STAT4 Directs a Protective Innate Lymphoid Cell Response to Gastrointestinal Infection Sarah J

STAT4 Directs a Protective Innate Lymphoid Cell Response to Gastrointestinal Infection Sarah J. Dulson, Emily E. Watkins, David K. Crossman and Laurie E. Harrington This information is current as of September 26, 2021. J Immunol published online 27 September 2019 http://www.jimmunol.org/content/early/2019/09/26/jimmun ol.1900719 Downloaded from

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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 © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 27, 2019, doi:10.4049/jimmunol.1900719 The Journal of Immunology

STAT4 Directs a Protective Innate Lymphoid Cell Response to Gastrointestinal Infection

Sarah J. Dulson,* Emily E. Watkins,* David K. Crossman,†,‡ and Laurie E. Harrington*

Innate lymphoid cells (ILCs) are strategically positioned at mucosal barrier surfaces where they respond quickly to infection or injury. Therefore, we hypothesized that ILCs are key contributors to the early immune response in the intestine against Listeria monocytogenes. Using a modified strain of L. monocytogenes that mimics human gastrointestinal listeriosis in mice, we find ILCs to be essential for control of early replication of L. monocytogenes in the intestine as well as for restricted dissemination of bacteria to peripheral tissues. Specifically, group 1 ILCs (ILC1s) and group 3 ILCs (ILC3s) respond to infection with proliferation and IFN-g and IL-22 production. Mechanistically, we show that the transcription factor STAT4 is required for the proliferative and IFN-g effector response by ILC1s and ILC3s, and loss of STAT4 signaling in the innate immune compartment results in an inability

to control bacterial growth and dissemination. Interestingly, STAT4 acts acutely as a transcription factor to promote IFN-g Downloaded from production. Together, these data illustrate a critical role for ILCs in the early responses to gastrointestinal infection with L. monocytogenes and identify STAT4 as a central modulator of ILC-mediated protection. The Journal of Immunology, 2019, 203: 000–000.

isteriosis is a serious foodborne infection that affects over Innate lymphoid cells (ILCs) are tissue-resident immune pop-

20,000 people each year and carries a 20–30% mortality ulations that are enriched at barrier surfaces, such as the gastro- http://www.jimmunol.org/ L rate in high risk individuals, including immunocompro- intestinal tract, and are responsive to infection and injury (8). ILC mised persons and pregnant women (1, 2). Listeria monocytogenes, subpopulations parallel T cells because of similarities in tran- the causative agent of listeriosis, is a Gram-positive, intracellular scriptional regulation and effector function (9). However, unlike bacteria that infects intestinal epithelial cells and subsequently T cells, ILCs lack specific AgR and are epigenetically poised to disseminates via the bloodstream to seed the spleen and liver respond at early stages of an immune response (9, 10). Five (3, 4). Productive infection with L. monocytogenes requires in- subpopulations of ILCs have been identified based on distinct teraction between the internalin A protein of the bacterium and developmental trajectories and transcriptional profiles: conven- the integrin E-cadherin expressed by the intestinal epithelial cell tional NK cells, which mirror cytotoxic CD8+ T cells; lymphoid (5). However, because of a single amino acid difference between tissue inducer cells, which are essential for secondary lymphoid by guest on September 26, 2021 human and mouse E-cadherin, this critical interaction does not organogenesis; and three subpopulations of helper-like ILCs akin occur in mice, limiting our understanding of the immune response to the CD4 helper T cell populations (11). Group 1 ILCs (ILC1s) during this early phase of intestinal infection (6). Therefore, a are analogous to Th1 CD4 T cells in that ILC1s require Tbet mutant L. monocytogenes strain with a modified internalin A for development and respond to IL-12, IL-18, and IL-15 with protein (Lm-InlAm) was developed that allows modeling of gas- the production of IFN-g and TNF-a. ILC2s express high levels trointestinal listeriosis in the mouse (7). Using this model, we are of the transcription factor GATA3 and produce Th2-associated now able to investigate the local immune responses in the gas- cytokines, IL-4 and IL-13, to mediate protective responses to trointestinal tract and delineate mechanisms by which distinct cell helminthic infections. Group 3 ILCs (ILC3s) are the most het- populations contribute to protection. erogeneous subpopulation of ILCs, all of which rely on RORgt for their development and phenotypically and functionally resemble Th17 CD4 T cells (12). ILC3 subpopulations include IL-17A, *Department of Cell, Developmental, and Integrative Biology, University of IL-22, and GM-CSF–producing cells that function in early response Alabama at Birmingham, Birmingham, AL 35294; †Department of Genetics, Uni- ‡ to infection, maintenance of mucosal barriers, and induction of versity of Alabama at Birmingham, Birmingham, AL 35294; and Heflin Center for Genomic Science, University of Alabama at Birmingham, Birmingham, AL 35294 tolerance to benign Ags. Importantly, ILC3s have been shown ORCIDs: 0000-0002-1965-0543 (S.J.D.); 0000-0002-0981-169X (D.K.C.); 0000- to have a plastic characteristic with the ability to upregulate 0002-6832-9529 (L.E.H.). Tbet and downregulate RORgt in response to inflammatory Received for publication June 25, 2019. Accepted for publication September 3, 2019. cytokine signals. In doing so, ILC3 gain surface expression of This work was supported by National Institutes of Health Grants R01 AI113007 (to NKp46 and the ability to produce IFN-g (13–15). Each of the L.E.H.), R21 AI142641 (to L.E.H.), and 2T32AI007051-39 (to S.J.D.). ILC populations have demonstrated roles in mounting early Address correspondence and reprint requests to Dr. Laurie E. Harrington, Depart- immune responses to infection; however, whether distinct ILC ment of Cell, Developmental, and Integrative Biology, University of Alabama at subpopulations can prevent the establishment or dissemination of Birmingham, 845 19th Street South, BBRB 471, Birmingham, AL 35294. E-mail address: [email protected] L. monocytogenes during enteric infection remains unknown. The online version of this article contains supplemental material. The transcription factor STAT4 is activated through the Jak2 Abbreviations used in this article: BHI, Brain Heart Infusion; cLP, colonic LP; ILC, and Tyk2 in response to IL-12 or IL-23R binding (16). STAT4 innate lymphoid cell; ILC1, group 1 ILC; ILC3, group 3 ILC; Lm-InlAm, mutant functions in driving the differentiation program of CD4 helper L. monocytogenes strain with a modified internalin A protein; LN, lymph node; LP, T cells, and STAT4 deficiency results in an inability of naive lamina propria. CD4 T cells to acquire a Th1 phenotype (16, 17). Importantly, Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 chromatin immunoprecipitation assays show that STAT4 directly

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900719 2 STAT4 MEDIATES ILC RESPONSES TO ENTERIC INFECTION interacts with the Ifng, Il18ra, and Tbx21 gene loci in developing For CFU assays, spleen, liver, mesenteric, and colonic lymph nodes Th1 cells (18–20), thereby not only dictating the expression of the (LNs) (pooled) and colon were collected into Molecular Biology Grade hallmark Th1 cytokine IFN-g but also influencing the expression Water (Thermo Fisher Scientific), weighed, and homogenized for 0.5– 1 min. Serial dilutions were performed and plated on BHI Agar (Thermo of the IL-18R a-chain as well as the Th1 lineage-defining tran- Fisher Scientific) supplemented with 15 g/l Lithium Chloride (Thermo scription factor, Tbet. Moreover, STAT4 has been demonstrated Fisher Scientific) and 10 g/l Glycine (Thermo Fisher Scientific) as de- to function in Th17 cells; STAT4-deficient Th17 cells have re- scribed previously (28). Colonies were enumerated after 24–36 h of induced the capacity of IL-17A production and lack certain plastic cubation at 37˚C. To enumerate CFU in colon, contents were removed with forceps, characteristics, such as the ability to produce IFN-g (21, 22). and tissue was flushed with 10 ml Molecular Biology Grade Water, cut The role of the STAT family proteins, including STAT4, in ILCs longitudinally, and weighed. The washed tissue was processed as is still not well understood, but recent studies show notably described above. distinct functions from T cells. For instance, STAT3 is required In vivo Ab treatment for Th17 differentiation but is dispensable for the generation of 2 2 intestinal ILC3s (23). To add complexity, STAT5 impacts the CD90.2+ cells were depleted from Rag1 / mice using anti-Thy1.2 mAb development of intestinal ILC1s to a much greater extent than (clone 30H12; 150 mg/mouse i.p.; Bio X Cell) or PBS control on day 23, 21, +1, +4, +7, +10, and +13 of infection. IFN-g was neutralized liver ILC1s, highlighting tissue-specific requirements of STAT in vivo using anti–IFN-g mAb (clone XMG1.2; 200 mg/mouse i.p.; Bio molecules in the regulation of ILC populations (24). Recent X Cell) or PBS control on day 21, +1, and +3 of infection. studies have identified STAT4 as an important regulator of IFN-g production in liver- and adipose-resident ILC1s (25, 26), but the Lymphocyte isolation role of STAT4 in intestinal ILC1s remains ill defined. LNs were mechanically dissociated and filtered through a 100-mm cell Downloaded from In this study, we report a critical role for ILCs in controlling strainer in complete media (RPMI 1640 with 1% FCS, 2 mM L-glutamine, early responses to gastrointestinal infection with L. monocytogenes. 100 IU/ml penicillin, 100 mg/ml streptomycin, and 5 mM 2-ME) to make a single-cell suspension. Mice lacking ILCs show increased intestinal bacterial burden as Lymphocytes from the colonic and small intestine LP were isolat- well as uncontrolled dissemination to the spleen and liver as early ed using the LP Dissociation Kit (Miltenyi Biotec) according to the as 2 d postinfection, culminating in increased overall mortality. manufacturer’s protocol. Cells were filtered through a 100-mmcell strainer in PBS containing 0.5% FBS. Cells were then resuspended in We find that L. monocytogenes induces proliferative and cytokine http://www.jimmunol.org/ 30% Percoll (Thermo Fisher Scientific) and centrifuged at 25˚C at 800 responses specifically by the ILC1 and ILC3 populations in the relative centrifugal force for 20 min without brake. Cells were collected colonic lamina propria (LP) (cLP). Importantly, STAT4 signaling at the bottom of the gradient and filtered through a 70-mmcellstrainer during the innate response to infection is protective. Although in PBS containing 0.5% FBS. Cells were resuspended in complete media dispensable for the development of all intestinal ILC subpopula- (RPMI 1640 with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, 3 tions, we establish that STAT4 directly influences the proliferation 100 mg/ml streptomycin, 1 nonessential amino acids, 1 mM sodium pyruvate, and 5 mM2-ME). and IFN-g production by intestinal ILC1s and NKp46+ ILC3s by regulating transcription of this cytokine. This is vital for host Flow cytometry protection against enteric L. monocytogenes as neutralization of Isolated cells were stained in PBS for 20–30 min on ice. Fc receptor IFN-g exacerbates intestinal bacterial burden and dissemination blockade was performed with anti-CD16/32 mAb prior to surface staining. by guest on September 26, 2021 to peripheral tissues. Altogether, this study reports a central role Dead cells were excluded from analysis using LIVE/DEAD Fixable Aqua for STAT4 signaling in driving protective ILC-mediated responses Dead cell stain (Invitrogen). For the detection of transcription factors to gastrointestinal infection by L. monocytogenes. and cytokines, cells were stimulated for 4 h with 50 ng/ml PMA (Sigma Aldrich) and 750 ng/ml ionomycin (Calbiochem); IL-12 (50 ng/ml) with or without IL-18 (50 ng/ml); or IL-23 (50 ng/ml) and IL-1b (50 ng/ml). Materials and Methods GolgiPlug (10 mg/ml; BD Biosciences) was added for the last 3 h of Mice stimulation. Cells were fixed and permeabilized for intracellular staining using the Foxp3 Transcription Factor Staining Buffer Kit (eBioscience) C57BL/6 and B6.129S7-Rag1tm1Mom/J (Rag12/2) mice were purchased according to the manufacturer’s protocol. Samples were acquired using from The Jackson Laboratory and bred at the University of Alabama at an LSRII flow cytometer (BD Biosciences), and data were analyzed Birmingham. B10;B6-Rag2tm1Fwa Il2rgtm1Wjl (Rag22/2gc2/2)and using FlowJo software (Tree Star). B6.129S6-Rag2tm1Fwa N12 (Rag22/2) were purchased from Taconic Biosciences and cohoused for at least 7 d prior to use. B6. STAT42/2 Abs and ILC identification (STAT42/2) were provided as a generous gift from M. Kaplan and bred 2 2 2 2 Except where otherwise indicated, ILC1s were identified as lineage at the University of Alabama at Birmingham. B6.Rag1 / STAT4 / 2/2 2/2 2/2 +/2 2/2 +/2 (TCRb,TCRgd,CD8a,CD8b, CD19, CD11b, DX5, Gr-1, Ter119)- (Rag1 STAT4 ) and B6.Rag1 STAT4 (Rag1 STAT4 ) were Thy1.2+CD127+NK1.1+NKp46+ Tbet+Eomes2. ILC2s were identified Ifng + + + generated at the University of Alabama at Birmingham. B6. /Thy1.1 as lineage-Thy1.2 CD127 KLRG1 . ILC3s were identified as lineage- knock-in mice were generated at the University of Alabama at Birmingham + + 2 2 +/2 + Tg Yfp/+ Thy1.2 CD127 KLRG1 NK1.1 NKp46 RORgt . NK cells were identi- Rorc(gt) 3 Rosa26R gt + + + + (27). -Cre (ROR -fatemap) mice were pur- fied as NK1.1 NKp46 Tbet Eomes . chased from The Jackson Laboratory and bred at the University of For lineage staining, all markers included in the lineage panel were Alabama at Birmingham. Both male and female mice were used for this biotinylated. The following mAb toward lineage markers were used: study. All animals were bred and maintained under specific pathogen- TCRb (H57-597), TCRgd (UC7-13D5; eBioscience), CD8a (53-6.7), free conditions at the University of Alabama at Birmingham according CD8b (YTS156.7.7), CD19 (6D5), CD11b (M1/70), CD49b (DX5), to Institutional Animal Care and Use Committee regulations. Gr-1 (RB6-8C5), and Ter119. A secondary stain was performed using Bacterial infection and burden determination a streptavidin-allophycocyanin-eFluor780 (eBioscience) to stain lineage- positive cells. L. monocytogenes strain EGDe carrying a recombinant internalin A The following Abs were used in this study: anti-CD90.2/Thy1.2 FITC/PE protein (Lm-InlAm) was a generous gift from B. Sheridan (Stony Brook Cy7/eFluor 450 (clone 53-2.1; eBiosciences), anti-CD90.2/Thy1.2 PerCP University, Stony Brook, NY). All mice were food deprived for ∼16 h (clone 53-2.1; BioLegend), anti-CD90.1/Thy1.1 FITC/PerCP Cy5.5 (clone prior to infection. Bacteria were grown overnight in Brain Heart In- HIS51; eBioscience), anti-CD127 PE Cy7 (clone A7R34; eBioscience), fusion (BHI) Broth (Thermo Fisher Scientific) shaking at 37˚C. Culture anti-KLRG1 FITC (clone 2F1; eBioscience), anti-NK1.1 PE (clone was diluted 1:10 in fresh BHI Broth and incubated shaking at 37˚C PK136; eBioscience), anti-CD335/NKp46 Alexa Fluor 647 (clone 29A1.4; with bacterial density measured frequently by OD. At OD 0.8–0.9 (with BioLegend), anti-CD335/NKp46 PerCP eFluor 710 (clone 29A1.4; OD = 0.8 corresponding to 2 3 109 CFU/ml), bacteria were suspended eBioscience), anti–IL-18Ra PE (clone P3TUNYA; eBioscience), anti– in PBS. Mice were administered 0.5–1 3 109 CFU L. monocytogenes via IL-17A PE (clone TC11-18H10; BD Biosciences), anti–IL-17A eFluor oral gavage. 450/PerCP Cy5.5/Alexa Fluor 647 (clone eBio17B7; eBioscience), The Journal of Immunology 3 anti–IL-22 PE (clone 1H8PWSR; eBioscience), anti–IFN-g PerCP mice would harbor a higher bacterial burden in intestinal tissues as Cy5.5/eFluor 450 (clone XMG1.2; eBioscience), anti-RORgt allophycocyanin/ well as experience increased bacterial dissemination to peripheral PerCP e710 (clone B2D; eBioscience), anti-GATA3 PE (clone TWAJ; tissues. Similar to the ILC-depleted Rag12/2 mice, infection of eBioscience), anti-Tbet PerCP Cy5.5 (clone 4B10; eBioscience), anti- 2/2 2/2 Eomes eFluor 450 (clone Dan11mag; eBioscience), anti-pSTAT4 (pY693) Rag2 gc mice resulted in 1.5 log-fold higher bacterial PE/Alexa Fluor 488 (BD Biosciences, clone 38/pSTAT4), and anti-Ki67 burden in colonic tissue as well as significantly increased dis- eFluor 450 (clone SolA15; eBioscience). semination to the periphery (Fig. 1C). Strikingly, these differ- Phospho-STAT4 staining ences were observed as early as 2 d postinfection. Together, these data support a central role of ILCs in early protection against For the detection of pSTAT4 protein, lymphocytes isolated from the cLP gastrointestinal infection and dissemination of Lm-InlAm. were stained for lineage markers and stimulated for 30 min with IL-12 (50 ng/ml) at 37˚C. Live/Dead staining was performed following a brief Intestinal ILC1s and ILC3s are activated by L. fixation step in 0.4% paraformaldehyde. Surface, intracellular, and monocytogenes infection pSTAT4 staining was performed following fixation with 4% paraformaldehyde and permeabilization with 100% methanol. ILCs are stimulated to proliferate and produce effector cytokines Serum cytokine analysis by activated myeloid and nonhematopoietic cell populations (26, 32, 33). ILC1s, group 2 ILCs, and ILC3s were defined using Protein levels of cytokine biomarkers were simultaneously measured in the gating strategy outlined in Supplemental Fig. 1A, and their serum using the mouse-specific MTH17MAG-47K MILLIPLEX mag- netic bead–based multianalyte panel (MilliporeSigma, Burlington, MA) identities were confirmed by transcription factor expression and following the manufacturer’s instructions. Protein quantification was RORgt fate-mapping mice (Supplemental Fig. 1B, 1C). ILC1s performed using the Luminex MAGPIX instrument platform and re- and NK cells share expression of NK1.1, NKp46, and Tbet (34). Downloaded from lated xPONENT software (Luminex, Austin, TX). The readouts were an- Hence, the populations are best distinguished using the tran- alyzed with the standard version of MILLIPLEX Analyst 5.1 software scription factor Eomes, which is expressed by NK cells but not (MilliporeSigma). by ILC1s. Using a gating strategytospecificallyinterrogate Whole colon transcript analysis these populations (Supplemental Fig. 1D), we found that CD127 Whole colon tissue was collected in RNAlater solution, and RNA was and CD49b are expressed by intestinal ILC1s and NK cells,

isolated using the RNA Lipid Tissue Mini Kit (QIAGEN) and quantified respectively. Therefore, we included CD49b in the lineage panel http://www.jimmunol.org/ using the DeNovix DS-11 Spectrophotometer (DeNovix, Wilmington, to exclude NK cells and further specified ILC1s by gating on . . DE). Samples of high purity (A260/A280 2, A260/A230 1.4) were CD127+ cells. Together, this allowed us to evaluate helper ILC- processed on the NanoString nCounter Flex system (NanoString Tech- nologies, Seattle, WA) using the mouse PanCancer Immune premade specific responses to L. monocytogenes infection. panels according to the manufacturer’s protocol. Analysis was performed To determine which population(s) of ILCs are responding to using nSolver 4.0 software (NanoString Technologies). Expression levels gastrointestinal infection with Lm-InlAm in C57BL/6 mice, pro- were normalized to housekeeping genes that were not discarded by the liferation was measured by intracellular Ki67 expression. By day gNorm program in the advanced analysis module. 4 postinfection, a significant increaseinKi67expressionwas Statistics measured in ILC1s and ILC3s from the cLP (Fig. 2A). In con-

m by guest on September 26, 2021 Statistical significance was calculated as indicated in figure legends using trast, ILC2s did not proliferate in response to Lm-InlA infec- Prism software (GraphPad). All t tests performed are two tailed, and p tion. Overall, this identified intestinal ILC1 and ILC3 as values , 0.05 were considered significant unless specifically indicated responding cell populations during early infection with Lm-InlAm. otherwise in the text. ILCs are poised for robust and rapid effector cytokine production upon activation. To determine the L. monocytogenes–responsive Results ILC subpopulations, colonic tissues and sera of cohoused ILC- ILCs protect against enteric L. monocytogenes infection sufficient Rag22/2 and ILC-deficient Rag22/2gc2/2 mice were ILCs are enriched in gastrointestinal tissues where they are poised analyzed 2 d postinfection. Importantly, systemic effector cyto- geographically and epigenetically to respond rapidly to pathogenic kine responses were dependent on ILCs because ILC1-associated infection (10, 29). Therefore, we hypothesized that ILCs exert a IFN-g and ILC3-associated IL-22 levels in the sera were increased protective role early in enteric infection with L. monocytogenes. at day 2 postinfection only in mice with an intact ILC population To test this, we depleted Rag1-deficient mice of ILCs using an (Fig. 2B). In accordance with the systemic responses, the analy- established anti-Thy1.2 Ab-mediated depletion model. We then sis of total colonic mRNA revealed a tissue-level increase in the 2 2 infected ILC-depleted Rag12/2 mice, nondepleted Rag12/2 ILC-associated transcripts Ifng, Tnf, and Il22 only in Rag2 / littermates, and C57BL/6 mice by oral gavage with Lm-InlAm. colons (Supplemental Fig. 2). Consistent with these data, increased At 5 d postinfection, the bacterial burden in the colon and the transcription of the IFN-g–responsive gene, Nos2, was detected 2 2 mesenteric and colonic LNs (draining LNs) was enumerated as in the Rag2 / colons. Notably, these transcripts were not 2 2 2 2 was the disseminated bacterial burden in the spleen and liver. induced in ILC-deficient Rag2 / gc / , highlighting the role ILC-depleted mice harbored increased bacterial burden in all of ILCs in directing these responses at both the local and sys- analyzed tissues compared with nondepleted Rag12/2 mice and temic levels. C57BL/6 mice (Fig. 1A). Importantly, no significant differences Upon infection, epithelial and myeloid cell populations re- in burden were detected between C57BL/6 and nondepleted spond to pathogen-associated proteins with the production of Rag12/2 mice, indicating that adaptive immune responses were lymphocyte-activating cytokines. To verify that our findings are 2 2 2 2 dispensable for control at this early stage of infection. Ultimately, due to ILCs and not differences in upstream signals in Rag2 / gc / and in accordance with increased bacterial burden, ILC-depleted mice, we analyzed the serum levels of key cytokines known Rag12/2 mice succumbed to infection more rapidly than their to activate type 1 and type 3 immune responses. We found no ILC-sufficient littermates (Fig. 1B). significant differences in circulating IL-23, IL-1b, IL-12p70, or To corroborate these findings, we employed a mouse model of IL-15 at day 2 postinfection (Supplemental Fig. 3A). Furthermore, genetic ILC deficiency, Rag22/2gc2/2 mice. Rag22/2gc2/2 mice colonic expression of Il18 transcripts were similar in the presence show increased susceptibility to enteric infection with wild-type or absence of ILCs (Supplemental Fig. 3B). Therefore, differ- L. monocytogenes (30, 31), and we posited that Rag22/2gc2/2 ences in ILC-associated effector molecules in Rag22/2 versus 4 STAT4 MEDIATES ILC RESPONSES TO ENTERIC INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 1. ILCs protect against enteric L. monocytogenes infection. (A and B) C57BL/6 and Rag12/2 mice treated with anti-Thy1.2 mAb or PBS were infected with Lm-InlAm.(A) On day 5 postinfection, the indicated organs were collected, and bacterial burden was assessed (three independent experiments). (B) Survival was monitored for the ﬁrst 15 d of infection (n = 7–9 mice per group; two independent experiments). (C) Rag22/2 and Rag22/2gc2/2 mice were infected with Lm-InlAm intragastrically and analyzed at day 2 postinfection for bacterial burden of indicated tissues (three independent experiments). (A and C) Outlier test and unpaired t test. Graphs show mean 6 SD. (B) Log rank test. *p , 0.05, **p , 0.01. ns, not signiﬁcant.

Rag22/2gc2/2 mice are due to the absence of ILCs rather than a cytokine responses were ablated, supporting an essential role for defect in upstream immune cell–activating cytokines. ILCs in an early response to gastrointestinal L. monocytogenes The absence of ILCs resulted in a defective cytokine response infection. Kinetically, ILC-dependent cytokine responses can following enteric Lm-InlAm infection at the local and systemic be measured systemically as early as 2 d postinfection and are levels, so next, we sought to determine whether intestinal ILCs followed by a proliferative phenotype of intestinal ILC1 and ILC3 were actively producing IFN-g in response to infection. We by day 4. employed an IFN-g reporter mouse model that identifies IFN-g– producing cells with the surface expression of the Thy1.1 Innate STAT4 signaling protects against molecule (27). This model allowed us to interrogate IFN-g L. monocytogenes infection production by ILCs directly ex vivo without needing to restim- STAT4 signaling occurs downstream of IL-12 and IL-23, activating ulate. At baseline, between 10 and 20% (average 15%) of ILC1 cytokines of ILC1 and ILC3, respectively (8, 16). Furthermore, and NKp46+ ILC3 express Thy1.1. By 3 d postinfection, the IL-12 deficiency enhances susceptibility in models of systemic frequency of Thy1.1-expressing ILC1 and NKp46+ ILC3 had L. monocytogenes infection, supporting a role for STAT4 in pro- significantly increased and reached over 40% in many animals tection in the liver and spleen (35). Therefore, we hypothesized (Fig. 2C). As expected, ILC2s did not express Thy1.1 in response that STAT4 signaling in ILCs is essential for control of gastroin- to infection. Because NK cells are known to respond to systemic testinal L. monocytogenes infection. To test this, we infected infection with L. monocytogenes, we sought to characterize the C57BL/6 and STAT4-deficient (STAT42/2) mice intragastrically NK cell IFN-g response. We found that Thy1.1 expression was with Lm-InlAm. We found significantly increased susceptibility not significantly induced on NK cells at 3 d postinfection. Taken to Lm-InlAm in STAT42/2 mice compared with C57BL/6 mice together, these data present a dominant ILC1 and ILC3 response (Fig. 3A). By day 4 postinfection, STAT42/2 mice exhibited a 1.9 to gastrointestinal Lm-InlAm infection that is mounted prior to and 1.7 log-fold change higher bacterial burden in the colon and the NK cell response. Importantly, in the absence of ILCs, key draining LN, respectively (Fig. 3B). We previously noted that The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Intestinal ILC1s and ILC3s are activated by L. monocytogenes infection. (A) C57BL/6 mice were infected intragastrically with Lm-InlAm. Representative flow cytometry plots show Ki67 expression by cLP ILC populations from uninfected mice and on days 2 and 4 postinfection. Graphs depict frequency of Ki67+ ILCs of indicated ILC subpopulation (four independent experiments). (B) Rag22/2 and Rag22/2gc2/2 mice were infected intragastrically with Lm-InlAm and analyzed at day 2 for serum cytokine levels by Luminex (four independent experiments). (C) B6.Ifng/Thy1.1 knock-in mice were infected with Lm-InlAm intragastrically, and Thy1.1 expression by ILC1s, ILC2s, NKp46+ ILC3s, and NK cells was analyzed by flow cytometry at 3 d postinfection. Representative flow cytometry plots show Thy1.1 expression. Graphs depict frequency of Thy1.1+ ILCs of indicated subpopulation (one to five independent experiments). Graphs shown are mean 6 SD. (A) One-way ANOVA. ***p , 0.001. (B and C)Unpairedt test. **p , 0.01, ***p , 0.005. ns, not significant 6 STAT4 MEDIATES ILC RESPONSES TO ENTERIC INFECTION

FIGURE 3. Innate STAT4 signaling pro- Downloaded from tects against L. monocytogenes infection. (A–C) C57BL/6 and STAT42/2 and (D–F) Rag12/2STAT4+/2 and Rag12/2STAT42/2 littermates were infected intragastrically with Lm-InlAm, and (A and D) survival was monitored (n = 9–14 mice per genotype, three independent experiments). (B and E) http://www.jimmunol.org/ On day 4 of infection, bacterial burden of the colonic tissue and draining LN was determined (three independent experiments). (C and F)Day2serumIFN-g concentration was measured by Luminex (two to three independent experiments). (A and D) Log rank test. (B, C, E, and F) Graphs show mean 6 SD. Outlier test and unpaired t test. *p , 0.05, **p , 0.01. by guest on September 26, 2021

ILC-deficient mice could not mount a systemic IFN-g response in innate immune responses, we generated Rag12/2STAT42/2 (Fig. 2B), so we sought to determine if STAT4 dictated this mice and infected these as well as Rag12/2STAT4+/2 littermates response. Serum analysis at day 2 postinfection revealed a robust with Lm-InlAm. Compared with Rag12/2STAT4+/2 littermates, IFN-g response by C57BL/6 mice (Fig. 3C). In contrast, serum Rag12/2STAT42/2 mice showed increased susceptibility to Lm- IFN-g was below the limit of detection in STAT42/2 animals at InlAm infection (Fig. 3D). Whereas 50% of the Rag12/2STAT4+/2 this same time point, thus STAT4 signaling is essential to elicit mice survived through day 14, the majority of the Rag12/2STAT42/2 a systemic IFN-g response to infection. mice succumbed to infection within the first 7 d. Innate STAT4 de- Notably, all STAT42/2 micesuccumbedinthefirstweek ficiency also resulted in increased bacterial burden in the colon of infection and before a strong adaptive response would be and the draining LN at day 4 postinfection (Fig. 3E). Similar mobilized (36), indicating its function in the innate immune to Rag22/2gc2/2 and STAT42/2 mice, Rag12/2STAT42/2 mice response. To directly interrogate the role of STAT4 specifically showed a defective serum IFN-g response at day 2 postinfection The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. STAT4 is dispensable for intestinal ILC development and maintenance. ILC subpopulations of the (A, D, and G) mesenteric LN, (C, F, and I) small intestinal LP (siLP), and (B, E, and H) cLP were compared between uninfected C57BL/6 and STAT42/2 mice. (A–C) Representative ﬂow cytometry plots of total ILCs (gated on live singlets). Graphs depict total ILC number. (D–F) ILC subpopulation frequencies of total ILCs are shown. (G–I) ILC3 subpopulation frequencies of total ILC3s are shown. (A–I) Combined from two to three independent experiments. Graphs show mean 6 SD. (A–C) Unpaired t test. (D–I) Two-way ANOVA with Holm–Sidak multiple comparisons test.

(Fig. 3F). Altogether, these data link STAT4 signaling to a robust To interrogate if STAT4 is required for the development or main- and protective innate immune response to Lm-InlAm. tenance of intestinal ILC populations, we analyzed the number and To ensure differences between the groups were due to effects phenotypes of ILCs in several intestinal compartments of C57BL/6 downstream of STAT4, we measured ILC-activating cytokines, and STAT42/2 mice. We found no difference in total ILC numbers including IL-12 and IL-23, in the serum at day 2 postinfection. in the mesenteric LNs nor in the LP of the small or large in- Importantly, no discernable difference in ILC-activating cytokines testine (Fig. 4A–C). Upon further subsetting, all ILC populations were detected in the serum of Rag12/2STAT42/2 compared with developed to normal frequencies in the measured intestinal tis- Rag12/2STAT4+/2 littermates at 2 d postinfection (Supplemental sues (Fig. 4D–F). Importantly, this includes the Th1-like ILC Fig. 3C). Collectively, these data highlight the role of STAT4 in subpopulation, ILC1s. Moreover, similar to previous studies, we a robust innate immune response against L. monocytogenes infec- also found that the subpopulations of ILC3s, identified based tion and identify STAT4 signaling as essential for early protection. on CD4 and NKp46 expression, were unaffected in number and frequency by STAT4 deficiency (Fig. 4G–I) (13, 37). Alto- STAT4 is dispensable for intestinal ILC development gether, these data demonstrate a profound distinction in the role and maintenance of STAT4 in regulating the development and plasticity of ILCs + STAT4 is essential for the differentiation of naive CD4 T cells into compared with its known role in T cell populations. the Th1 phenotype (17), but the role of STAT4 in the development of intestinal ILCs has not been fully elucidated. Because of the STAT4 directs a proliferative ILC response to enteric observation that STAT4 signaling in the innate immune response L. monocytogenes infection is important for early control of Lm-InlAm, we posited that STAT4 Because intestinal ILCs develop normally in the absence of STAT4, deficiency may result in an altered ILC population in the intestine. we postulated that STAT4 is required to elicit ILC effector functions. 8 STAT4 MEDIATES ILC RESPONSES TO ENTERIC INFECTION

FIGURE 5. STAT4 directs a proliferative ILC response to enteric L. monocytogenes infection. C57BL/6 and STAT42/2 mice were infected intragastrically with Lm-InlAm. Representative ﬂow cytometry plots show Ki67 expression by cLP ILC populations on day 4 postinfection. Graphs depict frequency of Ki67+ cells of indicated ILC subpopulation (three independent experiments). Graphs shown are mean 6 SD. Unpaired t test. Downloaded from *p , 0.05, ***p , 0.005. http://www.jimmunol.org/

First, we ascertained whether STAT4 influences the proliferative and IL-18 and assessed IFN-g production. As expected, ILC1s by guest on September 26, 2021 response of ILCs following enteric L. monocytogenes infection. and NKp46+ ILC3s from C57BL/6 mice responded to stimulation As previously noted, intestinal ILC1s and ILC3s from C57BL/6 with an average of 36 and 27% of these populations producing mice upregulated Ki67 on day 4 postinfection (Fig. 5). In contrast, IFN-g, respectively (Fig. 6C). In contrast, STAT42/2 ILC pop- STAT4-deficient ILC1s and ILC3s exhibited significantly blunted ulations from the cLP were unable to produce IFN-g in response Ki67 expression at this time point. Therefore, the proliferative to IL-12 and IL-18 signals. The NKp46+ ILC3 population consists response of ILC1s and ILC3s to intestinal L. monocytogenes in- of both RORgt+ ILC3s and RORgt2 ex-ILC3s. Strikingly, segre- fection is dependent on STAT4 signaling. These data support an gation of these populations by RORgt expression reveals that influence of STAT4 on ILC responses to gastrointestinal infection only the RORgt2 ex-ILC3s from C57BL/6 mice produce IFN-g with L. monocytogenes and indicate that STAT4 may direct other in the 4-h stimulation period (data not shown). Overall, these protective functions of ILC populations. data reveal an essential role for STAT4 in intestinal ILC production of IFN-g. STAT4 is required for IFN-g production by ILC1s + In addition to its function in Th1 differentiation and cytokine and NKp46 ILC3s secretion, STAT4 also supports cytokine production and plas- Both IL-12 and IL-23 drive STAT4 phosphorylation because of the ticity of Th17 cells, the adaptive counterparts to ILC3s (21, 22, use of a shared receptor subunit, IL-12Rb1 (16, 38), and ILC1s and 39). Therefore, we analyzed the production of additional ILC3 NKp46+ ILC3s have been shown to respond to IL-12 stimulation effector cytokines, IL-17A and IL-22, by activated C57BL/6 and with cytokine production (13, 34). Therefore, we tested the ability STAT42/2 ILC3s. We found no significant defect in IL-17A or of ILC populations to activate STAT4 signaling in response to IL-22 production by STAT42/2 ILC3s in response to IL-23/IL-1b IL-12 stimulation. We measured potent phosphorylation of STAT4 or PMA/ionomycin stimulation (Supplemental Fig. 4A–C). In in ILC1s following 30 min of IL-12 stimulation, with an average support of this ex vivo data, we also saw no difference in IL-17A of 60% of the cells responding. In contrast to a previous report or IL-22 serum concentrations from STAT42/2 or Rag12/2STAT42/2 (37), we detect rapid phosphorylation of STAT4 in .50% of the miceatday2post-Lm-InlAm infection when compared with their NKp46+ ILC3 population (Fig. 6A, 6B). wild-type counterparts (Supplemental Fig. 4D and data not STAT4 regulates IFN-g production by several immune cell shown). Altogether, these data support a principal role for STAT4 populations, including adipose- and liver-resident ILC1s (19, 25, in mediating IFN-g but not IL-17A or IL-22 production by ILCs. 26). However, the role of STAT4 in functional responses by in- The production of IFN-g is important for early control of sys- testinal ILCs has not been confirmed, and because of tissue- temic and enteric L. monocytogenes infection (31, 40–42). As specific ILC regulation, it is necessary to determine if STAT4 in ILC-deficient mice, STAT42/2 and Rag12/2STAT42/2 mice functions similarly in intestinal ILC1s. Therefore, we stimulated showed an inability to produce detectable levels of IFN-g in serum cLP lymphocytes from C57BL/6 and STAT42/2 mice with IL-12 atday2ofL. monocytogenes infection (Fig. 3C, 3F). Thus, we The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 6. STAT4 is required for IFN-g production by ILC1s and NKp46+ ILC3s. (A and B) cLP lymphocytes from Rag12/2 mice were stimulated with IL-12 for 30 min. Representative flow cytometry plots of (A) ILC1 and (B) NKp46+ ILC3 show phosphorylation of STAT4 (pSTAT4) without stimulation (gray) and with IL-12 stimulation (black). Graphs show paired values (n = 6 mice; two independent experiments). (C) cLP lymphocytes from C57BL/6 and STAT42/2 mice were stimulated with IL-12 and IL-18 for 4 h. Representative flow cytometry plots of IL-12/IL-18–stimulated ILC1 and NKp46+ ILC3 show intracellular IFN-g. Graphs indicate average frequency of IFN-g+ ILCs (n = 5 mice per genotype; two independent experiments). (D) Rag12/2 mice treated with anti–IFN-g mAb or PBS control were infected with Lm-InlAm intragastrically, and bacterial burden of indicated organs was determined on day 5 postinfection (three independent experiments). Graphs show mean 6 SD. (A and B) Paired t test. (C) Two-way ANOVA with Holm–Sidak multiple comparisons test. (D) Unpaired t test. *p , 0.05, **p , 0.01, ***p , 0.005, ****p , 0.001. posited that IFN-g is critical for protection against L. monocytogenes this is dependent on STAT4 (25, 26). The literature on Th1 cells infection. To test this, we neutralized IFN-g in Rag12/2 mice reveals that STAT4 functions in multiple aspects of IFN-g regu- (anti–IFN-g) and measured intestinal bacterial burden and dis- lation (19). Through IL-12–mediated STAT4 activation in Th1 semination at 5 d postinfection. Neutralization of IFN-g resulted cells, IL-18Ra expression is upregulated, resulting in enhanced in a 1.23 and 0.92 log-fold change higher bacterial burden in the sensitivity to IL-18 stimulation and directed IFN-g production colon and draining LN, respectively, compared with PBS-treated (19). In fact, the gene encoding IL-18Ra, Il18r1, is directly bound control mice (Fig. 6D). Dissemination of bacteria to the spleen by STAT4 in Th1 cells and is one of the most differentially and liver was also significantly increased in mice treated with expressed transcripts in STAT4-deficient Th1-polarized cells anti–IFN-g. Altogether, these data reveal STAT4 as a principal compared with wild-type Th1 cells (18, 19, 43). Therefore, we regulator of IFN-g by intestinal ILC populations and demonstrate analyzed the expression of this established STAT4 target gene a role for IFN-g in protection against enteric Lm-InlAm infection. on ILC populations of the cLP. Surprisingly, we observed that most intestinal ILC1s and ILC3s expressed high levels of STAT4 transcriptionally regulates IFN-g production by IL-18Ra at steady-state and that this was independent of STAT4 intestinal ILCs (Fig. 7A). Intriguingly, evaluation of ILC3s based on the expres- The data of our laboratory and others demonstrates that IL-12 and sion of NKp46 and IL-18Ra revealed three distinct populations: IL-18 signaling together drive ILC production of IFN-g and that NKp462IL-18Ra2, NKp462IL-18Ra+, and NKp46+IL-18Ra+. 10 STAT4 MEDIATES ILC RESPONSES TO ENTERIC INFECTION Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 7. STAT4 transcriptionally regulates IFN-g production by intestinal ILCs. (A) ILC1 and ILC3 from the cLP of C57BL/6 and STAT42/2 mice were analyzed for surface expression of IL-18Ra. Representative flow cytometry plots show IL-18Ra expression directly ex vivo. Graphs depict the frequency of IL-18Ra+ ILCs of total population (three independent experiments, n = 6–8 mice per genotype). (B) NKp46+ ILCs from C57BL/6 and STAT42/2 cLP were analyzed for intracellular expression of Tbet directly ex vivo. Representative flow cytometry plots are shown (two independent experiments, n = 6 mice per genotype). Graph depicts mean fluorescence intensity of Tbet+ ILCs. (C) Total cLP lymphocytes were preincubated with AZD1480 or DMSO (vehicle) and then stimulated with IL-12. Representative flow cytometry plots show IFN-g production gated on ILC1s (combined from two independent experiments). Graphs depict mean 6 SD. (A and C) Unpaired t test. *p , 0.05, ***p , 0.005. ns, not significant.

STAT4 is also known to regulate the expression of the tran- have been shown to directly bind the Ifng gene and activate scription factor Tbet (encoded by Tbx21), which binds and directs transcription, including but not limited to Tbet, STAT4, and transcription of Ifng (44–47). Hence, we examined Tbet expres- NF-kB (18, 47, 48). Because we observed that STAT42/2 ILCs sion between C57BL/6 and STAT42/2 ILC1s to test whether were unable to produce IFN-g despite unaltered Tbet and IL-18Ra STAT4-dependent IFN-g production is a result of decreased Tbet (which activates NF-kB) expression, we hypothesized that STAT4 levels. We found a modest but NS decrease in the Tbet expression is necessary as an acute transcription factor upon cytokine stim- at a per-cell level (Fig. 7B). These data contrast the requirement ulation to induce IFN-g production. To test this, we used a small of STAT4 in directing CD4 T cell expression of IL-18Ra and Tbet molecule inhibitor of Jak2 (AZD1480), which blocks the phos- and also indicate that the lack of IFN-g production by STAT4- phorylation of Jak2 (49), thereby preventing STAT4 activation and deficient ILCs is not a result of altered expression of these function as an acute transcription factor. cLP lymphocytes from upstream regulatory factors. C57BL/6 mice were incubated with AZD1480 or DMSO (vehicle) Finally, we tested whether STAT4 is required at the time of prior to stimulation with IL-12. An average of 14% of vehicle-treated activation to direct IFN-g production. Several transcription factors ILC1s responded to IL-12 stimulation with IFN-g production, The Journal of Immunology 11 but AZD1480 treatment reduced this frequency 3-fold to 4.6% on lymphocytes that share markers with NK cells requires re- average (Fig. 7C). These data indicate that ILC1s require STAT4 examination of the distinct roles played by each cell pop- as a direct transcription factor at the time of activation. ulation. Interestingly, we and others find that the intestine harbors a larger population of ILC1s than NK cells (25). In this Discussion study, we find that ILC1s and NKp46+ ILC3s are the primary As sentinel cells enriched at barrier surfaces, the protective role of producers of IFN-g, supporting an essential role for helper ILCs helper ILCs against viral, bacterial, and fungal infection are in- in early responses to enteric L. monocytogenes. This suggests creasingly appreciated. ILC deficiency in both depletion and ge- that early studies reporting the role of NK cells in systemic netic models results in increased intestinal burden, increased L. monocytogenes infection may have been describing contri- dissemination to peripheral tissues, and ultimately increased sus- butions of a heterogeneous population of innate lymphocytes. ceptibility to infection. By analyzing proliferative and functional Therefore, future work delineating these populations is of ut- properties of ILC subpopulations, we identified ILC1s and ILC3s, most interest. but not ILC2s, as L. monocytogenes responsive. To address the ILCs have been identified to stably populate the majority of mechanism of protection by ILCs, we measured protection in tissues throughout the body (29, 53). Life-long residency in these mice lacking innate STAT4 signaling and found that STAT4 is tissues results in transcriptional distinctions between similar ILC required for early protection. Intestinal ILC populations developed populations from disparate organs (54). For instance, non-NK cell and were maintained independently of STAT4. However, both ILC1 populations found in the liver rely on the transcription ILC1s and NKp46+ ILC3s required STAT4 to mount an IFN-g factor Hobit for development and/or maintenance; however, response. Mechanistically, STAT4 functioned as an acute tran- Hobit-deficient mice retain ILC1s in other compartments such as Downloaded from scription factor at the time of cytokine activation. Altogether, our the gastrointestinal tract (26). Similarly, ILC2 intrinsic expression data suggest a critical role of STAT4 for ILC-mediated protection of the tyrosine-protein kinase Itk is required for the establishment against entry and dissemination of intracellular bacteria. of gut ILC2s, yet Itk-deficient ILC2s can be found normally in the The immune events that occur upon gastrointestinal infection lung and adipose (55). In regard to the STAT family of molecules, with L. monocytogenes are largely unknown because of a lack of STAT5 has been demonstrated to impact the development of

appropriate tools until recently. Now, with the development of ILC1s in the intestine more so than the liver (24). In this study, http://www.jimmunol.org/ the Lm-InlAm mutant strain, we can better dissect key players in we found the transcription factor STAT4 to be dispensable in protection against intestinal listeriosis. Notably, it requires a rel- the development, maintenance, or plasticity of ILC populations in atively high dose of L. monocytogenes inoculum to elicit a pro- the LP and draining LNs of the intestine. This is consistent with ductive infection when it is administered via the oral route as previous publications that show STAT4 to be dispensable for the opposed to systemic injection. This implicates robust and ef- development of adipose- and liver-resident ILC1 populations (25, fective protective mechanisms are present in the intestinal tract. 26). Altogether, we can conclude that although some transcription A recent report demonstrates the commensal microbiota in me- factors have tissue-specific requirements for ILC development and diating this first line of defense against gastrointestinal infection maintenance, others may play similar roles regardless of tissue with L. monocytogenes (31). However, once the bacteria translo- environment. It will be of interest to better understand the func- by guest on September 26, 2021 cates across the intestinal barrier, the underlying immune cell tional implications of the distinct profiles in maintaining tissue- populations must be prepared to mount a protective response. In specific protective homeostasis. Also, identifying the signals that this study, we have identified ILC1s and ILC3s as essential drive the disparate transcriptional profiles of each compartment sentinel cells that respond to L. monocytogenes infection and will be important future work. inhibit the growth and dissemination of L. monocytogenes in the Helper ILCs have been defined as innate counterparts to CD4 intestinal tract in a STAT4-dependent manner. Future studies are helper T cells because of parallels in transcription factor and ef- needed to characterize the targets of STAT4-mediated ILC cy- fector cytokine profiles (56). In this study, we uncovered important tokine responses, and it will be of interest to delineate the distinctions between these immune populations. Although STAT4 myeloid populations that are recruited and activated by intestinal is essential for the differentiation of Th1 cells and the plasticity of ILCs in response to Lm-InlAm. Another important difference be- Th17 cells (17, 21), we find normal development and maintenance tween the oral and systemic L. monocytogenes inoculations is the of intestinal ILC1 and ILC3 subpopulations in the environment kinetics by which infection of the liver and spleen occur and how of STAT4 deficiency. Similarly, although STAT4 is a known this impacts innate and adaptive immune priming. Dissemination contributor to the production of IFN-g and IL-17A in CD4+ helper of L. monocytogenes from the intestine to the periphery occurs T cells (19, 22), we demonstrate that only IFN-g production was with delayed kinetics compared with the introduction of a large disrupted in ILCs lacking STAT4. In contrast to our findings, a bolus of bacteria to the liver and spleen via the bloodstream (50). recent report shows evidence that NKp46+ ILC3s are unable to Still, we were able to measure ILC-derived circulating cytokines, produce IFN-g prior to IL-23–dependent epigenetic remodeling specifically IFN-g and IL-22, following intragastric infection as of the Ifng gene locus (37). We observe ex-ILC3 IFN-g production early as day 2 postinfection. These systemic cytokines can prime to occur with similar kinetics as ILC1s. These differences could be immune responses in distant sites prior to bacterial dissemination, due to the exclusion in the previous study of ex-ILC3s that had permitting containment of the infection. Hence, the gastrointesti- downregulated RORgt. Using RORgt fate-mapping mice, it is nal route of infection is a useful tool to better understand host- known that ex-ILC3s can lose expression of RORgtasthey pathogen interaction at both local and systemic levels. Future upregulate Tbet and NKp46 (13, 34); therefore, we did not in- studies comparing the immune responses to distinct infection clude RORgt in our identification of ex-ILC3s. It is quite likely routes will further identify protective cell populations that can that as ILC3s transition into ex-ILC3s, the Ifng locus is remodeled be harnessed to enhance therapeutic design. and the resulting RORgt2 ex-ILC3s gain response kinetics similar The role of NK cells in systemic infection with L. monocytogenes to those of ILC1s. Altogether, these data support both redundant is extensively characterized, and this cell population is known and distinct roles for STAT4 in the ILC population compared with to directly kill L. monocytogenes–infected hepatocytes (51, 52). CD4 helper T cells and suggest differential regulation of fate Nevertheless, the recent identification of ILC1s and other innate decisions and effector gene expression between these populations. 12 STAT4 MEDIATES ILC RESPONSES TO ENTERIC INFECTION

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