Typhoid-like Disease Mutants Confer Susceptibility to Acute Induced ...

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of April 5, 2018. This information is current as Typhoid-like Disease Mutants Confer Susceptibility to Acute Induced STAT4 - -Nitrosourea N -Ethyl- N Transcriptional Expression Patterns in Mediated Immunity and - γ Altered IFN- Danielle Malo Paquet, Mark Lathrop, Jacek Majewski, Silvia M. Vidal and Jeremy A. Schwartzentruber, Michal Pyzik, Marilène Megan M. Eva, Kyoko E. Yuki, Shauna M. Dauphinee, http://www.jimmunol.org/content/192/1/259 doi: 10.4049/jimmunol.1301370 November 2013; 2014; 192:259-270; Prepublished online 27 J Immunol Material Supplementary 0.DC1 http://www.jimmunol.org/content/suppl/2013/11/27/jimmunol.130137 References http://www.jimmunol.org/content/192/1/259.full#ref-list-1 , 25 of which you can access for free at: cites 76 articles This article average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved. Copyright © 2013 by The American Association of 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on April 5, 2018 http://www.jimmunol.org/ Downloaded from by guest on April 5, 2018 http://www.jimmunol.org/ Downloaded from

Transcript of Typhoid-like Disease Mutants Confer Susceptibility to Acute Induced ...

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of April 5, 2018.This information is current as Typhoid-like Disease

Mutants Confer Susceptibility to Acute Induced STAT4−-NitrosoureaN-Ethyl-

NTranscriptional Expression Patterns in Mediated Immunity and−γAltered IFN-

Danielle MaloPaquet, Mark Lathrop, Jacek Majewski, Silvia M. Vidal andJeremy A. Schwartzentruber, Michal Pyzik, Marilène Megan M. Eva, Kyoko E. Yuki, Shauna M. Dauphinee,

http://www.jimmunol.org/content/192/1/259doi: 10.4049/jimmunol.1301370November 2013;

2014; 192:259-270; Prepublished online 27J Immunol 

MaterialSupplementary

0.DC1http://www.jimmunol.org/content/suppl/2013/11/27/jimmunol.130137

Referenceshttp://www.jimmunol.org/content/192/1/259.full#ref-list-1

, 25 of which you can access for free at: cites 76 articlesThis article

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2013 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Altered IFN-g–Mediated Immunity and TranscriptionalExpression Patterns in N-Ethyl-N-Nitrosourea–InducedSTAT4 Mutants Confer Susceptibility to Acute Typhoid-likeDisease

Megan M. Eva,*,† Kyoko E. Yuki,*,† Shauna M. Dauphinee,*,†

Jeremy A. Schwartzentruber,*,‡ Michal Pyzik,*,† Marilene Paquet,x Mark Lathrop,*,‡

Jacek Majewski,*,‡ Silvia M. Vidal,*,†,{ and Danielle Malo*,†,{

Salmonella enterica is a ubiquitous Gram-negative intracellular bacterium that continues to pose a global challenge to human

health. The etiology of Salmonella pathogenesis is complex and controlled by pathogen, environmental, and host genetic factors. In

fact, patients immunodeficient in genes in the IL-12, IL-23/IFN-g pathway are predisposed to invasive nontyphoidal Salmonella

infection. Using a forward genomics approach by N-ethyl-N-nitrosourea (ENU) germline mutagenesis in mice, we identified the

Ity14 (Immunity to Typhimurium locus 14) pedigree exhibiting increased susceptibility following in vivo Salmonella challenge. A

DNA-binding domain mutation (p.G418_E445) in Stat4 (Signal Transducer and Activator of Transcription Factor 4) was the causative

mutation. STAT4 signals downstream of IL-12 to mediate transcriptional regulation of inflammatory immune responses. In mutant

Ity14mice, the increased splenic and hepatic bacterial load resulted from an intrinsic defect in innate cell function, IFN-g–mediated

immunity, and disorganized granuloma formation. We further show that NK and NKT cells play an important role in mediating

control of Salmonella in Stat4Ity14/Ity14 mice. Stat4Ity14/Ity14 mice had increased expression of genes involved in cell–cell interactions

and communication, as well as increased CD11b expression on a subset of splenic myeloid dendritic cells, resulting in compromised

recruitment of inflammatory cells to the spleen during Salmonella infection. Stat4Ity14/Ity14 presented upregulated compensatory

mechanisms, although inefficient and ultimately Stat4Ity14/Ity14 mice develop fatal bacteremia. The following study further elucidates

the pathophysiological impact of STAT4 during Salmonella infection. The Journal of Immunology, 2014, 192: 259–270.

Acute foodborne bacterial infections remain a major publichealth problem associated with high morbidity and mor-tality worldwide. The intracellular bacterium Salmonella

enterica continues to pose a global challenge to human health (1).In humans, Salmonella infections cause a range of foodborne and

waterborne illnesses, from a self-limiting, localized gastroenteritisto the more severe, potentially fatal systemic disease of typhoidfever (2). Certain infected individuals (1–4%) may further developrecurrent infections or become asymptomatic chronic carriers actingas a reservoir for pathogen persistence and dissemination in thepopulation (3, 4). Typhoid fever is strictly caused by an infectionwith the human-restricted S. enterica serovars Typhi and Paratyphi.It is primarily endemic in developing areas of the world where poorsanitation and lack of access to clean drinking water are common(5). In contrast, nontyphoidal Salmonella (NTS) serovars, such asSalmonella Typhimurium and Salmonella Enteritidis, are capable ofinfecting a broad spectrum of hosts, resulting in intestinal and di-arrheal disease (salmonellosis). NTS serovars are the second leadingcause of foodborne illnesses in North America. Approximately 5%of patients with salmonellosis are at increased risk of further de-veloping invasive transient bacteremia and sepsis (6). Indeed, dis-ease manifestation of Salmonella-related infections in humans isdependent on the complex interaction between environmental fac-tors, bacterial serotype, and host genetic factors.The outcome of S. enterica infection relies on the activation of

both early innate functions and adaptive humoral and cell-mediatedimmune responses of the host (7, 8). During systemic Salmonellainfection, rapid neutrophilic infiltration and phagocytosis by tissuemacrophages are crucial in limiting hepatosplenic infection. Acti-vated macrophages limit Salmonella replication and disseminationto new foci by phagolysosomal bacterial killing and secretion of in-flammatory chemokines and cytokines, including TNF-a, IL-12,IL-18, and IFN-g (7). The inflammatory environment results inthe rapid recruitment of leukocytes that increase the ability for

*Department of Human Genetics, McGill University, Montreal, Quebec H3G 0B1,Canada; †Complex Traits Group, McGill University, Montreal, Quebec H3G 0B1,Canada; ‡McGill University and Genome Quebec Innovation Centre, Montreal, Que-bec H3B 1S6, Canada; xDepartement de Pathologie et de Microbiologie, Faculte deMedecine Veterinaire, Universite de Montreal, Saint-Hyacinthe, Quebec J2S 5B5,Canada; and {Department of Medicine, McGill University, Montreal, Quebec H3G0B1, Canada

Received for publication May 23, 2013. Accepted for publication October 25, 2013.

This work was supported by funds from the Team Program of the Canadian Institutesof Health Research (to S.M.V. and D.M.). M.M.E. was recipient of a studentship fromthe Research Institute of the McGill University Health Centre. K.E.Y. received a Fac-ulty of Medicine Internal Studentship award. S.M.D. received a Faculty of MedicineDavid Lin fellowship, a Research Institute of the McGill University Health Centrefellowship, and a Fonds de la Recherche du Quebec fellowship. S.M.V. holds a Can-ada Research chair and D.M. is a McGill Dawson scholar.

The sequences presented in this article have been submitted to the ArrayExpressdatabase (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-1931.

Address correspondence and reprint requests to Dr. Danielle Malo, Department ofHuman Genetics and Department of Medicine, McGill University Life SciencesComplex, Bellini Building, 3649 Promenade Sir-William-Osler, Montreal, QCH3G 0B1, Canada. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this article: DC, dendritic cell; ENU, N-ethyl-N-nitrosourea;IPA, Ingenuity pathway analysis; KO, knockout; MFI, mean fluorescence intensity;NTS, nontyphoidal Salmonella; PMN, polymorphonuclear cell; SNP, single nucleo-tide polymorphism.

Copyright� 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00

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intracellular pathogen killing and formation of granulomas. Im-portantly, IL-12 induces IFN-g secretion from NK cells, which iscritical in activating macrophages in the early response againstsystemic Salmonella infections (9, 10). Protective immunity isfurther mediated by IFN-g production from activated CD4+ Th1cells as well as the cytotoxic response of CD8+ T lymphocytes(11). Indeed, immunodeficient mice lacking IFN-g or IFN-gR failto resolve primary infection with an attenuated Salmonella strain(12). Moreover, neutralization of IL-12 in a mouse model of ty-phoid fever abrogates host immunity to primary Salmonella in-fection (13). In addition, clinical reports have shown that patientsimmunocompromised as a consequence of HIV infection, chronicgranulomatous disease, or functional genetic defects in the IL-12/IL-23 (IL-12b, IL-12Rb1) and IFN-g (IFN-gR1, IFN-gR2,STAT1) pathways are predisposed to Mendelian susceptibility tomycobacterial disease and/or disseminated Salmonella infection(14–19). The following highlights the importance of systemicimmunity to control invasive S. enterica infections.In mice, S. Typhimurium infection is a recognized experimental

model for studying acute systemic disease resembling clinicalfeatures of typhoid fever in humans (20, 21). Alternatively, the useof humanized mice (Rag22/2gc

2/2; [NOD]-scid IL2rgnull) and,more recently, Tlr112/2 mice, which have been shown to be ef-ficiently colonized by S. Typhi, are providing novel tools to studytyphoid fever (22–25). Following oral infection of mice, Salmo-nella invade the microfold cells in the intestinal epithelium andare taken up by dendritic cells (DCs) and macrophages in theunderlying Peyer’s patches before infecting the mesenteric lymphnodes and eventually disseminating via the circulation to replicatein resident phagocytes of the spleen and liver (26, 27). Previously,the genetic and molecular basis of several mutations, includingNramp1/Slc11a1, Tlr4, and Pklr, has been shown to be importantin resistance to Salmonella infection in mice (28–33). However, asthe mouse genome has a low frequency of naturally occurringspontaneous mutations, new approaches to identify host suscep-tibility or resistance genes in the mouse are essential to study thehost response to infectious diseases. As such, we have used anunbiased forward genomics approach by N-ethyl-N-nitrosourea(ENU) germline mutagenesis to screen and identify novel muta-tions critical in antibacterial immunity, as well as to investigategene function in vivo in the context of Salmonella infection.In this article, we report the identification of the Ity14 (Immu-

nity to Typhimurium locus 14) pedigree carrying an ENU-inducedmutation in Stat4 (Signal Transducer and Activator of Tran-scription Factor 4) conferring increased susceptibility to sublethalprimary Salmonella infection. The transcription factor STAT4 isspecifically expressed in myeloid and lymphoid cells and isa critical mediator of IL-12 signaling in development of inflam-matory immune responses (34, 35). IL-12 production from acti-vated macrophages and DCs activates TYK2 and JAK2 receptor–associated kinases, resulting in dimerization, phosphorylation, andactivation of STAT4. STAT4 has been shown to be involved inboth innate and adaptive immunity by regulating the transcriptionof target genes, including IFN-g; NK cell cytotoxicity; Th1 celldifferentiation from naive CD4+ T cells; and Ig isotype switchingto IgG1. Consistent with these findings, STAT4 is a central deter-minant in host resistance to various bacterial, viral, and protozoaninfections while playing a critical role in regulating inflammatoryimmune-mediated diseases (36).The physiological function of STAT4 in vivo in response to

S. Typhimurium infection remains poorly characterized. In this pa-per, we elucidate the impact of Stat4Ity14/Ity14 on innate immunityduring Salmonella infection. We show that Ity14 mice with a hy-pomorphic mutation in Stat4 have an increased innate susceptibility

following sublethal invasive S. Typhimurium challenge. Overall,Stat4Ity14/Ity14 mice have increased mortality following infection,with a concomitant progressive increase in splenic and hepaticbacterial load. Using genome-wide expression microarrays in spleentissue from wild-type and Ity14 mutants, we studied the impact ofStat4 on the inflammatory immune response to Salmonella infec-tion. We validated the importance of IFN-g–mediated immunityduring systemic Salmonella infection. We further show that NK andNKT cells play an important early role in controlling Salmonella inStat4Ity14/Ity14 mice. During oral infection using a model of intestinaltyphlitis, mice lacking STAT4 also develop early dissemination ofS. Typhimurium and systemic disease. In addition, increased ex-pression of genes involved in cell–cell interactions and communi-cation, as well as increased CD11b expression on a subset of splenicmyeloid DCs, shows compromised recruitment of inflammatorycells to the spleen during Salmonella infection in Stat4 mutants.

Materials and MethodsMice

All animal experiments were conducted following specific conditionsoutlined by the Canadian Council on Animal Care and protocols approvedby the McGill University Animal Care Committee. The 129S1/SvImJ,DBA/2J, C57BL/6J, and C.129S2-Stat4tm1Gru/J mice were purchased fromThe Jackson Laboratory (Bar Harbor, ME). Experiments were performedusing mice between 7 and 15 wk of age of both sexes.

Chemical mutagenesis (ENU)

Generation 0 (G0) males on a 129S1 background between 8 and 12 wk ofagewere i.p. injected with a single ENU dose of 150 mg per kg body weight.Efficiently mutagenized G0 males temporarily experienced a period ofinfertility and only regained fertility after 11 wk. ENU-induced mutationswere brought to homozygosity using a three-generation breeding scheme.N3 progeny on a resistant 129S1-129X1 background were initially screenedfor susceptibility to S. Typhimurium infection.

DNA preparation, genetic mapping, and genotyping

DNAwas extracted from a tail piece of mice by proteinase K digestion andphenol-chloroform extraction. A genome scan was performed on DNA sam-ples from 21 mice (9 susceptible, 12 resistant) and a panel of 708 singlenucleotide polymorphisms (SNPs) for 129 and DBA/2 strains of mice(MediumDensity SNP Panel; Illumina GoldenGate, The Centre for AppliedGenomics, University of Toronto, Toronto, ON, Canada). Mice were furthergenotyped by SNP sequencing (McGill University and Genome Quebec).The mutation in Stat4 in Ity14 mice (c.1335+5 G . A) was genotypedusing a Custom TaqMan SNP Genotyping Assay (Applied Biosystems,Streetville, ON, Canada).

Whole-exome sequencing

We followed standard manufacturer protocols to perform target capturewiththe SureSelectMouse All ExonKit (Agilent Technologies, Santa Clara, CA)and sequencing of 100-bp paired end reads on Illumina HiSEquation 2000.This process generated. 8 Gb of sequence for each of the two susceptibleIty14 samples. Reads were aligned to mm9 with BWA (37) and coveragewas assessed with BEDTools, showing an average of 58.9 reads coveringeach base of the consensus coding sequence genes for the mouse genome(38). Single nucleotide variants and short insertions and deletions werecalled using samtools pileup and varFilter (39) with the base alignmentquality adjustment disabled, and were then quality filtered to require $ 20%of reads supporting the variant call. Variants were annotated using bothAnnovar (40) and custom scripts to identify whether they affected theprotein coding sequence, and whether they had previously been seen inmouse dbSNP128 or in any of seven mouse exomes sequenced in parallel.To detect splice site mutations, the threshold of detection was increased to6 bp instead of the standard 2-bp flanking exons.

In vivo Salmonella mouse infections and tissue bacterial load

Mice were challenged i.v. in the caudal vein with an infectious dose of 53103 CFUs S. Typhimurium strain Keller. Dose preparation and method ofinfections have been previously described (41, 42). For the complemen-tation assay, mice were infected with a lower dose of 2.5 3 103 CFUs,given that the C.129S2-Stat4tm1Gru/J mice are on a BALB/c background

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(33). Infected mice were monitored over the course of 14 d, and suscep-tible mutants defined by a body score index , 2 were euthanized by CO2

asphyxiation. To determine bacterial load in organs at specific time pointspostinfection, aseptically harvested organs were weighed and homoge-nized in 0.9% saline using a Polytron (Kinematica, Bohemia, NY). Serialdilutions of organ homogenates were then plated on trypticase soy agar todetermine bacterial counts.

In vivo per os Salmonella infection

At 1 d prior to infection, mice were fasted for 6 h. At 4 h into the fast, micewere gavaged with 20 mg streptomycin sulfate diluted in 100 mL sterilewater. An overnight culture of S. Typhimurium SL1344 was prepared in5 ml TSB supplemented with 50 mg/ml streptomycin sulfate and incubatedat 37˚C. The day of the infection, 2 ml of the overnight culture was in-oculated in 50 ml TSB containing streptomycin, and grown to an OD of 0.9at 600 nm. Streptomycin-pretreated mice were each gavaged with 5 3 107

CFU bacteria resuspended in 100 ml sterile saline. For CFU enumeration,see the detailed method above.

Intestinal histopathological scoring

H&E-stained cecum and colon slides were analyzed in duplicate andblinded for genotype. A histopathological scoring system was adaptedfrom a previously described model (43). Briefly, the score evaluated thesubmucosal edema (0 = 0, 1 # 10%, 2 = 10–40%, 3 = .40%), thepolymorphonuclear cell (PMN) infiltration of the lamina propria (0 = # 5PMN, 1 = 5–20, 2 = 21–60, 3 = 61–100, 4 = . 100), the goblet cell count(0 = .10, 1 = 6–10, 2 = 1–5, 3 = 0 [per high-power field]), and the epi-thelial integrity (0 = no change, 1 = desquamation, 2 = erosion of theepithelial surface, 3 = ulceration) of cecum cross-sections (43).

In vivo bioluminescence imaging

Mice were injected i.v. with 6 3 104 CFUs S. Typhimurium strain XEN26.Whole-body imaging of mice was performed daily, beginning on day 3 today 8 postinfection on the xenogen IVIS Spectrum System (Caliper, Al-ameda, CA). Mice were anesthetized with isoflurane and anteriorly shavedprior to imaging. Bioluminescent images were acquired with an openemission filter, binning factor of 16, and exposure time ranging from 2 to5 min. The regions of interest were selected, normalized across timepoints, and quantified using Living Image software v.4.3.1 (Caliper).

Preparation of protein extracts for Western blot analysis

Protein extracts from spleen were prepared by homogenizing tissue usinga Polytron (Kinematica) in lysis buffer (1 M Tris-HCl, pH = 8; 0.5 M KCl;0.1 M MgCl2; 0.1% Triton X-100; 10% glycerol) with protease inhibitor(P2714; Sigma-Aldrich) and further sonicated for 15 s on ice at 60%amplitude. Protein extracts were centrifuged at 13,000 rpm at 4˚C for 15min. Supernatants were collected and protein concentration quantified byBradford assay (500-0006; Bio-Rad). Whole-cell lysates were extracted inLaemmli buffer and probed by immunoblot using Abs for STAT4 andGAPDH (Cell Signaling). The STAT4 Ab recognizes an epitope aroundlysine 151 upstream of the Ity14 mutation.

Splenocyte preparation, Abs, and flow cytometry

Spleens were harvested and collected from uninfected and day 4S. Typhimurium–infected mice in 4 ml PBS under sterile conditions. Spleenswere macerated with 70-mm cell strainers into single-cell suspensions,treated with ACK lysis buffer, and washed in cold PBS. Cell counts weredetermined using a Coulter Z2 particle counter (Beckman Coulter). Forsplenocyte stimulation, 5 3 105 cells were plated in 96-well round-bottomplates and stimulated with recombinant mouse IL-12 (20 ng/ml; R&DSystems) or LPS (1 mg/ml; Sigma-Aldrich) for 2, 4, 8, and 24 h at 37˚C,5% CO2. For surface staining, 5 3 106 splenocytes were plated in 96-wellround-bottom plates and stained for various cell surface markers, usingfluorochrome-labeled mAbs (eBioscience). The following anti-mouse Abswere purchased from eBioscience: CD49b (clone DX5), CD4 (GK1.5),CD8a (53-6.7), CD45R (RA3-6B2), Ly-6G (RB6-8C5), F4/80 (BM8),CD11c (HL3), and CD11b (M1/70). CD3e (500A2) was purchased fromBD. Fixable Viability Dye (eBioscience) to irreversibly stain dead cellswas used in accordance with the manufacturer’s protocol. Cells were ac-quired on a FACSCanto II (BD) flow cytometer, and results were analyzedusing FlowJo (v9.4.10) software.

Intracellular cytokine production

For intracellular cytokine staining, 10 3 106 splenocytes were plated incomplete RPMI 1640 media in six-well tissue culture–treated plates. Cells

were stimulated and activated ex vivo with 50 mg/ml PMA and 1 mg/mlionomycin (Sigma-Aldrich) for 4 h in the presence of GolgiStop (BD).IFN-g (XMG1.2) and IL-4 (eBioscience) were stained according to themanufacturer’s protocol (Cytofix/Cytoperm Plus Fixation/PermeabilizationKit with GolgiStop; BD). Cells were acquired on a FACSCanto II, andresults were analyzed as described above. IL-6, TNF-a, and IL-10 cyto-kines in the supernatant of splenocytes, as well as IFN-g and IL-12p70 inserum, were measured by sandwich ELISA (eBioscience).

Genome-wide expression microarrays

Spleens of Ity14 mice were collected for microarrays and frozen at280˚C.Total RNA was extracted from ∼ 50 mg splenic tissue, using TRIzol re-agent (Invitrogen Canada, Burlington, ON, Canada). RNA yield was de-termined using a NanoDrop spectrophotometer (ThermoFisher Scientific,Waltham, MA) and the overall quality assessed by denaturing agarose gelelectrophoresis. Four uninfected and four day 4 S. Typhimurium–infectedage-matched mice (two females, two males) per genotype were used forwhole-genome expression profiling on Illumina Mouse-Ref-8 v2.0 Bead-Chip (Illumina, San Diego, CA). The quality control, hybridization, andarray analysis were performed at the McGill University and GenomeQuebec Innovation Centre (Montreal, QC, Canada).

Microarray data analysis

Raw data are available through the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-1931. Preliminary ex-pression data analysis was done using FlexArray 1.4.1 software, as pre-viously described (44, 45) (Genome Quebec, Montreal, QC Canada)(http://genomequebec.mcgill.ca/FlexArray). Principal component analysiswas generated in FlexArray. Briefly, gene lists for each sample group weregenerated by selecting for genes with a false discovery rate p, 0.05 by theunpaired Student t test and . or , 2-fold change. The canonical pathwayanalyses were generated through the use of Ingenuity pathway analysis(IPA; Ingenuity Systems, www.ingenuity.com). Heatmaps were generatedusing R package (46).

Validation by quantitative real-time PCR and list of primersused

RNA quantification by the SYBR Green–based detection kit was performedusing the Applied Biosystem StepOnePlus (Applied Biosystems, Carlsbad,CA). Complementary DNAs were amplified by PCR using the followingprimer pairs: Usp: 18 59-AGTGCTGTCTAGAGACCTCTGC-39 and 59-GGAGTTAAGGAACACGTCTG-39; Il4: 59-TGAGAGAGATCATCGG-CATTT-39 and 59-GTGAGGACGTTTGGCACATC-39; Il10: 59-AGTGG-AGCAGGTGAAGAGTGA-39 and 59-ATGCAGTTGATGAAGATGTCA-AA-39; Ifng: 59-ACTGGCAAAAGGATGGTGAC-39 and 59-ATCCTTTT-TCGCCTTGCTGT-39; Lcn2: 59-CAGAAGGCAGCTTTACGATGT-39 and59-TGTTCTGATCCAGTAGCGACA-39; Mpo: 59-ATGCTTCAGACCTC-CAATGGT-39 and 59-CTCTGTCCACTAGCTGCTTGG-39; Gbp5: 59-C-AGGCAAATCCTACCTGATGA-39 and 59-ACCAAAGTGTGGTCTGG-CTTT-39; Stat4: 59-GCGTCCATTGACAAGAATGTT-39 and 59-CCTTG-GGTTGCAAATGTCTAA; Stat1: 59-ACAACATGCTGGTGACAGAGC-C-39 and 59-TGAAAACTGCCAACTCAACACCTC-39; and normalized tothe reference Hprt gene: Hprt 59-GTTGGATACAGGCCAGACTTTGTTG-39and 59-GATTCAACTTGCGCTCATCTTAGGC-39. The data obtained wereexpressed as 22DDCt.

Statistical analysis

Statistical analyses were done using GraphPad Prism v5.0 (GraphPadSoftware, La Jolla).

ResultsIdentification of the ENU-induced S. Typhimuriumsusceptibility locus, Ity14

The Ity14 pedigree was identified in an in vivo recessive screen ofENU-mutagenized mice for innate susceptibility to Salmonellainfection, as measured by survival analysis (Fig. 1). Mutagenized129S1 G0 males were crossed to wild-type 129X1 females to gen-erate G1 mice. G1 males were further outcrossed to 129X1 femalesto produce G2 females. For each G1 pedigree, two G2 daughterswere backcrossed to generate N3 offspring (Fig. 1A). We initiallyscreened N3 mice on a resistant 129S1/129X1 mixed backgroundwith a sublethal dose of S. Typhimurium and monitored mice

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for 14 d for development of clinical disease. We observed 25%mortality in Ity14 N3 progeny by day 6 postinfection (data notshown). To map and identify the mutation responsible for theSalmonella-susceptibility phenotype, we outcrossed both theG1 male and the G2 female to DBA/2J mice in an F2 cross. Weobserved 25% mortality by day 5 postinfection (Fig. 1B), con-sistent with the survival phenotype observed in N3 animals. Initialgenotyping was performed using a total of 21 F2 mice (9 sus-ceptible, 12 resistant) and 708 SNPs (polymorphic between 129and DBA/2J). We detected linkage to a 25.6-Mb region on chro-mosome 1 with a maximum LOD score of 6.23 at RS1347566.Fine mapping refined the Ity14 locus to a 2.7-Mb interval (Fig.1C). At the peak marker, F2 mice homozygous for the 129S1allele were highly susceptible to S. Typhimurium infection by day6 postinfection, whereas heterozygous and mice homozygous forthe DBA/2J allele were significantly more resistant (Fig. 1D).Overall, we identified the Ity14 pedigree with increased suscep-tibility to Salmonella infection contributed by a 2.7-Mb intervalon proximal chromosome 1.

A causal mutation in Stat4 underlies the Ity14Salmonella-susceptibility locus

Whole-exome sequencing of coding exons and flanking splicejunctions in two susceptible Ity14 mice identified a novel ENU-induced mutation in Stat4. The average coverage across theexome was 83-fold. The Stat4 mutation was the only one bothvalidated and segregated with the survival phenotype from a listof predicted missense and splicing variants identified by exomesequencing (Supplemental Table II). The mutation consisted ofa guanosine to adenosine substitution within the splice donorsite of exon 15, at position +5 of intron 15 (c.1335+5G . A) andwas confirmed by Sanger sequencing (Fig. 2A). This guanosine is ahighly conserved nucleotide involved in the splice donor sequence

recognition by the splicesome U1 subunit, and the maximumentropy score predicts significantly weakened splice site recog-nition (from 7.07 to 1.48) (http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq.html). Therefore, it was predicted that themutation would interfere with normal splicing function. PCRamplification of cDNA isolated from wild-type, heterozygous, andmutant spleens using primers located in flanking exons 14 and 16resulted in a smaller PCR product in mutant mice (Fig. 2B) as aresult of an 84-bp deletion corresponding to exon 15. The muta-tion resulted in a 28-aa deletion in the DNA binding domain ofSTAT4 (p.G418_E445) (Fig. 2C). We observed decreased ex-pression of a smaller STAT4 protein product at days 0 and 4postinfection in Stat4Ity14/Ity14 splenic tissue, compared with litter-mate controls (Fig. 2D). In contrast, at the transcript level, Stat4 geneexpression was downregulated to a lesser extent in Stat4Ity14/Ity14

mice compared with wild-type at day 4 postinfection (Fig. 2E).We further validated STAT4 as the candidate gene responsible forthe susceptibility phenotype in the Ity14 pedigree by alleliccomplementation assays. Stat4Ity14/+ mice were crossed to Stat42/2

mice, and susceptibility to infection was assessed by survivalanalysis in F2 progeny. We observed a lack of complementationin Stat4Ity14/2 mice with a mean survival time equivalent to thatin Stat4Ity14/Ity14 and Stat42/2 animals, thereby confirming thatthe mutation within Stat4 was responsible for the Ity14 phenotype(Fig. 2F).

Impaired systemic IFN-g secretion and microabscessformation contribute to increased bacterial burden in spleenand liver of Salmonella-infected Stat4Ity14/Ity14 mice

Increased mortality following S. Typhimurium infection inStat4Ity14/Ity14 mutants paralleled the significantly higher bacterialload in target organs, specifically in the spleen and liver at day 4postinfection. At day 4 following infection, we observed a 17.2-

FIGURE 1. Identification of the Ity14 Salmonella-susceptible pedigree. (A) The recessive breeding scheme used to screen for susceptibility to

S. Typhimurium infection in N3 mice and further confirm phenotypic heritability in an F2 cross. White represents mice homozygous for the mutant allele,

gray stands for the heterozygous allele, and black denotes mice homozygous for the wild-type allele, for both females (circle) and males (square). (B)

Cumulative survival analysis of Ity14 F2 mice (n = 50); DBA/2J and 129S1 controls infected i.v. with 5000 CFUs S. Typhimurium. (C) Fine mapping of the

Ity14 locus to a 2.7-Mb region. The 129S1 allele is represented by white boxes, and the heterozygous and DBA/2 alleles are represented by black boxes.

Susceptible mice were classified based on early mortality, prior to day 6 postinfection. (D) Survival curve according to Stat4 genotype at peak chromosome

1 marker; Stat4Ity14/Ity14 (n = 26), Stat4+/Ity14 (n = 41), and Stat4+/+ (n = 11). ***p , 0.0001 by log-rank Mantel–Cox test.

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fold change in Salmonella replication in the spleen and a 87.3-foldchange in the liver in Stat4Ity14/Ity14, compared with wild-typelittermate controls (Fig. 3A). In vivo imaging using luminescentS. Typhimurium strain XEN26 validated progressive increasedbacterial load in Ity14 mutant mice over the course of infection(Supplemental Fig. 1A, 1B). The in vivo systemic bacterial rep-lication observed in Stat4Ity14/Ity14 at day 4 after Salmonella in-fection parallels the low levels of circulating IFN-g measured inthe serum of these mice (Fig. 3B). Moreover, we observed in-creased IL-12p70 levels in the serum of Stat4Ity14/Ity14 mice at day 4postinfection, suggesting a lack of negative feedback (Fig. 3C).Consistent with the essential function of STAT4 in IFN-g inductiondownstream of IL-12 signaling, we observed significantly lowerlevels of IFN-g production in the supernatant of Stat4Ity14/Ity14

splenocytes following 24-h stimulation with recombinant mouseIL-12 (Fig. 3D). IFN-g is an important mediator of granulomaformation, critical to control and prevent intracellular bacterialdissemination. Consequently, reduced levels of IFN-g are con-sistent with the increased bacterial load observed in Stat4Ity14/Ity14

mice. Although we did not observe any significant differences inproinflammatory cytokine production, IL-6 and TNF-a, or anti-inflammatory cytokine IL-10 following LPS stimulation in ex-planted splenocytes (Fig. 3E–G), histopathological H&E staining ofinfected spleen and liver sections further demonstrates that Ity14mutants have impaired immune cell–mediated recruitment to thesite of infection. During Salmonella infection, Stat4Ity14/Ity14 haveboth fewer and smaller multifocal microabscesses of inflammatory

infiltrates composed of neutrophilic granulocytes and macro-phages in liver, as well as in both white and red pulps of spleen,compared with wild-type controls (Supplemental Fig. 1C).

STAT4 controls systemic dissemination of Salmonella duringintestinal disease

To further demonstrate the importance of STAT4 in controllingsystemic S. Typhimurium infection, streptomycin-pretreated wild-type and Ity14 mutant mice were infected per os to study intestinalpathological changes in the cecum, as well as bacterial dissemi-nation to the spleen and liver at days 1 and 4 postinfection. Nohistopathological differences in the cecum were observed at day 1postinfection; however, at day 4 postinfection we observed lesssubmucosal edema, less PMN cell infiltration of the lamina propria,increased goblet cell count, and decreased epithelial integrity inIty14 mutants, compared with wild-type (Fig. 4E). In contrast, Stat4knockout (KO) mice had increased pathological features at day 1postinfection, with no differences at day 4 postinfection (Sup-plemental Fig. 1D). We can clearly detect an effect of the back-ground on the severity of the pathological lesions: control mice ona BALB/cJ background were more affected than control mice ona mixed 129S1:DBA/2J background at day 1 postinfection, whereasthe lesions were more prominent in Ity14 mutants compared withStat4 KO at day 4. These data are consistent with our previousobservation that inbred strains present different clinicopathologicalfeatures of Salmonella-induced typhlitis (45). Despite these dif-ferences in kinetics and amplitude of the histopathological scores

FIGURE 2. ENU-induced mutation in Stat4 increases susceptibility to S. Typhimurium. (A) A guanosine to adenosine substitution (arrow) within the

splice donor site of exon 15, at position +5 of intron 15, was identified in Ity14 mutant mice. (B) PCR amplification of flanking exon 15 region in cDNA

isolated from Stat4+/+, Stat4+/ty14, and Stat4Ity14/Ity14 spleens. A shorter PCR product in Stat4Ity14/Ity14 mice corresponds to deletion of exon 15 (84 bp). (C)

The ENU-induced Stat4 mutation is found within the DNA binding domain, as shown in the representation of the STAT4 protein. (D) Western blot analysis

of protein extracts from uninfected or infected spleens probed with anti-STAT4 and anti-GAPDH. (E) Stat4 gene expression by quantitative real-time RT-

PCR in spleen tissue of wild-type and Stat4Ity14/Ity14 mice both uninfected (day 0) and infected (day 4) with 5000 CFUs S. Typhimurium. *p # 0.05. (F)

Survival curves of Stat4Ity14/2 (n = 17), Stat42/2 (n = 14), Stat4Ity14/Ity14 (n = 3), as well as control Stat4+/+ (n = 7), Stat4Ity14/+ (n = 4), and Stat42/+ (n = 7)

mice infected i.v. with 2500 CFUs S. Typhimurium confirm Stat4 underlies Ity14 susceptibility locus. HET, Heterozygous; MUT, mutant; WT, wild-type.

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between Ity14 mutants and Stat4 KO, we observed disseminationof Salmonella to systemic sites in both mutants. Indeed, Ity14mutant mice had a significantly higher bacterial load in the spleenat day 4 postinfection (Fig. 4A), as well as higher bacterial burdenin the liver compared with wild-type littermates (Fig.4B). Asexpected, Ity14 mutants had significantly decreased serum IFN-glevels and increased IL-12p70 levels in serum at day 4 postin-fection compared with wild-type littermates, as observed duringsystemic infection (Fig. 4C, 4D). The above data are consistent withwhat we observed in Stat4 KO mice (Supplemental Fig. 1E–G). In

the context of the oral Salmonella infection model, this set ofexperiments demonstrates that STAT4 is important in controllingbacterial dissemination to the spleen and liver in the presence ofintestinal disease.

Genome-wide expression profiles in the spleen of Stat4Ity14/Ity14

versus Stat4+/+ mice during acute S. Typhimurium infection

To assess the impact of the ENU-induced Stat4 mutation in Ity14mice on innate immunity to Salmonella infection, we performedexpression microarrays. Genome-wide expression analysis was

FIGURE 3. Decreased systemic

IFN-g secretion results in increased

bacterial load. (A) Bacterial load in

spleen and liver at day 4 following

Salmonella infection: Stat4+/+ (square),

Stat4+/Ity14 (triangle), and Stat4Ity14/Ity14

(circle). (B) IFN-g and (C) IL-12p70

production measured in serum at day

0 and day 4 postinfection in Stat4+/+

and Stat4Ity14/Ity14 mice. n = 5 per

genotype from two experiments for

IFN-g and n = 2 (day 0) and n = 5

(day 4) per genoytpe for IL-12p70.

(D) IFN-g measured in supernantant

of Stat4+/+ and Stat4Ity14/Ity14 ex-

planted splenocytes stimulated with

rIL-12 for 2, 4, 8, and 24 h. n = 2–3

per genotype from two experiments.

(E) IL-6, (F) TNF-a, and (G) IL-10

measured in supernatant of Stat4+/+

and Stat4Ity14/Ity14 explanted spleno-

cytes were stimulated with LPS for 2,

4, 8, and 24 h. n = 2–3 per genotype

from two experiments. *p = 0.0373,

**p = 0.0027, ***p , 0.0001 by un-

paired Student t test.

FIGURE 4. STAT4 controls sys-

temic dissemination of Salmonella

during intestinal disease. Bacterial

load in (A) spleen and (B) liver of

Stat4+/+ and Stat4Ity14/Ity14 mice at

day 1 and day 4 postinfection. (C)

Serum IFN-g levels and (D) IL-

12p70 levels at day 1 and 4 postin-

fection in Stat4+/+ and Stat4Ity14/Ity14

mice. (E) Pathology score in cecum

of Stat4+/+ and Ity14 mice at day 1

and day 4 postinfection; edema (black),

PMN infiltration (gray), goblet cell

count (light gray), and epithelial

layer integrity (white). Pathology

score analyzed by two-way ANOVA

between wild-type (WT) and Ity14

mutant (MUT) mice at day 4 postin-

fection, Statistical significances by

unpaired Student t test are indicated

as follows: ***p , 0.0001.

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done on Stat4+/+ and Stat4Ity14/Ity14 mRNA isolated from thespleen of uninfected mice versus day 4 Salmonella-infected mice.Venn diagram results showed that 111 genes were transcription-ally modulated at least 2-fold in a similar way in both wild-typeand Ity14 mutants (76 genes upregulated and 35 genes downreg-ulated) at day 4 postinfection. In addition, subsets of genes weredifferentially regulated uniquely in wild-type (46 genes upregu-lated and 40 genes downregulated) and Ity14 mutants (50 genes

upregulated and 29 genes downregulated) (Fig. 5A, SupplementalTable IA–C). By looking at overall expression patterns of genessignificantly up- or downregulated in wild-type or Ity14 mutantsafter Salmonella infection, we identified two clusters of genes thateither are unique to wild-type or Ity14 mutants, which we furtherfocused on (Fig. 5B).The transcriptional signature common to both wild-type and

Stat4Ity14/Ity14 consisted of genes critical in inflammation. Clusters

FIGURE 5. Genome-wide expression profiles in spleen of Ity14 mice during Salmonella infection. (A) Venn diagram results identify genes transcrip-

tionally modulated in spleen at least 2-fold between Stat4+/+and Stat4Ity14/Ity14 mice at day 4 postinfection. (B) Heatmaps showing patterns of gene ex-

pression unique to wild-type or Stat4Ity14/Ity14 mice. (C) Select subset of genes in the IFN-g resistome and (D) innate immune cell–cell interactions. (E) List

of canonical pathways enriched in wild-type or Stat4Ity14/Ity14 mice and associated statistical 2log (p value).

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of genes identified were involved in cell recruitment and cytokinesignaling (Ccl3, Ccl4, Cxcl1, Cxcl9, Cxcl10, Ccl21a, Ccl21c,Socs3), type 1 IFN pathway (Usp18, Upp1, Stat1, Irf1, Ifi27, Ifi47,Irgm1, Oasl2), acute phase response (Saa3), proinflammatory sig-naling (Il1b, Il1rn, Casp1, Casp4), cell growth (Slfn1), and innateimmunity to bacterial infections (Lcn2, Cebpb, Cd14, Usp18, Mpo).Consistent with the role of STAT4 in IFN-g–mediated immunity,

the global gene expression pattern observed solely in wild-typemice over the course of Salmonella infection was predominantlyIFN-g driven. In addition to IFN-g, a significant number of genesthat make up the IFN-g resistome were differentially regulatedfollowing infection, including the guanylate-binding proteins(Gbp1, Gbp2, Gbp5, Gbp6, Aif1, Fcgr3, Igfbp4, Ligp2, Mpo, Mt1,Psmb10, Scd1, Sepp1, Timp2, Trafd1, Ubd, Wars) (Fig. 5C).Furthermore, we observed upregulation of genes critical in co-stimulatory signal transduction and lymphocyte activation (Slamf8,Nkg7), neutrophil recruitment and cytotoxicity (S100a8, Plac8), Agpresentation (Psmb10), cell adhesion (Emilin2, Lgals9), apoptosis(Batf2, Mfge8), and serine protease activity (Prtn3, Ctsc, Ctsg,Serpinaf). The shift toward development of a Th1 immune responsetriggered by Salmonella infection in wild-type mice coincided withdownregulation of certain genes implicated in Th2 immunity(Sepp1, Ltbp4, Fcna). Although significantly upregulated in bothStat4+/+ and Stat4Ity14/Ity14, a number of genes were upregulated atday 4 postinfection but to a lesser extent in Stat4Ity14/Ity14 micecompared with Stat4+/+ littermates. The following includedT lymphocyte chemoattractants (Cxcl9 and Cxcl10), genes in-volved in IFN signaling (Irf1, Irgm1, Ifi47, Stat1) and lymphocyteactivation and maturation (Ly6c, Ly6a). Overall, the canonicalpathways enriched [2log(p value)] in wild-type mice comparedwith Ity14 mutants consisted of communication between innateand adaptive immunity, cell-mediated apoptosis, costimulationand activation of T cell response, as well as cytokine/chemokinesignaling (IPA) (Fig. 5E).Of interest, various pathogen recognition receptors critical in

innate immune signaling and activation of host defense mecha-nisms were significantly upregulated in Stat4Ity14/ty14 mice, but notin Stat4+/+ mice, upon Salmonella infection. The following in-cluded members of the C-type lectin family (Clec4a3, Clec4n),scavenger receptor family (Marco), SIRP family (Sirpb1), TLRfamily (Tlr2), and paired Ig-like receptors (Pira3, Pira4, Pira11)(Fig. 5D). There was significant enrichment [2log(p value)] ofinnate immune recognition canonical pathways in Ity14 mutantsversus wild-type controls (IPA) (Fig. 5E). The NF-kB pathway haspreviously been reported to be induced during Salmonella infec-tion. We observed significant upregulation of transcripts clusteringin the NF-kB pathway, including Irak3, Irf7, Myd88, and Nfkbia,as well as downstream antiapoptotic transcripts (Ier3, Bcl2a1c)unique to Stat4Ity14/Ity14 mice. In addition, Ity14 mutants upregu-lated expression of proinflammatory mediators (Il1a, Iftm2, Iftm6,Mmp3, Mmp9, Mmp14), cell growth factors (Anxa2, Anxa3), andinhibitors of cathepsins (Cstb, Stfa1). Although significantly up-regulated in both Stat4+/+ and Stat4Ity14/Ity14, a set of transcriptswere upregulated to a greater extent in Stat4Ity14/Ity14 mice com-pared with wild-type mice. The following included genes involvedin cytokine and chemokine signaling (Ccl3, Ccl4, Cxcl1, Socs3),innate immunity (Cd14, Cebpb, Chi3l1, Fcgr4, Timp1), type 1IFN signaling (Oas2), acute phase response (Saa3), and IL-1signaling (Il1b, Il1rn). A subset of transcripts were further se-lected, including previously known inflammatory mediators withIFN-g–independent regulation following Salmonella infection(Lcn2, Mpo, Usp18), anti-inflammatory cytokine Il10, and IFN-g–regulated genes (Gbp5, Stat1), to validate the expression datausing quantitative RT-PCR (Supplemental Fig. 2). Taken together

with the enhanced susceptibility to Salmonella infection in Ity14mutants, upregulation of various innate immune recognition recep-tors suggests a possible coping strategy for impaired immunity inthese mice.

Impaired IFN-g secretion primarily from NK and NKT cellscontributes to impaired Salmonella control in Stat4Ity14/Ity14

mice

Given the heterogeneity of IFN-g–producing lymphocytes thatcontribute to the control of S. Typhimurium infection, we exam-ined cell-specific IFN-g contribution by multistain flow cytometry(47–49). Upon Salmonella infection, total spleen cell numbersincrease similarly both in wild-type [226.1 6 71.4 (3106)] and inmutant [195.3 6 71.6 (3106)] mice (Fig. 6A). We did not observedifferences in the percentages of CD3-DX5+ NK cells, CD3+

DX5+ NKT cells, CD4+ or CD8+ T lymphocytes, and CD45R+

B lymphocytes between wild-type and mutants prior to and atday 4 after Salmonella infection (data not shown). We furtherconfirmed decreased IFN-g and increased IL-4 expression at thetranscript level in the spleen at day 4 following Salmonella in-fection in Stat4Ity14/Ity14 mice (Fig. 6B, 6C). To study intracellularcell-specific cytokine secretion, splenocytes from uninfected andday 4 Salmonella-infected mice were stimulated ex vivo withPMA and ionomycin. Salmonella-infected NK and NKT cellsisolated from Stat4Ity14/Ity14 mice produced significantly lessIFN-g [15.8%6 9.4 (NK) and 12.6+1.9% (NKT)], compared withlittermate controls [45.9%610.6 (NK) and 41%68.2 (NKT)] (Fig.6D, 6E). Of interest, we did not observe any difference in thepercentage of IFN-g secreted from CD4+ or CD8+ T cells inSalmonella-infected splenocytes. However, Ity14 mutants hadsignificantly lower IFN-g mean fluorescence intensity (MFI) inCD4+ T cells (Fig. 6F). Furthermore, a trend toward lower IFN-gMFI from CD8+ T cells from Ity14 mutants was observed inSalmonella-infected splenocytes (data not shown). In addition,there was increased IL-4 secretion and IL-4 expression by MFI inCD4+ T cells from Stat4Ity14/Ity14 mice after Salmonella infection(Fig. 6G). Previously, IFN-g+CD4+ and IFN-g+CD8+ T lympho-cytes were reported to acquire an activated phenotype (CD44hi,CD62Llow), and reach maximal IFN-g production, only about 2–3 wk following virulent S. Typhimurium (SL1344) infection inmice (50). Therefore, early susceptibility in Stat4Ity14/Ity14 mice,prior to day 5 postinfection, likely explains the observed minimalT lymphocyte IFN-g response. The following suggests that theimpairment of IFN-g secretion in Stat4Ity14/Ity14 mice upon Sal-monella infection was primarily contributed by NK and NKT celldeficiency.

Increased expression of innate immune signaling genes andCD11b on a subset of DCs in Stat4Ity14/Ity14 mice followingSalmonella infection

Several of the genes identified in the microarray analysis that areupregulated in Stat4Ity14/Ity14 mice during infection are reported inBioGPS to be expressed in myeloid CD8a2 DCs and/or gran-ulocytes, as well as macrophages stimulated with LPS (Clec4a3,Clec4n, Il1b, Oas2, Fcgr4, Sirpb1a) (51). Another subset of theabove genes is mainly expressed only in macrophages stimulatedwith LPS (Tlr2, Pira3, Pira4, Pira11, Myd88, Mmp14, Anxa2,Anxa3, Cstb, Ccl3, Ccl4, Cxcl1). We further characterized dif-ferent myeloid cell populations in the spleen of infected wild-typeand Stat4Ity14/Ity14 mutants by surface staining markers and flowcytometry. We did not observe any differences in the percentage ofsplenic macrophages, granulocytes, or DC populations duringSalmonella infection (data not shown, Fig. 7A). Of note, we ob-served significantly greater expression of CD11b (by MFI) in

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a CD32CD8a2CD45R2Gr12CD11clowCD11b+ splenic myeloidDC subset in Salmonella-infected Stat4Ity14/Ity14 versus Stat4+/+

mice (Fig. 7B). Expression of CD11b is involved in leukocyte

adhesion and migration, as well as complement-mediated op-sonization of bacteria. These results provide insight into innateimmune regulation in Stat4Ity14/Ity14 mice and suggest that altered

FIGURE 6. Flow cytometry analysis in uninfected and Salmonella-infected Ity14mice. (A) Total spleen cell counts at day 0 and day 4 postinfection in Stat4+/+

and Stat4Ity14/Ity14mice. (B) IFN-g and (C) IL-4 mRNA expression in spleen at day 0 and day 4 postinfection. Flow cytometry histograms with corresponding bar

graphs show percentage of (D) NK cells, (E) NKT cells, and (F) CD4+ T cells producing IFN-g, and MFI of IFN-g in the above cell types in uninfected and day

4 Salmonella-infected splenocytes from Stat4+/+and Stat4Ity14/Ity14 mice. (G) Flow cytometry histograms with corresponding bar graphs show percentage of

CD4+ T cells producing IL-4, and MFI of IL-4, in uninfected and day 4 Salmonella-infected splenocytes from Stat4+/+ and Stat4Ity14/Ity14 mice. For NK cells at

day 0, n = 8 per genotype; day 4 postinfection, n = 14 per genotype from at least three separate experiments. For NKT cells at day 0 postinfection n = 6 per

genotype; day 4 postinfection n = 11 per genotype from three separate experiments. For CD4+ cells at day 0, n = 2 per genotype; day 4 postinfection n = 3 per

genotype from two different experiments. Statistical significances by unpaired Student t test are indicated as follows: *p, 0.05, **p# 0.01, and ***p, 0.005.

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myeloid cell receptor expression mediates pathogen recognitionand limits Salmonella replication.

DiscussionThe use of ENU chemical mutagenesis in the mouse has proved aneffective means to identify and study mutations in genes that affectthe host immunological response to microbial challenge (52–54).We used an ENU mutagenesis approach to screen for recessivegermline mutations conferring increased mortality followingS. Typhimurium infection, a globally relevant human pathogen(55). In this article, we report the identification of the Ity14 de-viant pedigree. A combination of mapping and exome sequencingwas used to identify a splice site point mutation in STAT4(p.G418_E445). The STAT4 mutation was the only homozygousmutation that segregated in all susceptible mice and within the2.7-Mb mapped interval. This mutation likely interferes with thenormal DNA-binding function of STAT4 and in regulating tran-scription of target genes. Stat4 is expressed at the transcript levelin Ity14 mutants, but is a hypomorph with significantly decreasedSTAT4 expression at the protein level, as shown by Western blot inuninfected and infected splenic tissue, suggesting that the proteinis either rapidly degraded or its translation is inhibited. The Ity14allele is a recessive loss of function of Stat4 that was confirmed bycomplementation assays wherein Stat4Ity14/Ity14, Stat4 KO, andcompound heterozygous mice present similar survival curves fol-lowing Salmonella infection.Intact IFN signaling is essential in the host immune response to

viral, bacterial, and fungal disease. The importance of IFN sig-naling has been highlighted by patients carrying mutations in genesin the IFN-a/b and IFN-g pathways, which present with increasedsusceptibility to infection. Individuals with a defect in productionor response to IFN-g have been shown to have increased sus-ceptibility to Mendelian susceptibility to mycobacterial disease,viral disease, or Salmonella infection. These genetic deficiencieshave been identified in the following genes: IFNGR1, IFNGR2,STAT1, IL12B, IL12RB1, NEMO, and TYK2 (15, 56, 57). DeficientIL-12 signaling results in impaired Th1 differentiation and IFN-gproduction. In fact, multiple episodes of salmonellosis are re-portedly frequent in IL-12Rb1–deficient patients, illustrating theimportance of the IL-12/IFN-g axis in mounting an efficient im-mune response to primary and secondary infections (17). However,no mutations in STAT4 have been identified in humans to date.We have previously reported another ENU-induced mouse

mutant, Usp18Ity9, a negative regulator of type 1 IFN signaling,which emphasizes the importance of IFN signaling in suscepti-bility to Salmonella infection (42, 45). We have shown in this modelthat transient suppression of STAT4-induced IFN-g production

contributes in part to the pathogenesis of the disease. In USP18mutants, disease susceptibility was mainly driven by hyperactiva-tion of the type 1 IFN pathway, resulting in increased levels of IL-6and IFN-b in circulation and development of septic shock. In thecurrent article, we specifically demonstrate the impact of Stat4 inSalmonella disease susceptibility.Over the past decade, studies in Stat4 KO mice have illustrated

the importance of functional STAT4 in both infectious and non-infectious diseases (36, 58, 59). Stat4 KO mice have increasedsusceptibility to infections primarily driven by a Th1 immuneresponse, including those caused by Mycobacterium tuberculosis,Leishmania major, Trypanosoma cruzi, Toxoplasma gondii, Ba-besia, and Listeria monocytogenes (36). In contrast, STAT4 defi-ciency has generally been shown to protect from T cell–mediatedautoimmune diseases, including experimental allergic encephalo-myelitis, a model for multiple sclerosis and collagen-induced ar-thritis, and a model for rheumatoid arthritis (60, 61). Furthermore,Stat4 KO mice injected with LPS were shown to be somewhatprotected from endotoxemia (62).STAT4 is a transcription factor and has been shown to bind to

the promoter and/or drive expression of several target genes(63–65). Recent genomic approaches by chromatin immuno-precipitation–on–chip and chromatin immunoprecipitation–se-quencing have elucidated STAT4 binding sites, target genes, andSTAT4-dependent epigenetic modifications on a genome-widelevel (65–67). These studies have been done in activated CD4+

T cells cultured under Th1 cell conditions. As such, comparisonsbetween our microarray data done in uninfected and day 4 post-infected spleen, and previous studies, must be interpreted withcaution. The above potentially explains why we did not detect otherknown STAT4 targets, including Furin, Il18r1, and Il12rb2, andsignature Th1 cell genes, including Tbx21. These discrepancieshighlight the difference between in vitro and in vivo studies, and theimportance of studying the impact of STAT4 at the whole-organismlevel.The transcriptional signature was different in Stat4Ity14/Ity14 mice

compared with Stat4+/+ following Salmonella infection, whichcorrelates with the histopathological results. As expected, IFN-gand IFN-g–regulated gene expressions were downregulated inIty14 mutants, including a subset of guanylate-binding proteins(Gbp1,2,5,6) previously reported to protect against bacterialinfections (68, 69). We also observed impaired upregulation ofStat1, consistent with STAT1 as a target of STAT4. IFN-g has beenreported to have anti-inflammatory properties in suppressingIL-1a,b production by myeloid cells during Mycobacteriumtuberculosis infection (70). Consistent with these findings, wedetected increased expression of genes in the IL-1 signaling

FIGURE 7. Increased expression of CD11b receptors on a subset of DCs in Stat4Ity14/Ity14 mice following Salmonella infection. (A) Dot plots of

splenocytes gated on CD11clow and CD11b+ having gated for surface markers CD32CD8a2CD45R2Gr12. Bar graph representations of percentage and (B)

CD11b MFI of the CD11clow CD11b+ subpopulation. At day 0 n = 2 per genotype repeated in two separate experiments; day 4 postinfection n = 3 per

genotype repeated in three different experiments. Statistical significances by unpaired Student t test are indicated as follows: **p , 0.01.

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pathway (Il1a, Il1b, Ilrn) in Stat4Ity14/Ity14 mice that have lowlevels of circulating IFN-g.Stat4Ity14/Ity14 mice had significantly lower levels of IFN-g in

circulation, which contributed to impaired microabscess formationand increased systemic bacterial burden in the spleen and liver.This finding is consistent with previous data showing increasedbacterial burden in STAT4 KO mice compared with littermatecontrols in spleen and liver following S. Typhimurium infection(42). The following further extends to a model of intestinal dis-ease, wherein the absence of STAT4 leads to bacterial dissemi-nation to the spleen and liver with intestinal disease. It haspreviously been shown that positive feedback regulation existsbetween IL-12 and IFN-g (71). However, excessive amplificationof the IL-12/IFN-g axis can lead to immunopathological changes,and therefore several mechanisms are involved in regulating theIL-12 pathway (72). In fact, increased IFN-g correlated with lowerIL-12p70 serum levels in wild-type mice. In contrast, Ity14mutants with impaired IFN-g expression had significantly in-creased IL-12p70 levels in serum at day 4 following Salmonellainfection, consistent with the lack of negative feedback regulation.Previous studies have suggested differing roles of neutrophils,macrophages, T lymphocytes, and NK and NKT cells in con-tributing to IFN-g production during primary Salmonella infection(47–49, 73, 74). Recently, Kupz et al. (75) showed that Thy1-expressing precursor and immature NK cells are critical in anti-bacterial immunity via IFN-g–dependent control of S. Typhimu-rium. In addition, NK and NKT cells are reported to be earlyproducers of IFN-g from healthy human adult blood lymphocytesstimulated ex vivo with NTS contributing to protection frombacteremia (76). In the current study, we demonstrate that a defi-ciency in NK and NKT cell IFN-g secretion correlates with earlysusceptibility to S. Typhimurium infection in Stat4Ity14/Ity14 mice.In Stat4Ity14/Ity14 mice, we observed upregulation of various

genes following Salmonella infection involved in pathogen rec-ognition. For example, we observed an increase in expression ofPira3, Pira4, and Pira11 genes. The murine paired Ig-likereceptors PIR-A and PIR-B, activating and inhibitory receptors,respectively, are maintained in balance to regulate the host in-flammatory response. PirB2/2 mice have been reported to haveincreased susceptibility to Salmonella infection (77). These micealso have impaired DC maturation, increased Th2 immunity, andupregulation of PIR-A (78). In Stat4Ity14/Ity14 mutants, upregula-tion of the PirA group of genes suggests a potential shift in in-flammatory response. Indeed, lymphocytes from Stat4 KO micecultured under Th1 conditions produce higher levels of Th2cytokines (59). Consistent with these data, Stat4Ity14/Ity14 mice hadincreased IL-4 mRNA expression following Salmonella challenge.Furthermore, we also observed upregulation of innate immune

cell receptors like C-type lectins, macrophage scavenger receptors(Marco), TLR signaling (Tlr2), proinflammatory mediators (Il1a,Mmp3, Mmp9, Mmp14), and NF-kB signaling. A proportion ofthese upregulated genes in Stat4Ity14/Ity14 mutants clustered to-gether, showing high expression levels in myeloid CD8a2 DCsand/or granulocytes, and in LPS-stimulated macrophages. In ad-dition, CD11b is a b2 integrin involved in regulating leukocyteadhesion and migration during inflammation. CD11b is also a re-ceptor of C3bi that coats bacteria to drive complement-mediatedopsonization. Increased expression of CD11b on DCs in Stat4Ity14/Ity14

mice suggests a possible overwhelming and compensatory mech-anism in the mutants to facilitate complement-mediated Sal-monella opsonization and killing. Overall, the upregulation ofexpression of immune signaling genes suggests that Stat4Ity14/Ity14

mutants have adapted alternative mechanisms to counteract thelack of STAT4-induced IFN-g–mediated immunity. Ultimately,

the lack of IFN-g is detrimental to the host, as Stat4Ity14/Ity14

mutants develop fatal bacteremia following Salmonella infection.

AcknowledgmentsWe thank Genevieve Perreault, Nadia Prud’homme, Vanessa Guay, Marie

Chevenon, Lei Zhu, and Line Lariviere for technical assistance, and Sean

Wiltshire and Sean Beatty for help with R code.

DisclosuresThe authors have no financial conflicts of interest.

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