IL-12 and type I IFN response of neonatal myeloid DCto human CMV infection

Joelle Renneson1, Binita Dutta1, Stanislas Goriely1, Benedicte Danis1,

Sandra Lecomte1, Jean-Franc-ois Laes2, Zsuzsanna Tabi3,

Michel Goldman1 and Arnaud Marchant1

1 Institute for Medical Immunology, Universite Libre de Bruxelles, Charleroi, Belgium2 DNAVision SA, Charleroi, Belgium3 Department of Oncology and Palliative Medicine, Velindre Hospital, Whitchurch, Cardiff, UK

Following congenital human CMV (HCMV) infection, 15–20% of infected newborns develop

severe health problems whereas infection in immunocompetent adults rarely causes

illness. The immaturity of neonatal antigen presenting cells could play a pivotal role in

this susceptibility. Neonatal myeloid DC were shown to be deficient in IFN-b and IL-12

synthesis in response to TLR triggering. We studied the response of cord and adult blood-

derived myeloid DC to HCMV infection. Neonatal and adult DC were equally susceptible to

in vitro HCMV infection. Among immunomodulatory cytokines, IL-12, IFN-b and IFN-k1

were produced at lower levels by neonatal as compared with adult DC. In contrast,

neonatal and adult DC produced similar levels of IFN-a and IFN-inducible genes. Micro-

array analysis indicated that among the more than thousand genes up- or down-regulated

by HCMV infection of myeloid DC, 88 were differently regulated between adult and

neonatal DC. We conclude that neonatal and adult DC trigger a partly different response to

HCMV infection. The deficient IL-12 and mature IFN-a production by neonatal DC exposed

to HCMV are likely to influence the quality of the T lymphocyte response to HCMV

infection in early life.

Key words: Cytokines . DC . IFN . Infectious diseases . Neonate immunity

Supporting Information available online

Introduction

Neonates are highly susceptible to viral infection, as a result of the

immaturity of their immune system [1]. Deficiencies of both innate

and adaptive immune responses contribute to the impaired

neonatal host defense [2, 3]. Human CMV (HCMV) is the most

common cause of congenital infection, affecting between 5 and 20

newborns per thousand births worldwide [4]. Although primary

infection in immunocompetent adults rarely causes serious illness,

HCMV is responsible of severe health problems (deafness,

psychomotor retardation and death) in 10–20% of infected

neonates [5, 6]. Following primary infection, newborns are able

to develop a mature and functional CD81 T-cell response to HCMV

[7]. In contrast, anti-HCMV CD41 T-lymphocyte response is

defective in early life [8, 9]. This defect could be involved in the

prolonged viruria observed in young children [10].

DC play a pivotal role in the generation of antiviral response

and many viruses have evolved to interfere with DC functions

[11]. Myeloid cells are important targets for HCMV infection and

replication. The permissiveness of cells is related to their state of

differentiation, monocytes are non-permissive whereas differ-

entiated macrophages and immature DC support productiveCorrespondence: Dr. Arnaud Marchante-mail: arnaud.marchant@ulb.ac.be

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Eur. J. Immunol. 2009. 39: 2789–2799 DOI 10.1002/eji.200939414 Immunity to infection 2789

infection [5]. Cytokines and chemokines are critical for the host

to mount an effective antiviral immune response. Two essential

immunomodulatory cytokines can drive the differentiation of

T cells: IL-12 and type I IFN. IL-12 has a central role in the

generation of Th-1 responses [12], acting as a potent inducer of

IFN-g from T cells [13, 14]. Type I IFN (IFN-a/b) are a family of

cytokines specialized in coordinating immunity to viruses, both

directly and indirectly [15]. The cross-linking of type I IFN

receptor stimulates the expression of hundreds of IFN-stimulated

genes (ISG) promoting an antiviral state [16]. IFN-a/b induces a

program that limits viral spreading and activates cytotoxic

response against infected cells. Type III IFN (IFN-l or IL-28/29)

are novel antiviral cytokines that display very similar activities to

type I IFN [17], although they act through a distinct receptor

complex [18, 19]. Several studies have shown that the production

of IL-12 p70 and IFN-b by neonatal monocyte-derived DC

(moDC) stimulated with the TLR-4 ligand LPS is lower than that

of adult moDC [20, 21]. The defective production of IL-12 p70

was shown to involve a lower transcription of the IL-12 p35

subunit [21]. The capacity of neonatal DC to produce T-cell

immunoregulatory cytokines in response to viral infection has not

been characterized. The immaturity of neonatal DC could play an

important role in the susceptibility of young infant to HCMV and

other viral infections [22].

The aim of this study was to compare the immunomodulatory

cytokine response of cord and adult blood-derived DC to infection

with a clinical isolate of HCMV (TB40/E strain). We observed that

infected neonatal moDC produced lower concentrations of IL-12,

IFN-b and IFN-l1 as compared with adult moDC. In contrast,

neonatal and adult moDC produce high and similar concentra-

tions of IFN-a and express similar levels of IFN-stimulated/

inducible genes (ISG).

Results

Neonatal and adult moDC are equally susceptible toHCMV infection

Before investigating the response of neonatal moDC to HCMV

infection, we determined their susceptibility to infection by

comparing the infection rate of adult and neonatal moDC

exposed to HCMVTB40/E at a MOI of 10, for 6–48 h. Proportions

of infected moDC were evaluated by their expression of

immediate early viral proteins (Fig. 1A). Viral protein-positive

moDC were detected from 6 h post-infection and the percentage

of infected cells increased with time, until 24 h infection

(Fig. 1B), and with amounts of virus, to reach a maximum at

MOI of 5 (Fig. 1C). The proportion of infected moDC and the

kinetics of infection were similar in adult and neonatal moDC and

were similar among donors in each group (data not shown).

These results indicated that adult and neonatal moDC are equally

susceptible to infection with HCMVTB40/E. We examined the

influence of HCMV on the expression of antigen presentation

molecules HLA-A, B, C by adult and neonatal infected moDC

(Fig. 1A). As previously described [23], HCMV-infected moDC

expressed lower levels of HLA-A, B, C molecules than mock-

infected cells. The down-regulation of HLA-A, B, C was similar in

adult and neonatal moDC (data not shown).

Defective IL-12 p70 production in HCMV-infectedneonatal moDC

Deficient production of IL-12 by neonatal myeloid DC has been

described in response to TLR ligands, but the response of moDC

71.3%

MOI = 0

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-A,B

,C

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HCMV Ag

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MOI = 10A

B

C

Figure 1. Neonatal and adult moDC are equally susceptible to HCMVinfection. (A) Adult and neonatal moDC were either mock-infected(MOI 5 0) or infected with HCMV at a multiplicity of infection of 10(MOI 5 10). Cells were stained with Ab against HLA-A, B, C andintracellularly stained with anti-HCMV IE protein Ab at 24 h afterinfection. One representative of seven independent experiments, withadult moDC, showing the original flow cytometry data. Percentages ofinfected moDC were evaluated as HCMV Ag1 cells (right side of dotplots). (B) Adult (white bars) and neonatal moDC (black bars) wereeither mock-infected (MOI 5 0) or infected with HCMV at an MOI of 20for the indicated time. One representative of three independentexperiments. (C) Adult (white bars) and neonatal moDC (black bars)were either mock-infected (MOI 5 0) or infected with HCMV at MOIfrom 0.5 to 20, for 24 h. Mean7SEM are indicated.

Eur. J. Immunol. 2009. 39: 2789–2799Joelle Renneson et al.2790

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

to viral infection has not been reported. We measured the

secretion of IL-12 p70 and p40 in supernatants 24 h after HCMV

infection of adult and neonatal moDC, at two different MOI (0.5

or 10 plaque forming unit (PFU)/cell). We observed a significant

production of those cytokines following HCMV infection of moDC

(adult mean: 12.15 pg/mL for MOI 0.5, 85.23 pg/mL for MOI 10)

(Fig. 2A and B). Neonatal moDC infected with HCMV at an MOI

of 10 produced significantly lower concentrations of IL-12 p70

than adult moDC. As observed in response to TLR ligation, we

observed at the mRNA level that neonatal moDC infected with

HCMV produced lower levels of IL-12 p35 subunit mRNA and

similar levels of IL-12 p40 subunit mRNA as compared with adult

moDC. These results show that the deficient IL-12 p70 production

by neonatal myeloid DC activated by TLR ligation is also observed

in response to a complex viral infection.

Mature IFN-a and defective IFN-b and IFN-k1production by HCMV-infected neonatal moDC

We then analyzed the production of type I/III IFN that are essential

cytokines for antiviral responses. Previous studies have shown that

neonatal moDC stimulated by TLR ligands produce lower concen-

trations of IFN-b than adult moDC [20]. We observed that HCMV

infection induced the production of high concentrations of IFN-a by

adult moDC even at a low MOI (Fig. 3A). Similar concentrations

were produced by neonatal and adult moDC. Quantitative RT-PCR

confirmed that the IFN-a2 gene was similarly expressed upon

infection by neonatal and adult moDC. IFN-b and IFN-l1 proteins

were also detected in infected adult moDC supernatants, with

higher type I IFN concentrations being induced by increasing MOI

(Fig. 3B and C). In contrast, neonatal moDC infected with HCMV at

an MOI of 10 showed lower production of the two early IFN as

compared with adult cells. The low IFN-b production was associated

with a low accumulation of IFN-b mRNA in neonatal moDC. We

then measured the production of CXCL-9, a typical IFN-inducible

chemokine. As shown in Fig. 3D, the induction of CXCL-9 secretion

was similar in adult and neonatal infected moDC. These experi-

ments indicate that although neonatal moDC produce lower

concentrations of IFN-b and IFN-l1 than adult cells, they develop

mature IFN-a and IFN-dependent responses to HCMV infection.

Gene expression analysis of HCMV-infected adultand neonatal moDC

In order to further compare the response of adult and neonatal

moDC to HCMV infection, we analyzed their transcriptome by

oligonucleotide microarrays. Adult and neonatal moDC were either

mock-infected or infected with HCMV at an MOI of 10 for 6 or 16 h

and total RNA was harvested. The time points were chosen to

include an early and a late time period in the first phase of HCMV

life cycle associated with the expression of immediate-early genes

(Fig. 1 and [24]). RNA samples were hybridized to microarrays

IL-12 p70 protein125

IL-12 p35 mRNA30000

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A

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Figure 2. IL-12 production in HCMV-infected neonatal and adult moDC. Adult (white bars) and neonatal moDC (black bars) were either mock-infected or infected with HCMV at MOI of 0.5 or 10. Supernatants were collected after 24 h for cytokines detection. (A) IL-12 p70 (n 5 7 adults and 9neonates), (B) IL-12 p40 (n 5 10 adults and 8 neonates). mRNA were extracted after 3, 6 and 16 h and real-time RT-PCR was performed for mRNAquantification. Peak timepoint at 6 h is represented for IL-12 p35 (A) and IL-12 p40 (B). Mean7SEM of one representative of three independentexperiments involving three donors in each group is shown. ��po0.01 between adult and neonatal moDC as assessed by the Mann–Whitney test.

Eur. J. Immunol. 2009. 39: 2789–2799 Immunity to infection 2791

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

designed to detect 54 676 transcripts. We first compared the genes

expressed by resting adult and neonatal moDC and identified 23

genes differentially expressed by the two study groups at basal level

(Table 1, Supporting Information). These genes are marked by

asterisks in the following tables. Among them, IL-1R2, epiregulin

and IGF-II mRNA-binding protein 3 (IMP-3) could be involved in

the activation or maturation of DC (see the Discussion section). We

then analyzed the ratios of intensities in gene expression between

HCMV-infected and mock-treated DC. Genes changing expression

by four-fold or greater, with a p value less or equal to 0.001, were

considered significantly regulated. Six hours after infection, adult

and neonatal moDC increased the expression of 245 and 215 genes,

and decreased the expression of 28 and 13 genes, respectively. After

16 h infection, 662 and 854 genes were up-regulated and 478 and

315 genes were down-regulated in adult and neonatal moDC,

respectively (Fig. 4). A large fraction of the regulated genes were

different in adult and newborn cells (36.3% of up-regulated and

46.9% of down-regulated genes after 16 h infection). Further, the

statistical comparison of gene regulation magnitudes in adult and

neonatal moDC revealed that gene modulation was significantly

different in adult and neonatal cells for 20 genes after 6 h infection

(Table 2, Supporting Information) and 88 genes after 16 h infection

(Table 3, Supporting Information). Half of the genes that were

differentially modulated at 6 h were still differentially modulated at

16 h. Interestingly, most of the genes differentially regulated after

16 h infection were more up-regulated in neonatal than in adult

moDC. Together, these data indicate that HCMV infection triggers a

gene expression program that is partly different in adult and

neonatal moDC.

Induction of type I IFN and IFN-dependent genes inadult and neonatal moDC by HCMV

To confirm the ability of neonatal moDC to develop a mature IFN-

dependent response, we examined the expression of type I and

IFN-α protein3000 IFN-α2 mRNA2000

Adults

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Figure 3. Production of type I/III IFN upon HCMV infection in neonatal as compared with adult moDC. Adult (white bars) and neonatal moDC(black bars) were either mock-infected or infected with HCMV at MOI of 0.5 or 10. Supernatants were collected after 24 h for cytokines detection.(A) IFN-a (n 5 10), (B) IFN-b (n 5 10), (C) IFN-l1 (n 5 9), (D) CXCL-9 (n 5 5). mRNA were extracted after 3, 6 and 16 h and real-time RT-PCR wasperformed for mRNA quantification. Peak timepoint at 6 h is represented for (A) IFN-a2 and (B) IFN-b. Mean7SEM of one representative of threeindependent experiments involving three donors in each group is shown. �po0.05 between adult and neonatal moDC as assessed by theMann–Whitney test.

Eur. J. Immunol. 2009. 39: 2789–2799Joelle Renneson et al.2792

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

type III IFN, as well as a series of ISG in the microarray data set. As

shown in Fig. 5A, the expression of early-type IFN genes, including

IFN-a1, IFN-b and IFN-l1, was induced by 6 h infection of adult

moDC with HCMV. The induction of IFN-b and IFN-l1 gene

expression was lower in neonatal than in adult moDC. This defect

was still present after 16 h infection. In contrast, delayed-type IFN

genes, which include other IFN-a subtypes and IFN-o1 were

induced at similar levels in adult and neonatal infected cells 16 h

post-infection. Furthermore, adult and neonatal moDC infected

with HCMV for 6 and 16 h showed a similar up-regulation of a set

of 20 genes known to be IFN-inducible (Fig. 5B) [20]. Among this

series of identified ISG, expression of only two genes (GBP-1 and

EXT-1) was lower in neonatal as compared with adult HCMV-

infected moDC, and one gene (IFI-44) was significantly more up-

regulated by neonatal cells. We next analyzed the expression of

other cytokines and chemokines involved in the induction of an

adaptive immune response (Fig. 5C). IL-12 gene expression was

not detected by microarray analysis. The induction of only few

genes coding for proinflammatory and T-cell activating cytokines

was detectable following HCMV infection of moDC. Among them,

IL-15 mRNA was expressed at a lower level in neonatal than in

adult moDC. Chemokine genes were induced similarly by HCMV in

the two cell populations, except CCL-4, which was more up-

regulated in neonatal moDC.

Discussion

This study demonstrates that moDC infected with HCMV produce

significant amounts of IFN-a. Interestingly, cord blood-derived

DC expressed a large number of IFN-a subfamily genes at similar

levels than adult DC. This observation may be important for our

understanding of immunity against viruses in early life. Although

the secretion of IFN-a in the early phase of viral infections is

considered to be largely dependent on plasmacytoid DC [25],

myeloid DC have been shown to produce type I IFN in response to

viruses [26–28]. A previous study showed that CD11c1 myeloid

DC exposed to HCMV produced IFN-a [29]. Neonatal plasmacy-

toid DC were shown to be defective in their capacity to produce

IFN-a in response to TLR9 ligation [30, 31]. More recently, we

observed that neonatal plasmacytoid DC exposed to HCMV

produced lower concentrations of IFN-a than adult cells [32].

Together these results suggest that myeloid DC could represent

an important source of IFN-a production during HCMV infection

in early life. In contrast to IFN-a produced during the relatively

late stage of infection, we observed that the production of two

early-stage IFN genes, IFN-b and IFN-l1, was lower in neonatal

as compared with adult moDC infected by HCMV. However,

HCMV infection of neonatal DC induced many ISG at similar

levels than adult DC. This mature IFN-dependent response of

infected neonatal DC may be dependent on the low concentra-

tions of IFN-b and IFN-l1 produced or may be predominantly

dependent on IFN-a.

The differential production of IFN-a and IFN-b/l observed in

this study could be related to the involvement of different cell

activation pathways. This hypothesis is supported by the different

kinetics of gene expression observed in our study and by results

obtained in other experimental models. Following influenza A

infection, DC produce IFN-b and IFN-l1 but do not produce IFN-a[27]. The absence of IFN-a induction could involve the inter-

ference of the influenza A virus protein NS1, which has recently

been shown to form a complex with and block RIG-1, an RNA

helicase enzyme recognizing viral RNA [33]. Moreover, ligation

of TLR, including TLR3 and TLR4, has been shown to stimulate

the production of IFN-b and IFN-l by moDC whereas IFN-a is not

induced [34]. Although the receptors involved in HCMV recog-

nition by DC have not been identified, both TLR-dependent and

independent pathways are likely to be activated. Fibroblast acti-

vation by HCMV was shown to involve the TLR2/TLR1 hetero-

dimer [35]. In our study, 24% of the genes induced in adult DC

by infection with HCMV are involved in TLR signaling pathways

(data not shown). We propose that TLR could play an important

role in the induction of IFN-b and IFN-l gene expression

following HCMV infection of myeloid DC. The pathways inducing

the production of IFN-a in response to HCMV infection remain

unclear but might involved cytoplasmic viral sensors like RNA

helicase enzymes (RIG-1, MDA5) or viral double-stranded DNA

as-yet-unknown detectors [36].

The low IFN-b and IFN-l1 production by HCMV-infected

neonatal moDC was associated with a low production of IL-12

p70. This impaired IL-12 p70 secretion was related to a reduced

capacity to produce the IL-12 p35 subunit. The lower production

of IL-12p35 and IFN-b observed following HCMV infection is in

line with the response of neonatal moDC to TLR3 and TLR4

ligation [20, 21]. Aksoy et al. have shown that the defective IL-12

400

500

600 Common

Adult specific

Neonate specific

100

200

300

UP0

16 hrs pi6 hrs pi

DOWN UP DOWN

Num

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egul

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es

Figure 4. Gene expression analysis of HCMV-infected adult andneonatal moDC. Adult and neonatal moDC were either mock-infectedor infected with HCMV at MOI of 10 for 6 and 16 h. Total RNA wasprepared and processed for hybridization to Affymetrix genomearrays. Results from three independent experiments involving adultand neonatal moDC are shown. Statistical analysis was performed asdescribed in the Material and methods. M value cutoffs were 2rMr�2and p value r0.001. Numbers of genes that were regulated by HCMVinfection exclusively in adult (white bars) or in neonatal moDC (blackbars) are represented. Hatched bars represent numbers of regulatedgenes in both adult and neonatal cells (up-regulated (UP) and down-regulated (DOWN)).

Eur. J. Immunol. 2009. 39: 2789–2799 Immunity to infection 2793

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

p35, IFN-b and ISG synthesis by LPS-stimulated neonatal DC is

associated with decreased IRF (IFN regulatory factor)-3/CBP

(CREB-binding protein) interaction [20]. Another study sugges-

ted that IFN-l1 gene expression could also be dependent on IRF-3

[27]. We suggest that the low IL-12, IFN-b and IFN-l1 gene

expression by HCMV-infected neonatal DC may also involve a

reduced activity of IRF-3, whereas IFN-a gene expression by

HCMV-infected myeloid DC may not be dependent on IRF-3. IRF-

7 is a key regulator of type I IFN during viral infections [37]. IRF-

7 efficiently activates IFN-a and IFN-b gene expression and

represents a positive feedback loop as it is induced by type I IFN

[37]. It will be important to evaluate the role of IRF-7 in the

induction of IFN-a by HCMV-infected adult and neonatal moDC.

The mechanisms involved in the reduced capacity of neonatal

moDC to produce IL-12 and IFN-b in response to LPS stimulation

and HCMV infection remain to be identified. Aksoy et al. detected

similar levels of the signal transduction molecules TRIF, TRAM,

IRF-3, IKKe and TBK1 in immature neonatal and adult moDC by

Western blotting [20]. The comparison of gene expression

profiles of uninfected neonatal and adult moDC did not reveal

differences involving specific pathways but identified several

genes coding for proteins that could be involved in DC matura-

tion and function. Adult moDC expressed higher levels of IL-1R2

that could participate in an autocrine loop amplifying DC acti-

vation. Adult DC also expressed higher levels of epiregulin, a

member of epidermal growth factor superfamily involved in

Type I and III IFNs (6h)60 Type I and III IFNs (16h)300

Adults

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Figure 5. Induction of type I/III IFN, IFN-inducible and cytokine/chemokine genes in adult and neonatal moDC by HCMV. Adult (white bars) andneonatal moDC (black bars) were either mock-infected or infected with HCMV at MOI of 10 for 6 and 16 h. Total RNA was prepared and processedfor hybridization to Affymetrix genome arrays. M value cutoffs were 2rMr�2 and p value r0.001. Fold change in gene expression induced byHCMV infection are represented for (A) type I and III IFN genes, (B) IFN-inducible genes and (C) cytokine-chemokine genes. �po0.05 between adultand neonatal moDC using the statistical methods described in the Materials and methods section.

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& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

proinflammatory cytokine production in mouse macrophages

[38]. Neonatal moDC expressed higher levels of IMP-3. IMP-3 is a

translational activator of IGF-II, a molecule that could be

involved in the maturation of mouse DC [39, 40]. Differences in

IL-1R2 and IMP-3 between neonatal and adult immature moDC

were also observed in an independent study by Aksoy et al. [20].

Further studies are required to evaluate the role of these mole-

cules in the response of neonatal and adult DC to HCMV infection

and TLR ligation.

Microarray analysis showed that HCMV infection has a major

impact on the transcriptional activity of neonatal and adult

moDC. Sixteen hours after infection of adult DC, 1140 genes were

found to be up-regulated or down-regulated by at least four-fold.

Multiple pathways are affected by the regulation of those genes,

particularly TLR and cytokine receptor signaling, Jak-STAT

signaling, cell cycle and apoptosis (data not shown). IFN-

dependent response was strongly induced in HCMV-infected DC,

as previously described in infected fibroblasts [41]. Recently,

HCMV has been shown to induce a transcriptional program

similar to IFN-treated cells, suggesting that a majority of genes

are regulated coordinately by viral infection and type I IFN

treatment [42]. Comparing adult and neonatal response to

HCMV, we demonstrated that HCMV infection triggers a partly

different gene expression program in neonatal and adult moDC.

More than a third of the genes regulated by HCMV infection were

different in adult and newborn cells. The 88 genes that were

differentially regulated in adult and neonatal cells after 16 h

infection belong to multiple families and no unique pathway

could be identified (data not shown). However, important

differences were observed in the expression of immunomodula-

tory genes between adult and neonatal moDC. In addition, the

expression of IMP-3, which was expressed more by uninfected

neonatal DC than by adult DC, was significantly up-regulated

following HCMV infection of adult but not neonatal moDC. These

results suggest that IMP-3 may play an important role in the

regulation of the biology of neonatal and adult moDC. Together,

our results indicate that neonatal DC infected with HCMV show

defective production of the immunomodulatory cytokines

IL-12p70, IFN-b and IFN-l1 but are able to produce adult-like

concentrations of IFN-a and IFN-dependent genes. These defects

could be involved in the differential induction of CD81 and CD41

T-cell responses observed following congenital HCMV infection

[7, 8]. IL-12 is an essential cytokine for development of effector

functions and cytotoxic responses. Viruses have been shown to

induce IL-12 p70 release by DC, including influenza [43] and

dengue virus [28]. IFN-g production by antigen-specific CD41

T cells requires IL-12 and STAT-4 activation, whereas CD81

T cells can produce IFN-g independently of IL-12 [44]. In addi-

tion, both IL-12 and type I IFN signaling were shown to be

required for IFN-g production by antigen-specific CD41 T cells in

Listeria monocytogenes infection [45]. The defective IL-12

production by HCMV-infected neonatal moDC observed in our

study could therefore be involved in the deficient CD41 T-cell

responses in congenitally infected newborns. Defective CD41

T-cell responses were also observed following infection of infants

with HIV or HSV [46]. These defective responses are associated

with a poorer control of viral replication and more severe

outcome. To our knowledge, the responses of neonatal moDC to

infection with HSV and HIV have not been studied. In order to

increase our understanding of the pathogenesis of viral infections

in early life, it will be important to extend the study we have

conducted with HCMV to other pathogens causing severe infec-

tions in young infants. On the other hand, type I IFN contribute to

cytotoxic CD81 T-cell response by diverse mechanisms, indirect

action through induction of DC maturation [47] but also more

direct action [48]. In mice infected with lymphocytic chor-

iomeningitis virus, the expansion and the differentiation of CD81

T cells as well as their production of IFN-g was shown to be

dependent on endogenous IFN-a/b but not on IL-12 [49, 50]. The

preferential production of IFN-a by HCMV-infected neonatal

moDC could therefore play an important role in the functional

CD81 T-cell responses to this virus during fetal life. The TB40/E

strain that was used in the study was selected because it has the

property of inducing a full replicative cycle in DC. Indeed, human

DC are not permissive to conventional laboratory strains of

HCMV, such as the AD169 strain. Genetic variability of HCMV

strains, in particular of clinical isolates, could influence the

response of DC. However, the fact that the defect in IL-12 and

IFN-b production is also observed following activation of

neonatal moDC with LPS suggests that it is not specific to the

HCMV strain used in the study.

In conclusion, our study shows that neonatal moDC respond

differently to HCMV infection than adult moDC by producing

lower levels of specific immunomodulatory cytokines. These

findings have important implications for our understanding of the

pathogenesis of viral infections in early life and for the develop-

ment of vaccines protecting young infant from intracellular

pathogens.

Materials and methods

Generation and culture of moDC

Cord blood was obtained from normal full-term deliveries at the

obstetric departments of CHU Tivoli, La Louviere, CHU, Charleroi

and Clinique Notre Dame, Charleroi, Belgium. Fresh adult blood

was obtained from healthy laboratory staff volunteers. HCMV

seropositive and seronegative donors were included. This study

was approved by the institutional scientific and ethics commit-

tees. Mononuclear cells were isolated by centrifugation over

Lymphoprep density gradient (Nycomed, Oslo, Norway) and

monocytes were purified by positive selection using anti-CD14-

conjugated magnetic microbeads (Miltenyi, Germany). moDC

were generated by culturing monocytes for 5–6 days in RPMI

1640 (Cambrex, Verviers, Belgium) supplemented with 2 mM

L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, 1%

non-essential amino acids, 1% sodium pyruvate, 50 mM

b2-mercaptoethanol and 10% FBS (Hyclone, Perbio, Erembode-

Eur. J. Immunol. 2009. 39: 2789–2799 Immunity to infection 2795

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

gem, Belgium), plus GM-CSF (500 U/mL) and IL-4 (1000 U/mL).

GM-CSF was obtained from Leucomax (Shering-Plough, France)

and IL-4 was from R&D Systems (Abingdon, UK).

Virus

HCMVTB40/E is an endothelial cell-propagated HCMV strain

isolated from a bone marrow transplant recipient [51]. Human

foreskin fibroblasts HFF-1 (ATCC SCRC-1041, LGC Promochem,

Middlesex, UK) were cultured in DMEM (Cambrex) supplemented

with 100 U/mL penicillin, 100mg/mL streptomycin and 15% FBS.

Cells were grown to about 90% confluence and infected at low

MOI (0.1–1 PFU/cell) of HCMVTB40/E in a low volume of DMEM for

3 h at 371C. Complete DMEM with 15% FBS was then added and

cells cultured until the first cytopathic effects appeared (6–7 days).

Supernatants of infected HFF-1 were collected every 3–4 days,

until the cytopathic effects became advanced. Each supernatant

was centrifuged at 1200 g for 10 min to remove cell debris and the

supernatant was stored at �801C. Concentrated virus stock was

obtained by ultracentrifugation at 80 000 g for 120 min at room

temperature. The pellet containing viral particles from 20–25 mL

supernatant was resuspended in 1 mL RPMI, titrated and frozen at

�801C. Viral titers were determined by immunofluorescence on

MRC-5 cells and varied between 107 to 108 PFU/mL.

Infection of moDC

moDC (5� 105/mL) were either infected with HCMVTB40/E at

MOI between 0.5 and 20 PFU/cell or mock-infected with 0.5 mL

MEM 5% FCS for 3 h at 371C. After removal of the virus or

medium, moDC were washed and cultured in complete RPMI

containing 10% FBS for a total time indicated in the figure

legends.

Flow cytometry and detection of infection

Cells were harvested, washed in PBS supplemented with 0.5%

BSA and labeled for surface molecules for 20 min at 41C with the

following Ab: anti-CD80 PE, anti-CD86 APC, anti-HLA-A, B, C

APC and anti-HLA-DR PerCp (all from BD Biosciences, Erembo-

degem, Belgium). moDC were also stained with corresponding

isotype-control monoclonal Ab. The cells were then fixed in 4%

paraformaldehyde and permeabilized with 0.025% Triton-X100

(20 min each step at room temperature). After wash, cells were

stained intracellularly with anti-HCMV IE protein Ab (Argene,

France). The samples were analyzed on a CyAnTM

ADP flow

cytometry analyzer (DakoCytomation) using Summit Software

(version 4.2).

Cytokines quantification

Supernatants from moDC were collected after 24 h of incubation

and analyzed using specific ELISA for IFN-a, IFN-b and IL-12 p40

(all from Biosource, Nivelles, Belgium) with a detection limit of

19.5 pg/mL, 3.1 U/mL, and 9.8 pg/mL, respectively. IFN-l1 and

CXCL-9 levels were determined using ELISA kits (R&D Systems)

with a detection limit of 62.5 and 7.8 pg/mL, respectively. IL-12

p70 levels were measured using Quantikine HS ELISA (R&D

Systems) with a detection limit of 0.6 pg/mL. Statistical analyses

were performed using the Mann–Whitney test. Differences were

considered statistically significant when p value was less than or

equal to 0.05.

RNA purification and real-time RT-PCR

Cells were lyzed and mRNA was extracted using a MagNA Pure

LC RNA Isolation Kit-High Performance (Roche Diagnostics,

Brussels, Belgium). Reverse transcription and real-time PCR were

then carried out using LightCycler-RNA Master Hybridisation

Probes (one-step procedure). Primer sequences are listed in

Table s4 (Supporting Information).

RNA preparation and Affymetrix GeneChiphybridization

Adult and neonatal moDC (three donors in each group) were

either mock-infected or infected with HCMV at an MOI

of 10 PFU/cell for 6 and 16 h. Total RNA was isolated using

TRIzol reagent (Invitrogen, Merelbeke, Belgium) and RNeasy

Micro kit (Qiagen, The Netherlands). Samples were checked

for integrity and concentration using the RNA 6000 Pico

LabChip kit on the Agilent Bioanalyzer 2100 (Agilent Technolo-

gies, Palo Alto, CA, USA). All RNA used met the quality

criteria defined by Agilent and were processed according to

Affymetrix’s (Santa Clara, CA, USA) instructions. Briefly, probes

were generated using the Affymetrix GeneChip Expression

30 Amplification Two-cycle Target Labeling and Controls Reagents

kit . Two rounds of T7 polymerase-based linear RNA amplification

were performed by reverse transcription of RNA using

a T7-(dT) 24 primer and a random hexamer primer, as

described in the expression technical manual from Affymetrix

(http://www.affymetrix.com). Then, biotinylated cRNA was

synthesized from the double stranded cDNA using T7 RNA

polymerase and a biotin-conjugated pseudo-uridine containing

nucleotide mixture provided in the kit. Prior to hybridization, the

cRNA was purified and fragmented. About 10mg from each

experimental sample along with Affymetrix eukaryotic hybridiza-

tion controls were hybridized for 16 h to Human U 133 Plus 2.0

genome arrays, containing 54 676 transcripts. Arrays were washed

and stained with streptavidin-phycoerythrin (Molecular Probes,

Eugene, OR, USA), biotinylated anti-streptavidin (Vector Labora-

tories, Burlingame, CA, USA). Arrays were then scanned with an

Affymetrix GeneChips Scanner 3000 at 570 nm. The Affymetrix

eukaryotic hybridization control kit and Poly-A RNA control kit

were used to ensure efficiency of hybridization and cRNA

amplification. All cRNA were synthesized at the same time.

Eur. J. Immunol. 2009. 39: 2789–2799Joelle Renneson et al.2796

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

Microarray data analysis

Each array images (‘‘DAT’’ files) were visually screened to

discount for signal artifacts, scratches or debris and each

of the Genes Chips (‘‘rpt’’ files) was checked for the control

of hybridization defined by Affymetrix GeneChip Manual

(like the uniformity of the signal, the good position of

the grid, the scale factor, the background, the percent of

presence, the house keeping genes and the spikes control).

The data were first analyzed using Affymetrix Microarray

Suite 5.0 software. Various Bioconductor packages for R

(www.bioconductor.org) were used for data analysis. In brief,

Simpleaffy package was used to pre-process the raw data for

background correction, normalization and probe summarization

by the RMA method [52, 53]. Differentially expressed genes

were defined using a statistical tool called LIMMA package,

which is part of the Bioconductor statistical analysis software

[54]. Three statistical approaches, all part of BioConductor

software, were used to identify differentially expressed genes:

(i) significance of analysis for microarrays [55], (ii) a paired t test

controlling the false discovery rate using Benjamini and

Hochberg correction [56], and (iii) the moderated t test in linear

models for microarray data [57] also encompassing the Benja-

mini and Hochberg correction. Data were first filtered based

on ‘‘MA plot’’, where M 5 log2 (fluorescent dye intensity of

infected DC/uninfected DC) and A 5 log2 Ofluorescent dye

intensity of (infected DC)� (uninfected DC), as described by

Yang et al. for cDNA microarray [58]. In the first comparison

between infected and uninfected DC, a gene was called

differentially expressed if M value was more than 2 or less than

�2 and p value was less than or equal to 0.001. In the second

comparison between differentially regulated genes in adult

and neonatal DC, a gene was called differentially regulated

if M value was more than 1 or less than �1 and p value was

less than or equal to 0.05. From the previous gene lists, we

analyzed whether some ontologies or pathways are linked with

those lists. We scanned the Gene Ontology database as well as the

pathways from KEGG. We have used the same methodology

developed by Sorin Draghici for his Onto-Express algorithm

(http://vortex.cs.wayne.edu/ontoexpress/), which calculates

that an ontology or a pathway is statistically associated with a

list of genes according to the number of genes present on the

GeneChips, the number of genes associated with the ontology or

pathway and the number of genes associated with this ontology

or pathways from our gene list. From this information, the

algorithm calculates a p-value that will be corrected for multiple

testing to provide the information that the ontology or pathway

founded is not due to chance.

Acknowledgements: This work was supported by a grant from

the Fonds National de la Recherche Scientifique (FNRS, Belgium)

and the Televie program. The Institute for Medical Immunology

is supported by GlaxoSmithKline Biologicals and the government

of the Walloon Region. Stanislas Goriely is a postdoctoral

research fellow at the FNRS, Belgium. Arnaud Marchant is a

senior research associate at the FNRS, Belgium. Arnaud Marchant

and Michel Goldman have served as consultants and received

financial research support from GlaxoSmithKline Biologicals.

Conflict of interest: The Institute for Medical Immunology is

supported by GlaxoSmithKline Biologicals. Arnaud Marchant and

Michel Goldman have served as consultants and received finan-

cial research support from GlaxoSmithKline Biologicals. The

authors declare no other financial or commercial conflict of

interest.

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Abbreviations: HCMV: human CMV � IMP-3: IGF-II mRNA-binding

protein 3 � IRF: IFN-regulatory factor � ISG: IFN-stimulated genes �moDC: monocyte-derived DC � PFU: plaque forming unit

Full correspondence: Dr. Arnaud Marchant, Institute for Medical

Immunology, Universite Libre de Bruxelles, Rue Adrienne Bolland 8,

B-6041 Charleroi, Belgium

Fax: 132-2-650-9563

e-mail: arnaud.marchant@ulb.ac.be

Supporting Information for this article is available at

www.wiley-vch.de/contents/jc_2040/2009/39414_s.pdf

Received: 12/3/2009

Revised: 2/7/2009

Accepted: 17/7/2009

Eur. J. Immunol. 2009. 39: 2789–2799 Immunity to infection 2799

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu