Altered IFN- −γ Mediated Immunity and Transcriptional Expression Patterns in N -Ethyl-N-Nitrosourea−Induced STAT4 Mutants Confer Susceptibility to Acute This information is current as Typhoid-like Disease of September 25, 2021. Megan M. Eva, Kyoko E. Yuki, Shauna M. Dauphinee, Jeremy A. Schwartzentruber, Michal Pyzik, Marilène Paquet, Mark Lathrop, Jacek Majewski, Silvia M. Vidal and

Danielle Malo Downloaded from J Immunol 2014; 192:259-270; Prepublished online 27 November 2013; doi: 10.4049/jimmunol.1301370 http://www.jimmunol.org/content/192/1/259 http://www.jimmunol.org/

Supplementary http://www.jimmunol.org/content/suppl/2013/11/27/jimmunol.130137 Material 0.DC1 References This article cites 76 articles, 24 of which you can access for free at: http://www.jimmunol.org/content/192/1/259.full#ref-list-1 by guest on September 25, 2021

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Altered IFN-g–Mediated Immunity and Transcriptional Expression Patterns in N-Ethyl-N-Nitrosourea–Induced STAT4 Mutants Confer Susceptibility to Acute Typhoid-like Disease

Megan M. Eva,*,† Kyoko E. Yuki,*,† Shauna M. Dauphinee,*,† Jeremy A. Schwartzentruber,*,‡ Michal Pyzik,*,† Marile`ne 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 Downloaded from fact, patients immunodeficient in 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 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

Ity14 mice, the increased splenic and hepatic bacterial load resulted from an intrinsic defect in innate cell function, IFN-g–mediated http://www.jimmunol.org/ 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.

cute foodborne bacterial infections remain a major public waterborne illnesses, from a self-limiting, localized gastroenteritis by guest on September 25, 2021 health problem associated with high morbidity and mor- to the more severe, potentially fatal systemic disease of typhoid A tality worldwide. The intracellular bacterium Salmonella fever (2). Certain infected individuals (1–4%) may further develop enterica continues to pose a global challenge to human health (1). recurrent infections or become asymptomatic chronic carriers acting In humans, Salmonella infections cause a range of foodborne and as a reservoir for pathogen persistence and dissemination in the population (3, 4). Typhoid fever is strictly caused by an infection *Department of Human Genetics, McGill University, Montreal, Quebec H3G 0B1, with the human-restricted S. enterica serovars Typhi and Paratyphi. Canada; †Complex Traits Group, McGill University, Montreal, Quebec H3G 0B1, It is primarily endemic in developing areas of the world where poor Canada; ‡McGill University and Genome Quebec Innovation Centre, Montreal, Que- x sanitation and lack of access to clean drinking water are common bec H3B 1S6, Canada; De´partement de Pathologie et de Microbiologie, Faculte´ de Me´decine Ve´te´rinaire, Universite´ de Montre´al, Saint-Hyacinthe, Quebec J2S 5B5, (5). In contrast, nontyphoidal Salmonella (NTS) serovars, such as { Canada; and Department of Medicine, McGill University, Montreal, Quebec H3G Salmonella Typhimurium and Salmonella Enteritidis, are capable of 0B1, Canada infecting a broad spectrum of hosts, resulting in intestinal and di- Received for publication May 23, 2013. Accepted for publication October 25, 2013. arrheal disease (salmonellosis). NTS serovars are the second leading This work was supported by funds from the Team Program of the Canadian Institutes cause of foodborne illnesses in North America. Approximately 5% of Health Research (to S.M.V. and D.M.). M.M.E. was recipient of a studentship from the Research Institute of the McGill University Health Centre. K.E.Y. received a Fac- of patients with salmonellosis are at increased risk of further de- ulty of Medicine Internal Studentship award. S.M.D. received a Faculty of Medicine veloping invasive transient bacteremia and sepsis (6). Indeed, dis- David Lin fellowship, a Research Institute of the McGill University Health Centre Salmonella fellowship, and a Fonds de la Recherche du Que´bec fellowship. S.M.V. holds a Can- ease manifestation of -related infections in humans is ada Research chair and D.M. is a McGill Dawson scholar. dependent on the complex interaction between environmental fac- The sequences presented in this article have been submitted to the ArrayExpress tors, bacterial serotype, and host genetic factors. database (http://www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-1931. The outcome of S. enterica infection relies on the activation of Address correspondence and reprint requests to Dr. Danielle Malo, Department of both early innate functions and adaptive humoral and cell-mediated Human Genetics and Department of Medicine, McGill University Life Sciences immune responses of the host (7, 8). During systemic Salmonella Complex, Bellini Building, 3649 Promenade Sir-William-Osler, Montreal, QC H3G 0B1, Canada. E-mail address: [email protected] infection, rapid neutrophilic infiltration and phagocytosis by tissue The online version of this article contains supplemental material. macrophages are crucial in limiting hepatosplenic infection. Acti- Abbreviations used in this article: DC, dendritic cell; ENU, N-ethyl-N-nitrosourea; vated macrophages limit Salmonella replication and dissemination IPA, Ingenuity pathway analysis; KO, knockout; MFI, mean fluorescence intensity; to new foci by phagolysosomal bacterial killing and secretion of in- NTS, nontyphoidal Salmonella; PMN, polymorphonuclear cell; SNP, single nucleo- flammatory chemokines and cytokines, including TNF-a, IL-12, tide polymorphism. IL-18, and IFN-g (7). The inflammatory environment results in Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 the rapid recruitment of leukocytes that increase the ability for www.jimmunol.org/cgi/doi/10.4049/jimmunol.1301370 260 ROLE OF STAT4 IN SALMONELLA INFECTION intracellular pathogen killing and formation of granulomas. Im- following sublethal invasive S. Typhimurium challenge. Overall, portantly, IL-12 induces IFN-g secretion from NK cells, which is Stat4Ity14/Ity14 mice have increased mortality following infection, critical in activating macrophages in the early response against with a concomitant progressive increase in splenic and hepatic systemic Salmonella infections (9, 10). Protective immunity is bacterial load. Using genome-wide expression microarrays in spleen further mediated by IFN-g production from activated CD4+ Th1 tissue from wild-type and Ity14 mutants, we studied the impact of cells as well as the cytotoxic response of CD8+ T lymphocytes Stat4 on the inflammatory immune response to Salmonella infec- (11). Indeed, immunodeficient mice lacking IFN-g or IFN-gR fail tion. We validated the importance of IFN-g–mediated immunity to resolve primary infection with an attenuated Salmonella strain during systemic Salmonella infection. We further show that NK and (12). Moreover, neutralization of IL-12 in a mouse model of ty- NKT cells play an important early role in controlling Salmonella in phoid fever abrogates host immunity to primary Salmonella in- Stat4Ity14/Ity14 mice. During oral infection using a model of intestinal fection (13). In addition, clinical reports have shown that patients typhlitis, mice lacking STAT4 also develop early dissemination of immunocompromised as a consequence of HIV infection, chronic S. Typhimurium and systemic disease. In addition, increased ex- granulomatous disease, or functional genetic defects in the IL-12/ pression of genes involved in cell–cell interactions and communi- IL-23 (IL-12b,IL-12Rb1) and IFN-g (IFN-gR1, IFN-gR2, cation, as well as increased CD11b expression on a subset of splenic STAT1) pathways are predisposed to Mendelian susceptibility to myeloid DCs, shows compromised recruitment of inflammatory mycobacterial disease and/or disseminated Salmonella infection cells to the spleen during Salmonella infection in Stat4 mutants. (14–19). The following highlights the importance of systemic immunity to control invasive S. enterica infections. Materials and Methods In mice, S. Typhimurium infection is a recognized experimental Downloaded from Mice model for studying acute systemic disease resembling clinical features of typhoid fever in humans (20, 21). Alternatively, the use All animal experiments were conducted following specific conditions 2/2 2/2 null outlined by the Canadian Council on Animal Care and protocols approved of humanized mice (Rag2 gc ; [NOD]-scid IL2rg ) and, 2/2 by the McGill University Animal Care Committee. The 129S1/SvImJ, more recently, Tlr11 mice, which have been shown to be ef- DBA/2J, C57BL/6J, and C.129S2-Stat4tm1Gru/J mice were purchased from ficiently colonized by S. Typhi, are providing novel tools to study The Jackson Laboratory (Bar Harbor, ME). Experiments were performed

typhoid fever (22–25). Following oral infection of mice, Salmo- using mice between 7 and 15 wk of age of both sexes. http://www.jimmunol.org/ nella invade the microfold cells in the intestinal epithelium and Chemical mutagenesis (ENU) are taken up by dendritic cells (DCs) and macrophages in the underlying Peyer’s patches before infecting the mesenteric lymph Generation 0 (G0) males on a 129S1 background between 8 and 12 wk of age were i.p. injected with a single ENU dose of 150 mg per kg body weight. nodes and eventually disseminating via the circulation to replicate Efficiently mutagenized G0 males temporarily experienced a period of in resident phagocytes of the spleen and liver (26, 27). Previously, infertility and only regained fertility after 11 wk. ENU-induced mutations the genetic and molecular basis of several mutations, including were brought to homozygosity using a three-generation breeding scheme. Nramp1/Slc11a1, Tlr4, and Pklr, has been shown to be important N3 progeny on a resistant 129S1-129X1 background were initially screened in resistance to Salmonella infection in mice (28–33). However, as for susceptibility to S. Typhimurium infection. the mouse genome has a low frequency of naturally occurring DNA preparation, genetic mapping, and genotyping by guest on September 25, 2021 spontaneous mutations, new approaches to identify host suscep- DNA was extracted from a tail piece of mice by proteinase K digestion and tibility or resistance genes in the mouse are essential to study the phenol-chloroform extraction. A genome scan was performed on DNA sam- host response to infectious diseases. As such, we have used an ples from 21 mice (9 susceptible, 12 resistant) and a panel of 708 single unbiased forward genomics approach by N-ethyl-N-nitrosourea nucleotide polymorphisms (SNPs) for 129 and DBA/2 strains of mice (ENU) germline mutagenesis to screen and identify novel muta- (Medium Density SNP Panel; Illumina GoldenGate, The Centre for Applied Genomics, University of Toronto, Toronto, ON, Canada). Mice were further tions critical in antibacterial immunity, as well as to investigate genotyped by SNP sequencing (McGill University and Genome Quebec). function in vivo in the context of Salmonella infection. The mutation in Stat4 in Ity14 mice (c.1335+5 G . A) was genotyped In this article, we report the identification of the Ity14 (Immu- using a Custom TaqMan SNP Genotyping Assay (Applied Biosystems, nity to Typhimurium locus 14) pedigree carrying an ENU-induced Streetville, ON, Canada). mutation in Stat4 (Signal Transducer and Activator of Tran- Whole-exome sequencing scription Factor 4) conferring increased susceptibility to sublethal primary Salmonella infection. The transcription factor STAT4 is We followed standard manufacturer protocols to perform target capture with the SureSelect Mouse All Exon Kit (Agilent Technologies, Santa Clara, CA) specifically expressed in myeloid and lymphoid cells and is and sequencing of 100-bp paired end reads on Illumina HiSEquation 2000. a critical mediator of IL-12 signaling in development of inflam- This process generated . 8 Gb of sequence for each of the two susceptible matory immune responses (34, 35). IL-12 production from acti- Ity14 samples. Reads were aligned to mm9 with BWA (37) and coverage vated macrophages and DCs activates TYK2 and JAK2 receptor– was assessed with BEDTools, showing an average of 58.9 reads covering each base of the consensus coding sequence genes for the mouse genome associated kinases, resulting in dimerization, phosphorylation, and (38). Single nucleotide variants and short insertions and deletions were activation of STAT4. STAT4 has been shown to be involved in called using samtools pileup and varFilter (39) with the base alignment both innate and adaptive immunity by regulating the transcription quality adjustment disabled, and were then quality filtered to require $ 20% of target genes, including IFN-g; NK cell cytotoxicity; Th1 cell of reads supporting the variant call. Variants were annotated using both differentiation from naive CD4+ T cells; and Ig isotype switching Annovar (40) and custom scripts to identify whether they affected the coding sequence, and whether they had previously been seen in to IgG1. Consistent with these findings, STAT4 is a central deter- mouse dbSNP128 or in any of seven mouse exomes sequenced in parallel. minant in host resistance to various bacterial, viral, and protozoan To detect splice site mutations, the threshold of detection was increased to infections while playing a critical role in regulating inflammatory 6 bp instead of the standard 2-bp flanking exons. immune-mediated diseases (36). In vivo Salmonella mouse infections and tissue bacterial load The physiological function of STAT4 in vivo in response to Mice were challenged i.v. in the caudal vein with an infectious dose of 5 3 S. Typhimurium infection remains poorly characterized. In this pa- 3 Ity14/Ity14 10 CFUs S. Typhimurium strain Keller. Dose preparation and method of per, we elucidate the impact of Stat4 on innate immunity infections have been previously described (41, 42). For the complemen- during Salmonella infection. We show that Ity14 mice with a hy- tation assay, mice were infected with a lower dose of 2.5 3 103 CFUs, pomorphic mutation in Stat4 have an increased innate susceptibility given that the C.129S2-Stat4tm1Gru/J mice are on a BALB/c background The Journal of Immunology 261

(33). Infected mice were monitored over the course of 14 d, and suscep- were stimulated and activated ex vivo with 50 mg/ml PMA and 1 mg/ml tible mutants defined by a body score index , 2 were euthanized by CO2 ionomycin (Sigma-Aldrich) for 4 h in the presence of GolgiStop (BD). asphyxiation. To determine bacterial load in organs at specific time points IFN-g (XMG1.2) and IL-4 (eBioscience) were stained according to the postinfection, aseptically harvested organs were weighed and homoge- manufacturer’s protocol (Cytofix/Cytoperm Plus Fixation/Permeabilization nized in 0.9% saline using a Polytron (Kinematica, Bohemia, NY). Serial Kit with GolgiStop; BD). Cells were acquired on a FACSCanto II, and dilutions of organ homogenates were then plated on trypticase soy agar to results were analyzed as described above. IL-6, TNF-a, and IL-10 cyto- determine bacterial counts. kines in the supernatant of splenocytes, as well as IFN-g and IL-12p70 in serum, were measured by sandwich ELISA (eBioscience). In vivo per os Salmonella infection Genome-wide expression microarrays At 1 d prior to infection, mice were fasted for 6 h. At 4 h into the fast, mice were gavaged with 20 mg streptomycin sulfate diluted in 100 mL sterile Spleens of Ity14 mice were collected for microarrays and frozen at 280˚C. water. An overnight culture of S. Typhimurium SL1344 was prepared in Total RNA was extracted from ∼ 50 mg splenic tissue, using TRIzol re- 5 ml TSB supplemented with 50 mg/ml streptomycin sulfate and incubated agent (Invitrogen Canada, Burlington, ON, Canada). RNA yield was de- at 37˚C. The day of the infection, 2 ml of the overnight culture was in- termined using a NanoDrop spectrophotometer (ThermoFisher Scientific, oculated in 50 ml TSB containing streptomycin, and grown to an OD of 0.9 Waltham, MA) and the overall quality assessed by denaturing agarose gel at 600 nm. Streptomycin-pretreated mice were each gavaged with 5 3 107 electrophoresis. Four uninfected and four day 4 S. Typhimurium–infected CFU bacteria resuspended in 100 ml sterile saline. For CFU enumeration, age-matched mice (two females, two males) per genotype were used for see the detailed method above. whole-genome expression profiling on Illumina Mouse-Ref-8 v2.0 Bead- Chip (Illumina, San Diego, CA). The quality control, hybridization, and Intestinal histopathological scoring array analysis were performed at the McGill University and Genome ´ H&E-stained cecum and colon slides were analyzed in duplicate and Quebec Innovation Centre (Montreal, QC, Canada). blinded for genotype. A histopathological scoring system was adapted Microarray data analysis Downloaded from from a previously described model (43). Briefly, the score evaluated the submucosal edema (0 = 0, 1 # 10%, 2 = 10–40%, 3 = .40%), the Raw data are available through the ArrayExpress database (www.ebi.ac.uk/ polymorphonuclear cell (PMN) infiltration of the lamina propria (0 = # 5 arrayexpress) under accession number E-MTAB-1931. Preliminary ex- PMN, 1 = 5–20, 2 = 21–60, 3 = 61–100, 4 = . 100), the goblet cell count pression data analysis was done using FlexArray 1.4.1 software, as pre- (0 = .10, 1 = 6–10, 2 = 1–5, 3 = 0 [per high-power field]), and the epi- viously described (44, 45) (Genome Que´bec, Montreal, QC Canada) thelial integrity (0 = no change, 1 = desquamation, 2 = erosion of the (http://genomequebec.mcgill.ca/FlexArray). Principal component analysis epithelial surface, 3 = ulceration) of cecum cross-sections (43). was generated in FlexArray. Briefly, gene lists for each sample group were http://www.jimmunol.org/ generated by selecting for genes with a false discovery rate p , 0.05 by the In vivo bioluminescence imaging unpaired Student t test and . or , 2-fold change. The canonical pathway 4 analyses were generated through the use of Ingenuity pathway analysis Mice were injected i.v. with 6 3 10 CFUs S. Typhimurium strain XEN26. Whole-body imaging of mice was performed daily, beginning on day 3 to (IPA; Ingenuity Systems, www.ingenuity.com). Heatmaps were generated day 8 postinfection on the xenogen IVIS Spectrum System (Caliper, Al- using R package (46). ameda, CA). Mice were anesthetized with isoflurane and anteriorly shaved Validation by quantitative real-time PCR and list of primers prior to imaging. Bioluminescent images were acquired with an open emission filter, binning factor of 16, and exposure time ranging from 2 to used 5 min. The regions of interest were selected, normalized across time RNA quantification by the SYBR Green–based detection was performed points, and quantified using Living Image software v.4.3.1 (Caliper).

using the Applied Biosystem StepOnePlus (Applied Biosystems, Carlsbad, by guest on September 25, 2021 CA). Complementary DNAs were amplified by PCR using the following Preparation of protein extracts for Western blot analysis primer pairs: Usp: 18 59-AGTGCTGTCTAGAGACCTCTGC-39 and 59- Protein extracts from spleen were prepared by homogenizing tissue using GGAGTTAAGGAACACGTCTG-39; Il4:59-TGAGAGAGATCATCGG- a Polytron (Kinematica) in lysis buffer (1 M Tris-HCl, pH = 8; 0.5 M KCl; CATTT-39 and 59-GTGAGGACGTTTGGCACATC-39; Il10:59-AGTGG- AGCAGGTGAAGAGTGA-39 and 59-ATGCAGTTGATGAAGATGTCA- 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% AA-39; Ifng:59-ACTGGCAAAAGGATGGTGAC-39 and 59-ATCCTTTT- amplitude. Protein extracts were centrifuged at 13,000 rpm at 4˚C for 15 TCGCCTTGCTGT-39; Lcn2:59-CAGAAGGCAGCTTTACGATGT-39 and min. Supernatants were collected and protein concentration quantified by 59-TGTTCTGATCCAGTAGCGACA-39; Mpo:59-ATGCTTCAGACCTC- Bradford assay (500-0006; Bio-Rad). Whole-cell lysates were extracted in CAATGGT-39 and 59-CTCTGTCCACTAGCTGCTTGG-39; Gbp5: 59-C- Laemmli buffer and probed by immunoblot using Abs for STAT4 and AGGCAAATCCTACCTGATGA-39 and 59-ACCAAAGTGTGGTCTGG- GAPDH (Cell Signaling). The STAT4 Ab recognizes an epitope around CTTT-39; Stat4:59-GCGTCCATTGACAAGAATGTT-39 and 59-CCTTG- lysine 151 upstream of the Ity14 mutation. GGTTGCAAATGTCTAA; Stat1:59-ACAACATGCTGGTGACAGAGC- C-39 and 59-TGAAAACTGCCAACTCAACACCTC-3 9; and normalized to Splenocyte preparation, Abs, and flow cytometry the reference Hprt gene: Hprt 59-GTTGGATACAGGCCAGACTTTGTTG-39 and 59-GATTCAACTTGCGCTCATCTTAGGC-39. The data obtained were Spleens were harvested and collected from uninfected and day 4 expressed as 22DDCt. S. Typhimurium–infected mice in 4 ml PBS under sterile conditions. Spleens were macerated with 70-mm cell strainers into single-cell suspensions, Statistical analysis treated with ACK lysis buffer, and washed in cold PBS. Cell counts were determined using a Coulter Z2 particle counter (Beckman Coulter). For Statistical analyses were done using GraphPad Prism v5.0 (GraphPad splenocyte stimulation, 5 3 105 cells were plated in 96-well round-bottom Software, La Jolla). plates and stimulated with recombinant mouse IL-12 (20 ng/ml; R&D Systems) or LPS (1 mg/ml; Sigma-Aldrich) for 2, 4, 8, and 24 h at 37˚C, 6 Results 5% CO2. For surface staining, 5 3 10 splenocytes were plated in 96-well round-bottom plates and stained for various cell surface markers, using Identification of the ENU-induced S. Typhimurium fluorochrome-labeled mAbs (eBioscience). The following anti-mouse Abs susceptibility locus, Ity14 were purchased from eBioscience: CD49b (clone DX5), CD4 (GK1.5), CD8a (53-6.7), CD45R (RA3-6B2), Ly-6G (RB6-8C5), F4/80 (BM8), The Ity14 pedigree was identified in an in vivo recessive screen of CD11c (HL3), and CD11b (M1/70). CD3e (500A2) was purchased from ENU-mutagenized mice for innate susceptibility to Salmonella BD. Fixable Viability Dye (eBioscience) to irreversibly stain dead cells infection, as measured by survival analysis (Fig. 1). Mutagenized was used in accordance with the manufacturer’s protocol. Cells were ac- 129S1 G0 males were crossed to wild-type 129X1 females to gen- quired on a FACSCanto II (BD) flow cytometer, and results were analyzed using FlowJo (v9.4.10) software. erate G1 mice. G1 males were further outcrossed to 129X1 females to produce G2 females. For each G1 pedigree, two G2 daughters Intracellular cytokine production were backcrossed to generate N3 offspring (Fig. 1A). We initially For intracellular cytokine staining, 10 3 106 splenocytes were plated in screened N3 mice on a resistant 129S1/129X1 mixed background complete RPMI 1640 media in six-well tissue culture–treated plates. Cells with a sublethal dose of S. Typhimurium and monitored mice 262 ROLE OF STAT4 IN SALMONELLA INFECTION Downloaded from

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, http://www.jimmunol.org/ 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 1 marker; Stat4Ity14/Ity14 (n = 26), Stat4+/Ity14 (n = 41), and Stat4+/+ (n = 11). ***p , 0.0001 by log- Mantel–Cox test. for 14 d for development of clinical disease. We observed 25% recognition by the splicesome U1 subunit, and the maximum mortality in Ity14 N3 progeny by day 6 postinfection (data not entropy score predicts significantly weakened splice site recog- shown).Tomapandidentifythemutation responsible for the nition (from 7.07 to 1.48) (http://genes.mit.edu/burgelab/maxent/ by guest on September 25, 2021 Salmonella-susceptibility phenotype, we outcrossed both the Xmaxentscan_scoreseq.html). Therefore, it was predicted that the G1 male and the G2 female to DBA/2J mice in an F2 cross. We mutation would interfere with normal splicing function. PCR observed 25% mortality by day 5 postinfection (Fig. 1B), con- amplification of cDNA isolated from wild-type, heterozygous, and sistent with the survival phenotype observed in N3 animals. Initial mutant spleens using primers located in flanking exons 14 and 16 genotyping was performed using a total of 21 F2 mice (9 sus- resulted in a smaller PCR product in mutant mice (Fig. 2B) as a ceptible, 12 resistant) and 708 SNPs (polymorphic between 129 result of an 84-bp deletion corresponding to exon 15. The muta- and DBA/2J). We detected linkage to a 25.6-Mb region on chro- tion resulted in a 28-aa deletion in the DNA binding domain of mosome 1 with a maximum LOD score of 6.23 at RS1347566. STAT4 (p.G418_E445) (Fig. 2C). We observed decreased ex- Fine mapping refined the Ity14 locus to a 2.7-Mb interval (Fig. pression of a smaller STAT4 protein product at days 0 and 4 1C). At the peak marker, F2 mice homozygous for the 129S1 postinfection in Stat4Ity14/Ity14 splenic tissue, compared with litter- allele were highly susceptible to S. Typhimurium infection by day mate controls (Fig. 2D). In contrast, at the transcript level, Stat4 gene 6 postinfection, whereas heterozygous and mice homozygous for expression was downregulated to a lesser extent in Stat4Ity14/Ity14 the DBA/2J allele were significantly more resistant (Fig. 1D). mice compared with wild-type at day 4 postinfection (Fig. 2E). Overall, we identified the Ity14 pedigree with increased suscep- We further validated STAT4 as the candidate gene responsible for tibility to Salmonella infection contributed by a 2.7-Mb interval the susceptibility phenotype in the Ity14 pedigree by allelic on proximal . complementation assays. Stat4Ity14/+ mice were crossed to Stat42/2 mice, and susceptibility to infection was assessed by survival A causal mutation in Stat4 underlies the Ity14 analysis in F2 progeny. We observed a lack of complementation Salmonella-susceptibility locus in Stat4Ity14/2 mice with a mean survival time equivalent to that Whole-exome sequencing of coding exons and flanking splice in Stat4Ity14/Ity14 and Stat42/2 animals, thereby confirming that junctions in two susceptible Ity14 mice identified a novel ENU- the mutation within Stat4 was responsible for the Ity14 phenotype induced mutation in Stat4. The average coverage across the (Fig. 2F). exome was 83-fold. The Stat4 mutation was the only one both validated and segregated with the survival phenotype from a list Impaired systemic IFN-g secretion and microabscess formation contribute to increased bacterial burden in spleen of predicted missense and splicing variants identified by exome Ity14/Ity14 sequencing (Supplemental Table II). The mutation consisted of and liver of Salmonella-infected Stat4 mice a guanosine to adenosine substitution within the splice donor Increased mortality following S. Typhimurium infection in site of exon 15, at position +5 of intron 15 (c.1335+5G . A) and Stat4Ity14/Ity14 mutants paralleled the significantly higher bacterial was confirmed by Sanger sequencing (Fig. 2A). This guanosine is a load in target organs, specifically in the spleen and liver at day 4 highly conserved nucleotide involved in the splice donor sequence postinfection. At day 4 following infection, we observed a 17.2- The Journal of Immunology 263 Downloaded from http://www.jimmunol.org/

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 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) by guest on September 25, 2021 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. fold change in Salmonella replication in the spleen and a 87.3-fold infiltrates composed of neutrophilic granulocytes and macro- change in the liver in Stat4Ity14/Ity14, compared with wild-type phages in liver, as well as in both white and red pulps of spleen, littermate controls (Fig. 3A). In vivo imaging using luminescent compared with wild-type controls (Supplemental Fig. 1C). S. Typhimurium strain XEN26 validated progressive increased bacterial load in Ity14 mutant mice over the course of infection STAT4 controls systemic dissemination of Salmonella during (Supplemental Fig. 1A, 1B). The in vivo systemic bacterial rep- intestinal disease lication observed in Stat4Ity14/Ity14 at day 4 after Salmonella in- To further demonstrate the importance of STAT4 in controlling fection parallels the low levels of circulating IFN-g measured in systemic S. Typhimurium infection, streptomycin-pretreated wild- the serum of these mice (Fig. 3B). Moreover, we observed in- type and Ity14 mutant mice were infected per os to study intestinal creased IL-12p70 levels in the serum of Stat4Ity14/Ity14 mice at day 4 pathological changes in the cecum, as well as bacterial dissemi- postinfection, suggesting a lack of negative feedback (Fig. 3C). nation to the spleen and liver at days 1 and 4 postinfection. No Consistent with the essential function of STAT4 in IFN-g induction histopathological differences in the cecum were observed at day 1 downstream of IL-12 signaling, we observed significantly lower postinfection; however, at day 4 postinfection we observed less levels of IFN-g production in the supernatant of Stat4Ity14/Ity14 submucosal edema, less PMN cell infiltration of the lamina propria, splenocytes following 24-h stimulation with recombinant mouse increased goblet cell count, and decreased epithelial integrity in IL-12 (Fig. 3D). IFN-g is an important mediator of granuloma Ity14 mutants, compared with wild-type (Fig. 4E). In contrast, Stat4 formation, critical to control and prevent intracellular bacterial knockout (KO) mice had increased pathological features at day 1 dissemination. Consequently, reduced levels of IFN-g are con- postinfection, with no differences at day 4 postinfection (Sup- sistent with the increased bacterial load observed in Stat4Ity14/Ity14 plemental Fig. 1D). We can clearly detect an effect of the back- mice. Although we did not observe any significant differences in ground on the severity of the pathological lesions: control mice on proinflammatory cytokine production, IL-6 and TNF-a,oranti- a BALB/cJ background were more affected than control mice on inflammatory cytokine IL-10 following LPS stimulation in ex- a mixed 129S1:DBA/2J background at day 1 postinfection, whereas planted splenocytes (Fig. 3E–G), histopathological H&E staining of the lesions were more prominent in Ity14 mutants compared with infected spleen and liver sections further demonstrates that Ity14 Stat4 KO at day 4. These data are consistent with our previous mutants have impaired immune cell–mediated recruitment to the observation that inbred strains present different clinicopathological site of infection. During Salmonella infection, Stat4Ity14/Ity14 have features of Salmonella-induced typhlitis (45). Despite these dif- both fewer and smaller multifocal microabscesses of inflammatory ferences in kinetics and amplitude of the histopathological scores 264 ROLE OF STAT4 IN SALMONELLA INFECTION

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 Downloaded from 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, http://www.jimmunol.org/ **p = 0.0027, ***p , 0.0001 by un- paired Student t test.

between Ity14 mutants and Stat4 KO, we observed dissemination the context of the oral Salmonella infection model, this set of of Salmonella to systemic sites in both mutants. Indeed, Ity14 experiments demonstrates that STAT4 is important in controlling by guest on September 25, 2021 mutant mice had a significantly higher bacterial load in the spleen bacterial dissemination to the spleen and liver in the presence of at day 4 postinfection (Fig. 4A), as well as higher bacterial burden intestinal disease. in the liver compared with wild-type littermates (Fig.4B). As Genome-wide expression profiles in the spleen of Stat4Ity14/Ity14 expected, Ity14 mutants had significantly decreased serum IFN-g +/+ levels and increased IL-12p70 levels in serum at day 4 postin- versus Stat4 mice during acute S. Typhimurium infection fection compared with wild-type littermates, as observed during To assess the impact of the ENU-induced Stat4 mutation in Ity14 systemic infection (Fig. 4C, 4D). The above data are consistent with mice on innate immunity to Salmonella infection, we performed what we observed in Stat4 KO mice (Supplemental Fig. 1E–G). In expression microarrays. Genome-wide expression analysis was

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 ( 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. The Journal of Immunology 265 done on Stat4+/+ and Stat4Ity14/Ity14 mRNA isolated from the upregulated and 29 genes downregulated) (Fig. 5A, Supplemental spleen of uninfected mice versus day 4 Salmonella-infected mice. Table IA–C). By looking at overall expression patterns of genes Venn diagram results showed that 111 genes were transcription- significantly up- or downregulated in wild-type or Ity14 mutants ally modulated at least 2-fold in a similar way in both wild-type after Salmonella infection, we identified two clusters of genes that and Ity14 mutants (76 genes upregulated and 35 genes downreg- either are unique to wild-type or Ity14 mutants, which we further ulated) at day 4 postinfection. In addition, subsets of genes were focused on (Fig. 5B). differentially regulated uniquely in wild-type (46 genes upregu- The transcriptional signature common to both wild-type and lated and 40 genes downregulated) and Ity14 mutants (50 genes Stat4Ity14/Ity14 consisted of genes critical in inflammation. Clusters Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

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). 266 ROLE OF STAT4 IN SALMONELLA INFECTION of genes identified were involved in cell recruitment and cytokine with the enhanced susceptibility to Salmonella infection in Ity14 signaling (Ccl3, Ccl4, Cxcl1, Cxcl9, Cxcl10, Ccl21a, Ccl21c, mutants, upregulation of various innate immune recognition recep- Socs3), type 1 IFN pathway (Usp18, Upp1, Stat1, Irf1, Ifi27, Ifi47, tors suggests a possible coping strategy for impaired immunity in Irgm1, Oasl2), acute phase response (Saa3), proinflammatory sig- these mice. naling (Il1b, Il1rn, Casp1, Casp4), cell growth (Slfn1), and innate Impaired IFN-g secretion primarily from NK and NKT cells immunity to bacterial infections (Lcn2, Cebpb, Cd14, Usp18, Mpo). Ity14/Ity14 Consistent with the role of STAT4 in IFN-g–mediated immunity, contributes to impaired Salmonella control in Stat4 the global gene expression pattern observed solely in wild-type mice mice over the course of Salmonella infection was predominantly Given the heterogeneity of IFN-g–producing lymphocytes that IFN-g driven. In addition to IFN-g, a significant number of genes contribute to the control of S. Typhimurium infection, we exam- that make up the IFN-g resistome were differentially regulated ined cell-specific IFN-g contribution by multistain flow cytometry following infection, including the guanylate-binding (47–49). Upon Salmonella infection, total spleen cell numbers (Gbp1, Gbp2, Gbp5, Gbp6, Aif1, Fcgr3, Igfbp4, Ligp2, Mpo, Mt1, increase similarly both in wild-type [226.1 6 71.4 (3106)] and in Psmb10, Scd1, Sepp1, Timp2, Trafd1, Ubd , Wars) (Fig. 5C). mutant [195.3 6 71.6 (3106)] mice (Fig. 6A). We did not observe Furthermore, we observed upregulation of genes critical in co- differences in the percentages of CD3-DX5+ NK cells, CD3+ stimulatory signal transduction and lymphocyte activation (Slamf8, DX5+ NKT cells, CD4+ or CD8+ T lymphocytes, and CD45R+ Nkg7), neutrophil recruitment and cytotoxicity (S100a8, Plac8), Ag B lymphocytes between wild-type and mutants prior to and at presentation (Psmb10), cell adhesion (Emilin2, Lgals9), apoptosis day 4 after Salmonella infection (data not shown). We further

(Batf2, Mfge8), and serine protease activity (Prtn3, Ctsc, Ctsg, confirmed decreased IFN-g and increased IL-4 expression at the Downloaded from Serpinaf). The shift toward development of a Th1 immune response transcript level in the spleen at day 4 following Salmonella in- triggered by Salmonella infection in wild-type mice coincided with fection in Stat4Ity14/Ity14 mice (Fig. 6B, 6C). To study intracellular downregulation of certain genes implicated in Th2 immunity cell-specific cytokine secretion, splenocytes from uninfected and (Sepp1, Ltbp4, Fcna). Although significantly upregulated in both day 4 Salmonella-infected mice were stimulated ex vivo with Stat4+/+ and Stat4Ity14/Ity14, a number of genes were upregulated at PMA and ionomycin. Salmonella-infected NK and NKT cells Ity14/Ity14 Ity14/Ity14 day 4 postinfection but to a lesser extent in Stat4 mice isolated from Stat4 mice produced significantly less http://www.jimmunol.org/ compared with Stat4+/+ littermates. The following included IFN-g [15.8% 6 9.4 (NK) and 12.6+1.9% (NKT)], compared with T lymphocyte chemoattractants (Cxcl9 and Cxcl10), genes in- littermate controls [45.9%610.6 (NK) and 41%68.2 (NKT)] (Fig. volved in IFN signaling (Irf1, Irgm1, Ifi47, Stat1) and lymphocyte 6D, 6E). Of interest, we did not observe any difference in the activation and maturation (Ly6c, Ly6a). Overall, the canonical percentage of IFN-g secreted from CD4+ or CD8+ T cells in pathways enriched [2log(p value)] in wild-type mice compared Salmonella-infected splenocytes. However, Ity14 mutants had with Ity14 mutants consisted of communication between innate significantly lower IFN-g mean fluorescence intensity (MFI) in and adaptive immunity, cell-mediated apoptosis, costimulation CD4+ T cells (Fig. 6F). Furthermore, a trend toward lower IFN-g and activation of T cell response, as well as cytokine/chemokine MFI from CD8+ T cells from Ity14 mutants was observed in signaling (IPA) (Fig. 5E). Salmonella-infected splenocytes (data not shown). In addition, by guest on September 25, 2021 Of interest, various pathogen recognition receptors critical in there was increased IL-4 secretion and IL-4 expression by MFI in innate immune signaling and activation of host defense mecha- CD4+ T cells from Stat4Ity14/Ity14 mice after Salmonella infection nisms were significantly upregulated in Stat4Ity14/ty14 mice, but not (Fig. 6G). Previously, IFN-g+CD4+ and IFN-g+CD8+ T lympho- in Stat4+/+ mice, upon Salmonella infection. The following in- cytes were reported to acquire an activated phenotype (CD44hi, cluded members of the C-type lectin family (Clec4a3, Clec4n), CD62Llow), and reach maximal IFN-g production, only about 2– scavenger receptor family (Marco), SIRP family (Sirpb1), TLR 3 wk following virulent S. Typhimurium (SL1344) infection in family (Tlr2), and paired Ig-like receptors (Pira3, Pira4, Pira11) mice (50). Therefore, early susceptibility in Stat4Ity14/Ity14 mice, (Fig. 5D). There was significant enrichment [2log(p value)] of prior to day 5 postinfection, likely explains the observed minimal innate immune recognition canonical pathways in Ity14 mutants T lymphocyte IFN-g response. The following suggests that the versus wild-type controls (IPA) (Fig. 5E). The NF-kB pathway has impairment of IFN-g secretion in Stat4Ity14/Ity14 mice upon Sal- previously been reported to be induced during Salmonella infec- monella infection was primarily contributed by NK and NKT cell tion. We observed significant upregulation of transcripts clustering deficiency. in the NF-kB pathway, including Irak3, Irf7, Myd88, and Nfkbia, Increased expression of innate immune signaling genes and as well as downstream antiapoptotic transcripts (Ier3, Bcl2a1c) Ity14/Ity14 unique to Stat4Ity14/Ity14 mice. In addition, Ity14 mutants upregu- CD11b on a subset of DCs in Stat4 mice following lated expression of proinflammatory mediators (Il1a, Iftm2, Iftm6, Salmonella infection Mmp3, Mmp9, Mmp14), cell growth factors (Anxa2, Anxa3), and Several of the genes identified in the microarray analysis that are inhibitors of cathepsins (Cstb, Stfa1). Although significantly up- upregulated in Stat4Ity14/Ity14 mice during infection are reported in regulated in both Stat4+/+ and Stat4Ity14/Ity14, a set of transcripts BioGPS to be expressed in myeloid CD8a2 DCs and/or gran- were upregulated to a greater extent in Stat4Ity14/Ity14 mice com- ulocytes, as well as macrophages stimulated with LPS (Clec4a3, pared with wild-type mice. The following included genes involved Clec4n, Il1b, Oas2, Fcgr4, Sirpb1a) (51). Another subset of the in cytokine and chemokine signaling (Ccl3, Ccl4, Cxcl1, Socs3), above genes is mainly expressed only in macrophages stimulated innate immunity (Cd14, Cebpb, Chi3l1, Fcgr4, Timp1), type 1 with LPS (Tlr2, Pira3, Pira4, Pira11, Myd88, Mmp14, Anxa2, IFN signaling (Oas2), acute phase response (Saa3), and IL-1 Anxa3, Cstb, Ccl3, Ccl4, Cxcl1). We further characterized dif- signaling (Il1b, Il1rn). A subset of transcripts were further se- ferent myeloid cell populations in the spleen of infected wild-type lected, including previously known inflammatory mediators with and Stat4Ity14/Ity14 mutants by surface staining markers and flow IFN-g–independent regulation following Salmonella infection cytometry. We did not observe any differences in the percentage of (Lcn2, Mpo, Usp18), anti-inflammatory cytokine Il10, and IFN-g– splenic macrophages, granulocytes, or DC populations during regulated genes (Gbp5, Stat1), to validate the expression data Salmonella infection (data not shown, Fig. 7A). Of note, we ob- using quantitative RT-PCR (Supplemental Fig. 2). Taken together served significantly greater expression of CD11b (by MFI) in The Journal of Immunology 267 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 6. Flow cytometry analysis in uninfected and Salmonella-infected Ity14 mice. (A) Total spleen cell counts at day 0 and day 4 postinfection in Stat4+/+ and Stat4Ity14/Ity14 mice. (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)NKcells,(E) NKT cells, and (F)CD4+ T cells producing IFN-g,andMFIofIFN-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 =6per 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 =3per 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. a CD32CD8a2CD45R2Gr12CD11clowCD11b+ splenic myeloid adhesion and migration, as well as complement-mediated op- DC subset in Salmonella-infected Stat4Ity14/Ity14 versus Stat4+/+ sonization of bacteria. These results provide insight into innate mice (Fig. 7B). Expression of CD11b is involved in leukocyte immune regulation in Stat4Ity14/Ity14 mice and suggest that altered 268 ROLE OF STAT4 IN SALMONELLA INFECTION

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. myeloid cell receptor expression mediates pathogen recognition contributes in part to the pathogenesis of the disease. In USP18 and limits Salmonella replication. mutants, disease susceptibility was mainly driven by hyperactiva- Downloaded from tion of the type 1 IFN pathway, resulting in increased levels of IL-6 Discussion and IFN-b in circulation and development of septic shock. In the The use of ENU chemical mutagenesis in the mouse has proved an current article, we specifically demonstrate the impact of Stat4 in effective means to identify and study mutations in genes that affect Salmonella disease susceptibility. the host immunological response to microbial challenge (52–54). Over the past decade, studies in Stat4 KO mice have illustrated

We used an ENU mutagenesis approach to screen for recessive the importance of functional STAT4 in both infectious and non- http://www.jimmunol.org/ germline mutations conferring increased mortality following infectious diseases (36, 58, 59). Stat4 KO mice have increased S. Typhimurium infection, a globally relevant human pathogen susceptibility to infections primarily driven by a Th1 immune (55). In this article, we report the identification of the Ity14 de- response, including those caused by Mycobacterium tuberculosis, viant pedigree. A combination of mapping and exome sequencing Leishmania major, Trypanosoma cruzi, Toxoplasma gondii, Ba- was used to identify a splice site point mutation in STAT4 besia, and Listeria monocytogenes (36). In contrast, STAT4 defi- (p.G418_E445). The STAT4 mutation was the only homozygous ciency has generally been shown to protect from T cell–mediated mutation that segregated in all susceptible mice and within the autoimmune diseases, including experimental allergic encephalo- 2.7-Mb mapped interval. This mutation likely interferes with the myelitis, a model for multiple sclerosis and collagen-induced ar- normal DNA-binding function of STAT4 and in regulating tran- thritis, and a model for rheumatoid arthritis (60, 61). Furthermore, by guest on September 25, 2021 scription of target genes. Stat4 is expressed at the transcript level Stat4 KO mice injected with LPS were shown to be somewhat in Ity14 mutants, but is a hypomorph with significantly decreased protected from endotoxemia (62). STAT4 expression at the protein level, as shown by Western blot in STAT4 is a transcription factor and has been shown to bind to uninfected and infected splenic tissue, suggesting that the protein the promoter and/or drive expression of several target genes is either rapidly degraded or its translation is inhibited. The Ity14 (63–65). Recent genomic approaches by chromatin immuno- allele is a recessive loss of function of Stat4 that was confirmed by precipitation–on–chip and chromatin immunoprecipitation–se- complementation assays wherein Stat4Ity14/Ity14, Stat4 KO, and quencing have elucidated STAT4 binding sites, target genes, and compound heterozygous mice present similar survival curves fol- STAT4-dependent epigenetic modifications on a genome-wide lowing Salmonella infection. level (65–67). These studies have been done in activated CD4+ Intact IFN signaling is essential in the host immune response to T cells cultured under Th1 cell conditions. As such, comparisons viral, bacterial, and fungal disease. The importance of IFN sig- between our microarray data done in uninfected and day 4 post- naling has been highlighted by patients carrying mutations in genes infected spleen, and previous studies, must be interpreted with in the IFN-a/b and IFN-g pathways, which present with increased caution. The above potentially explains why we did not detect other susceptibility to infection. Individuals with a defect in production known STAT4 targets, including Furin, Il18r1,andIl12rb2,and or response to IFN-g have been shown to have increased sus- signature Th1 cell genes, including Tbx21.Thesediscrepancies ceptibility to Mendelian susceptibility to mycobacterial disease, highlight the difference between in vitro and in vivo studies, and the viral disease, or Salmonella infection. These genetic deficiencies importance of studying the impact of STAT4 at the whole-organism have been identified in the following genes: IFNGR1, IFNGR2, level. STAT1, IL12B, IL12RB1, NEMO, and TYK2 (15, 56, 57). Deficient The transcriptional signature was different in Stat4Ity14/Ity14 mice IL-12 signaling results in impaired Th1 differentiation and IFN-g compared with Stat4+/+ following Salmonella infection, which production. In fact, multiple episodes of salmonellosis are re- correlates with the histopathological results. As expected, IFN-g portedly frequent in IL-12Rb1–deficient patients, illustrating the and IFN-g–regulated gene expressions were downregulated in importance of the IL-12/IFN-g axis in mounting an efficient im- Ity14 mutants, including a subset of guanylate-binding proteins mune response to primary and secondary infections (17). However, (Gbp1,2,5,6) previously reported to protect against bacterial no mutations in STAT4 have been identified in humans to date. infections (68, 69). We also observed impaired upregulation of We have previously reported another ENU-induced mouse Stat1, consistent with STAT1 as a target of STAT4. IFN-g has been mutant, Usp18Ity9, a negative regulator of type 1 IFN signaling, reported to have anti-inflammatory properties in suppressing which emphasizes the importance of IFN signaling in suscepti- IL-1a,b production by myeloid cells during Mycobacterium bility to Salmonella infection (42, 45). We have shown in this model tuberculosis infection (70). Consistent with these findings, we that transient suppression of STAT4-induced IFN-g production detected increased expression of genes in the IL-1 signaling The Journal of Immunology 269 pathway (Il1a, Il1b, Ilrn)inStat4Ity14/Ity14 mice that have low the lack of IFN-g is detrimental to the host, as Stat4Ity14/Ity14 levels of circulating IFN-g. mutants develop fatal bacteremia following Salmonella infection. Stat4Ity14/Ity14 mice had significantly lower levels of IFN-g in circulation, which contributed to impaired microabscess formation Acknowledgments and increased systemic bacterial burden in the spleen and liver. We thank Genevie`ve Perreault, Nadia Prud’homme, Vanessa Guay, Marie This finding is consistent with previous data showing increased Chevenon, Lei Zhu, and Line Larivie`re for technical assistance, and Sean bacterial burden in STAT4 KO mice compared with littermate Wiltshire and Sean Beatty for help with R code. controls in spleen and liver following S. Typhimurium infection (42). The following further extends to a model of intestinal dis- Disclosures ease, wherein the absence of STAT4 leads to bacterial dissemi- The authors have no financial conflicts of interest. nation to the spleen and liver with intestinal disease. It has previously been shown that positive feedback regulation exists between IL-12 and IFN-g (71). However, excessive amplification References of the IL-12/IFN-g axis can lead to immunopathological changes, 1. Crump, J. A., S. P. Luby, and E. D. Mintz. 2004. The global burden of typhoid fever. Bull. 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