Virus-Plus-Susceptibility Gene Interaction Determines Crohn's Disease Gene Atg16L1 Phenotypes in...

Virus-Plus-Susceptibility GeneInteraction Determines Crohn’s DiseaseGene Atg16L1 Phenotypes in IntestineKen Cadwell,1 Khushbu K. Patel,1 Nicole S. Maloney,1 Ta-Chiang Liu,1 Aylwin C.Y. Ng,3,4 Chad E. Storer,5

Richard D. Head,5 Ramnik Xavier,3,4 Thaddeus S. Stappenbeck,1,* and Herbert W. Virgin1,2,6,*1Department of Pathology and Immunology2Department of Molecular Microbiology

Washington University School of Medicine, St. Louis, MO 63110, USA3Center for Computational and Integrative Biology and Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School,

Boston, MA 02114, USA4Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA5Inflammation and Immunology Research Unit, Pfizer Global Research and Development, St. Louis, MO 63017, USA6Midwest Regional Center of Excellence for Biodefense and Emerging Infectious Diseases Research, St. Louis, MO 63110, USA

*Correspondence: [email protected] (T.S.S.), [email protected] (H.W.V.)

DOI 10.1016/j.cell.2010.05.009

SUMMARY

It is unclear why disease occurs in only a smallproportion of persons carrying common risk allelesof disease susceptibility genes. Here we demon-strate that an interaction between a specific virusinfection and a mutation in the Crohn’s disease sus-ceptibility gene Atg16L1 induces intestinal patholo-gies in mice. This virus-plus-susceptibility geneinteraction generated abnormalities in granule pack-aging and unique patterns of gene expression inPaneth cells. Further, the response to injury inducedby the toxic substance dextran sodium sulfate wasfundamentally altered to include pathologies resem-bling aspects of Crohn’s disease. These pathologiestriggered by virus-plus-susceptibility gene interac-tion were dependent on TNFa and IFNg and wereprevented by treatment with broad spectrum antibi-otics. Thus, we provide a specific example of howa virus-plus-susceptibility gene interaction can, incombination with additional environmental factorsand commensal bacteria, determine the phenotypeof hosts carrying common risk alleles for inflamma-tory disease.

INTRODUCTION

Common genetic polymorphisms predispose to complex dis-

eases such as type I diabetes, multiple sclerosis, Crohn’s

disease, and ulcerative colitis (The Wellcome Trust Case Control

Consortium, 2007; Altshuler et al., 2008). It is not clear why some

individuals with a given polymorphism acquire disease whereas

others remain unaffected. The concept that environmental

factors including infections trigger disease in individuals with

certain genetic backgrounds is broadly recognized. In animal

models, autoimmune disease can be influenced by viral infec-

tions. For example, glomerulonephritis is exacerbated by lym-

phocytic choriomeningitis virus and polyoma virus infections in

certain inbred backgrounds (Tonietti et al., 1970), and lympho-

cytic choriomeningitis virus infection inhibits development of dia-

betes in NOD mice or BB rats (Dyrberg et al., 1988; Oldstone,

1988). The genes responsible for these differences in outcome

have not been defined, but these studies indicate that virus infec-

tion can alter disease in specific genetic backgrounds.

Crohn’s disease is a common type of inflammatory bowel

disease involving mucosal ulceration and inflammation occur-

ring in the distal small intestine (ileum) and variable discontin-

uous regions of the colon. A distinguishing feature of inflamma-

tion in Crohn’s disease is involvement of the entire thickness of

the bowel wall. This transmural inflammation leads to atrophy

of ileal villi, fibrosis, hypertrophy of smooth muscle and auto-

nomic nerve cells in the outer layer of the bowel wall, and an

inflammatory response including lymphoid aggregates and

granulomas (Day et al., 2003). Polymorphisms in over 30 loci

have been associated with increased risk of Crohn’s disease

(Barrett et al., 2008). Yet these genetic components individually,

or in combination (Weersma et al., 2008), confer limited risk.

Environmental factors such as exposure to pathogens are

potential cofactors for disease development, but the etiology

of Crohn’s disease remains a controversial topic (Packey and

Sartor, 2009).

One Crohn’s disease susceptibility allele is in the autophagy

gene ATG16L1 (The Wellcome Trust Case Control Consortium,

2007; Rioux et al., 2007; Hampe et al., 2007). The ATG16L1

disease variant is frequent (�50% in European-derived popula-

tions) and confers less than a 2-fold increase in susceptibility.

We previously established two mouse lines in which Atg16L1

expression is disrupted by gene trap mutagenesis (Cadwell

et al., 2008a). Mutant mice displayed hypomorphic (HM)

Atg16L1 protein expression and reduced autophagy (Cadwell

Cell 141, 1135–1145, June 25, 2010 ª2010 Elsevier Inc. 1135

mailto:[email protected]

mailto:[email protected]

et al., 2008a; Ju et al., 2009). Conventionally raised Atg16L1HM

mice display striking abnormalities in Paneth cells, epithelial cells

at the base of ileal crypts that are important in mucosal immunity

(Porter et al., 2002; Ouellette, 2006; Vaishnava et al., 2008).

In addition to aberrant packaging and exocytosis of antimicrobial

granules, Paneth cells from Atg16L1HM mice display a surprising

gain-of-function transcriptional profile in which transcripts asso-

ciated with lipid metabolism, proinflammatory cytokines, and

other pathways were enriched. Importantly, we observed similar

Paneth cell abnormalities in Crohn’s disease patients homozy-

gous for the risk allele of ATG16L1 but not in control patients

(Cadwell et al., 2008a). Therefore, these aspects of intestinal

pathology link Crohn’s disease patients carrying the suscepti-

bility allele of ATG16L1 to mice that are hypomorphic for

Atg16L1.

Here, we report a striking genetic interaction between Atg16L1

mutation and a specific strain of an enteric virus, murine norovi-

rus (MNV). This interaction determines multiple pathologic

abnormalities in the intestine including several similar to those

observed in Crohn’s disease patients. Noroviruses are positive-

sense encapsidated RNA viruses responsible for the majority of

epidemic nonbacterial gastroenteritis in humans (Mead et al.,

1999). Multiple strains of MNV are prevalent in mouse facilities

around the world (Hsu et al., 2005; Pritchett-Corning et al.,

2009; Goto et al., 2009), and some MNV strains persist for

months after initial infection (Thackray et al., 2007). We demon-

strate that Paneth cell abnormalities in Atg16L1HM mice are

triggered by infection with an MNV strain that establishes persis-

tent infection (persistent strain). This virus-plus-susceptibility

gene interaction alters the transcriptional signature of Paneth

cells and the nature of the inflammatory response in mice

treated with the toxic substance dextran sodium sulfate (DSS).

The mechanism for this pathologic response driven by the

virus-plus-susceptibility gene interaction involved the cytokines

TNFa and IFNg as well as commensal bacteria. Our results

provide evidence for a multi-hit model of inflammatory dis-

ease defined in a highly specific way by a viral infection only in

the presence of a mutation in a disease susceptibility gene.

This observation provides a basis for understanding how a

common allele can be linked to an infrequent severe disease,

and why mice carrying mutations in human disease susceptibil-

ity genes do not always spontaneously reproduce human

pathology.

RESULTS

Viral Infection Triggers Paneth Cell Abnormalitiesin Atg16L1HM MiceAtg16L1HM mice raised in a conventional barrier facility were

rederived into an enhanced barrier facility (Experimental Proce-

dures) by embryo transfer. Surprisingly, in contrast to observa-

tions made in mice from a conventional barrier (Cadwell et al.,

2008a), Paneth cells in rederived Atg16L1HM mice were indistin-

guishable morphologically from those in littermate wild-type

control mice (herein referred to as WT) (Figure 1A) and failed to

display aberrant packaging of the granule protein lysozyme

(Figure 1B). All subsequent experiments were performed in

mice raised in the enhanced barrier facility.

1136 Cell 141, 1135–1145, June 25, 2010 ª2010 Elsevier Inc.

These results suggest that Paneth cell abnormalities require

an exogenous factor present in the conventional barrier facility.

We previously identified the first murine norovirus (MNV) in

mice from this facility (Karst et al., 2003). We therefore consid-

ered the possibility that MNV triggers Paneth cell abnormalities

in Atg16L1HM mice. To test this, mice were orally inoculated

with an MNV strain that establishes persistent infection (MNV

CR6) (Thackray et al., 2007). Seven days postinoculation,

Atg16L1HM but not WT mice displayed Paneth cell abnormalities

including aberrant granule numbers, size, and distribution (Fig-

ure 1A). UV-inactivated virus did not induce these abnormalities

(Figure S1A, available online) indicating that productive virus

infection was required.

MNV CR6 infection for 7 days also induced abnormal lyso-

zyme distribution and abnormal ultrastructural morphology in

Paneth cells from Atg16L1HM mice (Figure 1B and Figures S1B

and S1C). We used a previously established scale to blindly

identify cells with increasingly abnormal lysozyme staining (Fig-

ure 1C) (Cadwell et al., 2008a). Only Atg16L1HM mice that were

infected with MNV CR6 displayed Paneth cells with the two

most severe abnormalities (Figure 1D). Analysis of Paneth cells

by transmission electron microscopy also revealed a substantial

proportion of cells that were depleted of secretory granules in

MNV CR6-infected Atg16L1HM mice (Figures S1B and S1C).

Moreover, many of the Paneth cells from these mice contained

distended rough endoplasmic reticulum (Figures S1B and

S1C). Other cytoplasmic organelles including mitochondria

were not obviously altered in virally-infected Atg16L1HM mice.

Thus, Paneth cell morphological and granule packaging abnor-

malities were induced by the combination of viral infection and

mutation in the Crohn’s disease susceptibility gene Atg16L1.

Hereinafter we will refer to this as a virus-plus-susceptibility

gene interaction.

Properties of MNV Associated with Paneth CellAbnormalitiesSince autophagy is important in innate immunity (Virgin and

Levine, 2009), a mutation in Atg16L1 might lead to enhanced

viral replication and cytopathicity in Paneth cells. When com-

paring WT and Atg16L1HM mice, we did not observe significant

differences in viral shedding in the stool or viral titers in organs

including the distal ileum where Paneth cell abnormalities were

observed (Figure S2A). Consistent with a previous report in

which MNV was detected in the lamina propria (Mumphrey

et al., 2007), we did not detect MNV in Paneth cells by immuno-

histochemistry, and viral RNA was not detected in Paneth cell

RNA procured by laser capture microdissection (data not shown,

Experimental Procedures). Thus, direct infection of Paneth cells

is not responsible for triggering the abnormalities described

above.

To determine if Paneth cell abnormalities develop after infec-

tion with any virus, we investigated MNV CW3 that does not

persist in immunocompetent mice despite sharing 95% amino

acid sequence identity across the genome with MNV CR6

(Mumphrey et al., 2007; Thackray et al., 2007). As in WT mice,

shedding of MNV CW3 in Atg16L1HM mice is initially high but

reduced or undetectable at later time points (Figure S2B). MNV

CW3 failed to trigger aberrant Paneth cell morphology 7 days

A WT Uninfected BWT Uninfected

HM Uninfected

WT MNV CR6

HM MNV CR6

WT + MNV CR6

HM Uninfected HM + MNV CR6 HM + MNV CR6

CD0 D1 D2 D3

D

H M + MN V CR6

W T

W T + MN V CR6H M

D 0 D 1 D 2 D 30

20

40

60

80

100

% P

anet

h C

ell

***

**

*

Degree of abnormality

Lysozyme Distribution Day 7 Post Infection

Figure 1. Murine Norovirus Infection Triggers Paneth Cell Abnormalities in Atg16L1 Mutant Mice

(A and B) Atg16L1 hypomorph (Atg16L1HM) and WT mice in the MNV-free barrier facility were orally inoculated with 3 3 107 particle forming units (pfu) of MNV CR6

for 7 days or left untreated (n > 6 mice for each group).

(A) Light microscopy images of ileal sections stained with PAS-Alcian blue. Dotted line denotes crypt unit containing several Paneth cells each, and arrowheads

indicate typical granules. For the two representative images of Atg16L1HM mice infected with MNV CR6, red dotted circle denotes aggregated granules and red

arrows indicate granules with abnormal staining and size. Scale bar represents 10 mm.

(B) Indirect immunofluorescence of ileal sections stained for lysozyme (red) and nuclei (blue). Dotted line denotes crypt unit. Scale bar represents 10 mm.

(C) Four types of lysozyme distribution patterns observed in Paneth cells: normal (D0), disordered (D1), depleted (D2), diffuse (D3).

(D) Percentage of Paneth cells displaying each of the four types of lysozyme distribution patterns from WT and Atg16L1HM mice that were uninfected or inoculated

with MNV CR6 (n > 5,700 cells from 3 mice/condition, *p < 0.05, **p < 0.01, ***p < 0.001, mean ± SEM).

See also Figure S1.

postinfection (Figure 2A). Whereas MNV CW3 induced some

changes in lysozyme distribution (increase in D2), the most

abnormal distribution pattern (D3) was not detected (Fig-

ure 2B). Since the lysozyme staining pattern seen after MNV

CW3 infection was similar in both WT and Atg16L1HM mice,

these mild changes may constitute a normal response to viral

infection. Nevertheless, induction of Paneth cell abnormalities

specific to Atg16L1HM mice is virus strain-dependent and asso-

ciated with viral persistence, a property of MNV CR6 but not

MNV CW3.

Since MNV CW3 shedding is reduced or undetected 7 days

postinfection, we also evaluated lysozyme distribution at day 3

postinfection when viral shedding is high. The majority of Paneth

cells were normal (D0 pattern; Figure S2C) in WT and Atg16L1HM

mice infected with either MNV CR6 or CW3. However,

Atg16L1HM mice infected with MNV CR6 for 35 days displayed

Paneth cell abnormalities similar to mice infected for 7 days

(data not shown). These results are consistent with a requirement

for the continual presence of virus past the first several days of

initial infection to induce Paneth cell abnormalities.

Virus-Plus-Susceptibility Gene Interaction Resultsin a Unique and Virus Strain-Specific TranscriptionalProfile in Paneth CellsWe previously reported aberrant gene expression in Paneth cells

of conventionally raised Atg16L1HM mice (Cadwell et al., 2008a).

To determine if gene expression is influenced by the virus-plus-

susceptibility gene interaction, we compared gene expression in

Paneth cells from WT and Atg16L1HM mice 7 days after infection

with MNV CR6 (Figure 3). Data were analyzed from three biolog-

ical replicate experiments. Among genes whose expression

were increased or decreased at least 1.5-fold (p < 0.05) in

response to MNV infection, 39 were shared between WT and

Atg16L1HM mice (Zones C and F in Venn diagram, Figure 3).

Of these, 27 exhibited expression changes of similar magnitude

and in the same direction in WT and Atg16L1HM mice (Figure S3).


D 0 D 1 D 2 D 30

20

40

60

80

100

W T + MN V C W 3H M + MN V C W 3

% P

anet

h C

ell

WT + MNV CW3

HM + MNV CW3

Lysozyme Distribution Day 7 Post InfectionA B Figure 2. Induction of Paneth Cell Abnor-

malities Is Virus Strain Specific

(A) Light microscopy of ileal sections stained with

PAS-Alcian blue of WT and Atg16L1HM mice

7 days postinfection with 3 3 107 pfu of MNV

CW3 (n = 6 mice/genotype). Dotted line denotes

crypt unit and arrowheads indicate Paneth cell

granules. Scale bar represents 10 mm.

(B) Percent Paneth cells displaying abnormal

lysozyme distribution from WT and Atg16L1HM

mice in (A) (n > 6100 cells from 3 mice/condition,

mean ± SEM).

See also Figure S2.

We next identified gene expression changes attributable to

virus-plus-susceptibility gene interaction employing a factorial

design approach incorporating ‘‘differential-of-differential’’ anal-

ysis that examined factor combinations of the two experimental

dimensions (infection states and genotypes). Seven hundred

fifty-nine genes were altered differentially between the two geno-

types as a result of MNV CR6 infection (Zones A–D in Venn

diagram, Figure 3). Enrichment analysis of these genes identified

a significant overrepresentation of the biological processes of

intracellular protein traffic, protein targeting and localization,

and amino acid metabolism (Figure 3). These results are consis-

tent with autophagy deficiency and Paneth cell ER and granule

packaging abnormalities observed in virally-infected Atg16L1HM

mice (Figure 1 and Figures S1B and S1C). We also identified tran-

scripts that were not significantly altered in response to infection

within a genotype, but displayed significant expression differ-

ences between genotypes (Zone A in Venn diagram, Figure 3).

These transcripts were enriched for those encoding transporters

and ligases, particularly those involved in processing ubiquitin.

Strikingly, when comparing the direction of gene expression

changes between WT and Atg16L1HM Paneth cells, entire sets

of genes were altered in opposite directions (Figure 3). For

example, changes in the expression of genes encoding ligases

or proteins associated with amino acid metabolism are regulated

in different directions in WT versus Atg16L1HM mice after MNV

CR6 infection. In some functional groups (intracellular protein

traffic and protein targeting and localization), changes in gene

expression are reciprocal with gene sets that increase in one

genotype decreasing in the other. Therefore, the virus-plus-

susceptibility gene interaction changes the fundamental nature,

and not just the extent, of transcriptional responses during

infection.

To determine if the transcriptional profile of Paneth cells from

Atg16L1HM versus WT mice was unique to MNV CR6 infection,

we defined the transcriptional profile of Paneth cells in mice

infected with MNV CW3 7 days postinfection. Infection with

MNV CW3 led to changes in 2226 transcripts in Atg16L1HM not

altered in WT mice (Figure S4). Of these, 2181 were unique to

MNV CW3 compared to MNV CR6 infection. Enrichment anal-


yses of the MNV CR6 and CW3 data sets supported the virus

strain-specific nature of this effect; there was no overlap in signif-

icantly enriched functional groups (Figure S4).

Virus-Plus-Susceptibility Gene Interaction Generatesan Aberrant Intestinal Injury ResponseSince changes in Paneth cells depended on a virus-plus-

susceptibility gene interaction, intestinal responses to an addi-

tional environmental ‘‘hit’’ might also be influenced by this inter-

action. Administration of dextran sodium sulfate (DSS) induces

intestinal injury. We chose a concentration of DSS (2.5%) that

causes mild pathology and no lethality 7 days posttreatment

in immunocompetent mice. Instead, this treatment induces a

reproducible programmed response in immunocompetent mice

including decreased epithelial cell proliferation, loss of epithelial

integrity, and generation of ulcers in specific colonic regions (Pull

et al., 2005). As expected, uninfected Atg16L1HM and both unin-

fected and MNV CR6-infected WT mice fed DSS developed

a typical response including focal ulcers in the transverse colon

and a single confluent ulcer adjacent to the ano-rectal junction,

whereas the ileum was not ulcerated (Figure 4A). Thus,

Atg16L1 mutation alone does not lead to altered responses to

DSS-induced intestinal injury.

Remarkably, Atg16L1HM mice infected with MNV CR6 for

1 week prior to DSS treatment exhibited aberrant responses to

injury in the colon (Figure 4). In ulcers adjacent to the ano-rectal

junction, infected Atg16L1HM mice unexpectedly exhibited mul-

tiple hallmarks of human Crohn’s disease including increased

inflammation in the muscularis and associated mesenteric fat

and blood vessels, increases in lymphoid aggregates, develop-

ment of subserosal fibrosis, hypertrophy of the muscularis prop-

ria, and hyperplasia of the proximal epithelium (Figure 4). These

aspects of intestinal pathology in our experimental system were

similar to the typical intestinal pathology of Crohn’s disease

patients (Roberts, 2009). Thus, virus-plus-susceptibility gene

interaction resulted in a significantly altered intestinal injury

response.

In contrast to MNV CR6, infection of Atg16L1HM mice with

the nonpersistent strain MNV CW3 did not induce inflammatory

Intra

cellu

lar p

rote

in tr

affic

1 2 3Snap91_17092_zoneDPpig_23052_zoneDCpne1_33947_zoneALmbr1_43122_zoneD3000004C01Rik_10080_zoneATubb3_10330_zoneAKif17_41701_zoneACyth4_12160_zoneBErc2_2621_zoneBStx4a_6372_zoneBVamp2_29950_zoneBAp2s1_34094_zoneBLamp2_2924_zoneBGosr2_1633_zoneBDock5_9683_zoneBScamp1_1924_zoneBCopz2_5871_zoneBMcfd2_26464_zoneBSnap23_10667_zoneAArf4_38889_zoneARab3a_661_zoneAPex11b_31481_zoneBTomm20_42034_zoneBWdr60_41476_zoneDMyo1b_37591_zoneDIpo7_5873_zoneASec31a_40230_zoneBArcn1_24886_zoneBArf3_1870_zoneAStx8_16034_zoneDSdcbp2_36639_zoneDRab14_13403_zoneDTubd1_25186_zoneDTmed7_4212_zoneDHook1_40583_zoneACtsa_29002_zoneASec61a1_23622_zoneADnajc5_6647_zoneASec23a_18961_zoneARab6b_39087_zoneAPtger4_6408_zoneBAp3m1_9279_zoneASurf2_17773_zoneBRabgef1_15202_zoneAClip1_37122_zoneBCltc_3384_zoneARab19_5386_zoneBCpne2_20265_zoneBSec61g_19552_zoneB

Probe_spotID_zone

WT infected

vs. uninfected

Atg16L1HM infected

vs. uninfected

‘Differential-of-differential’

Union of A,B,C,D

E (955) G (298)

A (374)

B (273) D (100)

C

(12)

F(27)

1 2

3

p < 0.05FC > 1.5x

1

1.5

2

2.5

nt sco

re, -L

og

(p

-valu

e)

Ligase (p=0.0086)

Transporter (p=0.047)

0

0.5

0 10 20 30 40 50

En

rich

men

Occurrence per Molecular Function category

p=0.05

Zone A1

1.5

2

2.5

3

nt s

co

re

, -L

og

(p

-v

alu

e)

Intracellular protein traffic (p=0.001)

Protein targeting & localization (p=0.038)Amino acid metabolism (p=0.044)

Carbohydrate metabolism (p=0.059)

0

0.5

0 20 40 60 80 100 120

En

ric

hm

en

Occurrence per Biological Process category

p=0.05

Liga

se

1 2 3Ankib1_41135_zoneAAss1_978_zoneAUbr3_17459_zoneATrim39_15653_zoneAMycbp2_13928_zoneARnf123_13432_zoneATrim13_3125_zoneAUbe2q2_5337_zoneAHuwe1_21857_zoneAUbe2z_38602_zoneASiah1b_15315_zoneALig3_20714_zoneAUbe3c_39050_zoneAFbxo4_34865_zoneA

Tran

spor

ter

Atp6v0a1_28251_zoneASlc4a10_17149_zoneAAbcg2_39085_zoneASlc35b1_36995_zoneARhbg_1639_zoneAReep3_7406_zoneASec61a1_23622_zoneASlc1a2_7267_zoneAFolr2_15224_zoneAAtp4a_14862_zoneASlc7a6_753_zoneASlc5a9_36251_zoneASlc34a2_36459_zoneATmem184b_791_zoneAIpo7_5873_zoneAAtp2a2_18361_zoneAAtp6v0c_24448_zoneA

Pro

tein

targ

etin

g&

loca

lizat

ion

Am

ino

acid

met

abol

ism

Car

bohy

drat

e m

etab

olis

m

Asns_591_zoneBSlc1a4_41108_zoneBSlc6a9_34570_zoneBAgxt2l1_38213_zoneCSlc7a5_34885_zoneDFasn_21615_zoneASlc7a6_753_zoneAAss1_978_zoneACth_1768_zoneASlc1a2_7267_zoneAGgt5_30965_zoneACtbp2_28562_zoneAFahd1_29999_zoneB

Fut2_30924_zoneDSorbs1_21810_zoneDPfkfb4_7123_zoneBPiga_818_zoneAHk2_26260_zoneASlc35b1_36995_zoneAPrkab2_35518_zoneCGbe1_16867_zoneASlc5a9_36251_zoneAPpp3cb_513_zoneBExtl2_88_zoneBReep3_7406_zoneAMdh2_2714_zoneAGyk_35186_zoneAEdem2_219_zoneAGalnt3_39270_zoneADlat_10210_zoneAPgm3_25259_zoneBNeu1_23174_zoneBAdh4_36408_zoneDSt8sia5_29142_zoneBMan2b1_19011_zoneAChi3l1_25223_zoneAGalnt13_22054_zoneAUgt2a1_25041_zoneAFoxq1_15988_zoneA

Stx4a_6372_zoneBIft57_24755_zoneBMpdz_40040_zoneAKif17_41701_zoneA3000004C01Rik_10080_zoneARrbp1_15922_zoneBZyx_6769_zoneBVps13c_11265_zoneBAkap8_38730_zoneBSurf2_17773_zoneBClip1_37122_zoneBSec61g_19552_zoneB

-4 1:1 4

Fold-change

1 2 3 Probe_spotID_zone

Probe_spotID_zone

Figure 3. MNV Infection Induces a Distinct Transcriptional Response in Paneth Cells from Atg16L1 Mutant Mice

Factorial design analysis was performed to identify gene sets showing differential gene expression in Paneth cells from WT and Atg16L1HM mice with or without

MNV CR6 infection. Venn diagram displays the subsets of genes found to be differentially expressed (p < 0.05 as defined using a linear model and exhibiting

a fold-change > 1.5) in the respective comparisons as indicated (circles 1 to 3). The ‘‘differential-of-differential’’ analysis in the factorial design identified genes

that respond differently to infection in Atg16L1HM mice compared to WT mice (Zones A, B, C, and D bounded by the red circle). These were found to be enriched

for genes associated with intracellular protein traffic, protein targeting and localization, and amino acid metabolism. Also noteworthy is the sizable representation

of genes associated with carbohydrate metabolism. The expression profiles for genes in these biological process categories are displayed as Log2-transformed

fold-changes in the heat maps. The ‘‘differential-of-differential’’ analysis also identified genes that were not significantly altered in response to infection when

compared within each genotype but exhibited significant differences when comparing responses to infection between genotypes as highlighted in Zone A. Genes

with transporter functions or those with ligase activity were significantly enriched in this subset. See also Figures S3 and S4.

hallmarks of Crohn’s disease after DSS treatment (Figures 4C

and 4D). Further, Atg16L1HM mice receiving MNV CR6 and

DSS concurrently did not develop inflammatory hallmarks of

Crohn’s disease and were indistinguishable from similarly

treated WT mice (Figures 4C and 4D). In contrast, Atg16L1HM

mice infected with MNV CR6 for 28 days before DSS treatment

developed similar inflammatory pathologies to those seen 7

days after infection (data not shown). These data are consistent

with a role for viral persistence. Taken together, these results

indicate that in the context of DSS treatment, virus strain speci-

ficity and timing of infection are important factors in virus-plus-

susceptibility gene interaction.

Unexpected Ileal Pathology Dependenton Virus-Plus-Susceptibility Gene InteractionAtg16L1HM mice infected with MNV CR6 prior to DSS adminis-

tration displayed ileal pathology not previously reported in

DSS-treated mice. In contrast to WT mice, Atg16L1HM mice

receiving DSS exhibited virus-dependent mucosal atrophy man-

ifested as blunting of villi (Figure 5) similar in degree to that

observed in Crohn’s disease (Roberts, 2009), celiac disease

(Chand and Mihas, 2006), or human norovirus-induced gastro-

enteritis (Agus et al., 1973; Dolin et al., 1975). Villus blunting in

response to DSS was inconsistent in Atg16L1HM mice infected

with the nonpersistent MNV CW3 strain (two out of six mice,


A

B

HM + MNV CR6

WT Uninfected

WT+ MNV CR6

HM Uninfected

C

D

-20

0

20

40

60

80

100

****

*

W T H M W T H M W T H M W T H M

Un in fe cte d M NV C R67 d ays

M NV C W 37 d ays

M NV C R60 d ays

% In

crea

se in

mu

scle

wid

th

0

1

2

3



M NV C W 37 d ays

M NV C R60 d ays

******

***

Lym

ph

oid

ag

gre

gat

es

N.D.

M N V C R 6 for 7 days D S S for 7 days

M N V CW 3 for 7 days D S S for 7 days

M N V C R 6 for 7 days

D S S for 7 daysS am e D ay

D S S for 7 days

In flam m atory H a llm arks o f C D

D S S Treatm ent P rotoco l W T H M

+M N V C R 6 for 7 days D S S for 7 days

M N V CW 3 for 7 days D S S for 7 days

M N V C R 6 for 7 days

D S S for 7 daysS am e D ay

D S S for 7 daysD S S for 7 days

In flam m atory H a llm arks o f C D

D S S Treatm ent P rotoco l W TW T H M

+

H M

+

Figure 4. Atg16L1 Mutant Mice Display

a Virus-Dependent Aberrant Response to

DSS in the Colon

(A) WT and Atg16L1HM mice were orally inoculated

with MNV CR6 for 7 days or uninfected, and

subsequently given 2.5% DSS for an additional

7 days at which point intestines were harvested.

Light microscopy images of H&E stained sections

of ulcerated regions located immediately adjacent

to the ano-rectal junction are shown. Yellow

double-headed arrows indicate the muscularis

propria thickness. In the MNV CR6-infected

Atg16L1HM sample, lymphoid aggregates are

indicated by yellow dashed circles, and the black

dashed region contains submucosal fibrosis and

inflammation. Scale bar represents 500 mm.

(B) Table summarizing the outcome of DSS treat-

ment. All intestines were harvested at the end of

DSS treatment for analysis. + and � refer to the

presence or absence of inflammatory hallmarks

of Crohn’s disease respectively (n R 6 mice/

condition).

(C) Quantification of the muscularis propria thick-

ness in the region adjacent to the ano-rectal junc-

tion from DSS-treated mice. Seven days and

0 days refer to the amount of time mice were inoc-

ulated with the virus prior to DSS treatment.

The increase in muscle thickness was normalized

to the average of uninfected WT mice treated with

DSS (*p < 0.05, ***p < 0.001, mean ± SEM).

(D) Number of lymphoid aggregates in the region

adjacent to the ano-rectal junction from DSS-

treated mice (***p < 0.001, mean ± SEM). N.D.

refers to not detected.

See also Figure S5.

Figure 5B). MNV CR6 infection for 1 week prior to DSS adminis-

tration was required for induction of villus blunting (Figure 5B).

One explanation for aberrant DSS-induced pathologies

observed in virus-infected Atg16L1HM mice would be uncon-

trolled viral replication. However, there were no significant differ-

ences in viral replication in the ileum and colon where aberrant

pathology is observed prior to or after DSS treatment (Figures

S2A and S5). Less MNV CR6 was detected in the mesenteric

lymph nodes from Atg16L1HM compared to WT mice receiving

DSS, indicating that virus-plus-susceptibility gene effects were

not mediated by increased viral replication.

TNFa, IFNg, and Commensal Bacteria MediateIntestinal Injury Responses Dependenton Virus-Plus-Susceptibility Gene InteractionTo identify mediators of DSS-induced pathology dependent on

virus-plus-susceptibility gene interaction, we examined the role

of cytokines. TNFa is a major mediator of inflammation in Crohn’s

disease; administration of blocking antibodies that interfere with


ly

IF

c

(S

D

o

m

to

(F

v

b

D

w

il

g

d

d

TNFa signaling is a powerful therapeutic

approach (Targan et al., 1997). Further-

more, excess TNFa induces villus blunt-

ing as observed in Figure 5 (Piguet et al.,

1999). Upon in vitro stimulation, intestinal

mphocytes from Crohn’s disease patients secrete increased

Ng (interferon-g) (Fuss et al., 1996; Fais et al., 1991), which

an function in concert with TNFa in inflammatory processes

uk et al., 2001; Lake et al., 1994), and IFNg may have a role in

SS-induced intestinal injury (Brem-Exner et al., 2008).

MNV CR6-infected, DSS-treated Atg16L1HM mice given TNFa

r IFNg blocking antibodies displayed dramatically reduced

uscular hypertrophy, fewer lymphoid aggregates adjacent

the ano-rectal junction, and decreased ileal villus blunting

igures 6A–6C). Importantly, only the pathologies specific to

irally-infected Atg16L1HM mice were altered as blocking anti-

odies had no effect on the superficial ulceration typical of

SS treatment present in all conditions. Thus, TNFa and IFNg

ere each necessary for the DSS-induced pathologies in both

eum and colon that were dependent on virus-plus-susceptibility

ene interaction.

Commensal bacteria are important in inflammatory bowel

isease (Sartor, 1997; Kang et al., 2008), and there is prece-

ence for a role of bacteria in disease triggered by viral infection

WT + MNV CR6

Villi

-10

0

10

20

30 *****

**



M NV C W 37 d ays

M NV C R60 d ays

n.s.

% R

edu

ctio

n in

Vill

us

Hei

gh

tHM + MNV CR6

Crypts

Villi

Crypts

A B Figure 5. Virus-Infected Atg16L1 Mutant

Mice Display Mucosal Atrophy in the Ileum

in Response to DSS

(A) Light microscopy of H&E stained ileal sections

from the same mice in Figure 4. Scale bar =

200 mm.

(B) Quantification of average villus height normal-

ized to uninfected WT mice treated with DSS.

Seven days and 0 days refer to the amount of

time mice were inoculated with the virus prior to

DSS treatment (an average of 286 total villi

from at least 5 mice/condition was measured,

**p < 0.01, ***p < 0.001, mean ± SEM).

See also Figure S5.

as in secondary infection with Streptococcus pneumoniae after

influenza (McCullers, 2006). To determine if the intestinal injury

response dependent on virus-plus-susceptibility gene interac-

tion requires commensal bacteria, Atg16L1HM mice were

infected with MNV CR6 and treated with both DSS and broad

spectrum antibiotics. Antibiotics uptake was similar across

groups and did not interfere with the administration of DSS

(Extended Experimental Procedures). Administration of antibi-

otics to virally-infected Atg16L1HM mice prevented abnormal

DSS-induced pathologies in both ileum and colon reported

above (Figures 6D–6F). Thus, aberrant injury responses depen-

dent on virus-plus-susceptibility gene interaction require com-

mensal bacteria.

DISCUSSION

An important goal is to identify specific genetic, infectious, or

environmental factors responsible for a given disease. When

such an etiologic smoking gun is detected, the full power of

medicine, chemistry, and vaccine development can be brought

to bear on the illness. However, if complex diseases are com-

binatorial in etiology, then an individual gene or pathogen

may display only poor association with disease incidence and

severity. This model of complex disease is widely recognized

but experimental evidence has not been reported.

In this study, we define a complex inflammatory disease in

mice that depends on an interaction between a specific enteric

virus strain and a single host disease susceptibility gene. Several

of the pathologic features observed in this model resemble

those in many Crohn’s disease patients. In particular, Paneth

cell structural and granule packaging abnormalities observed

in virus-infected Atg16L1HM mice are remarkably similar to those

in patients homozygous for the risk allele of ATG16L1. Since

Crohn’s disease patients homozygous for the nonrisk allele of

ATG16L1 do not display this pathology, virus-plus-susceptibility

gene interaction may contribute to heterogeneity between

patients. Importantly, Paneth cells from Atg16L1HM mice in-

fected with virus have a fundamentally different gene expression

profile than controls. These results provide a specific example of

how a genetic factor and an environmental agent, each innoc-

uous by itself, can have profound effects on the host when

combined. These results also provide one explanation for why

persons in clinical trials may display widely variable responses

to treatment for a single pathologically defined disease.

Mechanisms of Pathology Induced by Virus-Host

Susceptibility Gene InteractionDespite the importance of autophagy genes in regulating the

intracellular replication of pathogens, Atg16L1 mutant mice did

not display increased MNV replication, virus was not detected

in Paneth cells, and we found no role for Atg16L1 in control

of MNV replication in vitro using primary macrophages from

Atg16L1HM mice (data not shown). In this context, it is interesting

that Paneth cell abnormalities, the transcriptional response to

infection, and DSS-induced pathologies were dependent on

infection with MNV CR6, a strain capable of persistent infection.

Notably, the two strains of MNV used in this study generate sero-

logically indistinguishable antibody responses (Thackray et al.,

2007) despite the dramatic difference in ability to induce disease

in Atg16L1 mutant mice. Additionally, aberrant DSS-induced

pathologies were dependent on the timing of MNV infection.

The relationship between an infectious agent and a complex

disease is typically established through serology or the presence

of a pathogen in infected tissue. These conventional approaches

may dismiss etiologic agents if the disease is dependent on

a specific pathogen strain or the timing of the infection. Detection

of virus-plus-susceptibility gene interactions in humans may

therefore require advances in the capacity to identify viruses

present in tissues in an unbiased way, allowing a better under-

standing of the relationship between the human virome and

disease pathogenesis (Virgin et al., 2009). To be revealing,

such studies will have to be coupled to high power epidemiolog-

ical studies and analysis of when patients were exposed to

environmental factors.

The aberrant intestinal injury response we described in

virally-infected Atg16L1HM mice was dependent on three factors

associated with Crohn’s disease pathogenesis in humans—

TNFa, IFNg, and commensal bacteria. This common role of

two cytokines and commensal bacteria suggest that the human

disease and the observed pathologies in mice share an under-

lying mechanism, and supports the relevance of the virus-

plus-susceptibility gene interaction reported here. Consistent

with the efficacy of TNFa and IFNg blockade in our model, cyto-

kine-mediated injury after viral infection is receiving attention as

an attractive target for therapeutic intervention (Marsolais et al.,

2009; Brockman et al., 2009). There is also evidence for an

intersection between viral infection and commensal bacteria

(Brenchley et al., 2006; Xi et al., 2008). The antibiotic responsive-

ness of the DSS-induced disease in our model reveals an


A

B

D

-20

0

20

40

60

80

100 *

W T H M W T H M

+Antibiotics-Antibiotics

% In

crea

se in

mu

scle

wid

th

0

50

100*

*

WT H M WT H M WT H M

Is o typ e An ti-TN Fα An ti- IF Nγ

% In

crea

se in

mu

scle

wid

th

E

C F

-10

0

10

20

30*

***

W T H M W T H M W T H M

Isotype Anti-T N Fα Anti-IFN γ% R

edu

ctio

n in

Vill

us

Hei

gh

t

-10

0

10

20

30*

W T H M W T H M

-Antibiotics + Antibiotics% R

edu

ctio

n in

Vill

us

Hei

gh

t

0

1

2

3

4

WT H M WT H M WT H M

Is o typ e An ti-TN Fα An ti- IF N γ

******

Lym

ph

oid

ag

gre

gat

es

0

1

2

3

W T H M W T H M

+ Antibiotics-Antibiotics

***

Lym

ph

oid

ag

gre

gat

es

Figure 6. TNFa and IFNg Inhibition or Anti-

biotics Treatment Ameliorates DSS-

Induced Disease in Virus-Infected Atg16L1

Mutant Mice

(A) Isotype control or blocking antibodies against

TNFa and IFNg were administered to WT and

Atg16L1HM mice infected with MNV CR6 for

7 days followed by additional 7 days of DSS treat-

ment at which point intestines were harvested.

The muscularis propria thickness in the region

adjacent to the ano-rectal junction was quantified

and normalized to the average of WT mice

receiving isotype control antibodies (n R 6 mice/

condition, *p < 0.05, mean ± SEM).

(B) Number of lymphoid aggregates in the region

adjacent to the ano-rectal junction from mice in

(A) (*p < 0.001, mean ± SEM).

(C) Quantification of average villus height from

mice in (A) normalized to the average of WT mice

receiving isotype control antibodies (an average

of 263 total villi from at least 5 mice/condition

was measured, *p < 0.05, ***p < 0.001, mean ±

SEM).

(D) WT and Atg16L1HM mice were orally inoculated

with MNV CR6 for 7 days and then given 2.5%

DSS and broad spectrum antibiotics concurrently

for another 7 days at which point intestines were

harvested. The muscularis propria thickness was

normalized to the average of uninfected WT mice

treated with DSS from Figure 4D (n = 6 mice/

condition, *p < 0.05, mean ± SEM).

(E) Number of lymphoid aggregates from mice in

(D) (***p < 0.001, mean ± SEM).

(F) Quantification of average villus height from

mice in (D) normalized to the average of uninfected

WT mice treated with DSS from Figure 5B (an

average of 320 total villi from at least 5 mice/condi-

tion was measured, *p < 0.05, mean ± SEM).

See also Figure S5.

important bacterial component to the virus-plus-susceptibility

gene interaction that may be exploited therapeutically.

The gene expression and morphological changes in Paneth

cells we report are consistent with an important role of

Atg16L1 in the secretory pathway and the response of epithelial

cells to infection. These functions of Atg16L1 may be related to

the membrane trafficking component of autophagy. Consistent

with a cell autonomous role of Atg16L1 in Paneth cells, intestinal

epithelium-specific deletion of two other autophagy genes also

leads to Paneth cell granule defects (Cadwell et al., 2008a;

Cadwell et al., 2008b). Since Atg16L1 and its binding partner

Atg5 can limit cytokine production (Saitoh et al., 2008; Jounai

et al., 2007; Tal et al., 2009), viral infection may trigger an altered

cytokine response in Atg16L1 mutant mice. Although the

changes we see are in the epithelium, the complex inflammatory

phenotype reported here may also depend on Atg16L1 function

in other cell types. For example, the necessity of autophagy

genes for T-cell function (Pua et al., 2007; Nedjic et al., 2008)

may be relevant given the role of T cells in colitis after adoptive


transfer into Rag2-deficient mice (Barnes and Powrie, 2009);

the role of MNV has not been explored in that model.

It is tempting to speculate that Paneth cell abnormalities

contribute to aberrant DSS-induced pathologies. Local pro-

duction of inflammatory molecules by Paneth cells may drive

villus blunting in the ileum. Moreover, virally-infected Atg16L1HM

mice can have alterations in commensal bacteria since mice

with Paneth cells that have lost a-defensin activation develop

intestinal dysbiosis including the emergence of segmented fila-

mentous bacteria (Salzman et al., 2010) that stimulate the devel-

opment of Th17 lymphocytes (Ivanov et al., 2009). Paneth cells

are not present in the mouse colon where viral infection alters

injury responses in Atg16L1 mutant mice. However, the effects

of Paneth cells need not be restricted to the ileum since active

peptides from their granules can be detected in the distal colonic

lumen (Mastroianni and Ouellette, 2009). Since it is unclear how

diverse pathologies are related to one another in Crohn’s disease

patients, it will be important to address the relationship between

Paneth cells, the microbiome, and the virome in the context

of the aberrant injury response dependent on the virus-plus-

susceptibility gene interaction in our model.

Disease Penetrance in Mouse Modelsof Mucosal ImmunityBased on our observations in Atg16L1 mutant mice, we specu-

late that other traits attributed to specific mutations are also

dependent on viral infections. The presence of an infectious

agent can lead to different experimental outcomes between

laboratories. For example, experimental allergic encephalomy-

elitis correlates with mouse facility-specific pathogen exposure

(Goverman et al., 1993). Experimental discrepancies between

facilities are particularly germane for models of intestinal disease

since MNV is an enteric virus commonly detected in specific

pathogen free facilities (Hsu et al., 2005; Pritchett-Corning

et al., 2009; Goto et al., 2009).

The effect of viral infection can be remarkably specific. In our

model, the MNV CR6 strain altered experimental outcomes only

in Atg16L1HM mice. MNV CR6 infection does not alter immune

responses in C57/BL6 mice challenged with Friend leukemia

virus, influenza, vaccinia, or MCMV (Ammann et al., 2009; Hens-

ley et al., 2009; Doom et al., 2009). In contrast, MNV infection of

Mdra1�/�mice enhances immune responses to Helicobacter bilis

infection (Lencioni et al., 2008). Thus, the effect that a particular

pathogen has on the host cannot be generalized. Although we

focus on a single virus and gene, multiple interactions may exist

between different susceptibility genes and an array of commen-

sals and pathogens. Much remains to be learned about the inter-

actions between susceptibility genes and specific pathogens to

understand what may be a general ‘‘microbe plus susceptibility

gene’’ contribution to a range of complex diseases.

Understanding the Contributions of Geneticand Environmental Factors in Complex DiseasesEpidemiological studies have not identified a specific infectious

cause for inflammatory bowel disease. Reported associations

between disease risk and infectious gastroenteritis are correla-

tive (Porter et al., 2008; Garcia Rodriguez et al., 2006; Gradel

et al., 2009), but are of interest given our data since human

noroviruses related to MNV cause human gastroenteritis (Mead

et al., 1999). Since many bacteria and viruses cause gastroenter-

itis, the infectious trigger of a complex disease need not be

a single specific agent. Rather several agents that affect similar

immunologic pathways can be involved. Interestingly, the

Crohn’s disease associated gene NOD2 may recognize viral

RNA in addition to bacterial peptidoglycan (Sabbah et al.,

2009; Shapira et al., 2009), raising the possibility that a viral infec-

tion can interact with multiple susceptibility genes.

Although Atg16L1 mutant mice do not display all pathology

found in Crohn’s disease patients, we have reproduced several

disease hallmarks in a mouse with a mutation in a single suscep-

tibility gene. It is commonly assumed that the failure to recreate

the entirety of Crohn’s disease in various mouse models is due

to inherent differences between humans and mice. However, in

the combinatorial view of complex disease supported by our

results, reproducing full disease may require combinations of

specific alleles of multiple genes with certain environmental

agents. It is worth noting that not all Crohn’s disease patients

exhibit identical symptoms or pathologies, and the nature of

Crohn’s disease varies over time even within one individual.

In addition, therapeutic interventions that improve conditions for

some do not always alleviate disease in others. Therefore, com-

plex diseases may represent a combinatorial confluence of path-

ologic responses, each with overlapping but nonidentical genetic

and environmental causes and therefore therapeutic responses.

This paradigm has profound implications for how we view

the relationship between genetic heterogeneity and phenotype

in humans. We propose that studies examining associations

between disease susceptibility and genetic variation should

consider the history and current status of viral infections in the

individuals. Similarly, studies examining the correlation between

viral infections and disease would benefit from sorting individ-

uals based on genetic background. If we can improve our knowl-

edge in this area, the concept of personalized medicines may

become closer to clinical application.

EXPERIMENTAL PROCEDURES

Mice

Atg16L1HM mice were described (Cadwell et al., 2008a). The Atg16L1HM line

housed in a conventional barrier facility was rederived by embryo transfer

into the enhanced facility with the following specialized features: all cages,

bedding, chow, and water are autoclaved prior to use; access to breeding

mice in this facility is restricted to specially trained personnel; and sentinel

mice are routinely screened for common mouse pathogens including MNV.

Mice were generated by mating heterozygotes for the gene trap mutagenized

Atg16L1 locus. Progeny homozygous for the mutation and wild-type litter-

mates aged 7–15 weeks were used in experiments.

Viruses

Generation of concentrated stocks was described (Chachu et al., 2008).

Twenty-five microliters of concentrated stocks (3 3 107 pfu) of MNV CR6

and CW3 was orally inoculated into mice. For virus quantification, total RNA

was harvested per one stool pellet, 1 cm of intestine, or total mesenteric lymph

nodes. qRT-PCR for MNV genome copy numbers was as described (Thackray

et al., 2007). For detection of virus in Paneth cells, �300 crypts from three

mice/genotype were procured by laser capture microdissection (Extended

Experimental Procedures) 7 days postinfection with MNV CR6. As a positive

control, MNV was detected in whole gut RNA from similarly prepared tissue.

Statistical Analysis

Lysozyme distribution, muscle thickness, lymphoid aggregates, and villus

height were analyzed by two-tailed unpaired t tests. Viral genome copies

were analyzed with the nonparametric Mann-Whitney test. Analyses except

for microarray data used GraphPad Prism (version 5.00).

ACCESSION NUMBERS

Microarray files have been deposited in Array Express with accession codes

E-TABM-957 and E-TABM-958.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures and

five figures and can be found with this article online at doi:10.1016/j.cell.

2010.05.009.

ACKNOWLEDGMENTS

This research was supported by grant U54 AI057160 Project 5 and the Broad

Foundation (K.C., N.M., and H.W.V.), R01 AI084887 (T.S.S. and H.W.V.), the

Lallage Feazel Wall Fellowship DRG-1972-08 from the Damon Runyon Cancer


http://dx.doi.org/doi:10.1016/j.cell.2010.05.009

http://dx.doi.org/doi:10.1016/j.cell.2010.05.009

Research Foundation (K.C.), training grant NIH T32-AI007172 (N.M.), the Pew

Foundation (K.K.P. and T.S.S.), Washington University Digestive Diseases

Research Core Center DK52574, Pfizer biomedical agreement with Washing-

ton University, fellowship award from the Crohn’s and Colitis Foundation of

America (A.C.Y.N), and grants DK83756, DK086502, and DK043351 (R.J.X.).

Washington University holds U.S. patents 7,041,444 B2, 7,264,923, and US

7,455,972 related to growth and detection of MNV. Washington University

and H.W.V. receive income based on licenses for MNV technology.

Received: December 9, 2009

Revised: February 26, 2010

Accepted: April 19, 2010

Published: June 24, 2010

REFERENCES

Agus, S.G., Dolin, R., Wyatt, R.G., Tousimis, A.J., and Northrup, R.S. (1973).

Acute infectious nonbacterial gastroenteritis: intestinal histopathology. Histo-

logic and enzymatic alterations during illness produced by the Norwalk agent

in man. Ann. Intern. Med. 79, 18–25.

Altshuler, D., Daly, M.J., and Lander, E.S. (2008). Genetic mapping in human

disease 4. Science 322, 881–888.

Ammann, C.G., Messer, R.J., Varvel, K., Debuysscher, B.L., Lacasse, R.A.,

Pinto, A.K., and Hasenkrug, K.J. (2009). Effects from acute and chronic murine

norovirus infections on immune responses and recovery from Friend retrovirus

infection. J Virol. 83, 13037–13041.

Barnes, M.J., and Powrie, F. (2009). Regulatory T cells reinforce intestinal

homeostasis. Immunity 31, 401–411.

Barrett, J.C., Hansoul, S., Nicolae, D.L., Cho, J.H., Duerr, R.H., Rioux, J.D.,

Brant, S.R., Silverberg, M.S., Taylor, K.D., Barmada, M.M., et al. (2008).

Genome-wide association defines more than 30 distinct susceptibility loci

for Crohn’s disease. Nat. Genet. 40, 955–962.

Brem-Exner, B.G., Sattler, C., Hutchinson, J.A., Koehl, G.E., Kronenberg, K.,

Farkas, S., Inoue, S., Blank, C., Knechtle, S.J., Schlitt, H.J., et al. (2008).

Macrophages driven to a novel state of activation have anti-inflammatory

properties in mice. J. Immunol. 180, 335–349.

Brenchley, J.M., Price, D.A., Schacker, T.W., Asher, T.E., Silvestri, G., Rao, S.,

Kazzaz, Z., Bornstein, E., Lambotte, O., Altmann, D., et al. (2006). Microbial

translocation is a cause of systemic immune activation in chronic HIV infection.

Nat. Med. 12, 1365–1371.

Brockman, M.A., Kwon, D.S., Tighe, D.P., Pavlik, D.F., Rosato, P.C., Sela, J.,

Porichis, F., Le, G.S., Waring, M.T., Moss, K., et al. (2009). IL-10 is up-regu-

lated in multiple cell types during viremic HIV infection and reversibly inhibits

virus-specific T cells. Blood 114, 346–356.

Cadwell, K., Liu, J.Y., Brown, S.L., Miyoshi, H., Loh, J., Lennerz, J.K., Kishi, C.,

Kc, W., Carrero, J.A., Hunt, S., et al. (2008a). A key role for autophagy and the

autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature

456, 259–263.

Cadwell, K., Patel, K.K., Komatsu, M., Virgin, H.W., and Stappenbeck, T.S.

(2008b). A common role for Atg16L1, Atg5, and Atg7 in small intestinal Paneth

cells and Crohn’s disease. Autophagy 5, 250–252.

Chachu, K.A., LoBue, A.D., Strong, D.W., Baric, R.S., and Virgin, H.W. (2008).

Immune mechanisms responsible for vaccination against and clearance of

mucosal and lymphatic norovirus infection. PLoS Pathog. 4, e1000236.

Chand, N., and Mihas, A.A. (2006). Celiac disease: current concepts in diag-

nosis and treatment. J. Clin. Gastroenterol. 40, 3–14.

Day, D.W., Morson, B.C., and Williams, G.T. (2003). Morson and Dawson’s

Gastrointestinal Pathology (Malden, MA: Blackwell Publishing).

Dolin, R., Levy, A.G., Wyatt, R.G., Thornhill, T.S., and Gardner, J.D. (1975).

Viral gastroenteritis induced by the Hawaii agent. Jejunal histopathology and

serologic response. Am. J. Med. 59, 761–768.

Doom, C.M., Turula, H.M., and Hill, A.B. (2009). Investigation of the impact of

the common animal facility contaminant murine norovirus on experimental

murine cytomegalovirus infection. Virology 392, 153–161.


Dyrberg, T., Schwimmbeck, P.L., and Oldstone, M.B. (1988). Inhibition of

diabetes in BB rats by virus infection. J. Clin. Invest. 81, 928–931.

Fais, S., Capobianchi, M.R., Pallone, F., Di, M.P., Boirivant, M., Dianzani, F.,

and Torsoli, A. (1991). Spontaneous release of interferon gamma by intestinal

lamina propria lymphocytes in Crohn’s disease. Kinetics of in vitro response to

interferon gamma inducers. Gut 32, 403–407.

Fuss, I.J., Neurath, M., Boirivant, M., Klein, J.S., de La, M.C., Strong, S.A.,

Fiocchi, C., and Strober, W. (1996). Disparate CD4+ lamina propria (LP)

lymphokine secretion profiles in inflammatory bowel disease. Crohn’s disease

LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis

LP cells manifest increased secretion of IL-5. J. Immunol. 157, 1261–1270.

Garcia Rodriguez, L.A., Ruigomez, A., and Panes, J. (2006). Acute gastroen-

teritis is followed by an increased risk of inflammatory bowel disease. Gastro-

enterology 130, 1588–1594.

Goto, K., Hayashimoto, N., Yasuda, M., Ishida, T., Kameda, S., Takakura, A.,

and Itoh, T. (2009). Molecular detection of murine norovirus from experimen-

tally and spontaneously infected mice. Exp. Anim. 58, 135–140.

Goverman, J., Woods, A., Larson, L., Weiner, L.P., Hood, L., and Zaller, D.M.

(1993). Transgenic mice that express a myelin basic protein-specific T cell

receptor develop spontaneous autoimmunity. Cell 72, 551–560.

Gradel, K.O., Nielsen, H.L., Schonheyder, H.C., Ejlertsen, T., Kristensen, B.,

and Nielsen, H. (2009). Increased short- and long-term risk of inflammatory

bowel disease after salmonella or campylobacter gastroenteritis. Gastroenter-

ology 137, 495–501.

Hampe, J., Franke, A., Rosenstiel, P., Till, A., Teuber, M., Huse, K., Albrecht,

M., Mayr, G., De La Vega, F.M., Briggs, J., et al. (2007). A genome-wide asso-

ciation scan of nonsynonymous SNPs identifies a susceptibility variant for

Crohn disease in ATG16L1. Nat. Genet. 39, 207–211.

Hensley, S.E., Pinto, A.K., Hickman, H.D., Kastenmayer, R.J., Bennink, J.R.,

Virgin, H.W., and Yewdell, J.W. (2009). Murine norovirus infection has no

significant effect on adaptive immunity to vaccinia virus or influenza A virus.

J. Virol. 83, 7357–7360.

Hsu, C.C., Wobus, C.E., Steffen, E.K., Riley, L.K., and Livingston, R.S. (2005).

Development of a microsphere-based serologic multiplexed fluorescent

immunoassay and a reverse transcriptase PCR assay to detect murine norovi-

rus 1 infection in mice. Clin. Diagn. Lab. Immunol. 12, 1145–1151.

Ivanov, I.I., Atarashi, K., Manel, N., Brodie, E.L., Shima, T., Karaoz, U., Wei, D.,

Goldfarb, K.C., Santee, C.A., Lynch, S.V., et al. (2009). Induction of intestinal

Th17 cells by segmented filamentous bacteria. Cell 139, 485–498.

Jounai, N., Takeshita, F., Kobiyama, K., Sawano, A., Miyawaki, A., Xin, K.Q.,

Ishii, K.J., Kawai, T., Akira, S., Suzuki, K., and Okuda, K. (2007). The Atg5

Atg12 conjugate associates with innate antiviral immune responses. Proc.

Natl. Acad. Sci. USA 104, 14050–14055.

Ju, J.S., Miller, S.E., Jackson, E., Cadwell, K., Piwnica-Worms, D., and Weihl,

C.C. (2009). Quantitation of selective autophagic protein aggregate degrada-

tion in vitro and in vivo using luciferase reporters. Autophagy 5, 511–519.

Kang, S.S., Bloom, S.M., Norian, L.A., Geske, M.J., Flavell, R.A., Stappen-

beck, T.S., and Allen, P.M. (2008). An antibiotic-responsive mouse model of

fulminant ulcerative colitis. PLoS Med. 5, e41.

Karst, S.M., Wobus, C.E., Lay, M., Davidson, J., and Virgin, H.W. (2003).STAT1-

dependent innate immunity to a Norwalk-like virus. Science 299, 1575–1578.

Lake, F.R., Noble, P.W., Henson, P.M., and Riches, D.W. (1994). Functional

switching of macrophage responses to tumor necrosis factor-alpha (TNF

alpha) by interferons. Implications for the pleiotropic activities of TNF alpha.

J. Clin. Invest. 93, 1661–1669.

Lencioni, K.C., Seamons, A., Treuting, P.M., Maggio-Price, L., and Brabb, T.

(2008). Murine norovirus: an intercurrent variable in a mouse model of

bacteria-induced inflammatory bowel disease. Comp. Med. 58, 522–533.

Marsolais, D., Hahm, B., Walsh, K.B., Edelmann, K.H., McGavern, D., Hatta,

Y., Kawaoka, Y., Rosen, H., and Oldstone, M.B. (2009). A critical role for the

sphingosine analog AAL-R in dampening the cytokine response during influ-

enza virus infection. Proc. Natl. Acad. Sci. USA 106, 1560–1565.

Mastroianni, J.R., and Ouellette, A.J. (2009). Alpha-defensins in enteric innate

immunity: functional Paneth cell alpha-defensins in mouse colonic lumen.

J. Biol. Chem. 284, 27848–27856.

McCullers, J.A. (2006). Insights into the interaction between influenza virus and

pneumococcus. Clin. Microbiol. Rev. 19, 571–582.

Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C.,

Griffin, P.M., and Tauxe, R.V. (1999). Food-related illness and death in the

United States. Emerg. Infect. Dis. 5, 607–625.

Mi, H., Guo, N., Kejariwal, A., and Thomas, P.D. (2007). PANTHER version 6:

protein sequence and function evolution data with expanded representation

of biological pathways. Nucleic Acids Res. 35, D247–D252.

Mumphrey, S.M., Changotra, H., Moore, T.N., Heimann-Nichols, E.R., Wobus,

C.E., Reilly, M.J., Moghadamfalahi, M., Shukla, D., and Karst, S.M. (2007).

Murine norovirus 1 infection is associated with histopathological changes in

immunocompetent hosts, but clinical disease is prevented by STAT1-depen-

dent interferon responses. J. Virol. 81, 3251–3263.

Nedjic, J., Aichinger, M., Emmerich, J., Mizushima, N., and Klein, L. (2008).

Autophagy in thymic epithelium shapes the T-cell repertoire and is essential

for tolerance. Nature 455, 396–400.

Oldstone, M.B. (1988). Prevention of type I diabetes in nonobese diabetic mice

by virus infection. Science 239, 500–502.

Ouellette, A.J. (2006). Paneth cell alpha-defensin synthesis and function.

Curr. Top. Microbiol. Immunol. 306, 1–25.

Packey, C.D., and Sartor, R.B. (2009). Commensal bacteria, traditional and

opportunistic pathogens, dysbiosis and bacterial killing in inflammatory bowel

diseases. Curr. Opin. Infect. Dis. 22, 292–301.

Piguet, P.F., Vesin, C., Donati, Y., and Barazzone, C. (1999). TNF-induced

enterocyte apoptosis and detachment in mice: induction of caspases and

prevention by a caspase inhibitor, ZVAD-fmk. Lab. Invest. 79, 495–500.

Porter, C.K., Tribble, D.R., Aliaga, P.A., Halvorson, H.A., and Riddle, M.S.

(2008). Infectious gastroenteritis and risk of developing inflammatory bowel

disease. Gastroenterology 135, 781–786.

Porter, E.M., Bevins, C.L., Ghosh, D., and Ganz, T. (2002). The multifaceted

Paneth cell. Cell. Mol. Life Sci. 59, 156–170.

Pritchett-Corning, K.R., Cosentino, J., and Clifford, C.B. (2009). Contemporary

prevalence of infectious agents in laboratory mice and rats. Lab. Anim. 43,

165–173.

Pua, H.H., Dzhagalov, I., Chuck, M., Mizushima, N., and He, Y.W. (2007). A crit-

ical role for the autophagy gene Atg5 in T cell survival and proliferation. J. Exp.

Med. 204, 25–31.

Pull, S.L., Doherty, J.M., Mills, J.C., Gordon, J.I., and Stappenbeck, T.S.

(2005). Activated macrophages are an adaptive element of the colonic epithe-

lial progenitor niche necessary for regenerative responses to injury. Proc. Natl.

Acad. Sci. USA 102, 99–104.

Rioux, J.D., Xavier, R.J., Taylor, K.D., Silverberg, M.S., Goyette, P., Huett, A.,

Green, T., Kuballa, P., Barmada, M.M., Datta, L.W., et al. (2007). Genome-wide

association study identifies new susceptibility loci for Crohn disease and impli-

cates autophagy in disease pathogenesis. Nat. Genet. 39, 596–604.

Roberts, M.E. (2009). Inflammatory disorders of the small intestine. In Surgical

Pathology of the GI Tract, Liver, Biliary Tract and Pancreas, R.D. Odze and J.R.

Goldblum, eds. (Philadelphia, PA: Saunders Elsevier).

Sabbah, A., Chang, T.H., Harnack, R., Frohlich, V., Tominaga, K., Dube, P.H.,

Xiang, Y., and Bose, S. (2009). Activation of innate immune antiviral responses

by Nod2. Nat. Immunol. 10, 1073–1080.

Saitoh, T., Fujita, N., Jang, M.H., Uematsu, S., Yang, B.G., Satoh, T., Omori, H.,

Noda, T., Yamamoto, N., Komatsu, M., et al. (2008). Loss of the autophagy

protein Atg16L1 enhances endotoxin-induced IL-1beta production. Nature

456, 264–268.

Salzman, N.H., Hung, K., Haribhai, D., Chu, H., Karlsson-Sjoberg, J., Amir, E.,

Teggatz, P., Barman, M., Hayward, M., Eastwood, D., et al. (2010). Enteric

defensins are essential regulators of intestinal microbial ecology. Nat. Immu-

nol. 11, 76–83.

Sartor, R.B. (1997). The influence of normal microbial flora on the development

of chronic mucosal inflammation. Res. Immunol. 148, 567–576.

Scholtens, D., Miron, A., Merchant, F.M., Miller, A., Miron, P.L., Iglehart, J.D.,

and Gentleman, R. (2004). Analyzing factorial designed microarray experi-

ments. J. Multivariate Anal. 90, 19–43.

Schreiber, R.D., Hicks, L.J., Celada, A., Buchmeier, N.A., and Gray, P.W.

(1985). Monoclonal antibodies to murine gamma interferon which differentially

modulate macrophage activation and antiviral activity. J. Immunol. 134,

1609–1618.

Shapira, S.D., Gat-Viks, I., Shum, B.O., Dricot, A., de Grace, M.M., Wu, L.,

Gupta, P.B., Hao, T., Silver, S.J., Root, D.E., et al. (2009). A physical and regu-

latory map of host-influenza interactions reveals pathways in H1N1 infection.

Cell 139, 1255–1267.

Sheehan, K.C., Ruddle, N.H., and Schreiber, R.D. (1989). Generation and char-

acterization of hamster monoclonal antibodies that neutralize murine tumor

necrosis factors. J. Immunol. 142, 3884–3893.

Smyth, G.K. (2005). Limma: linear models for microarray data. In Bioinfor-

matics and Computational Biology Solutions Using R and Bioconductor, R.

Gentleman, V. Carey, S. Dudoit, R. Irizarry, and W. Huber, eds. (New York:

Springer).

Stappenbeck, T.S., Mills, J.C., and Gordon, J.I. (2003). Molecular features of

adult mouse small intestinal epithelial progenitors. Proc. Natl. Acad. Sci.

USA 100, 1004–1009.

Suk, K., Kim, S., Kim, Y.H., Kim, K.A., Chang, I., Yagita, H., Shong, M., and Lee,

M.S. (2001). IFN-gamma/TNF-alpha synergism as the final effector in autoim-

mune diabetes: a key role for STAT1/IFN regulatory factor-1 pathway in

pancreatic beta cell death. J. Immunol. 166, 4481–4489.

Tal, M.C., Sasai, M., Lee, H.K., Yordy, B., Shadel, G.S., and Iwasaki, A. (2009).

Absence of autophagy results in reactive oxygen species-dependent amplifi-

cation of RLR signaling. Proc. Natl. Acad. Sci. USA 106, 2770–2775.

Targan, S.R., Hanauer, S.B., Van Deventer, S.J., Mayer, L., Present, D.H.,

Braakman, T., DeWoody, K.L., Schaible, T.F., and Rutgeerts, P.J. (1997).

A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis

factor alpha for Crohn’s disease. Crohn’s Disease cA2 Study Group.

N. Engl. J. Med. 337, 1029–1035.

Thackray, L.B., Wobus, C.E., Chachu, K.A., Liu, B., Alegre, E.R., Henderson,

K.S., Kelley, S.T., and Virgin, H.W. (2007). Murine noroviruses comprising

a single genogroup exhibit biological diversity despite limited sequence diver-

gence. J. Virol. 81, 10460–10473.

The Wellcome Trust Case Control Consortium. (2007). Genome-wide associ-

ation study of 14,000 cases of seven common diseases and 3,000 shared

controls. Nature 447, 661–678.

Tonietti, G., Oldstone, M.B., and Dixon, F.J. (1970). The effect of induced

chronic viral infections on the immunologic diseases of New Zealand mice.

J. Exp. Med. 132, 89–109.

Vaishnava, S., Behrendt, C.L., Ismail, A.S., Eckmann, L., and Hooper, L.V.

(2008). Paneth cells directly sense gut commensals and maintain homeostasis

at the intestinal host-microbial interface. Proc. Natl. Acad. Sci. USA 105,

20858–20863.

Virgin, H.W., and Levine, B. (2009). Autophagy genes in immunity. Nat. Immu-

nol. 10, 461–470.

Virgin, H.W., Wherry, E.J., and Ahmed, R. (2009). Redefining chronic viral

infection. Cell 138, 30–50.

Weersma, R.K., Zhernakova, A., Nolte, I.M., Lefebvre, C., Rioux, J.D., Mulder,

F., van Dullemen, H.M., Kleibeuker, J.H., Wijmenga, C., and Dijkstra, G. (2008).

ATG16L1 and IL23R are associated with inflammatory bowel diseases but not

with celiac disease in the Netherlands. Am. J. Gastroenterol. 103, 621–627.

Xi, Z., Ramirez, J.L., and Dimopoulos, G. (2008). The Aedes aegypti toll

pathway controls dengue virus infection. PLoS Pathog. 4, e1000098.

Yang, Y.H., Dudoit, S., Luu, P., Lin, D.M., Peng, V., Ngai, J., and Speed, T.P.

(2002). Normalization for cDNA microarray data: a robust composite method

addressing single and multiple slide systematic variation. Nucleic Acids Res.

30, e15.


Supplemental Information

EXTENDED EXPERIMENTAL PROCEDURES

Administration of DSSAqueous solution of DSS (TDB Consultancy, Uppsala, Sweden) was filtered using a 0.22-mm cellulose acetate filter prior to admin-

istration through drinking water. For antibiotics experiments, 1 g/L ampicillin (Columbus Serum Company, Columbus, OH), 500 mg/L

vancomycin (Sigma, St. Louis, MO), 1 g/L neomycin sulfate (Sigma, St. Louis, MO), and 1 g/L metronidazole (Sigma, St. Louis, MO)

were dissolved in drinking water along with 2.5% DSS, then filtered using a 0.22-mm cellulose acetate filter. 20 mg/ml sugar-sweet-

ened grape Kool-Aid Mix (Kraft Foods) was included in the water to encourage consumption. All mice receiving antibiotics displayed

a similar degree of focal ulceration in the descending colon compared to mice receiving DSS alone, indicating that the presence of

antibiotics did not interfere with the administration of DSS through water and that bacteria are not essential for this aspect of DSS-

induced intestinal injury. Additionally, both WT and Atg16L1HM mice displayed epithelial hypo-proliferation in non-ulcerated regions

of the rectum that is similar to findings in DSS-treated germ-free mice (Pull et al., 2005), thus confirming successful antibiotic uptake.

For TNFa neutralization, mice received 1 mg of either hamster anti-TNFa (TN3–19.12) or hamster anti-PIP isotype control antibodies 1

day prior to DSS treatment, then received a second injection of 0.5 mg 5 days later (4 days into DSS treatment). For IFNg neutrali-

zation, mice received one injection of 0.2 mg of hamster anti-IFNg (H22) 1 day prior to DSS treatment. Blocking antibodies were kindly

provided by Dr. Robert Schreiber (Sheehan et al., 1989; Schreiber et al., 1985).

UV Inactivation of MNV CR6UV-inactivated virus was generated by placing 600 ml of 1.2x109 pfu/ml MNV CR6 in a 6 well plate and subjected to 1000 mJ of UV in

a UV Stratalinker 2400 (Stratagene). The inactivation was confirmed by qRT-PCR for the presence of viral genome in stool specimens

7 days post infection.

MicroscopyThe distal ileum and colon were removed, cut open along the length, and pinned on black wax. The ileum was fixed in 10% formalin

and the colon was fixed in Bouin’s fixative overnight at 4�C. 2 cm strips of intestinal tissues were embedded in agar to enrich for well-

oriented crypt-villus units. To determine villus blunting, villus height was quantified in ileal sections that were cut perpendicular to the

villus-crypt axis as defined by the presence of a visible crypt lumen from the orifice to the base of the crypt, thereby minimizing tissue

orientation-based artifacts of villus height measurements (Stappenbeck et al., 2003). Images were taken on an Olympus BX51 micro-

scope. Immunohistochemistry for lysozyme was previously described (Cadwell et al., 2008). Sections were viewed with a Zeiss Ax-

iovert 200 inverted fluorescence microscope and quantified on an Olympus AX70 epi-fluorescence microscope. Electron micros-

copy of ileal crypts was previously described (Cadwell et al., 2008).

Laser Capture MicrodissectionThe distal ileum was removed, cut open along the length, pinned on black wax, and fixed in methacarn. The tissue was blocked and

embedded as described above and 0.6 mm serial sections were cut and deparaffinized. The crypt-base epithelial cells enriched for

Paneth cells were captured and RNA was isolated by previously described techniques (Stappenbeck et al., 2003; Pull et al., 2005). 25

ng of total RNA from each sample was amplified using the Transplex Complete Whole Transcriptome Amplification Kit (Sigma, St

Louis MO), labeled using a modified protocol with the ULS aRNA Fluorescent Cy5 labeling kit (Kreatech Diagnostics, The

Netherlands); and purified using DNA Clean and Concentrate spin columns according to manufacturer’s protocol (Zymo Research

Corporation, Orange CA).

MicroarraycDNA was hybridized to 4x44K Mouse Whole Genome Microarrays (Agilent) for 18 hr following the manufacturer’s protocol. Micro-

arrays were washed in 0.01x SSC/0.005% Triton X-102 at 40�C, dried with HEPA filtered compressed nitrogen and scanned on an

Agilent Technologies DNA Microarray Scanner at 5 mm resolution. Data were extracted from the scanned image using Agilent Tech-

nologies Feature Extraction Software, background subtracted, and normalized by the Lowess method (Yang et al., 2002). Microarray

files have been submitted to Array Express, accession numbers E-TABM-957 (WT + MNV CR6 and Atg16L1HM + MNV CR6) and E-

TABM-958 (WT uninfected, Atg16L1HM uninfected, WT + MNV CW3, and Atg16L1HM + MNV CW3). A factorial design approach

(Scholtens et. al., 2004) was implemented in the R programming language, in which a linear model (Smyth, 2005) was fitted with

a coefficient for each of the 4 factor combinations and then simultaneously extracted as contrasts for each of the three comparisons

(summarized in the Venn diagram of Figure 3) including the interaction (‘differential-of-differential’) term. Sets of differentially ex-

pressed genes (p < 0.05 and fold-change > 1.5) for each comparison were examined for enrichment in terms of biological process

or molecular function categories compared to their representation in the whole genome using the Panther classification system (Mi

et al., 2007). Enrichment p-values were computed using the hypergeometric distribution and implemented in the R language.

Cell 141, 1135–1145, June 25, 2010 ª2010 Elsevier Inc. S1

SUPPLEMENTAL REFERENCES

Cadwell, K., Liu, J.Y., Brown, S.L., Miyoshi, H., Loh, J., Lennerz, J.K., Kishi, C., Kc, W., Carrero, J.A., Hunt, S., et al. (2008). A key role for autophagy and the

autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature 456, 259–263.

Mi, H., Guo, N., Kejariwal, A., and Thomas, P.D. (2007). PANTHER version 6: protein sequence and function evolution data with expanded representation of bio-

logical pathways. Nucleic Acids Res. 35 (Database issue), D247–D252.

Pull, S.L., Doherty, J.M., Mills, J.C., Gordon, J.I., and Stappenbeck, T.S. (2005). Activated macrophages are an adaptive element of the colonic epithelial progen-

itor niche necessary for regenerative responses to injury. Proc. Natl. Acad. Sci. USA 102, 99–104.

Scholtens, D., Miron, A., Merchant, F.M., Miller, A., Miron, P.L., Iglehart, J.D., and Gentleman, R. (2004). Analyzing Factorial Designed Microarray Experiments. J.

Multivariate Anal. 90, 19–43.

Schreiber, R.D., Hicks, L.J., Celada, A., Buchmeier, N.A., and Gray, P.W. (1985). Monoclonal antibodies to murine gamma-interferon which differentially modulate

macrophage activation and antiviral activity. J. Immunol. 134, 1609–1618.

Sheehan, K.C., Ruddle, N.H., and Schreiber, R.D. (1989). Generation and characterization of hamster monoclonal antibodies that neutralize murine tumor

necrosis factors. J. Immunol. 142, 3884–3893.

Smyth, G.K. (2005). Limma: linear models for microarray data. In Bioinformatics and Computational Biology Solutions using R and Bioconductor, R. Gentleman,

V. Carey, S. Dudoit, R. Irizarry, and W. Huber, eds. (New York: Springer).

Stappenbeck, T.S., Mills, J.C., and Gordon, J.I. (2003). Molecular features of adult mouse small intestinal epithelial progenitors. Proc. Natl. Acad. Sci. USA 100,

1004–1009.

Yang, Y.H., Dudoit, S., Luu, P., Lin, D.M., Peng, V., Ngai, J., and Speed, T.P. (2002). Normalization for cDNA microarray data: a robust composite method ad-

dressing single and multiple slide systematic variation. Nucleic Acids Res. 30, e15.

S2 Cell 141, 1135–1145, June 25, 2010 ª2010 Elsevier Inc.

A

B

Normal Abnormal

Granules

Abnormal

ER

Abnormal

ER + Granules

0

20

40

60

80

100

NormalAbnormal ERAbnormal GranulesAbnormal ER + Granules

WT H M WT H M

U ninfe cte d M N V C R 6

% P

anet

h C

ell

Day 7 post-inoculation withUV-inactivated MNV CR6

WT HM

Structural changes detected in Atg16L1HM mice infected with MNV CR6 for 7 days

C

Quantification of strucutral changes

Figure S1. MNV CR6 Infection Triggers Paneth Cell Abnormalities, related to Figure 1

(A) Atg16L1HM and WT mice in the enhanced barrier facility were orally inoculated with 3x107 particle forming units (pfu) of MNV CR6 inactivated by UV-irradiation.

Ileal sections were stained with PAS-alcian blue to detect Paneth granules by light microscopy (n = 5 mice/genotype). The dotted line denotes crypt unit and

arrowheads indicate typical Paneth cell granules. Scale bar represents 10 mm.

(B) Atg16L1HM and WT mice infected with MNV CR6 for 7 days or uninfected were analyzed by transmission electron microscopy (n = 3 mice/condition). Paneth

cells were categorized as having a normal appearance, distended endoplasmic reticulum (ER), diminished granules (%2 granules present), or a combination of

these ER and granule defects. Representative images of each category are shown. Bar = 2 mm.

(C) Quantification of the percentage of Paneth cells corresponding to the categories defined in (B).


A B

0

2

4

6

8

WT HM WT HM WT HM WT HM

n.s.

n .s.

n .s.

n .s.

S to o l Ileu m C o lo n M L Ns

Lo

g V

iral

Gen

om

e C

op

ies

0

2

4

6

8

10

12n.s .

WT HM WT HM WT HM

3 d p i 7 d p i 35 d p i

Lo

g V

iral

Gen

om

e C

op

ies

C

D 0 D 1 D 2 D 30

20

40

60

80

100

W T + MN V C R 6

H M + MN V C R 6

W T + MN V C W 3H M + MN V C W 3%

Pan

eth

Cel

l

**

Lysozyme Distribution Day 3 Post Infection

MNV CR6 Titre (Day 7) MNV CW3 Titre in Stool

Figure S2. Properties of MNV CR6 and CW3 Associated with Paneth Cells, Related to Figure 2

(A) Viral genome copy numbers were determined by qRT-PCR in various tissues harvested from Atg16L1HM and WT mice 7 days after oral inoculation with MNV

CR6 (n = 11 mice/genotype). MLNs indicate mesenteric lymph nodes. Dotted line represents a limit of detection of 600 genome copies.

(B) Stool pellets were collected from WT and Atg16L1HM mice 3, 7, and 35 days post oral inoculation with 3x107 pfu MNV CW3. Viral genome copy numbers were

determined by qRT-PCR (n = 5 mice/genotype for day 3 and day 7 samples, n = 4 mice/genotype for day 35 samples). Dotted line represents a limit of detection of

600 genome copies.

(C) Atg16L1HM and WT mice infected with 3x107 pfu of either MNV CR6 or MNV CW3 for 3 days. The percentage of Paneth cells displaying each of the four types of

lysozyme distribution patterns were quantified (n = 3 mice/condition, mean +/�SEM). A slight increase in the D3 population was detected in Atg16L1HM compared

to WT mice infected with MNV CR6 (p = 0.069), but otherwise Paneth cells were similar to uninfected WT mice (Figure 1D). No granule abnormalities were de-

tected by light microscopy in these conditions (n = 4 mice/genotype, not shown).


4932438A13Rik_29881_zoneFAbca13_17292_zoneFOas1a_17392_zoneFOasl2_39261_zoneFD5Wsu178e_19585_zoneFMgll_17073_zoneFPrr8_14020_zoneFRmi1_36673_zoneFCamta1_40861_zoneFEct2_9211_zoneFCcpg1_40046_zoneFAcsl1_21776_zoneFFam3c_6701_zoneFPrps2_6911_zoneFFnip2_14653_zoneFKlhl22_13959_zoneFNcoa4_35500_zoneFDnm1l_28024_zoneFHspbap1_22892_zoneFPkn2_25979_zoneF1110038D17Rik_29024_zoneFRnf139_365_zoneFDok7_36970_zoneFShisa2_6111_zoneF5730601F06Rik_26834_zoneFPapola_23141_zoneFSipa1l3_7324_zoneF

1 2 3

-4 1:1 4

Fold-change

WT infected vs. uninfected

Atg16L1HM infected vs. uninfected

‘Differential-of-differential’(Atg16L1HM infected vs. uninfected)

vs. (WT infected vs. uninfected)

Figure S3. Genes that Display Similar Changes in Expression in Paneth Cells from WT and Atg16L1 Mutant Mice in Response to MNV CR6,

Related to Figure 3Heatmap of genes represented in Zone F of the Venn diagram from Figure 3. Changes in gene expression within each comparison are represented as Log2-trans-

formed fold-changes (p < 0.05, fold-change > 1.5).


0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 10 20 30 40 50 60

En

rich

men

t sco

re

,-L

og

(p

-va

lu

e)


0

1

2

3

4

5

6

7

8

0 100 200 300 400 500

En

ric

hm

en

t s

co

re

,-L

og

(p

-valu

e)


00.20.40.60.8

11.21.41.61.8

2

0 10 20 30 40 50

En

ric

hm

en

t s

co

re

,-L

og

(p

-va

lu

e)


00.5

11.5

22.5

33.5

44.5

5

0 50 100 150 200 250

En

ric

hm

en

t s

co

re

,-L

og

(p

-va

lu

e)


CW3 (Atg16L1HM vs. WT) CR6 (Atg16L1HM vs. WT)

Enrichment analysis of gene set (i) Enrichment analysis of gene set (ii)

Receptor (p=4.36e-5)

Cell adhesion molecule (p=8.59e-5)

Ion channel (p=0.00012)

Extracellular matrix (p=0.00076)

Signaling molecule (p=0.0046)Transcription factor (p=0.011)

Signal transduction (p=7.94e-8)

Sensory perception (p=4.64e-5)

Neuronal activities (p=5.82e-5)

Cell adhesion (p=0.001)

Muscle contraction (p=0.015)

Developmental processes (p=0.003)

Amino acid metabolism (p=0.037)

Membrane traffic protein (p=0.016)

Transporter (p=0.04)Oxidoreductase (p=0.05)

(i) (ii)

(a’)

p=0.05

p=0.05

p=0.05

p=0.05

(b’) (c’)

(d’) (e’)

Figure S4. Comparison of Gene Expression Changes in Response to MNV CR6 versus CW3, Related to Figure 3

Comparison of respective genes that were altered in mRNA abundance in Atg16L1HM versus WT mice infected with MNV CW3 (i) and MNV CR6 (ii) is illustrated by

the Venn diagram (p < 0.05, fold-change > 1.5) (a0). A significant enrichment of the biological processes categories of signal transduction, sensory perception,

neuronal activities, cell adhesion, developmental processes, and muscle contraction was observed among genes uniquely differentially expressed when infected

with MNV CW3 (b0 ). This is distinct from infection with MNV CR6 which showed enrichment for amino acid metabolism among the differentially expressed genes

(c0). A significant enrichment of the molecular functions categories receptor, cell adhesion, ion channel, extracellular matrix, signaling molecule, and transcription

factor was observed in Atg16L1HM versus WT mice infected with MNV CW3 (d0). In contrast, membrane traffic protein and transporter were associated with MNV

CR6 and not MNV CW3 (e0).


0

2

4

6

8

WT HM WT HM

n.s.

n .s.

Ileu m C o lo n

p = 0.0101

WT HM

M L Ns

Log

Vira

l Gen

ome

Cop

ies

MNV CR6 titre after DSS treatment(day 14 post infection)

Figure S5. MNV CR6 Replication Is Similar in WT and Atg16L1HM Mice Treated with DSS, Related to Figures 4, 5, and 6Ileum, colon, and mesenteric lymph nodes (MLNs) were harvested from WT and Atg16L1HM mice orally inoculated with 3x107 pfu of MNV CR6 for 7 days prior to

receiving DSS for another week. Viral genome copy numbers were determined in these tissues by qRT-PCR (n = 10 mice/genotype). Dotted line represents a limit

of detection of 600 genome copies.


Virus-Plus-Susceptibility Gene Interaction Determines Crohn's Disease Gene Atg16L1 Phenotypes in...

Documents

Transcript of Virus-Plus-Susceptibility Gene Interaction Determines Crohn's Disease Gene Atg16L1 Phenotypes in...