-hybridised with respectively a

trachea or lung reference sample. These reference samples consisted of pooled RNA

extracted from tracheas or lungs of 4 chickens that were not included in this experiment.

Microarray arrays were analysed as previously described (Reemers et al., 2009b). Ensembl

Gallus gallus, (assembly: WASHUC2, May 2006, genebuild: Ensembl, Aug 2006, database

version: 47.2e) was used for gene names and description. For analysis of gene lists and

Gene Ontology (GO) analysis Database for Annotation, Visualization and Integrated

Discovery (DAVID) 2008 was used. Primary data are available in the public domain

through Expression Array Manager at http://www.ebi.ac.uk/microarray-as/aer/?#ae-main[0]

under accession number E-TABM-771 for the lung and E-TABM-772 for the trachea.

Results

Age-dependent gene expression in control birds

Before we compared virus-induced host responses between 1- and 4-wk-old birds we

determined the effect of age on gene expression in control birds. Differences would affect

virus-induced gene profiles and a direct comparative study between the age groups would

not be possible. The number of genes in the lung that differed significantly between 1- and

4-wk-old control birds at 0, 8, 16 and 24 h.p.i. were respectively 230, 167, 74 and 227

genes, and in the trachea 58, 104, 47 and 19 genes. On each set of genes Gene Ontology

(GO) analysis was performed using DAVID and the resulting top three functional groups of

every gene set were depicted in Table S1 in the supplementary data. These functional

groups were mainly related to tissue development, but most functional groups contained

genes that also play a role in immune responses. Furthermore, several functional groups

relating to immune related processes like immune system response, lymphocyte activation

and chemotaxis were also significantly differentially expressed between 1- and 4-wk old

control birds, but did not belong to the top 5 of functional groups found. The number of

immune related genes that were significantly differentially expressed between 1- and 4-wk-

old control birds was determined. The immune related category was based on the GO

annotations host-pathogen interaction, external stimulus and immune response. The number

of immune related genes at 0, 8, 16, and 24 h.p.i. were in the lung respectively 34, 33, 13

and 38 genes and in the trachea 7, 17, 4 and 4 genes (Supplementary data Table S2 and S3).

In the lung these genes were involved in antigen presentation/binding, apoptosis, cell


141

differentiation and proliferation, chemotaxis, innate immune response, protein folding and

binding and signal transduction. In the trachea less genes were differentially expressed than

in the lung, but the ones expressed were involved in similar biological processes. Most

genes were expressed at a higher rate in 4-wk-old birds in both the lung and trachea. Genes

higher expressed in the lung and trachea of 1-wk-old birds were mostly involved in

apoptosis, cell adhesion and proliferation, innate immune responses, protein binding and

folding, and signal transduction.

These results indicated that gene expression levels differed significantly between 1- and 4-

wk-old control birds due to development of the tissue itself and maturation of the immune

system. Therefore, host responses induced by H9N2 inoculation in 1- and 4-wk-old birds

could only be compared indirectly by comparing expression patterns between control and

H9N2 inoculated birds within an age group.

Early gene expression patterns after H9N2 inoculation

In the lung and trachea of all H9N2 inoculated birds viral RNA was detected using qRT-

PCR (Fig. 1A). There was no significant difference in viral RNA levels between 1-wk and

4-wk-old birds in both the lung and trachea at any time point.

To determine the effect of H9N2 inoculation at transcriptional level over time, we

compared gene expression rates in H9N2 inoculated birds to age matched control birds

within a time point. Differences in gene expression rates were given as fold change and

depicted over time generating gene expression patterns. For the lung and trachea gene

expression patterns were generated for global genes (Fig. 1B) and immune related genes

(data not shown, but patterns similar to those of global genes). After inoculation, genes

were mostly up regulated and not down regulated in both the lung and trachea independent

of age. In the lung more genes were up regulated than in the trachea, but gene expression

patterns in the lung and trachea were similar. The biggest difference was found between the

age groups. In 1-wk-old birds gene expression was most affected by H9N2 inoculation at

16 h.p.i. and the amplitude of change declined at 24 h.p.i. In 4-wk-old birds the effect of

H9N2 inoculation on gene expression increased over time and gene expression was affected

most at 16 and 24 h.p.i. Therefore the overall gene expression pattern in response to H9N2

inoculation differed between 1- and 4-wk-old birds. Genes involved in development (based

on GO annotation terms developmental process, developmental maturation, multicellular

organismal development, anatomical structure development) did not follow this expression

pattern and did not differ in expression between H9N2 inoculated birds and age matched

control birds in both age groups (data not shown).

Chapter 7

142

A.

B.

Figure 1. A) Viral RNA expression in lung and trachea of 1- and 4-wk-old H9N2 inoculated birds. Viral RNA

expression was determined using qRT-PCR (n = 4) and data were expressed as means with standard error of the

mean (SEM). B) Gene expression patterns of global genes induced after H9N2 inoculation in lung and trachea.

Gene expression of global genes was determined using microarray analysis. Gene expression rates in 1- and 4-wk-

old H9N2 inoculated birds were compared to gene expression rates in age and time matched control birds (n = 4).

Red indicated up regulated genes and green down regulated genes.

Early gene expression after H9N2 inoculation

In order to determine early responses to H9N2 inoculation in the respiratory tract we

compared gene expression in H9N2 inoculated birds to age matched control birds within a

time point. The number of global and immune related genes significantly differentially

induced after H9N2 inoculation at 8, 16 and 24 h.p.i. in the lung and trachea of 1- and 4-

wk-old birds were depicted in Figure 2A. In the lung and trachea more genes were

differentially expressed after H9N2 inoculation in 4- compared to 1-wk-old birds except for

global genes at 16 h.p.i. Immune related genes significantly differentially expressed after

H9N2 inoculation were depicted in Table 1 for the lung and Table 2 for the trachea. Since

basal gene expression levels differed between 1- and 4-wk-old birds in both the lung and

trachea, a direct comparison between host responses to H9N2 inoculation could not be

0

5

10

15

20

8h 16h 24h 8h 16h 24h

1wk 4wk

0

5

10

15

20

24 2416 1688

1 wk 4 wk

Cor

rect

40-

Ct +

SE

M

Lung

Age

h.p.i.0

5

10

15

20

8h 16h 24h 8h 16h 24h

1wk 4wk

0

5

10

15

20

24 2416 1688

1 wk 4 wk

Cor

rect

40-

Ct +

SE

M

Lung

Age

h.p.i.0

5

10

15

20

8h 16h 24h 8h 16h 24h

1wk 4wk

0

5

10

15

20

Cor

rect

40-

Ct +

SE

M

Trachea

24 2416 1688

1 wk 4 wk Age

h.p.i.0

5

10

15

20

8h 16h 24h 8h 16h 24h

1wk 4wk

0

5

10

15

20

Cor

rect

40-

Ct +

SE

M

Trachea

24 2416 1688

1 wk 4 wk Age

h.p.i.

1

10

100

0.1

0.018 16 24 8 16 24 h.p.i.

1 wk 4 wk

Gen

e ex

pre

ssio

n r

ate

(fol

d c

han

ge)

Lung

Age

1

10

100

0.1

0.018 16 24 8 16 24 h.p.i.

1 wk 4 wk

Gen

e ex

pre

ssio

n r

ate

(fol

d c

han

ge)

Lung

Age

h.p.i.

1

10

100

0.1

0.018 16 24 8 16 24

1 wk 4 wk

Gen

e ex

pre

ssio

n r

ate

(fol

d c

han

ge)

Trachea

Age

h.p.i.

1

10

100

0.1

0.018 16 24 8 16 24

1 wk 4 wk

Gen

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pre

ssio

n r

ate

(fol

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Trachea

Age


143

performed. Instead we performed an indirect comparison by comparing both gene lists.

However, the comparison of gene lists obtained from gene expression in H9N2 inoculated

birds and age matched control birds could still contain genes that are differentially

expressed due to age and not to virus inoculation. To distinguish between gene expression

induced by the virus and gene expression only resulting from aging of the birds we made

venn diagrams (Fig. 2B). Within a diagram one circle contains genes from the comparison

of gene expression in H9N2 inoculated birds to the age matched control birds; H9N2

related genes in 1-wk old or in 4-wk-old birds. The other circle contains genes from the

comparison of gene expression in 1- to 4-wk-old control birds, combined with the

comparison of gene expression in 1- to 4-wk-old H9N2 inoculated birds; age related genes

linked to development and age related genes after virus inoculation. The overlap between

H9N2 related and age related gene expression in the venn diagram was analyzed, and this

overlap indicated the genes that pollute the true H9N2 related genes since they were

differentially expressed between the age groups independent of H9N2 inoculation. There

were few genes found in the overlap between H9N2 related and age related gene expression

in both age groups and tissues. No genes were shared between the overlaps in the trachea

and lung. The overlaps in 1- and 4-wk-old birds shared only 2 genes (PDPN, HSPA4L) in

the lung and 1 gene (STAT4) in the trachea. Genes within the overlaps were not part of

similar functional groups. Thus differences found in host responses after H9N2 inoculation

between 1- and 4-wk-old birds were more related to H9N2 inoculation and hardly affected

by differences in age related genes expression in control birds.

Therefore an indirect comparison of host responses between the age groups by comparing

the lists of genes significantly differentially expressed after H9N2 inoculation could be

performed (Table 1 and 2). In both the lung and trachea no large differences in gene

expression rates between both age groups were found, only more genes were significantly

differentially expressed and relatively more genes were down regulated in 4-wk-old birds

after H9N2 inoculation. Furthermore, more genes involved in innate response like

complement and TLRs were induced in 4-wk-old birds after H9N2 inoculation. In contrast,

in control birds more genes involved in innate response were expressed in 1-wk-old birds.

The difference in number of genes induced after H9N2 inoculation between the age groups

is larger in the lung than in the trachea. In the lung, functional groups containing most

genes in both infected groups were apoptosis, response to virus, transcription, signal

transduction and protein folding. Functional groups containing more than 1 gene that were

mainly expressed in 1-wk-old birds were antigen presentation/binding at 8 h.p.i. and anti-

apoptosis at 24 h.p.i. Functional groups containing more than 1 gene that were mainly

expressed in 4-wk-old birds were cell proliferation from 8 to 24 h.p.i., and chemotaxis and

cytokine activity and inflammatory responses at 16 h.p.i.

Chapter 7

144

Tab

le 1

. Im

mun

e re

late

d ge

nes

indu

ced

afte

r H

9N2

inoc

ulat

ion

in lu

ng o

f 1-

and

4-w

k-ol

d bi

rds

at 8

, 16

and

24 h

.p.i.

1-

wk-

old

4-

wk-

old

8

h.p.

i

16 h

.p.i.

24 h

.p.i.

8 h.

p.i

16

h.p

.i.

24

h.p

.i.

Func

tion

al g

roup

G

ene

Rat

io

G

ene

Rat

io

G

ene

Rat

io

G

ene

Rat

io

G

ene

Rat

io

G

ene

Rat

io

H

SP70

2.

06

H

SP90

B1

2.67

HSP

90B

1 0.

63

Ant

i-ap

opto

sis

H

SPA

5 2.

86

C

TSB

1.

48

A

ntig

en p

rese

ntat

ion/

bind

ing

M

HC

II

0.

62

B

CA

P29

1.82

DA

P 0.

75

F

ASL

1.

68

IAP

3.58

IAP

2.33

IAP

2.43

P

AK

2 1.

60

PD

CD

1 0.

64

TG

FB2

0.66

Apo

ptos

is

TG

FB3

0.57

Cal

cium

ion

bind

ing

F

AM

20C

0.

67

CT

NN

A1

0.76

M

SL

N

0.56

SD

C4

0.65

Cel

l adh

esio

n

SD

C4

2.13

Cel

l div

isio

n

SGO

L1

0.67

PB

EF1

2.

70

PB

EF1

2.

25

PCN

A

0.71

PCN

A

0.65

PD

L1

3.42

PDL

1 2.

66

PD

PN

0.54

PDPN

0.

64

PDPN

0.

47

Cel

l pro

life

rati

on

T

PX2

0.61

TPX

2 0.

56


145

C

CL

i7

2.81

C

CR

L1

2.05

Che

mot

axis

and

cyt

okin

e ac

tivit

y

IL

18

3.06

C

1QB

2.

52

C

1S

1.52

Com

plem

ent

C

3AR

1 1.

87

Def

ense

res

pons

e

BP

I 1.

44

Dev

elop

men

t

IFIT

M5

1.66

M

YD

88

1.93

T

LR

1 1.

88

Infl

amm

ator

y re

spon

se

T

LR

3 2.

11

Inna

te i

mm

une

resp

onse

C

MA

P27

1.52

D

GK

E

2.99

Intr

acel

lula

r si

gnal

ling

SO

CS

1 6.

68

Man

nose

bin

ding

BS

G

1.53

Prot

ein

a.a.

dep

hosp

hory

lati

on

DU

SP1

0.62

FLN

29

2.46

FL

N29

2.

54

FL

N29

1.

82

PHF

11

3.43

PHF

11

2.82

PHF

11

2.60

Prot

ein

bind

ing

SH3Y

L1

0.64

D

NA

JA1

2.06

H

SC

70

2.38

HS

C70

1.

70

H

SP60

1.

85

H

SP60

1.

59

Prot

ein

fold

ing

H

SPA

4L

1.75

HSP

A4L

1.

44

Prot

ein

mod

ific

atio

n

O

AS

L

7.58

OA

SL

7.

89

O

AS

L

5.17

Prot

eoly

sis

C

TSL

1.

56

Chapter 7

146

M

MP2

0.

54

MM

P2

0.49

Res

pira

tory

bur

st

N

CF1

2.

23

Res

pons

e to

DN

A d

amag

e

FAN

CL

0.

70

F

AN

CL

0.

73

FA

NC

L

0.65

H

SP10

5 1.

78

H

SP10

5 1.

63

Res

pons

e to

str

ess

HSP

25

3.61

HSP

25

4.65

IFI3

5 2.

51

IF

I35

2.81

IFI3

5 1.

86

ISG

12-2

5.

52

IS

G12

-2

6.64

MD

A5

3.93

MD

A5

2.78

MD

A5

3.20

Res

pons

e to

vir

us

MX

3.

86

M

X

5.79

RN

A b

indi

ng

RA

LY

0.

69

LE

PR

2.54

LE

PR

2.

84

L

EPR

2.

28

L

Y6E

1.

91

L

Y96

3.

30

RG

S18

1.88

SI

RP-

B1

1.39

SP

RY

3 0.

71

Sign

al tr

ansd

uctio

n

T

NFR

SF11

B

0.66

T

NFR

SF11

B

2.08

Suga

r bi

ndin

g

Gal

ecti

n C

G-1

6 0.

67

IRF1

2.

85

IR

F1

2.39

IRF1

2.

23

IR

F10

3.29

IRF1

0 2.

59

IR

F2

1.61

IR

F3

1.49

IRF8

1.

75

IR

F8

1.83

NM

I 2.

59

N

MI

2.50

NM

I 1.

86

Tra

nscr

ipti

on

ST

AT

4 4.

88

ST

AT

4 4.

25

Tra

nspo

rt

L

APT

M5

1.58

Ubi

quit

in-d

ep c

atab

olic

pro

cess

U

SP18

3.

20

U

SP18

3.

07

U

SP18

2.

14


147

Imm

une

rela

ted

gene

s si

gnif

ican

tly

diff

eren

tial

ly in

duce

d af

ter

H9N

2 in

ocul

atio

n in

lung

of

1- a

nd 4

-wk-

old

bird

s at

8, 1

6 an

d 24

h.p

.i. w

ere

dete

rmin

ed u

sing

MA

AN

OV

A. A

p-va

lue

< 0

.05

was

con

side

red

sign

ific

ant.

Rat

io r

epre

sent

s th

e fo

ld c

hang

e ex

pres

sion

rat

e in

H9N

2 in

ocul

ated

bir

ds c

ompa

red

to a

ge a

nd ti

me

mat

ched

con

trol

bir

ds. G

enes

are

divi

ded

into

fun

ctio

nal g

roup

s ba

sed

on G

O a

naly

sis

usin

g D

AV

ID.

Tab

le 2

. Im

mun

e re

late

d ge

nes

indu

ced

afte

r H

9N2

inoc

ulat

ion

in tr

ache

a of

1-

and

4-w

k-ol

d bi

rds

at 8

, 16

and

24 h

.p.i.

1-

wk-

old

4-w

k-ol

d

8

h.p.

i

16 h

.p.i.

24 h

.p.i.

8 h.

p.i

16

h.p

.i.

24

h.p

.i.

Fun

ctio

nal

gro

up

G

ene

Rat

io

G

ene

Rat

io

G

ene

Rat

io

G

ene

Rat

io

G

ene

Rat

io

G

ene

Rat

io

Act

ivat

ion

of M

AP

K a

ctiv

ity

C

1QT

NF

2 0.

58

H

SP

70

2.26

HS

P70

2.

05

H

SP

90B

1 1.

70

H

SP

A5

2.34

HS

PA

5 2.

89

BC

L2A

1 1.

81

C

IDE

A

0.

51

TR

AIL

1.

70

B c

ell m

arke

r

B

u-1

1.50

PCP4

0.

69

Cal

cium

ion

bind

ing

BR

I3B

P 0.

74

M

HC

B-G

0.

31

TN

IP3

1.85

Mis

cell

aneo

us

T

RA

F3IP

3 3.

04

Chapter 7

148

CD

34

0.60

C

D47

1.

34

CD

H28

0.

57

LG

AL

S3B

P

1.70

SDC

4 1.

83

Cel

l adh

esio

n

TIN

AG

0.

55

B

TG

1 1.

34

P

BE

F1

1.56

PBE

F1

2.26

Cel

l pro

life

rati

on

PDPN

0.

61

CC

Li7

8.

07

CC

Li7

2.

45

C

MT

M3

0.61

Che

mot

axis

and

cyt

okin

e ac

tivit

y

TR

AIL

-lik

e 0.

67

Com

plem

ent

C1S

1.

58

Def

ense

res

pons

e

L

YG

2.

35

Dev

elop

men

t

IF

ITM

5 2.

95

LY

86

0.59

MY

D88

1.

93

Infl

amm

ator

y re

spon

se

TL

R3

1.92

Intr

acel

lula

r si

gnal

ling

A

SB

9 0.

67

AS

B9

0.69

Lys

ozom

e or

gani

satio

n

PSA

P

1.40

Mis

cell

aneo

us

IGS

F3

0.55

Pro

tein

bin

ding

P

HF

11

2.71

PH

F11

2.

41

DN

AJA

1 1.

83

DN

AJB

9 1.

33

H

SC

70

2.04

HS

C70

2.

10

HS

P60

1.

76

Pro

tein

fol

ding

HS

PA

4L

1.63

Pro

tein

mod

ific

atio

n

O

AS

L

7.88


149

Res

pira

tory

bur

st

NC

F1

1.82

H

SP10

5 1.

61

H

SP10

5 2.

24

HSP

25

3.22

Res

pons

e to

str

ess

RPS

6KA

1.

61

IFI3

5 2.

25

IRF3

1.

43

MD

A5

3.55

M

DA

5 2.

70

Res

pons

e to

vir

us

MX

3.

26

A

SB

2 0.

63

LE

PR

1.99

L

EP

R

1.69

LY

6E

2.36

MA

RC

O

1.59

Sign

al tr

ansd

uctio

n

SPR

Y2

0.71

Suga

r bi

ndin

g

CD

69

1.69

PD

L2

0.64

T c

ell

prol

ifer

atio

n

TIM

D4

1.37

IRF

1 2.

64

IRF

10

2.29

NA

RG

1 1.

54

NM

I 2.

50

SOC

S3

2.69

SO

CS

3 4.

51

Tra

nscr

ipti

on

STA

T4

4.20

ST

AT

4 3.

35

Ubi

quiti

n-de

p ca

tabo

lic

proc

ess

USP

18

3.32

Ves

icle

traf

fick

ing

CL

EC

3B

0.57

Mis

cell

aneo

us

IGSF

3 0.

55

Imm

une

rela

ted

gene

s si

gnif

ican

tly

diff

eren

tial

ly in

duce

d af

ter

H9N

2 in

ocul

atio

n in

trac

hea

of 1

- and

4-w

k-ol

d bi

rds

at 8

, 16

and

24 h

.p.i.

wer

e de

term

ined

usi

ng M

AA

NO

VA

.

A p

-val

ue <

0.0

5 w

as c

onsi

dere

d si

gnif

ican

t. R

atio

rep

rese

nts

the

fold

cha

nge

expr

essi

on r

ate

in H

9N2

inoc

ulat

ed c

ompa

red

to a

ge a

nd ti

me

mat

ched

con

trol

bir

ds. G

enes

are

divi

ded

into

fun

ctio

nal g

roup

s ba

sed

on G

O a

naly

sis

usin

g D

AV

ID.

Chapter 7

150

A.

B.

Figure 2. A) The number of global and immune related genes significantly differentially expressed after H9N2

inoculation in lung and trachea. Gene expression of H9N2 inoculated birds were compared to age and time

matched control birds (n = 4) using microarray analysis and significance was determined with MAANOVA

(p<0.05). B) Overlap between age related and H9N2 related gene expression in trachea and lung of both age

groups depicted in venn diagrams. The age related gene set consists of genes significantly differentially expressed

0 100 200 300

24

16

8

24

16

8

Tra

chea

Lung

No. genes significantly differentially expressed after infection

Global genes

0 100 200 300

24

16

8

24

16

8

Tra

chea

Lung

No. genes significantly differentially expressed after infection

Global genesh.p.i.

Lung 1 wk

66 7 37

Lung 1 wk

66 7 37

Lung 4 wk

64 9 59

Lung 4 wk

64 9 59

Trachea 1 wk

35 4 35

Trachea 1 wk

35 4 35

Trachea 4 wk

33 6 39

Trachea 4 wk

33 6 39

Age related

H9N2 related

Age related

H9N2 related

A+ B

CorD

A+ B

CorD

1 wk control 1 wk H9N2 infected


A B

C

D



A B

C

D

Venn diagram legend

0 20 40 60

24

16

8

24

16

8

Tra

chea

Lung

No. genes significantly differentially expressed after to infection

Immune related genes

0 20 40 60

24

16

8

24

16

8

Tra

chea

Lung

No. genes significantly differentially expressed after to infection

Immune related genesh.p.i.

Age:

1 wk

4 wk

Age:

1 wk

4 wk


151

between 1- and 4-wk-old birds within a time point and treatment group in lung and trachea (comparison A+B; n =

4). The H9N2 related gene set consists of genes significantly differentially expressed between control and H9N2

inoculated birds within a time point and age group in lung and trachea (comparison C or D; n = 4). Gene sets were

obtained with microarray analysis and significance was determined using MAANOVA (p<0.05).

In the trachea, functional groups containing most genes in both 1- and 4-wk-old birds were

signal transduction and cell adhesion, but genes were expressed at different time points

(Table 2).

A functional group containing more than 1 gene that was mainly expressed in 1-wk-old

birds was response to virus at 16 h.p.i. However, at 16 h.p.i. no genes were significantly

differentially expressed after H9N2 inoculation in 4-wk-old birds, which seemed to be

caused by large variation within this group. Up regulation of genes related to response to

virus were seen at 16 h.p.i., but in individual birds and not consistent in the whole group. A

functional group containing more than 1 gene that was mainly expressed in 4-wk-old birds

was protein folding at 24 h.p.i.

Cytokine mRNA expression levels

Microarray analysis suggested IL-1β, IL-8 and IL-18 mRNA expression was up regulated

and IFN-α expression was not affected after H9N2 inoculation in 1- and 4-wk-old birds.

Although up regulation of IL-1β, IL-8 and IL-18 genes was rarely significant because of the

very strict microarray statistics that was applied, we did see an up regulation from 8 to 24

h.p.i. of up to 4-fold change compared to age matched control birds. The qRT-PCR data did

show a significant up regulation in H9N2 inoculated compared to control birds for IL-1β,

IL-8 and IL-18 in the lung and trachea of 1- and 4-wk-old birds at several time points (Fig.

3). In both the lung and trachea IFN-α mRNA expression was not significantly different

between H9N2 inoculated and control birds in both qRT-PCR and microarray data. The

difference in significances between microarray and qRT-PCR data are caused by

differences in the statistics that were used. Based on qRT-PCR and microarray analysis, IL-

1β, IL-8 and IL-18 mRNA expression in the lung and trachea was up regulated in a pattern

similar over time (data not shown). mRNA expression in 1-wk-old birds peaked at 16 h.p.i.,

whereas it peaked in 4-wk-old birds at 24 h.p.i.

To determine whether mRNA expression was related to viral RNA expression, correlation

coefficients were calculated between viral RNA expression and cytokine mRNA expression

in both the lung and trachea. We first determined this correlation for all cytokines per age

group over time, because if a correlation between cytokine mRNA and viral RNA

expression is found, this means they correlate at every time point. In 1-wk-old birds there

was a significant strong positive correlation between viral RNA expression and mRNA

expression of IL-1β, IL-8, IL-18 and IFN-α in the lung and mRNA expression of IL-1β,

Chapter 7

152

Figure 3. Cytokine mRNA expression in lung and trachea of 1- and 4-wk-old birds in response to H9N2

inoculation. IL-1β, IL-8, IL-18 and IFN-α mRNA expression was depicted as fold change in H9N2 inoculated

compared to time and age matched control birds in lung and trachea. Cytokine mRNA expression was determined

using qRT-PCR (n = 4) and data were expressed as means with standard error of the mean (SEM) with asterisk (*)

indicating a significant difference (p<0.05) in cytokine expression between H9N2 inoculated birds and age and

time matched control bird.

IL-8 and IFN-α in the trachea (Table 3). In 4-wk-old birds there was no correlation between

viral RNA expression and expression of these cytokines mRNAs over time in both the lung

and trachea. Therefore we determined correlations within a time point. In 4-wk-old birds

there was a significant strong positive correlation in the trachea at 24 h.p.i. between viral

RNA expression and IL-1β and IL-8 mRNA expression. In the lung at 16 h.p.i. a significant

strong negative correlation was found for IFN-α mRNA expression (Table 3).

Host gene expression hijacking by influenza

To determine the effect of H9N2 inoculation on mRNA expression of host factors that

interact with viral factors we compared their gene expression rate in H9N2 inoculated with

control birds within a time point. Results were depicted in Table 4. Mini chromosome

maintenance complex (MCM) related genes were down regulated after H9N2 inoculation

0

5

10

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

10

5

0

Fol

d c

hang

e

IFN-a

0

5

10

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

10

5

0

Fol

d c

hang

e

IFN-a

0

50

100

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

100

50

0

Fol

d ch

ang

e

IL-18

**

*

*

*0

50

100

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

100

50

0

Fol

d ch

ang

e

IL-18

**

*

*

*

0

250

500

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

500

250

0

Fol

d c

hang

e

IL-8

*

* *

*

*

*

0

250

500

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

500

250

0

Fol

d c

hang

e

IL-8

*

* *

*

*

*

0

75

150

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

150

75

0

Fol

d ch

ang

e

IL-1ß

*

*

* *

* *

*

0

75

150

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

150

75

0

Fol

d ch

ang

e

IL-1ß

*

*

* *

* *

*

IFN-aIL-18

IL-8IL-1ß

Age

Age Age

Age

8 h.p.i. 16 h.p.i. 24 h.p.i.8 h.p.i. 16 h.p.i. 24 h.p.i.

0

5

10

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

10

5

0

Fol

d c

hang

e

IFN-a

0

5

10

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

10

5

0

Fol

d c

hang

e

IFN-a

0

50

100

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

100

50

0

Fol

d ch

ang

e

IL-18

**

*

*

*0

50

100

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

100

50

0

Fol

d ch

ang

e

IL-18

**

*

*

*

0

250

500

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

500

250

0

Fol

d c

hang

e

IL-8

*

* *

*

*

*

0

250

500

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

500

250

0

Fol

d c

hang

e

IL-8

*

* *

*

*

*

0

75

150

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

150

75

0

Fol

d ch

ang

e

IL-1ß

*

*

* *

* *

*

0

75

150

1wk 4wk 1wk 4wk

Lung Trachea

1 wk 4 wk 1 wk 4 wk

Lung Trachea

150

75

0

Fol

d ch

ang

e

IL-1ß

*

*

* *

* *

*

IFN-aIL-18

IL-8IL-1ß

Age

Age Age

Age

8 h.p.i. 16 h.p.i. 24 h.p.i.8 h.p.i. 16 h.p.i. 24 h.p.i.


153

while other genes affected by H9N2 inoculation were up regulated. MCM2 and MCM4

were down regulated after H9N2 inoculation at an early stage at 8-16 h.p.i. in the lung of

both 1- and 4-wk-old birds and the trachea of 1-wk-old birds. Interferon-induced GTP-

binding protein Mx (MX) was up regulated at 16 h.p.i. in the lung of 1- and 4-wk-old birds

and in the trachea of 1-wk-old birds. HSP70 and HSC70 were both up regulated in a later

stage after H9N2 inoculation at 24 h.p.i. in both the lung and trachea of 1- and 4-wk-old

birds. The effect of H9N2 inoculation on expression of these genes was independent of age

and organ. DEAD box helicase related genes DDX3, DDX18 and DDX50 were only up

regulated in the trachea of 4-wk-old birds at 24 h.p.i., while in 1-wk-old birds or in the lung

expression of DDX genes were not affected by H9N2 inoculation. Expression of other

genes coding for host factors known to bind to viral factors and being involved in influenza

virus replication like importins (or karyopherin), SFPQ/CPSF, core histones, RACK I and

ERK were not significantly differentially expressed. Genes coding for other host factors

that play a role in influenza virus replication like CRM1 (exportin-1), BAT1/UAP56

(DEAD-box helicase), NXP-2, RanBP5, eIF-4GI, PAB II, Tat-SF1 were not annotated on

this microarray.

Discussion

Previous studies have shown age-dependent development of resistance to infection in both

mammals and birds (Hume et al., 1998, Mukiibi-Muka et al., 1999, Velilla et al., 2006). In

mammals neonatal immune responses seem to be dominated by T helper cell type 2

(Adkins, 1999), with for example reduced numbers of dendritic cells and impaired antigen-

presenting cell (APC) function (Velilla et al., 2006). In birds the genetic background also

plays an important role in susceptibility to pathogens like Salmonella enterica. However, at

young age both susceptible and resistant lines were highly susceptible to infection (Beal et

al., 2005). Here we describe the differences in gene expression in early host responses to

AIV in the respiratory tract between 1- and 4-wk-old birds and investigated the effect of

age on gene expression of cellular host factors that interact with viral factors of which some

are needed for viral replication.

Before we compared virus-induced host responses between 1- and 4-wk-old birds we

determined the effect of age on gene expression in control birds. Differences in gene

expression between 1- and 4-wk-old control birds mainly related to tissue developmental

processes and immune related functional groups. Most of these immune related genes were

expressed in a higher rate in 4- compared to 1-wk-old control birds and most likely related

to the higher number of leukocytes present in the respiratory tract of 4-wk-old birds. Genes

expressed at higher rate in 1-wk-old birds are likely an indication for the ongoing

development of the respiratory immune system. Genes involved in innate immune

Chapter 7

154

IL

-1β

IL

-8

IL

-18

IF

N-α

Tis

sue

Age

T

ime

(h.p

.i.)

r p

r

p

r p

r

p

1 w

k 8,

16,

24

0.

95

1.92

E-0

6

0.94

5.

90E

-06

0.

87

2.40

E-0

4

0.59

0.

042

Lun

g 4

wk

16

-

-

-

-0

.96

0.03

4

1 w

k 8,

16,

24

0.

58

0.04

7

0.65

0.

023

-

0.61

0.

036

Tra

chea

4

wk

24

0.99

0.

014

0.

97

0.03

4

-

-

Cor

rela

tion

s be

twee

n vi

ral R

NA

and

cyt

okin

e m

RN

A e

xpre

ssio

n w

ere

base

d on

the

Pea

rson

cor

rela

tion

coe

ffic

ient

(r)

and

det

erm

ined

usi

ng S

PSS

15.

0 so

ftw

are.

A p

-val

ue f

or

2-ta

iled

sig

nifi

canc

e (p

) <

0.0

5 w

as c

onsi

dere

d si

gnif

ican

t.

Tab

le 4

. Eff

ect o

f H9N

2 in

ocul

atio

n on

gen

e ex

pres

sion

of

prot

eins

that

can

inte

ract

wit

h in

flue

nza

viru

s in

lung

and

trac

hea

of 1

- and

4-w

k-ol

d bi

rds

at 8

, 16

and

24 h

.p.i.

Lun

g T

rach

ea

1 w

k 4

wk

1 w

k 4

wk

Age

H

ost

fact

or

Inte

ract

ing

vira

l fa

ctor

P

ropo

sed

func

tion

H

ost

fact

or

chic

ken

8 16

24

8

16

24

8 16

24

8

16

24

h.p

.i.

Kar

yoph

erin

-α 2

-β

NP

Nuc

lear

impo

rt v

RN

P K

PNA

2 -

- -

- -

- -

- -

- -

-

MxA

N

P In

hibi

t nuc

lear

impo

rt v

RN

P M

X

- ↑

- -

↑ -

- ↑

- -

- -

ER

K

M1

E

RK

-

- -

- -

- -

- -

- -

-

RA

CK

1 M

1 M

1 ph

osph

oryl

atio

n R

AC

K1

- -

- -

- -

- -

- -

- -

Hsp

70

NP/

RN

P N

ucle

ar e

xpor

t vR

NP

H

sp70

-

- ↑

- -

- -

- ↑

- -

↑

Hsc

70

M1

Inhi

bit n

ucle

ar e

xpor

t, vR

NP,

M1,

NP

Hsc

70

- -

↑ -

- ↑

- -

↑ -

- ↑

CPS

F4

- -

- -

- -

- -

- -

- -

CPS

F5

- -

- -

- -

- -

- -

- -

SFPQ

/CPS

F N

S1

Inhi

bits

nuc

lear

exp

ort c

ellu

lar

mR

NA

s, n

ot v

iral

mR

NA

s

CPS

F6

- -

- -

- -

- -

- -

- -

Tab

le 3

. Sig

nifi

cant

cor

rela

tion

s be

twee

n vi

ral R

NA

exp

ress

ion

and

cyto

kine

mR

NA

exp

ress

ion

in lu

ng a

nd tr

ache

a of

1-

and

4-w

k-ol

d bi

rds

at 8

, 16

and

24 h

.p.i.


155

MC

M2

↓ -

- ↓

↓ -

↓ -

- -

- -

MC

M3

- -

- -

- -

- -

- -

- -

MC

M4

- -

- ↓

↓ -

↓ -

- -

- -

MC

M5

- -

- -

- -

- -

- -

- -

MC

M

PA

R

epli

cati

on

MC

M6

- -

- -

- -

- -

- -

- -

H2A

FZ

-

- -

- -

- -

- -

- -

-

H2B

-VII

I -

- -

- -

- -

- -

- -

-

H2B

-V

- -

- -

- -

- -

- -

- -

H3-

IX

- -

- -

- -

- -

- -

- -

His

tone

pro

tein

s M

1, R

NP

Nuc

lear

exp

ort v

RN

P

H4-

I -

- -

- -

- -

- -

- -

-

DD

X3

- -

- -

- -

- -

- -

- ↑

DD

X18

-

- -

- -

- -

- -

- -

↑

DD

X50

-

- -

- -

- -

- -

- -

↑

DE

AD

box

RN

A

heli

case

(D

DX

3,

DD

X5)

Poly

mer

ase

com

plex

N

ucle

ar e

xpor

t vR

NP

DD

Xa

- -

- -

- -

- -

- -

- -

Dif

fere

nces

in g

ene

expr

essi

on b

etw

een

cont

rol b

irds

and

H9N

2 in

ocul

ated

bir

ds o

f pr

otei

ns th

at c

an in

tera

ct w

ith

infl

uenz

a vi

rus

wer

e id

enti

fied

usi

ng M

AA

NO

VA

wit

hin

tim

e

and

age

in lu

ng a

nd tr

ache

a. A

p-v

alue

< 0

.05

was

con

side

red

sign

ific

ant. S

igni

fica

nt d

iffe

rent

ial e

xpre

ssio

n in

H9N

2 in

ocul

ated

com

pare

d to

con

trol

bir

ds is

indi

cate

d w

ith

(↑)

for

up r

egul

atio

n an

d (↓

) fo

r do

wn

regu

lati

on. G

enes

exp

ress

ion

that

did

not

dif

fer

sign

ific

antl

y af

ter

H9N

2 in

ocul

atio

n w

as in

dica

ted

wit

h (-

). D

DX

a sta

nds

for

gene

DD

X10

,

DD

X24

, DD

X25

, DD

X26

, DD

X29

, DD

X31

, DD

X32

, DD

X41

, DD

X42

, DD

X46

, DD

X55

, DD

X59

.

Chapter 7

156

responses were also higher expressed in 1-wk-old control birds and may indicate that

protective responses in young birds are more dependent on innate responses (Levy, 2007).

In the trachea less genes were significantly differentially expressed between 1- and 4-wk-

old control birds compared to the lung. For the trachea constitutive lymphoid tissue has not

been described unlike for the lung (Kothlow et al., 2008), which would explain the lower

number of differentially expressed genes between 1- and 4-wk-old control birds.

Difference in virus deposition within the respiratory tract has an influence on gene

expression (Baas et al., 2006, Reemers et al., 2009b). Since no significant differences in

viral RNA expression were found between 1- and 4-wk-old birds, differences in gene

expression after H9N2 inoculation are caused by differences in host response between the

age groups. We showed that these differences were not influenced by differences in gene

expression between the age groups in control birds and are thus directly related to H9N2

inoculation.

Although genes involved in innate responses were expressed at higher rate in 1-wk-old

control birds, after H9N2 inoculation genes involved in innate responses were more

induced in 4-wk-old birds. In the lung, more genes related to cell proliferation, chemotaxis

and cytokine activity, inflammatory response and transcription were expressed in 4-wk-old

birds. This suggests a different host response at transcriptional level in 1- compared to 4-

wk-old birds likely relating to age-related differences in immune responses. APC play an

important role during innate immune responses. Neonatal APC from mice and humans have

been reported to be less effective in supporting proliferation of T cells (Petty et al., 1998)

probably due to lower expression of MHCI, MHCII and costimulatory molecules (Hunt et

al., 1994). Furthermore, they are defective in cytokine production upon LPS stimulation or

influenza virus infection (Chelvarajan et al., 2004, Zhou et al., 2006) and require a higher

level of activation than adult APC (Petty et al., 1998), which has been proposed to be due to

defects in TLR signalling (De Wit et al., 2003, Velilla et al., 2006). In our study MHCI and

MHCII were less expressed in 1-wk-old birds which might suggest a similar immaturity of

APC as found in mice. Cytokine genes and TLR signalling related genes were more

expressed in 4-wk-old birds, while MHCII was down regulated in 1-wk-old birds after

H9N2 inoculation relating to functional impairment described for mammalian APC.

In the trachea the largest and clearest differences between 1- and 4-wk-old birds were the

time points of expression and the number of genes expressed, but no large differences

between functional groups were found, unlike in the lung. This possibly indicates that

maturity of the respiratory immune system has less effect on the trachea than on the lung

correlating with the lack of constitutive lymphoid tissue which has not been described in

the avian trachea so far.

Elevated expression levels of inflammatory cytokines and chemokines due to influenza

infection have been reported as early as 6 h.p.i. depending on the influenza strain (Julkunen


157

et al., 2001, Chan et al., 2005). Also in this study up regulation of IL-1β, IL-8 and IL-18

mRNA expression was found after H9N2 inoculation in both age groups. The level of viral

RNA expression correlated with the mRNA expression levels of IL-1β, IL-8, IL-18 and

IFN-α over time in 1-wk-old birds, but for 4-wk-old birds correlation was only seen at later

time points after H9N2 inoculation. Although the mean IFN-α mRNA expression was not

significantly up regulated, there were correlations between viral RNA en IFN-α mRNA

expression for individual birds at both 1- and 4-wk of age. These data indicate that

induction of gene expression of inflammatory cytokines and chemokines after H9N2

inoculation is more related to viral load in 1-wk-old birds compared to 4-wk-old birds.

Influenza viral proteins bind to several mammalian host proteins which promote viral

replication or induce host responses (Engelhardt et al., 2006, Naffakh et al., 2008, Nagata et

al., 2008). MCM2 and MCM4 are part of the minichromosome maintenance complex

which is proposed to activate virus genome replication at the early phase of infection when

there is no newly synthesised NP present (Nagata et al., 2008). These genes were down

regulated early after H9N2 inoculation which may be an attempt of the host to block viral

replication. At 16 h.p.i. blocking of viral replication is possibly enhanced by up regulation

of MX, which for humans binds to viral NP and this binding likely prevents nuclear import

of incoming vRNP (Turan et al., 2004, Naffakh et al., 2008). In the chicken some MX

proteins are found to have a similar inhibitory function while others do not depend on breed

and virus strain, for which a possible explanation is the high polymorphism in the chicken

MX gene (Ko et al., 2002, Benfield et al., 2008). For chickens HSP70 and HSC70 were up

regulated at 24 h.p.i. possibly preventing assembly of new virions, since HSP70 prevents

binding of viral M1 to vRNP (Hirayama et al., 2004) and HSC70 binds to viral M1 (Nagata

et al., 2008) resulting in inhibition of nuclear export. A common response of the host to

block viral replication seemed to occur, because gene expression of MCMs, MX, HSP70

and HSC70 was regulated independent of age and tissue. Interestingly DDX3, DDX18 and

DDX50 were only up regulated in the trachea of 4-wk-old birds at 24 h.p.i. when newly

assembled RNPs are exported to be packaged into progeny virions. The influenza

polymerase complex is known to interact with DDX3, which plays an important role in

RNA nuclear export and cytoplasmic mRNA localisation (Jorba et al., 2008), promotes

export of HIV-1 RNAs from the nucleus to cytoplasm (Ishaq et al., 2008) and is required

for HCV RNA replication (Ariumi et al., 2007). This may indicate that DDX is needed for

influenza virus replication at a later stage after virus inoculation when newly assembled

RNPs are exported to be packaged into progeny virions and expression is possibly age and

tissue related.

In summary, gene expression in control birds and host responses to AIV inoculation in the

trachea and especially the lung are correlated with the development and maturation of the

respiratory immune system. Differences in immune related gene expression after H9N2

Chapter 7

158

inoculation in the lung can be related to the higher levels of stimulation needed to activate

neonatal host responses and age-dependent functionality of leukocytes. However,

expression of most cellular host factors that block viral replication by interacting with viral

factors is independent of age. These findings suggest that the strength of virus-induced host

responses is affected by maturation of the respiratory immune system and may be a key

factor in age-dependent host responses to infection. However, the differences found at

transcriptional level were not yet translated to differences in viral load between the age

groups, due to the time frame in which we measured the responses. This study shows

multiple factors could be involved in neonatal impaired response, such as functional

impairment APC, NK cells and T cells and more research into the contribution of these

factors is needed to get a better understanding of the functional capability of the neonatal

immune system and the relation to susceptibility.

Acknowledgements

We thank Christine Jansen and Eveline de Geus from the department of Infectious Diseases

and Immunology of the Faculty of Veterinary Medicine Utrecht for their technical

assistance, Cheuk Ko, Sander van Hooff and Patrick van Kemmeren from the Genomics

Laboratory, UMC Utrecht for performing the microarrays and bioinformatical assistance.

This work was supported by a BSIK VIRGO consortium grant (Grant no. 03012), The

Netherlands.

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41. Zhou, J., Law, H.K., Cheung,, C.Y., Ng, I.H., Peiris, J.S., Lau, Y.L., 2006. Differential expression of

chemokines and their receptors in adult and neonatal macrophages infected with human or avian

influenza viruses. J. Infect. Dis. 194, 61-70.


161

Supplementary data

Tab

le S

1. T

op th

ree

func

tion

al g

roup

s ba

sed

on G

O th

at w

ere

sign

ific

antly

dif

fere

ntia

lly

expr

esse

d be

twee

n 1-

and

4-w

k-ol

d co

ntro

l bir

ds in

lung

and

trac

hea

at 0

, 8, 1

6 an

d 24

h.p

.i..

Lun

g

Tra

chea

Tim

e (h

.p.i.

) T

op

Fun

ctio

nal g

roup

N

umbe

r of

gen

es

invo

lved

Fun

ctio

nal g

roup

N

umbe

r of

gen

es

invo

lved

0 1

Mul

tice

llula

r or

gani

smal

pro

cess

39

Dev

elop

men

tal p

roce

ss

15

2

Dev

elop

men

tal p

roce

ss

36

M

ulti

cellu

lar

orga

nism

al p

roce

ss

14

3

Mul

tice

llula

r or

gani

smal

dev

elop

men

t 30

Mul

tice

llula

r or

gani

smal

dev

elop

men

t 12

8 1

Mul

tice

llula

r or

gani

smal

pro

cess

37

Cel

lula

r ca

rboh

ydra

te m

etab

olic

pro

cess

5

2

Dev

elop

men

tal p

roce

ss

35

C

arbo

hydr

ate

met

abol

ic p

roce

ss

5

3

Cel

l com

mun

icat

ion

30

A

lcoh

ol m

etab

olic

pro

cess

5

16

1 Si

gnal

tran

sduc

tion

14

M

ulti

cellu

lar

orga

nism

al p

roce

ss

10

2

Ana

tom

ical

str

uctu

re d

evel

opm

ent

10

A

nato

mic

al s

truc

ture

dev

elop

men

t 7

3

Sys

tem

dev

elop

men

t 8

S

yste

m p

roce

ss

6

24

1 C

ellu

lar

proc

ess

108

G

luco

se m

etab

olic

pro

cess

2

2

Bio

logi

cal r

egul

atio

n 51

Alc

ohol

met

abol

ic p

roce

ss

2

3

Cel

l com

mun

icat

ion

45

C

ellu

lar

carb

ohyd

rate

met

abol

ic p

roce

ss

2

Dif

fere

nces

in g

ene

expr

essi

on in

lung

and

trac

hea

of 1

- and

4-w

k-ol

d co

ntro

l bir

ds a

t 8, 1

6 an

d 24

h.p

.i. w

ere

dete

rmin

ed u

sing

MA

AN

OV

A w

ithi

n a

tim

e po

int.

A p

-val

ue <

0.05

was

con

side

red

sign

ific

ant.

Gen

es s

igni

fica

ntly

dif

fere

ntia

lly

expr

esse

d w

ere

subj

ecte

d to

GO

ana

lysi

s us

ing

DA

VID

, red

ucin

g ge

ne li

sts

into

fun

ctio

nall

y re

late

d ge

ne

grou

ps. T

he to

p 3

func

tion

al g

roup

s an

d th

e nu

mbe

r of

gen

es s

igni

fica

ntly

dif

fere

ntia

lly

expr

esse

d w

ithi

n th

is g

roup

are

sho

wn.

Chapter 7

162

Tab

le S

2.

Imm

une

rela

ted

gene

s di

ffer

enti

ally

exp

ress

ed b

etw

een

1- a

nd 4

-wk-

old

cont

rol b

irds

in lu

ng a

t 0, 8

, 16

and

24 h

.p.i.

0 h.

p.i.

8

h.p.

i.

16 h

.p.i.

24 h

.p.i.

Fun

ctio

nal g

roup

Gen

e ID

R

atio

Gen

e ID

R

atio

Gen

e ID

R

atio

Gen

e ID

R

atio

Ant

i-ap

opto

sis

H

SP70

E

1171

5 0.

70

C

D74

E

0459

4 0.

59

C

D74

E

0459

4 0.

51

D

MA

E

0015

8 0.

73

IF

I30

E03

389

0.69

IG

J E

1155

1 0.

53

IG

J E

1155

1 0.

47

IG

J E

1155

1 0.

45

IG

LC

E

2113

9 0.

16

IG

LC

E

2113

9 0.

22

IG

LC

E

2113

9 0.

23

IG

LC

E

2113

9 0.

13

M

HC

I

E02

614

0.21

M

HC

I a

E

2435

0 0.

43

M

HC

II

b E

0014

1 0.

51

M

HC

II

b E

0014

1 0.

54

M

HC

II

b E

0014

1 0.

65

Ant

igen

pre

sent

atio

n/bi

ndin

g

Y

FV

E24

348

0.31

B

NIP

3L

E00

231

1.70

C

ASP

7 E

0893

3 0.

79

C

ASP

9 E

0136

6 1.

39

D

AP

E13

002

1.36

PA

K2

E06

426

2.36

PAK

2 E

0642

6 1.

87

Apo

ptos

is

PD

CD

1 E

0745

4 2.

06

PD

CD

1 E

0745

4 1.

72

B

CL

6 E

0735

7 0.

59

B

CL

6 E

0735

7 0.

65

B c

ell d

iffe

rent

iatio

n

B

LN

K

E06

973

0.77

B c

ell m

arke

r

Bu-

1 E

1546

1 0.

66

B c

ell m

atur

atio

n

BT

K

E04

958

0.69

BT

K

E04

958

0.70

C

AD

11

E05

278

1.49

C

ell a

dhes

ion

C

D44

-lik

e E

0784

9 0.

74

C

D44

-lik

e E

0784

9 0.

69

Cel

l dif

eren

tiat

ion

IF

RD

1 E

0944

8 1.

67


163

PD

GFD

E

1717

9 0.

70

PD

PN

E04

095

1.70

Cel

l pro

life

rati

on

S

PAR

C

E04

184

1.54

C

X3C

R1

E11

955

0.77

IL

16

E06

388

0.68

IL16

E

0638

8 0.

64

IL

16

E06

388

0.57

X

CL

1 E

1523

5 0.

51

X

CL

1 E

1523

5 0.

45

Che

mot

axis

X

CR

1 E

1173

5 0.

74

Coa

gula

tion

F3

E05

619

0.68

C

1QB

E

2156

9 0.

76

Com

plem

ent

C

4BP

A

E01

175

1.35

Cyt

okin

e re

cept

or a

ctiv

ity

G

HR

E

1485

5 1.

56

Dev

elop

men

t

IFIT

M5

E04

243

2.25

IFIT

M5

E04

243

1.83

Foca

l adh

esio

n

LIM

S2

E01

995

1.41

GT

P bi

ndin

g

GIM

AP

7 E

0513

6 0.

64

Imm

une

resp

onse

cyto

kine

E

0232

9 0.

69

cy

toki

ne

E02

329

0.67

C

MA

P27

E

1969

6 1.

65

G

AL

6 E

1666

8 1.

62

Inna

te im

mun

e re

spon

se

T

LR

7 E

1659

0 0.

74

Man

nose

bin

ding

BS

G

E01

328

1.87

Prot

ein

amin

o ac

id

deph

osph

oryl

atio

n

DU

SP1

E03

706

0.60

A

NX

A6

E04

357

1.79

AN

XA

6 E

0435

7 1.

81

K

BT

BD

8 E

0756

9 1.

39

L

CP

1 E

1698

6 0.

67

T

AR

P g

2 E

1960

2 0.

52

T

AR

P g

2 E

1960

2 0.

47

T

AR

P g2

E

1960

2 0.

58

T

IMP4

E

0496

9 0.

65

T

IMP4

E

0496

9 0.

65

T

XK

E

1412

7 0.

77

T

XK

E

1412

7 0.

76

SL

A

E16

234

0.64

Prot

ein

bind

ing

SU

SD3

E05

231

1.56

Chapter 7

164

H

SPA

4L

E10

185

0.74

Pr

otei

n fo

ldin

g

H

SPB

2 E

0794

2 2.

14

H

SPB

2 E

0794

2 1.

75

Prot

ein

mat

urat

ion

H

SP47

E

1121

4 1.

66

C

APN

3 E

0903

6 1.

85

C

APN

3 E

0903

6 1.

50

Prot

eoly

sis

C

TSD

E

0661

3 1.

45

IG

SF2

E15

467

1.53

IGSF

2 E

1546

7 1.

43

L

Y96

E

1564

8 0.

60

PR

KA

R2B

E

0806

0 1.

46

R

ASS

F2

E00

206

0.65

R

ASS

F3

E09

842

1.31

SI

RP-

B1

E06

171

2.67

SIR

P-B

1 E

0617

1 2.

24

SI

RP-

B1

E06

171

1.86

SP

R3

E07

448

2.31

SPR

3 E

0744

8 1.

72

Sign

al tr

ansd

uctio

n

SO

CS

3 E

0718

9 0.

65

Sign

al tr

ansd

uctio

n in

hibi

tor

R

GS1

E

0254

9 0.

47

C

D3D

E

0741

8 0.

54

C

D3D

E

0741

8 0.

56

C

D3D

E

0741

8 0.

62

C

D3D

E

0741

8 0.

55

C

D3E

E

0741

6 0.

74

C

D3E

E

0741

6 0.

64

C

D3E

E

0741

6 0.

75

T c

ell a

ctiv

atio

n

C

D7

E01

631

0.71

CD

7 E

0163

1 0.

66

C

D7

E01

631

0.74

T c

ell d

iffe

rent

iati

on

IL

2RG

E

0563

8 0.

69

T c

ell p

roli

fera

tion

TIM

D4

E03

876

0.71

M

AFF

E

1227

7 1.

42

Tra

nscr

ipti

on

T

ITF1

E

2042

3 1.

33


165

B

RI3

BP

E02

913

1.51

M

HC

B-G

E

2435

7 1.

84

M

IM1

E06

323

2.17

E06

699

0.38

E07

735

1.54

E17

614

2.48

E

1761

4 2.

11

Mis

cell

aneo

us

E21

142

0.59

E

2114

2 0.

49

E21

142

0.40

mm

une

rela

ted

gene

s si

gnif

ican

tly

diff

eren

tial

ly e

xpre

ssed

in lu

ng b

etw

een

1- a

nd 4

-wk-

old

cont

rol b

irds

at 8

, 16

and

24 h

.p.i.

wer

e de

term

ined

usi

ng M

AA

NO

VA

. A p

-val

ue

< 0

.05

was

con

side

red

sign

ific

ant.

Rat

io r

epre

sent

s th

e fo

ld c

hang

e ex

pres

sion

rat

e in

1-

com

pare

d to

4-w

k-ol

d ti

me

mat

ched

con

trol

bir

ds. T

he E

nsem

bl I

D n

umbe

r is

dep

icte

d

n th

e ID

col

umn

in w

hich

E s

tand

s fo

r E

NSG

AL

G00

0000

. Gen

es a

re d

ivid

ed in

to f

unct

iona

l gro

ups

base

d on

GO

ana

lysi

s us

ing

DA

VID

.

Tab

le S

3. I

mm

une

rela

ted

gene

s di

ffer

entia

lly

expr

esse

d be

twee

n 1-

and

4-w

k-ol

d co

ntro

l bir

ds in

trac

hea

at 0

, 8, 1

6 an

d 24

h.p

.i.

0

h.p.

i.

8 h.

p.i.

16

h.p

.i.

24

h.p

.i.

Fun

ctio

nal g

roup

Gen

e ID

R

atio

Gen

e ID

R

atio

Gen

e ID

R

atio

Gen

e ID

R

atio

IG

LC

E

2113

9 0.

19

IG

LC

E

2113

9 0.

16

IG

J E

1155

1 0.

35

IG

J E

1155

1 0.

37

C

D74

E

0459

4 0.

51

M

HC

II β

E00

141

0.51

Ant

igen

pre

sent

atio

n/bi

ndin

g

L

Y75

E

1115

3 0.

66

Apo

ptos

is

R

BM

5 E

0462

6 0.

58

C

D34

E

0117

7 0.

66

C

D44

-lik

e E

0784

9 0.

72

Cel

l adh

esio

n

C

D36

E

0843

9 1.

54

Coa

gula

tion

F3

E05

619

0.63

Com

plem

ent

C

D93

E

0835

9 1.

40

Intr

acel

lula

r si

gnal

ing

casc

ade

A

SB

9 E

1656

3 0.

73

Chapter 7

166

Lip

id tr

ansp

orte

r ac

tivi

ty

SF

TP

A1

E02

503

2.32

Prot

ein

amin

o ac

id d

epho

spho

ryla

tion

DU

SP26

E

0163

3 1.

66

T

IMP4

E

0496

9 0.

62

Prot

ein

bind

ing

T

AK

1L

E15

822

1.85

Pro

tein

fol

ding

HSP

B2

E07

942

1.39

Sign

al tr

ansd

uctio

n

LY

6E

E16

152

0.72

Supe

roxi

de d

ism

utas

e ac

tivi

ty

SO

D3

E18

557

0.57

Tra

nscr

ipti

on

ST

AT

4 E

0765

1 0.

67

Ves

icle

traf

fick

ing

C

LE

C3B

E

1188

3 0.

44

C

LE

C3B

E

1188

3 0.

48

IG

SF3

E15

469

0.61

E21

142

0.45

ev

a E

0741

2 0.

68

E13

378

1.70

E

1337

8 1.

70

E13

378

1.80

E03

338

1.93

Mis

cell

aneo

us

E11

796

6.52

Imm

une

rela

ted

gene

s si

gnif

ican

tly

diff

eren

tial

ly e

xpre

ssed

in tr

ache

a be

twee

n 1-

and

4-w

k-ol

d co

ntro

l bir

ds a

t 8, 1

6 an

d 24

h.p

.i. w

ere

dete

rmin

ed u

sing

MA

AN

OV

A. A

p-

valu

e <

0.0

5 w

as c

onsi

dere

d si

gnif

ican

t. R

atio

rep

rese

nts

the

fold

cha

nge

expr

essi

on r

ate

in 1

- co

mpa

red

to 4

-wk-

old

tim

e m

atch

ed c

ontr

ol b

irds

. The

Ens

embl

ID

num

ber

is

depi

cted

in th

e ID

col

umn

in w

hich

E s

tand

s fo

r E

NSG

AL

G00

0000

. Gen

es a

re d

ivid

ed in

to f

unct

iona

l gro

ups

base

d on

GO

ana

lysi

s us

ing

DA

VID

.

General discussion

Chapter 8

General discussion

169

Upon entry of the respiratory tract, avian influenza virus (AIV) triggers early immune

responses in the host that are aimed to prevent or control this infection. Although much

research is performed to elucidate the course of events that follow after AIV infection, the

interaction between the virus and the host at molecular and cellular level at an early stage of

infection is unclear. More insight into mechanisms underlying innate and adaptive immune

responses leading to pathogenesis or elimination of AIV may contribute to a better

understanding of AIV induced pathogenesis and new concepts for vaccine development.

Early innate responses blocking virus entry

Although adaptive responses are important for the control and clearance of AIV infection,

innate responses are critical for blocking of virus entry and virus replication, and the

induction of virus specific adaptive response. In the airways, virus entry is blocked via

several mechanisms such as mucus, secretory IgA (Tamura et al., 2004), complement

molecules (Favoreel et al., 2003) or antimicrobial peptides such as gallinacins (Xiao et al.,

2004) that neutralize the virus. Also part of the innate immune system are the collectins,

which belong to a family of pattern recognition receptors. Mammalian collectins are

described to have a strong neutralizing activity against influenza A virus and enhance

complement mediated phagocytosis by macrophages (Hartshorn et al., 1997; Gil et al.,

2009). For chicken several collectins have been identified (Hogenkamp et al., 2006), but

whether these collectins have the same functional properties as their mammalian

counterparts was elusive. We showed that chicken collectins were regulated in expression

during AIV infection and might play a role in the host response to AIV in the respiratory

tract of chickens (chapter 3 and 4). Further research using recombinant collectin proteins

and collectin specific antibodies will give more insight into the functional properties of

chicken collectins and their function during early host responses to AIV.

Early innate responses upon infection

When virus entry is not successfully blocked, AIV infects the epithelial cells resulting in

the production of pro-inflammatory cytokines, chemokines and interferons (Julkunen et al.,

2000). This attracts macrophages and DC, which upon activation or AIV infection also start

producing cytokines and chemokines, attracting more APC and lymphocytes to the place of

infection. Between the different strains of influenza A virus varying levels of cytokine

production are reported. In mammals higher cytokine responses are seen for strains causing

lethal infections (Sandbulte et al., 2008; Cillóniz et al., 2009). Hypercytokinemia is

proposed to be related to virulence of the strain and the outcome of the infection (De Jong

et al., 2006; Kobasa et al., 2007). In chicken the level of cytokine expression is also related

to the virulence of the influenza virus strain and increased pathogenicity for H5N1 (Suzuki

Chapter 8

170

et al., 2009). A direct comparison between different strains is not reported, but in our study

we also saw a high level of cytokine expression in chickens primary infected with low

pathogenic H9N2 (chapter 7). Could this indicate that in chicken the virulence of the strain

is not the main factor in defining cytokine expression levels as in mammals? Between the

study of Suzuki et al. (2009) and our study the infection dose differed, which could have

caused difference in host response (chapter 5). Also in chicken differences in susceptibility

to infection between various breeds have been reported (Sironi et al., 2008) resulting in a

difference in host response. Furthermore, a difference in age affects responses (chapter 6).

A direct comparison between host responses induced by AIV strains varying in virulence

and pathogenesis is needed to answer the question whether virulence of the strain defines

cytokine responses in a similar manner as in mammals.

Macrophages play an important role during steady-state conditions and early responses

upon AIV entry in the respiratory tract. During steady-state conditions, mammalian alveolar

macrophages suppress induction of DC maturation (Holt et al., 1993; Bilyk et al., 1995) and

T cell proliferation (Strickland et al., 1993; Upham et al., 1997). Upon activation after AIV

entry, macrophages become highly phagocytic cells and start producing cytokines,

interferons, but also IL-18 stimulating IFN-γ production by NK cells and T cells, hereby

stimulating viral clearance. This indicates the importance of macrophages during early

defence responses to AIV infection and in activation of adaptive responses. However,

several reports also link macrophages to immunopathology. Influenza strains capable of

causing lethal influenza virus infection are known to induce larger and earlier macrophage

influxes in the lungs of mice compared to non-lethal strains and this excessive

inflammation is proposed to contribute to influenza virus pathogenesis (Perrone et al.,

2008). Influenza virus infected human macrophages were shown to up regulate TRAIL

production, which was related to their cytotoxicity towards influenza virus infected T cells

in vitro (Zhou et al., 2006; Korteweg et al., 2008). Furthermore, a direct role for

macrophages in promoting apoptosis of lung epithelial cells, lung leakage and decreased

survival upon influenza virus infection is shown in mice (Herold et al., 2009). However,

another study in mice demonstrates that although macrophages may contribute to

pathogenesis due to their large numbers in the airways they are crucial in controlling the

infection (Tumpey et al., 2005). We saw a massive influx of macrophages early during AIV

especially in unprotected birds (chapter 7), but down regulation of TRAIL in unprotected

birds. This suggests macrophages are important for virus clearance in the avian respiratory

tract, but likely play a role in immunopathology during AIV infection due to their large

numbers in the respiratory tract of chickens, which is not related to TRAIL expression as

described for mammals.

General discussion

171

Age-dependent innate responses are not reflected in early virus infection

As mentioned before a difference in age is reflected in host responses. The immune system

of neonates is not yet fully matured, which may be a cause of increased susceptibility to

infectious diseases in both chickens and humans (Raj et al., 1997, Gasparoni et al., 2003,

Beal et al., 2005). In chicken, age-dependent functionality is shown for activation,

phagocytosis and bactericidal activities of heterophils and macrophages (Kodama et al.,

1976, Wells et al., 1998, Kogut et al., 2002), proliferation and IFN-γ and IL-2 production of

T cells (Lowenthal et al., 1994) and antibody production (Mast et al., 1999). Maternal

antibodies that protect the neonate from disease in the early weeks of life are also proposed

to interfere with development of protective responses in both chickens and humans (Crowe

et al., 2003; Negash et al., 2004). We also detected differences in host responses at

transcriptional level within 24 hours after AIV inoculation between 1- and 4-week-old

chickens, which were likely related to functionality of macrophages, NK cells and T cells

(chapter 6). However, we did not see a difference in viral RNA expression between these

age groups indicating that age-dependent functionality of early innate responses has no

effect upon virus infection within 24 hours after inoculation. A possible explanation could

be that it was too early to measure age-related differences in host responses at protein level,

and therefore no difference in their effect on AIV infection could be seen. That would

suggest that there is no difference in functionality of early innate molecules that block viral

entry between the age groups upon AIV inoculation. If the effect of age-dependent

responses on AIV infection in relation to increased disease susceptibility in chicks is to be

elucidated, these responses and their effect upon AIV infection need to be studied for a

longer period of time.

The challenge route affects host responses

When studying host responses to respiratory virus infections in vivo, several aspects that

may have an influence on these responses should be kept in mind. A study in macaques

shows significant differences in gene expression in the lung of influenza virus infected

animals between sites containing viral RNA and sites lacking viral RNA (Baas et al., 2006).

We showed that in the avian lung airflow and anatomy caused differences in viral load

within the lung resulting in differences in host responses at transcriptional level (chapter 5).

This indicates that pathogens do not spread evenly through the avian lung and accurate

sampling is essential when investigating in vivo responses to respiratory virus infections.

If the airflow and anatomy influence virus deposition and subsequently host responses, the

challenge route most likely also affects host responses. Previous studies showed that the

size of particles affects the deposition of particles within the avian respiratory tract

(Corbanie et al., 2006). Therefore airborne particles administered via spray inoculation

Chapter 8

172

would likely penetrate deeper into the lungs than particles administered intratracheally in a

liquid. This causes higher viral RNA expression in lungs of spray inoculated birds

compared to birds inoculated intratracheally (unpublished data Degen et al.), resulting in

differences in host responses. Furthermore, spray inoculation in naïve birds causes less

variation in viral RNA expression and host responses at transcriptional level compared to

intratracheally inoculated birds (chapter 6 and 7). This means more differences in gene

expression due to inoculation can be measured and fewer birds would be needed to measure

these host responses when spray inoculation is used.

Microarray analysis for screening of new adjuvants and vaccines

For optimal efficacy a vaccine needs both an antigen and a strong immune activator as an

adjuvant. Adjuvants are able to steer the immune response in mammals into a Th1 mediated

response as described for CpG (Klinman, et al., 2004), a Th2 mediated response as reported

for aluminium salts (Marrack et al., 2009) or a combination of Th1 and Th2 mediated

response under certain condition as described for oil emulsions (Spickler et al., 2003). Due

to this ability, the adjuvant added to a vaccine has to be chosen carefully in order to induce

an optimal immune response leading to protection. Much research is being performed in

development of new vaccines and adjuvants for protection against AIV infection, especially

adjuvants that can be applied to mucosal surfaces. In chicken protective ability of vaccines

and adjuvants is tested using techniques that determine virus specific antibodies, viral load

and virus shedding, cytokine expression and clinical signs (Degen et al., 2006; Maas et al.,

2009; Wang et al., 2009). We immunised birds with three different adjuvants to gain more

insight into protective host responses to AIV infection in the chicken using microarray

analysis (chapter 7). Based on gene expression profiles it was clear that in 1-day-old birds,

Al(OH)3 and water-in-oil (w/o) were able to induce a protective response to challenge 2

weeks later while CpG was not. Microarray analysis is described to be a useful tool for

diagnosis of the metastatic state of cancer (Roepman et al., 2005) or for screening of

disease related biomarkers that could be used for early disease diagnosis, follow-up

progression or development of new treatment methods for Alzheimer’s disease (Ho et al.,

2009), inflammatory bowel diseases (Wu et al., 2007), HIV (Giri et al., 2006), influenza A

virus (Cameron et al., 2008) and other pathogenic virus or bacterial infections in mammals

(Ramilo et al., 2007; Yoo et al., 2009). These results show that microarray analysis could

be used as a tool to screen new vaccines and adjuvants for their ability to provide

protection. For the poultry industry this screening technique may be somewhat expensive

and is likely more suited for screening of human vaccines and adjuvants. However,

microarray analysis has the advantage that, besides a screening tool for the protective

ability of vaccines and adjuvants, it gives insight into how protection is provided in one

overview at genome level, which cannot be achieved with other screening techniques.

General discussion

173

Neutralizing antibodies or CTL?

Immunisation in presence of CpG as immunomodulator is described to provide protection

against influenza virus challenge in both chicken and mice (Wang et al., 2009; Wong et al.,

2009). We found that CpG was unable to induce a protective response in 1-day-old birds to

a homologues AIV challenge 2 weeks later (chapter 7). In mice addition of CpG to an

influenza virus M2e peptide vaccine is shown to enhance M2e peptide specific T cell

responses and antibody titers, but fails to provide protection. In combination with

aluminium salts and CpG, M2e peptide specific T cell responses and antibody titers are also

enhanced and mice are protected to a low challenge dose, but with a high challenge dose

protection is less strong than when only aluminium salts were added (Wu et al., 2009). CTL

are known to provide protection against influenza virus infection in mice (Kreijtz et al.,

2009). Age-related defects in antigen-presenting cell function are shown to lead to poor T

cell clonal expansion and function in mice (Plowden et al., 2004; Velilla et al., 2006 ).

These impaired responses may have caused the lack of protection in CpG immunised birds

(chapter 7). Furthermore, M2 peptide immunisation in pigs causes enhancement of disease

rather than protection, which is proposed to be caused by inability of M2 specific antibodies

to neutralize the virus and prevent viral entry (Heinen et al., 2002). These data indicate that

although CTL are known to provide protection against influenza virus infection,

neutralizing antibodies play a crucial role for providing early protection against AIV

infection by inhibiting viral entry and a lack in these antibodies results in less protection or

even enhancement of disease. In our study protected birds immunised with Al(OH)3 and

w/o did have high neutralizing antibody titers, while unprotected CpG immunised birds did

not (chapter 7), which shows the importance of neutralizing antibodies to prevent viral

entry resulting in protection.

Future perspectives

Influenza virus infection is one of the major causes of respiratory disease in both humans

and animals and vaccination against influenza virus is used to protect against infection and

control outbreaks depending on the country. Poultry vaccines preferably provide a broad

protection against different influenza virus strains especially HPAI strains, provide clinical

protection and prevent transmission to achieve efficient control of the disease. Currently

induction and maturation stages of innate and specific immune responses leading to

protection or pathogenesis are poorly understood. Much research is performed to get more

insight into these processes in order to obtain new leads for development of vaccines. In

this thesis we displayed several aspects of early host responses after primary AIV infection

and viral challenge in immunised chickens at host transcriptional level. The use of genome-

wide microarray analysis in combination with more classical techniques, such as histology

Chapter 8

174

and serology, allowed us to gain more insight into possible correlates of protection or

pathogenesis at cellular and transcriptional level.

For development of vaccines that can be used in young as well as older birds, it is crucial to

gain insight into age-related host responses. Age proved to have a major effect on early host

responses to AIV infection at transcriptional level, but no effects on virus RNA expression

were found. The effect of age on host responses has been described at later stage during

infection, but it is unknown to what extent and through which mechanisms the host

responses to AIV infection are affected by age. If age does not affect host responses at an

early, but at a later stage during infection, research into new concepts of age-independent

vaccines should therefore focus on induction stages of adaptive responses. Research into

age-related responses should therefore be performed over a longer time period so not only

effects on early innate responses, but also adaptive responses can be investigated.

Furthermore, besides virus RNA expression, shedding, transmission and clinical signs of

the birds should be monitored to gain insight into age-related disease susceptibility.

The effect of age may also relate to defects in the induction of protective responses induced

by CpG. While in our study birds immunised with CpG were not protected against AIV

challenge, previous studies did report protection against AIV infection in CpG immunised

birds (Degen et al., personal comment; Wang et al., 2009; Linghua et al., 2007). The birds

in these studies were older than our birds and age-related host responses may cause this

difference in protective immunity. CpG is known to act in a TLR-dependent manner (Ishii

et al., 2007) and likely age-related defects in antigen-presenting cell (APC) function leading

to poor T cell clonal expansion and function as described for mice (Plowden et al., 2004;

Velilla et al., 2006 ) are involved. To gain insight into this difference in immunity provided

by CpG between young and adult birds a comparative study between these age groups

needs to be performed. Preferably spray inoculation should be used, since this gives less

variation in virus RNA expression and host gene expression between the birds.

CpG, a TLR-dependent Th1 skewing adjuvant, did not provide protection in our study.

However, w/o and Al(OH)3, TLR-independent Th2 skewing adjuvants, did provide

protection against homologues challenge. Could this failure of CpG to perform also be

related to defects in functionality of APC as mentioned above resulting in differences in

induction of cell-mediated responses? This suggests that although CTL are known to

provide protection against influenza virus infection, in young birds a high concentration of

neutralizing antibodies is foremost important in providing protection. New concepts for

age-independent vaccines should therefore focus on adjuvants that can induce both a strong

humoral and cell-mediated response.

By comparing host responses after challenge in birds immunised with various adjuvants we

investigated whether correlates of protection could be found and how protection is

provided. Gene expression profiles, cellular influxes, HI and virus-specific IgG titers were

General discussion

175

found to be correlates of protection. These correlates of protection proved to be

independent of the adjuvant, but the mechanisms by which this protection was induced by

the different adjuvants remained elusive. Knowledge into mechanisms by which adjuvants

induce protection may help in the improvement of vaccine efficacy. By analyzing host

responses after immunization at the infection site, the spleen and lungs and after challenge

in the spleen and lungs a complete overview of correlates of protection and the mechanism

by which this protection is induced by the different adjuvants can be obtained. From our

results microarray analyses proved to be a useful tool for screening protective responses.

Combining microarray analyses with classical techniques as FACS analysis, histology and

serology will give the advantage of an overview of host responses at transcriptional, protein

and cellular level.

In conclusion

Early host responses are important in providing protection against AIV infection,

controlling infection and activating adaptive host responses. As discussed in this thesis

various factors influence these responses. While the challenge route and sampling method

influence investigation into host response, age and adjuvants directly affect host responses.

The direct effects of these factors on host responses are important in the development of

new vaccines against AIV infection. The findings presented in this thesis enhance our

knowledge on the course of events that follow early after AIV infection and lead to

pathogenesis or elimination of AIV, which will contribute to more insight into AIV induced

pathogenesis and new concepts for vaccine development.

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Summary

Summary

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Upon entry of the respiratory tract avian influenza virus (AIV) triggers early immune

responses in the host that are aimed to prevent or in case of already established infection

control this infection. Although much research is performed to elucidate the course of

events that follow after AIV infection, the interactions between the virus and the host at

molecular level at an early stage of infection are unclear. More insight in the mechanisms

underlying innate and adaptive immune responses leading to pathogenesis or elimination of

the AIV may contribute to a better understanding of AIV induced pathogenesis and new

concepts for vaccine development. In this thesis we unravelled several aspects of early host

responses after primary AIV infection and viral challenge in immunised chickens at host

transcriptional level. The use of genome-wide microarray analysis in combination with

more classical techniques allowed us to elucidate possible correlates of protection or

pathogenesis at cellular and transcriptional level. This last chapter summarises the findings

in this thesis.

Respiratory epithelial cells are the first target of AIV infection and their initial response

will affect the development of the host immune response. In mammals early host responses

to AIV infection have been investigated using respiratory epithelial cell lines and primary

epithelial cell cultures. Since chicken respiratory epithelial cell lines or primary cell

cultures were not available at the time, we decided to explore the use of tracheal organ

cultures (TOC) for this purpose as described in chapter 2. Although H9N2 AIV was able to

infect and replicate in TOC, virus specific gene expression profiles were masked by wound

healing responses that are induced by preparation of TOC independent of the virus

infection. Therefore only a small overlap was found in host response genes between

infected TOC and in vivo infected trachea. We were able to keep TOC in culture for a

longer time period, but histopathological changes occurred which would also affect host

responses at transcriptional level. This implies that although TOC is a suitable model for

culturing of virus and lectin or virus binding studies, it is not suitable for measuring early

immune responses upon viral infection at transcriptional level.

Upon entry of the respiratory tract AIV encounters several barriers that are able to block the

virus from entering epithelial cells. One of these barriers are collectins which in mammals

can bind and neutralize a wide range of pathogens including influenza A virus. Mammalian

collectins have been implicated to play an important role in the early defense against

influenza A virus infection, but for chicken collectins this is not yet clarified. In chapter 3

recombinant chicken Lung Lectin (cLL) was used to characterize structural and functional

properties of this collectin. Purified recombinant cLL has lectin activity, but failed to

neutralise influenza virus infectivity of the human isolates, while for an avian virus isolate

neutralisation was seen once. On the other hand recombinant cLL proved to have

haemagglutination inhibiting activity to at least one human influenza virus strain. How

Summary

183

expression of chicken collectins is affected by influenza virus infection in the chicken is

unknown and this was analyzed in chapter 4. Collectin mRNA expression was down

regulated in lung and up regulated in trachea after AIV inoculation, indicating tissue

specific expression. We also detected that the effect of AIV inoculation on collectin mRNA

expression was age specific in both the trachea and lung and viral RNA expression only

correlated with collectin mRNA expression in the lung of 1-wk-old but not in 4-wk-old

birds. Without knowing the exact function of chicken collectins in innate defenses, it is

difficult to relate the observed changes in gene expression to a biological effect. However,

these findings indicate that in lung collectins may play a role in limiting respiratory

infection in neonatal chickens.

Before we could start with the in vivo analysis of early host responses to AIV infection in

the respiratory tract at the molecular level, there was a simple question unanswered in

literature. It was described that differences in influenza virus deposition in the lung of

macaque had an affect on host responses at transcriptional level. Whether this uneven virus

deposition occurred in the chicken was unknown. In chapter 5 we investigated whether

differences in anatomy and airflow in the avian respiratory tract affected deposition of virus

and subsequent host responses. Although the upper trachea contained more viral RNA than

the lower trachea there were no significant differences in gene expression. Lung was

divided into 4 segments according to airflow and anatomy, which proved to affect virus

deposition in that lung segments containing the larger airways and the bifurcations to the

secondary bronchi contained highest viral RNA levels. In lung differences in viral RNA

distribution enhanced the differences in gene expression that were already seen in non-

infected birds. Host responses shared by trachea, lung L1 (cranial) and L4 (caudal) have

been previously described as common response to pathogens in mouse, rat, macaque and

human indicating that these are common responses independent of the amount of viral RNA

or the type of respiratory inflammatory disease. These common responses involved

chemokine activity and inflammatory responses correlating to massive KUL-01+

macrophage influxes. However, in trachea and L1 more genes were expressed due to

infection and larger KUL-01+, CD4+ and CD8α+ cell influxes were found compared to L4.

These findings suggest that an unequal deposition of pathogens throughout an organ will

induce localised responses and sampling at specific sites will affect the outcome of the

study.

The development of the avian immune system starts early during embryogenesis and

reaches maturity several weeks after hatching. It is unknown to what extent and through

which mechanisms the host responses to AIV infection are affected by age. For replication

and transcription of the influenza virus genome, the virus uses both viral and cellular host

Summary

184

factors. Whether age affects expression of these host factors and thereby AIV replication is

also unknown. In chapter 6 the effect of age on early host responses to AIV and on host

factors affecting replication in the respiratory tract were analyzed. Gene expression

between 1- and 4-wk-old birds was compared in PBS inoculated control birds and in H9N2

AIV inoculated birds early after inoculation. When comparing 1- and 4-wk-old control

birds, most genes were expressed at a higher rate in 4-wk-old birds, while genes related to

innate responses and development of the respiratory immune system were expressed at a

higher rate in 1-wk-old birds. These differences in gene expression between the age groups

related to differences in tissue development and maturation of the immune system. In AIV

inoculated birds gene expression was most affected at 16 h.p.i. in 1-wk-old birds, and at 16

and 24 h.p.i. in 4-wk-old birds. Furthermore, in 1-wk-old birds less genes were affected by

AIV inoculation than in 4-wk-old birds and which might be due to age-dependent reduced

functionality of APC, T cells and NK cells. Expression of cellular host factors that block

virus replication by interacting with viral factors was independent of age or tissue for most

host factors. These data show that differences in development are reflected in gene

expression and suggest that the strength of host responses at transcriptional level may be a

key factor in age-dependent susceptibility to infection, and the cellular host factors involved

in virus replication are not.

To gain more insight in the mechanisms leading to protection after vaccination with

different adjuvants, w/o, Al(OH)3 and CpG, we investigated correlates of protection after

AIV challenge, described in chapter 7. H9N2+w/o and H9N2+Al(OH)3 vaccinated birds

were protected based on low viral RNA expression. We found that protection after AIV

challenge correlated to a lower number of genes induced after challenge and gene

expression patterns with a low amplitude of change. The gene expression profile of

protected birds showed that expression was especially up regulated at 1 d.p.c., while in

unprotected birds higher and prolonged gene expression was found. Protected birds had

smaller cellular influxes en high neutralizing antibody titers. These findings suggest that

lack of immune activation is the most important correlate of protection after challenge with

AIV most likely due to high neutralizing antibody titers resulting in lower viral RNA

expression.

Early host responses to respiratory virus infections are complex processes providing the

first defense against AIV infection and are influenced by various factors as discussed in this

thesis. The research into host responses is influenced by the sampling method, because

airflow and anatomy affect virus distribution in the lung enhancing differences in host

responses between different segments in the lung already seen in uninfected birds. This

points out the importance of sampling approach which has to be taken into account when

Summary

185

investigating in vivo responses to respiratory virus infections in large organs. Host

responses themselves are directly affected by the age and adjuvants. Prolonged and

enhanced responses relate to the maturation of the respiratory immune system and may be

key factors in age-dependent susceptibility to infection and induction of protective

responses. A high neutralizing antibody titer is a know correlate of a protective response.

This correlate of protection relates to lower gene expression levels and less cellular influxes

indicating that a lack of immune activation is the most important correlate of protection

after AIV challenge. The findings presented in this thesis enhance our knowledge on the

course of events that follow early after AIV infection leading to pathogenesis or elimination

of AIV.

Nederlandse samenvatting


188

Influenza A virus

Vogelgriep werd voor het eerst beschreven in 1878 als een ziekte die kippen infecteert. Pas

in 1955 werd de veroorzaker van vogelgriep ontdekt, een influenza A virus. Tegenwoordig

zijn er vele varianten van influenza A virus bekend die in vogels, maar ook onder andere in

mensen, varkens, paarden, katachtigen, fretten en walvissen infecties veroorzaken vooral in

de luchtwegen.

De ziekteverschijnselen in kippen variëren van een lichte hoest tot de dood, wat

samenhangt met de het type influenza virus. Laag pathogeen aviair influenza virus (LPAI)

veroorzaakt geen of lichte ziekteverschijnselen en infecties zijn voornamelijk terug te

vinden in de luchtwegen. Hoog pathogeen aviair influenza virus (HPAI) veroorzaakt

ernstige ziekteverschijnselen zoals de dood binnen 2 dagen en veroorzaakt naast in de

luchtwegen ook infecties in andere organen, inclusief de hersenen.

De luchtwegen van vogels

De luchtwegen van de kip zijn anders dan die van zoogdieren zoals de mens. Kippen

hebben geen middenrif zoals zoogdieren, maar in plaats daarvan hebben ze luchtzakken die

ze gebruiken als blaasbalgen om verse lucht door de longen te laten stromen. De longen van

de kip zijn klein en star vergeleken met de grote, flexibele longen van zoogdieren die

uitzetten bij inademing. Daarnaast stroomt de lucht bij zoogdieren via dezelfde weg de

longen in en uit, terwijl bij kippen de lucht circulair door de longen stroomt en dus via een

andere weg de longen uitstroomt als dat het binnen gekomen is. Hierdoor heeft de kip altijd

zuurstofrijke lucht in de longen. Naast ademhaling spelen de longen ook een belangrijke rol

in het immuunsysteem van de luchtwegen, respiratoir immuunsysteem genaamd. In de

longen liggen naast de grote luchtwegen gebieden waar cellen van het immuunsysteem bij

elkaar liggen in een organiseerde structuur, zogenaamde lymfeknopen. In de longen worden

deze gebieden BALT (bronchus associated lymphoid tissue) genoemd. De BALT

ontwikkelt zich nadat een kuiken uit het ei is gekomen en is terug te vinden vanaf 2-3

weken na uitkomst. Volledig ontwikkelde BALT is terug te vinden vanaf 6-8 weken.

Daarnaast hebben factoren van buitenaf, zoals een infectie, ook een positief effect op de

ontwikkeling van de BALT.

De vroege immuunrespons in de luchtwegen

Voordat influenza virus cellen van de luchtwegen kan infecteren, moet het eerst voorbij een

eerste blokkade zien te komen. Deze blokkade bestaat uit slijm wat bovenop de

epitheelcellen ligt die de buitenste cellaag van de luchtwegen vormen. In dit slijm komen

van allerlei stoffen voor, geproduceerd door epitheelcellen en cellen van het

immuunsysteem, die ervoor zorgen dat het virus niet kan binden aan de epitheelcellen en


189

daarmee infectie blokkeren. Het slijm met daarin gevangen virus wordt door trilhaartjes op

de epitheelcellen omhoog, de luchtwegen uit bewogen. Als influenza virus voorbij de

slijmblokkade komt, kan het de epitheelcellen infecteren. De geïnfecteerde cellen scheiden

stoffen uit die de omgeving en het immuunsysteem alarmeren, de zogenaamde cytokines en

chemokines. Hierdoor worden allerlei processen op gang gebracht die er uiteindelijk voor

zorgen dat de infectie onder controle wordt gehouden en het virus en geïnfecteerde cellen

worden opgeruimd. Witte bloedcellen spelen hierin een belangrijke rol. Macrofagen,

dendritische cellen (DC) en natural killer (NK) cellen spelen een belangrijke rol in de

vroege immuunrespons, terwijl verschillende soorten T-cellen (CD4+ T-cellen en CD8+ T-

cellen) een belangrijke rol spelen in een later stadium van de immuunrespons. Macrofagen

en DC zijn zogenaamde antigeen presenterende cellen. Zij nemen lichaamsvreemde stoffen

(antigeen), wat in dit geval aviair influenza virus is, in zich op, breken het af tot kleine

stukjes en presenteren deze stukjes virus aan hun celoppervlak waarmee ze T-cellen kunnen

activeren. CD4+ T-cellen zijn helpende cellen, zij sturen andere cellen van het

immuunsysteem aan en ondersteunen deze cellen in het uitvoeren van hun functie in de

immuunrespons. CD8+ T-cellen en NK-cellen doden geïnfecteerde cellen of cellen die virus

hebben opgenomen. Cellen van het immuunsysteem worden aangestuurd door cytokines en

chemokines die worden uitgescheiden waardoor ze weten waar de infectie plaatsvindt en

wat ze moeten doen. Daarnaast zorgt cel-cel contact via eiwitten aan het celoppervlak, die

receptoren worden genoemd, ervoor dat cellen weten wat voor soort ziekteverwekker de

infectie veroorzaakt, welke cellen geïnfecteerd zijn en wat ze moeten doen om de infectie

onder controle te krijgen.

Controle van aviair influenza virus (AIV) uitbraken

Vaccins worden al decennia lang gebruikt om uitbraken van allerlei infectieziekten onder

controle te houden of te voorkomen, maar werken niet voor alle infectieziekten even

succesvol. Door vaccinatie wordt een immuunrespons opwekt tegen de ziekteverwekker in

het vaccin, in dit geval AIV, en een succesvolle immuunrespons leidt over het algemeen tot

een immunologisch geheugen tegen deze AIV variant. Bij een volgende infectie met deze

AIV variant zorgt het immunologisch geheugen voor een goede en snelle immuunrespons

om de infectie te stoppen. Een vaccin tegen AIV heeft tot doel een langdurige

immunologische bescherming te bieden tegen liefst zoveel mogelijk varianten AIV. In

kippen worden hiervoor geïnactiveerd virus of geselecteerde delen van het virus, zoals het

HA oppervlakte eiwit van het virus, gebruikt. Toedienen van geïnactiveerd virus of

geselecteerd virus eiwitten is echter onvoldoende om een immuunrespons te veroorzaken

die een goed immunologisch geheugen opwekt. Daarom wordt er nog een hulpstof aan het

vaccin toegevoegd, genaamd adjuvant. Er zijn verschillende adjuvants zoals olie emulsies,

aluminium zouten en bacterieel DNA die allemaal op hun eigen manier de immuunrespons


190

tegen een vaccin versterken zodat er een langdurig immunologisch geheugen wordt

opgebouwd.

Eén van de belangrijkste vragen in de bestrijding van influenza virus infecties is hoe een

sterke en veelzijdige bescherming tegen allerlei virus varianten kan worden geïnduceerd.

Hiervoor is het van belang te weten hoe een vroege immuunrespons tegen influenza eruit

ziet. Daarnaast kunnen bestaande vaccins een idee geven over de manier waarop

bescherming wordt geïnduceerd en hoe een dergelijk beschermende immuunrespons eruit

ziet. In dit proefschrift gaat het voornamelijk over de vroege immuunrespons tegen AIV

infectie in de luchtwegen van de kip, welke factoren invloed hebben op deze

immuunrespons en hoe een beschermende immuunrespons eruit ziet op genexpressie

niveau. Hierin geeft de mate van genexpressie de mate van aan- of uitschakelen van genen

aan.

In dit proefschrift

Immuunresponsen tegen AIV infectie kunnen worden bestudeerd in een dier, in vivo

genaamd, maar ook in cellen gekweekt in het lab, dit wordt in vitro genoemd. In vitro heeft

als voordeel dat het beter onder controle te houden is dan in vivo. Een nadeel van in vitro is

dat het vaak om 1 type cel gaat terwijl een organisme uit meerdere typen cellen bestaat die

onderling communiceren. Een tussenvorm is een orgaan cultuur waarin het controleerbare

van in vitro wordt gecombineerd met de multi-variëteit aan cellen van in vivo zonder dat de

orgaan structuur verloren gaat. In hoofdstuk 2 is onderzoek gedaan naar vroege

immuunresponsen tegen AIV infectie in een orgaan cultuur bestaande uit smalle ringetjes

van de luchtpijp (trachea), genaamd tracheale orgaan cultuur (TOC). Door het prepareren

van TOC uit de trachea bleken immuunresponsen te worden opgewekt die de

immuunresponsen tegen AIV infectie maskeerden. Door deze maskering kwamen de

immuunresponsen gemeten in vitro in TOC niet overeen met immuunresponsen tegen AIV

infectie gemeten in vivo in de trachea. Dit maakt TOC ongeschikt als model om

immuunresponsen tegen AIV infectie te meten in vitro op genexpressie niveau.

Een onderdeel van de vroege immuunrespons vormt een groep eiwitten genaamd

collectines. In zoogdieren is gebleken dat collectines de infectie voor een deel blokkeren

door te binden aan het influenza virus, maar voor kippen collectines is dit nog niet

aangetoond. In hoofdstuk 3 wordt de structuur en functionaliteit van een nieuw kippen

collectine dat voorkomt in de luchtwegen van de kip, genaamd chicken lung lectin (cLL),

onderzocht. De structuur van cLL kwam overeen met de karakteristieke structuur van

collectines. Daarnaast bleek cLL te kunnen binden aan menselijke varianten influenza virus

en de infectie van AIV in TOC af te remmen, hoewel dit niet herhaalbaar was. Hieruit blijkt

dat cLL een mogelijke rol speelt in het afremmen van AIV infecties, maar dat hiernaar nog

meer onderzoek moet worden gedaan om dit met zekerheid vast te kunnen stellen. Verder is


191

het onbekend of AIV infectie in de kip invloed heeft op de productie van kippen collectines

op genexpressie niveau en of een verschil in leeftijd hierin een rol speelt. Dit is bestudeerd

in hoofdstuk 4 in de trachea en longen van jonge en oudere kippen voor verschillende

kippen collectins. Wij vonden dat AIV infectie een effect had op de productie van

verschillende kippen collectines op genexpressie niveau en dat dit effect leeftijd en orgaan

afhankelijk is. Verder onderzoek naar de precieze functie die kippen collectines vervullen

tijdens de vroege immuunrespons moet uitwijzen wat deze verschillen voor invloed hebben

op de afweer tegen AIV in de luchtwegen van de kip.

Zoals eerder al beschreven zijn de luchtwegen van de kip anders dan van zoogdieren.

Kippen hebben een circulaire luchtstroom door de longen en andere receptoren waaraan

AIV kan binden die net als in zoogdieren niet gelijkmatig door het luchtwegen systeem zijn

verspreid. De AIV stam H9N2 is een LPAI die er de voorkeur aan geeft om hoog in de

luchtwegen infecties te veroorzaken. Uit onderzoek in makaken is gebleken dat een

ongelijke verdeling van virus in de longen een effect heeft op de immuunresponsen die

worden gemeten op genexpressie niveau. In hoofdstuk 5 is onderzocht of verschil in

luchtstroom en anatomie in de trachea en longen van de kip een effect heeft op de verdeling

van virus in de luchtwegen en de immuunrespons op genexpressie niveau. Er was weinig

verschil in immuunrespons tussen het bovenste en onderste deel van de trachea in zowel

ongeïnfecteerde als geïnfecteerde kippen. In de long van ongeïnfecteerde kippen kwam de

immuunrespons in de twee delen waarin de hoofdluchtweg of primaire bronchus loopt met

elkaar overeen en verschilde die van de immuunrespons in de twee delen waarin de kleinere

luchtwegen of tertiaire bronchi zich bevonden. In geïnfecteerde dieren bevond het meeste

virus RNA zich in de twee delen waarin de hoofdluchtweg zich bevond. Dit verschil in

virusverdeling versterkte het verschil in immuunrespons wat al te zien was in de long van

ongeïnfecteerde dieren. Dit geeft aan dat het verschil in luchtstroom en anatomie binnen de

kippenlong invloed heeft op de virusverdeling en de immuunrespons, waardoor bestuderen

van maar een klein deel van de long invloed heeft op het resultaat van de studie.

De luchtwegen, en vooral de longen, spelen een belangrijke rol in de immuunrespons tegen

AIV infectie. Het respiratoir immuunsysteem ontwikkelt zich nadat het kuiken uit het ei is

gekomen waardoor jonge dieren verschillen in immuunrespons van oudere dieren. In

hoofdstuk 6 is bestudeerd in welke mate de immuunrespons tegen AIV infectie in de

luchtwegen op genexpressie niveau wordt beïnvloed door een verschil in leeftijd. Daarvoor

werd de immuunrespons in 1 en in 4 weken oude ongeïnfecteerde en AIV geïnfecteerde

dieren met elkaar vergeleken. In ongeïnfecteerde dieren werden vooral verschillen gezien in

expressie van genen die een rol spelen bij ontwikkeling van weefsel en organen en

immunologische ontwikkeling. In geïnfecteerde dieren kwamen er minder immunologische

genen tot expressie en voor een kortere periode in jonge dieren dan in oudere dieren. Deze

verschillen lijken gekoppeld te kunnen worden aan de functionele ontwikkeling van T


192

cellen, NK cellen en APC. Deze resultaten tonen aan dat immunologische ontwikkeling

invloed heeft op de immuunrespons tegen AIV infectie in de luchtwegen van de kip en

suggereren dat de sterkte en duur van de immuunrespons leeftijdsafhankelijk zijn.

Expressie van genen die een rol spelen bij blokkering van virusvermeerdering was

leeftijdsonafhankelijk.

In hoofdstuk 7 werden immuunresponsen tegen AIV infectie in de luchtwegen van kippen

die gevaccineerd zijn met verschillende adjuvantia vergeleken. De vergelijking van de

immuunrespons in dieren die beschermd zijn tegen AIV infectie met dieren die

onbeschermd zijn kan een idee geven over de manier waarop bescherming wordt

geïnduceerd en hoe een dergelijk beschermende immuunrespons eruit ziet. In onze studie

werden dieren gevaccineerd met een geïnactiveerd H9N2 virus in combinatie met een olie

emulsie (w/o), een aluminium zout (Al(OH)3), bacterieel DNA (CpG) of zonder adjuvant.

Daarna werden gevaccineerde en ongevaccineerde dieren geïnfecteerd met levend H9N2

virus. Dieren gevaccineerd met w/o en Al(OH)3 waren beschermd. Beschermde dieren

hadden weinig virus RNA in de longen en trachea, lagere genexpressie niveaus, vooral

sterke genexpressie op dag 1 na infectie die daarna afnam, minder CD4+ T cellen en

macrofagen in de longen en veel virus-neutraliserende antilichamen in het bloed vergeleken

met onbeschermde dieren. Bij onbeschermde dieren was het tegenovergestelde te zien wat

duidde op een heftige immuunrespons die door zijn sterkte zelf schade leek te veroorzaken.

Deze data suggereren dat naast veel virus-neutraliserende antilichamen in het bloed en lage

genexpressie, de afwezigheid van een sterke immuunrespons de belangrijkste factor is die

samenhangt met bescherming tegen een herhaalde infectie.

Conclusie

Vroege immuunresponsen zijn belangrijk voor de blokkering en onder controle houden van

de infectie, maar ook voor het induceren van een specifieke immuunrespons. De studies in

dit proefschrift dragen in de eerste plaats bij tot een beter inzicht in de vroege respiratoire

immuunrespons tegen AIV in de kip en hoe een beschermende immuunrespons verloopt na

een herhaalde infectie. Daarnaast blijkt uit de studies beschreven in dit proefschrift dat

verschillende factoren invloed hebben op de vroege immuunrespons tegen AIV infectie in

de luchtwegen. Zo heeft de locatie waar monsters in de long worden genomen invloed op

de uitkomst van de studie. De immuunrespons zelf wordt direct beïnvloed door leeftijd van

de dieren en de adjuvantia in een vaccin. Voor de ontwikkeling van nieuwe vaccins tegen

AIV is het van belang dat het effect van deze factoren in ogenschouw wordt genomen.

Dankwoord

Dankwoord

194

Eindelijk is het dan zover, de hoofdstukken voor dit proefschrift zijn af! Dan is het nu tijd

voor het belangrijkste en meeste gelezen deel van een proefschrift: het dankwoord. Hier wil

ik graag de vele mensen bedanken die direct of indirect een bijdrage hebben geleverd aan

dit boekje, zonder jullie had het er nooit zo uit gezien! Een aantal van hen wil ik daarom

graag bij naam noemen.

Allereerst mijn co-promotor Lonneke Vervelde, aanstekelijk vaatje positiviteit en energie,

rots in de branding, “chicken killer queen”. De afgelopen 5 jaar lijken één lange achtbaanrit

met een geweldige finale (recordje papers submitten). Door jouw onwrikbare geloof in mij,

je aanmoediging en enthousiasme (de kip is ook absoluut een super cool beest!) vond ik bij

twijfel steeds de energie en het geloof om door te zetten. Ik heb zoveel van je geleerd en

vond onze samenwerking bijzonder en erg gezellig. Thanks, big hug!

Mijn promotor Willem van Eden. Dank je voor alle vrijheid die je me gegeven hebt binnen

dit project waardoor ik ruimte had om me te ontwikkelen en me op de microarray ik-zie-

door-de-stippen-het-bos-niet-meer wereld te storten. Ik waardeer de snelheid en je kritische

oog waarmee je mijn stukken nakeek op het eind van mijn AIO tijd (mijn kommafobie is al

aan het afnemen).

Alle mensen van de kippengroep. Toen ik begon als AIO was het nog maar een klein

groepje. Mijn kippen-AIO-voorbeeld Mark, altijd in voor een praatje of advies (zit je nog

steeds op de labvloer om eens goed na te kunnen denken?); Daphne met haar gouden

handjes die altijd voor je klaar staat; Evert, die discussiëren naar een kunstvorm heeft

verheven. Daarna groeide de groep snel. Eveline, die schreeuwen-in-de-wei sessies waren

heerlijk; Christine, boordevol positiviteit en geen probleem te groot, ik ben ook erg blij om

nu met je samen te werken in mijn nieuwe project; Peter, geen apparaat of computer

probleem wat je niet op kon lossen; Willem de onverstoorbare; kip/koe Ad (nieuw ras denk

ik), de rust zelve met praktische tips tijdens de werkbespreking; Stefan, gezellig babbelen in

de flowkast of achter de facs; Kjell, kort maar onvergeetbaar; Christoph, always in for a

quick chat. Ontzettend bedank voor al jullie hulp, maar zeker ook voor alle gezelligheid in

en buiten het lab. Wanneer is het volgende kippenetentje en gaan we na Oz nu Hongarije op

z’n kop zetten?

And of course the chicken students Manon, Marie-Luise (we had lots of fun together and I

still miss your contagious laughter), Tim (al andere kleuren in exel gevonden?), Pauline,

Hillary, Ad (lekker nuchter) and Alida who lightened up the lab in their own way.

Al mijn roomies. Huidige roomies Rachel, Wiebren, Josée, Sanne, Manoek en Jeroen, dank

je voor de stiltes als ik weer een paperdeadline probeerde te halen.

Dankwoord

195

Old roomies Daphne, Christine, Peter, Christoph, Kim (my writing course buddy) it was

short but fun!

Oud-oud roomies Lotte en Daniëlle, bij jullie kon ik altijd stoom afblazen en was er een

bemoedigend woord of advies, dank je, en later ook Marieke en Frank (kom op, de laatste

loodjes!).

Verder alle nog niet genoemde mede-AIOs de afgelopen jaren: Erwin (zo moeilijk waren je

4 vragen toch niet, maar wel leuk), Teun (hoe blijf je altijd zo kalm?), (sportie-) Aad en

Natascha, (regelneef) Marij, Martijn (waar blijven die bitterballen tijdens de journal club?),

Chantal, Bram en Annette. Voor degene die nog hard bezig zijn heel veel succes en ik kijk

uit naar jullie schitterende boekjes (onthoud: uiteindelijk komt alles goed, zo niet dan toch,

echt waar) en allen bedankt voor de gezelligheid.

Uiteraard wil ik ook alle mensen bedanken die door hun aanwezigheid de afdeling

immunologie tot een leerzame en gezellige werkplek hebben gemaakt de afgelopen 5 jaar.

Het record met zoveel mogelijk mensen om 1 lunchtafel proppen en het fameuze

koekjesrooster dragen daar zeker aan bij. Femke, Ildiko, Willemien, Dietmar, Alice, Ineke,

Victor, Ton, Ruurd, Mieke, Elles, Esther, Jeroen, Cornelis, Ger, Janine, Peter van Kooten,

Peter Reinink…..en alle andere bedankt! Marielle, dank je voor alle regeldingetjes.

Ook wil ik de mensen van de groep van Frank Holstege bedanken voor al hun hulp bij mijn

microarray experimenten, zonder hen was microarray data een wereld van gekleurde

stippen gebleven. In het bijzonder wil ik bedanken Marian Groot Koerkamp voor alle

brainstorm sessies, genespring tips en er altijd te zijn als ik weer eens binnen kwam

stormen voor advies en Dik van Leenen voor het oplossen van mijn vele vetes met de

bioanalyzer en de computer. Ook Tony Miles voor zijn praktische tips voor in het lab en

Engels schrijven (en cauliflower with cheese).

Ik wil graag alle mensen van Intervet die betrokken waren bij dit project bedanken.

Winfried Degen en Virgil Schijns bedankt voor de prettige samenwerking en het snel

doorloosden van mijn stukken door de patent afdeling, we waren een leuk kippenfront in de

muizen en humane wereld.

Natuurlijk ook al mijn andere Virgo-collega’s bedankt voor de samenwerking, jullie

enthousiasme en alle interessante discussies.

Henk Haagsman, Edwin Veldhuizen, Astrid Hogenkamp en Cherina Fleming dank je voor

de samenwerking en de introductie in de wereld van de collectines. Er valt daar nog zoveel

te ontdekken!

Dankwoord

196

Natuurlijk wil ik ook mijn familie en mijn vrienden buiten het lab bedanken en in het

bijzonder Bregje, Erwin (wat is de kaft mooi geworden!), Rudy en Anke. Dank je voor

jullie geduld en interesse, maar vooral voor jullie steun en de hoognodige ontspannende

momenten. Hopelijk heb ik nu weer wat meer tijd zodat ik weer wat vaker kan komen

binnenvallen.

De weg naar dit proefschrift was lang en soms zwaar. Ik mag me gelukkig prijzen dat ik

vijf mensen om me heen heb die er altijd voor me zijn en zonder hun aanmoediging was ik

nooit zover gekomen.

Mijn lieve schoonouders, Cor en Thea, dank je voor jullie opbeurende woorden alle steun

en adviezen en de heerlijke ontspannende weekendjes weg.

Lieve pap en mam, dank je voor jullie onvoorwaardelijke steun en vertrouwen in mij. Alle

interesse in “die DNA dingen” die er altijd is en voor alles wat jullie mij hebben

meegegeven.

Lieve Jeroen, wat hebben we veel meegemaakt samen. Dank je voor alle liefde, steun en

vertrouwen die je me altijd gegeven hebt, maar ook voor je geduld en de vele avondjes

koken vooral in het afgelopen jaar, en nog zoveel meer. Ik hoop dat we nog vele mooie

tijden zullen beleven samen. Zullen we beginnen bij een mooi duikseizoen?

Sylvia

List of abbreviations

198


AFC Antibody forming cells

Ag Antigen

AIV Avian influenza virus

Al(OH)3 Aluminium hydroxide

APC Antigen presenting cells

BALT Bronchus associated lymphoid tissue

cCL-1 Chicken collectin 1



cLL Chicken lung lectin

CpG Nonmethylated CpG oligonucleotides

CRD Carbohydrate recognition domains

cSP-A Chicken surfactant protein A

Ct threshold cycle value

CTL Cytotoxic T lymphocytes

d.p.c. Days post challenge

DAVID Database for Annotation, Visualization and Integrated Discovery

DC Dendritic cells

DDX DEAD box helicase

EID50 Embryo infectious dose 50

ELISA Enzyme-linked immunosorbent assay

FAE Follicle-associated epithelium

GO Gene Ontology

h.p.i. Hours post inoculation

HA Hemagglutinin

HAA-inhibition Haemagglutination inhibition

HG Harderian gland

HI titer Hemagglutinin inhibition titer

HPAI Highly pathogenic avian influenza

IAV Influenza A virus

IFN Interferon

IL Interleukin

LPAI Low pathogenic avian influenza

M1 Matrix protein 1

M2 Matrix protein 2

mAb Monoclonal antibody


199

MCM Mini chromosome maintenance complex

MDCK Madin-Darby canine kidney

MHC Major histocompatibility complex

MIAME Minimum Information About a Microarray Experiment

MФ Macrophage

NA Neuraminidase

NDV Newcastle Disease Virus

NK cells Natural killer cells

NP Nucleoprotein

NS1 Non-structural protein 1

NS2 Non-structural protein 2

p.i. Post inoculation

PAMP Pathogen-associated molecular patterns

qRT-PCR Real-time quantitative reverse transcription-PCR

r Pearson correlation coefficient

RNP Ribonucleoprotein

SEM Standard error of the mean

sIgA Secretory IgA

SP-A Surfactant protein A

SP-D Surfactant protein D

SPF Specific-pathogen-free

TLR Toll-like receptor

TOC Tracheal organ cultures

vRNA Viral RNA

vRNP Viral ribonucleoprotein

w/o Water-in-oil

wk Week

Curriculum Vitae

200

Curriculum Vitae

Sylvia Reemers werd op 22 augustus 1978 te Veldhoven geboren. In 1994 behaalde zij het

MAVO diploma aan het Koningshof te Veldhoven, in 1996 het HAVO diploma aan het

Sondervick College te Veldhoven en in 1998 het VWO diploma aan het Sondervick

College te Veldhoven, waarna zij met haar studie bioprocestechnologie aan Universiteit van

Wageningen begon. Haar eerste stage liep zij in het Laboratorium voor Virologie aan de

Universiteit van Wageningen onder leiding van Dr. ir. Marielle Van Hulten. Hier deed zij

onderzoek naar capside formatie en bepaling van de positie van de structurele eiwitten in

het virion van het White Spot Syndrome Virus (WSSV) en screenen van een

antilichamenbank op specifieke antilichamen met behulp van phage display. In haar

volgende stage deed zij onderzoek naar inductie van specifieke CD8+ T-lymfocyten tegen

twee verschillende HIV immunogenen tot expressie gebracht in een recombinant

adenovirus. Dit onderzoek werd uitgevoerd op de Human Immunology Unit van het

Weatherall Institute of Molecular Medicine, Oxford University, Oxford, United Kingdom

onder leiding van Dr. Joseph Nkolola en Dr. Tomas Hanke. Vervolgens deed zij een stage

bij de afdeling Virologie aan de Faculteit Diergeneeskunde van de Universiteit van Utrecht

onderleiding van Dr. Berend-Jan Bosch waar zij onderzoek deed naar het remmende effect

van bepaalde domeinen van het MHV en SARS coronavirus spike eiwit op virus-cel fusie

en cel-cel fusie. In maart 2004 werd het doctoraal diploma met veel genoegen behaald.

Vanaf maart 2005 begon zij als assistent in opleiding op de afdeling Immunologie aan de

Faculteit Diergeneeskunde van de Universiteit van Utrecht. Dit onderzoek uitgevoerd

onderleiding van Dr. ir. Lonneke Vervelde staat beschreven in dit proefschrift.

Vanaf september 2009 is zij werkzaam als post-docteraal onderzoeker bij Dr. Christine

Jansen op de afdeling Immunologie aan de Faculteit Diergeneeskunde van de Universiteit

van Utrecht.

Early host responses to avian influenza A virus are prolonged and enhanced at transcriptional level...

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