And Infection Control of IL-22 Production in Homeostasis IL-22

IL-22 Fate Reporter Reveals Origin and Control of IL-22 Production in Homeostasis and Infection

This information is current as Helena Ahlfors, Peter J. Morrison, João H. Duarte, Ying Li, of September 28, 2021. Judit Biro, Mauro Tolaini, Paola Di Meglio, Alexandre J. Potocnik and Brigitta Stockinger J Immunol 2014; 193:4602-4613; Prepublished online 26 September 2014;

doi: 10.4049/jimmunol.1401244 Downloaded from http://www.jimmunol.org/content/193/9/4602

References This article cites 37 articles, 8 of which you can access for free at: http://www.jimmunol.org/content/193/9/4602.full#ref-list-1 http://www.jimmunol.org/

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

IL-22 Fate Reporter Reveals Origin and Control of IL-22 Production in Homeostasis and Infection

Helena Ahlfors, Peter J. Morrison, Joa˜o H. Duarte, Ying Li, Judit Biro, Mauro Tolaini, Paola Di Meglio, Alexandre J. Potocnik,1 and Brigitta Stockinger

IL-22 is a cytokine that regulates tissue homeostasis at barrier surfaces. A variety of IL-22–producing cell types is known, but identification on the single-cell level remains difficult. Therefore, we generated a fate reporter mouse that would allow the identification of IL-22–producing cells and their fate mapping in vivo. To trace IL-22–expressing cells, a sequence encoding Cre recombinase was cloned into the Il22 locus, and IL22Cre mice were crossed with reporter mice expressing enhanced yellow fluorescence protein (eYFP) under control of the endogenous Rosa26 promoter. In IL22CreR26ReYFP mice, the fluorescent reporter permanently labels cells that have switched on Il22 expression, irrespective of cytokine production. Despite a degree of underreporting, eYFP expression was detectable in nonimmune mice and restricted to group 3 innate lymphoid cells (ILC3) in the gut Downloaded from and gd T cells in skin or lung. Upon skin challenge with imiquimod, eYFP+ gd and CD4 T cells expanded in the skin. Infection with Citrobacter rodentium initially was controlled by ILC3, followed by expansion of eYFP+ CD4 T cells, which were induced in innate lymphoid follicles in the colon. No eYFP expression was detected in small intestinal Th17 cells, and they did not expand in the immune response. Colonic eYFP+ CD4 T cells exhibited plasticity during infection with expression of additional cytokines, in contrast to ILC3, which remained largely stable. Single-cell quantitative PCR analysis of eYFP+ CD4 T cells confirmed their heterogeneity, suggesting that IL-22 expression is not confined to particular subsets or a dedicated Th22 subset. The Journal of http://www.jimmunol.org/ Immunology, 2014, 193: 4602–4613.

nterleukin-22 is a cytokine expressed by immune cells but Despite the undisputed biological importance of IL-22, it re- acting on nonhematopoietic cells. The receptor for IL-22 is mains difficult to follow its expression in vivo either in the steady- I expressed in barrier sites, such as skin, intestine, and lung, as state or during inflammatory responses because of technical well as in liver, pancreas, and kidney (1, 2). IL-22 production is problems with intracellular staining. An additional complication attributed to many immune cell types, such as CD4, CD8, and gd is the issue of effector cell plasticity, which makes it difficult to un- T cells, NK cells, and subsets of innate lymphoid cells (ILC) (3). equivocally assign IL-22 production to different subsets. We (14) Thus, the expression pattern of IL-22 and its receptor creates sig- addressed this issue for IL-17–producing cells by generating a fate by guest on September 28, 2021 naling directionality from the immune system to the tissues, in line reporter that marked cells that had initiated the IL-17 program with with the important function that IL-22 has in maintaining tissue enhanced yellow fluorescence protein (eYFP) expression. This allowed integrity. IL-22 plays an important role in the homeostasis of mu- easy identification of such cells ex vivo and further determination of cosal surfaces. During inflammation, IL-22 induces the expression their effector program, irrespective of ongoing IL-17 production. of acute-phase proteins, antimicrobial peptides, and chemokines (4), In this study, we used the same strategy to generate a knock-in which support resolution of the local inflammation, repair of injured mouse strain bearing a gene encoding Cre recombinase in the Il22 tissue, and re-establishment of homeostasis. IL-22 is required for locus and bred those mice with reporter mice expressing eYFP protective immune responses against certain extracellular bacteria from the Rosa26 promoter to monitor expression of IL-22 in the (5–9), and it prevents the dissemination of intestinal microbiota (10). steady-state and during infection with Citrobacter rodentium. Our In contrast, dysregulated production of IL-22 is associated with data demonstrate a substantial expansion of eYPF+ CD4 T cells in certain human autoinflammatory diseases, including rheumatoid the large intestinal lamina propria (LI LP) from day 5 postinocu- arthritis, inflammatory bowel disease, and psoriasis (2, 11–13). lation (p.i.), whereas eYFP+ ILCs are present in uninfected mice and do not expand substantially on infection. IL-22–expressing Division of Molecular Immunology, Medical Research Council National Institute for CD4 T cells predominantly associate with a Th17 profile but show Medical Research, London NW7 1AA, United Kingdom pronounced plasticity in the course of infection, in contrast to ILCs 1Current address: School of Biological Sciences, College of Science and Engineer- that remain committed to IL-22 production. Single-cell quantitative ing, University of Edinburgh, Edinburgh, U.K. PCR (qPCR) analysis of gene expression for CD4 T cell subsets Received for publication May 16, 2014. Accepted for publication August 26, 2014. indicates substantial heterogeneity, which suggests that IL-22 expres- This work was supported by Medical Research Council UK Grant U117512792. sion is not confined to particular subsets or a dedicated Th22 subset. Address correspondence and reprint requests to Dr. Brigitta Stockinger, Division of Molecular Immunology, Medical Research Council National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K. E-mail address: Materials and Methods [email protected] Mice Abbreviations used in this article: AhR, aryl hydrocarbon receptor; B6, C57BL/6; Codon-improved Cre recombinase (iCre) (15) was inserted into the first eYFP, enhanced yellow fluorescence protein; iCre, codon-improved Cre recombinase; ILC, innate lymphoid cell; ILF, isolated lymphoid follicle; iTreg, inducible exon of the Il22 locus by homologous recombination in B6/N mouse em- regulatory T cell; LI LP, large intestinal lamina propria; mLN, mesenteric lymph bryonic stem cells. The neocassette was removed via FLPe-mediated re- Cre node; qPCR, quantitative PCR; SI LP, small intestinal lamina propria; wt, wild-type. combination. To visualize Cre-mediated recombination, IL22 mice were intercrossed with R26ReYFP reporter mice (expressing eYFP from the Rosa26 Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 promoter) (16), generating IL22CreR26ReYFP reporter mice. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401244 The Journal of Immunology 4603

C57BL/6 (B6), B6.Rag22/2CD45.1, and IL22CreR26ReYFP reporter mice a 70-ml cell strainer to obtain single-cell suspensions. Cells from skin were were bred in the animal facility of the Medical Research Council National isolated as described above. Institute for Medical Research. All mice were kept under specific pathogen– Gut-resident lymphocytes were prepared by cutting the small intestine or free conditions. All animal experiments were approved by the local Ethical large intestine (colon and cecum) in small pieces and incubating in digestion Review panel at the National Institute for Medical Research in accordance buffer (IMDM supplemented with 3% [v/v] FCS, 5 mM EDTA, 25 mM with the Institutional Committees on Animal Welfare of the UK Home HEPES, penicillin/streptomycin, and 0.145 mg/ml DTT) for 20–40 min at Office (the Home Office Animals Scientific Procedures Act, 1986). 37˚C with shaking. The supernatant containing epithelial and intraepithelial cells was enriched for intraepithelial cells using 36.5% Percoll (Amersham) Generation of bone marrow chimeras density gradient centrifugation. The remaining tissue was digested further 7 with a mixture containing 400 mg/ml Liberase TL (Roche) and DNase I Bone marrow chimeras were generated by i.v. injection of 10 T cell– eYFP (10 U/ml; Sigma) for 30 min at 37˚C. For flow cytometry analysis, cells were depleted bone marrow cells from homozygous or heterozygous R26R reporter mice into B6.Rag22/2CD45.1 hosts that were sublethally irradi- stained for surface molecules (see list of Abs in Table I) in the presence of ated (5 Gy). Mice were used in experiments from 7 wk after reconstitution. 10% Fc block and acquired on a FACSCanto II (BD). Citrobacter rodentium infection In vitro T cell differentiation Naive T cells (CD4+CD252CD44low) were purified using flow cytometry C. rodentium strain DBS100 (ATCC 51459; American Type Culture Col- lection) was cultured in Luria–Bertani broth overnight, diluted 1:400 with from spleen and lymph nodes of fate reporter mice and activated with plate- fresh Luria–Bertani broth, and cultured for additional 3–4 h. Bacterial bound anti-CD3 (clone 2C11; eBioscience) and anti-CD28 (clone 37.51; m concentration was determined by measuring the OD at 600 nm. The cul- BioLegend) (coated with 0.5 and 5 g/ml, respectively) in the presence of tures were serially diluted and plated to confirm the inoculum. IL-22 fate lineage-polarizing cytokines as follows: IL-12 (5 ng/ml) for Th1 cells; IL-4 +/2 9 (10 ng/ml) for Th2 cells; IL-4 (10 ng/ml) and TGF-b (5 ng/ml) for Th9 reporter, Il22 , or wild-type (wt) mice were gavaged with 2 3 10 CFU cells; TGF-b (10 ng/ml) for inducible regulatory T cells (iTregs); and IL-6 in 200 ml PBS. Postinfection, mice were weighed daily, and weight loss Downloaded from (20 ng/ml), TGF-b (1 ng/ml), IL-1b (10 ng/ml), IL-23 (20 ng/ml) (all from was determined. Distal colon pieces were weighed and homogenized in + sterile PBS, and serial dilutions were plated in duplicates onto Brilliance R&D Systems), or FICZ (250 nM; Enzo) for Th17 cells. Alternatively, CD4 E. coli/coliform Selective Agar (Fisher Scientific) plates for measurement cells were purified using an EasySep Mouse CD4+ T Cell Enrichment Kit of CFU. C. rodentium colonies were identified based on morphology and (STEMCELL Technologies) from spleen of fate reporter mice and acti- g m color after 18–24 h of incubation at 37˚C. vated as described above. Cells were cultured with anti–IFN- (10 g/ml, clone XMG1.2; eBioscience), anti–IL-4 (10 mg/ml, clone 11B11), IL-6, Imiquimod-induced psoriasiform-like skin inflammation IL-1b, and FICZ, as well as with TGF-b for Th17 cells or with anti–TGF-

b (10 mg/ml, clone 1D11) for “Th22” cells. All cytokines were of mouse http://www.jimmunol.org/ Mouse dorsal skin was shaved, and Aldara cream containing 5% imiquimod origin. Cells were cultured in IMDM (Sigma) supplemented with 5% FCS, (Meda; 50 mg/mouse) was applied daily for five consecutive days to induce 0.002 M L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and psoriasiform skin inflammation. On day 5, an 8-mm biopsy punch (Kai 5 3 1025 M 2-ME and analyzed for intracellular cytokines on day 4. medical) was used to collect full-thickness skin biopsies of the treated area. Two skin biopsies were minced, digested with a mixture containing 400 mg/ml Cytokine measurement Liberase TL and 1 mg/ml collagenase D (both from Roche) in IMDM for Cells were stimulated for 4 h with PdBU (500 ng/ml) and ionomycin (500 m 2 h at 37˚C with shaking, and mashed through a 70- lcellstrainertoobtain ng/ml) in the presence of brefeldin A (1 mg/ml), fixed with 4% parafor- single-cell suspensions. Leukocytes were enriched using 36.5% Percoll maldehyde, permeabilized with 0.1% Nonidet P-40 or 0.1% saponin, and (Amersham) density gradient centrifugation. stained for intracellular cytokines or transcription factors (Abs in Table I).

Flow cytometry analysis of tissue-resident cells Cells were acquired on a FACSCanto II (BD). The Th cell culture super- by guest on September 28, 2021 natants (Fig. 1D) were analyzed for IL-22 using FlowCytomix, according Lung, pancreas, kidney, liver, and spleen were minced. Minced lung was in- to the manufacturer’s instructions (Bender MedSystems, Vienna, Austria). cubated with a digestion mixture containing 400 mg/ml Liberase TL (Roche) eYFP+ and eYFP2 CD4 T cells (CD45+CD4+TCRb+)wereFACSsorted in IMDM for 30 min at 37˚C with shaking. Lung, pancreas, kidney, liver, from LI LP of C. rodentium–infected fate reporter mice on day 14 p.i., and spleen, lymph nodes, thymus, and Peyer’s patches were mashed through 25,000 cells were cultured on anti-CD3–coated wells for 18 h. Medium-only

Table I. Abs used in ﬂow cytometry analysis

Ag Clone Fluorochrome Supplier CD3ε 145-2C11 Allophycocyanin BioLegend CD4 GK1.5 Allophycocyanin, V500 BioLegend, BD Biosciences CD8a 53-6.7 PerCP/Cy5.5, allophycocyanin BioLegend CD11b M1/70 Allophycocyanin BioLegend CD11c N418 Allophycocyanin BioLegend CD19 6D5 Allophycocyanin BioLegend CD45.2 104 PE/Cy7, allophycocyanin/Cy7 BioLegend cKit 2B8 PE BioLegend CD49b DX5 Allophycocyanin BioLegend Foxp3 150D Alexa Fluor 647 BioLegend gdTCR UC7-13D5 PE/Cy7, BV421, allophycocyanin BioLegend Ly-6G/Ly-6C (Gr1) RB6-8C5 Allophycocyanin BioLegend IFNg XMG1.2 PE BD Biosciences IL-17A TC11-18H10.1 Alexa Fluor 647, Pacific Blue BioLegend IL-22 AM-22 Allophycocyanin Gift from J.-C. Renauld IL-4 11B11 PE BioLegend IL-7R A7R34 PE, PE-Cy5 BioLegend, eBioscience IL-9 RM9A4 PE BioLegend NK1.1 PK136 PE, PerCP/Cy5.5 BioLegend NKp46 29A1.4 Brilliant Violet 421 BioLegend Sca-1 D7 Pacific Blue BioLegend TCRb H57-597 Allophycocyanin/Cy7, PerCP/Cy5.5, allophycocyanin BioLegend Ter119 TER-119 Allophycocyanin BioLegend CD90.2 (Thy1.2) 53-2.1 PE/Cy7 BioLegend Rorgt O31-378 PE BD Biosciences GATA-3 TWAJ PerCP–eFluor 710 eBioscience 4604 FATE MAPPING OF IL-22 PRODUCTION wells were included as controls. The culture supernatants were analyzed conditions with IL-1b had a similar effect as culture with FICZ, for IL-22, IL-17A, and IFN-g, as described above. whereas IL-23 had no enhancing effect on IL-22/eYFP expression. For serum IL-22 measurement, blood was collected from both Optimal expression was achieved by combining FICZ and IL-1b Citrobacter-infected animals and steady-state control mice and processed by centrifugation to isolate the serum. The concentration of IL- (Fig. 1E, 1F). There was only marginal induction of eYFP or IL- 22 in the serum was determined by capture ELISA, according to the 22 under “Th22” conditions using IL-6 and anti–TGF-b (Fig. 1G). manufacturer’s instructions (eBioscience). Only a small proportion of cells that stained for intracellular IL- + Single-cell gene-expression analysis 22 coexpressed eYFP; in addition, a fraction of eYFP cells did not stain for IL-22 (Table I). This suggests that there is a disconnect + eYFP CD4 T cells from LI LP were FACS sorted and subsequently deposited between the production of IL-22 cytokine and induction of the into 96-well PCR plates using an automated cell deposition unit on a MoFlo sorter (Becton Dickinson, Franklin Lakes, NJ) containing 10 ml reporter. We also had seen a degree, although less pronounced, of Cre lysis buffer. The sorter provided single cells in .99% of the wells, and no “underreporting” in the IL-17 fate reporter model. A likely ex- well contained more than one cell, as assessed by routinely sorting fluo- planation for incomplete reporting is the delay between cytokine rescent beads or cells prior to and after single-cell sorting. Cells were induction and production of Cre, which is necessary to reveal the processed for quantitative real-time PCR analysis using a Single Cell-to-CT fluorescent reporter. In the case of IL-22, cytokine production may qRT-PCR Kit (Ambion), according to the manufacturer’s protocol. A total of 175 single cells was analyzed (day 0 = 75, day 5 = 44, day 25 = 56). be of shorter duration, thus compromising effective Cre induction. The resultant cDNA served as a template for the amplification of the Furthermore, IL-22 expression, like that of the closely related cy- genes of interest by real-time PCR with TaqMan Gene Expression assays tokine IL-10, may be monoallelic and, therefore, more severely and universal PCR Master Mix on an ABI PRISM 7900 Sequence Analyzer compromised when combined with the disruption of one allele by Cre. (all from Applied Biosystems). Downloaded from + Immunohistology Detecting IL22-eYFP cells in vivo under steady-state conditions Frozen sections of large intestine were prepared at 8 mm thickness and fixed in 4% paraformaldehyde. Sections were counterstained with DAPI and To assess which cells express IL-22 in unchallenged mice, different mounted using Fluoromount-G (Southern Biotechnology). mAbs used are organs, including gut, lung, skin, lymph nodes, spleen, thymus, listed in Table I. Confocal images were obtained using a Leica TCS-SP2 liver, pancreas, kidney, and bone marrow, were analyzed for the microscope equipped with 405-, 488-, 594-, and 647-nm lasers, and image + presence of eYFP cells. In concordance with the predominant http://www.jimmunol.org/ analysis was done in Adobe Photoshop. + Skin was fixed in neutral buffered formalin (Sigma), and tissue sections function of IL-22 in maintaining epithelial barriers, eYFP cells were stained with H&E for histopathology analysis. Epidermal and scale were found only in tissues involved in barrier function, namely thickness were quantified as previously described (17). gut, mesenteric lymph nodes (mLNs), lung, and skin (Fig. 2A, Statistical analysis 2B). However, the cell type expressing eYFP was tissue dependent (Fig. 2C): in the gut, the majority of eYFP+ cells was ILCs, whereas Statistical analysis was performed using Prism version 6.0 (GraphPad in skin or lung the majority of eYFP+ cells was gdT cells. We did Software). If two groups were compared, significance was calculated using + the unpaired t test. If more than two groups were assessed, significance was not detect eYFP cells in the resident Th17 population of the small calculated using one-way ANOVA and the Dunnett or Sidak corrected intestinal lamina propria (SI LP) with the caveat that a small number p value for multiple comparisons. For weight loss data, significance was of such cells might have escaped detection because of underreport- by guest on September 28, 2021 calculated using two-way ANOVA and the Tukey multiple-comparisons test. ing. However, there were eYPF+ CD4 T cells in the intraepithelial lymphocyte population and Peyer’s patches, as well as CD8aa Results and gd T cells and ILCs (Fig. 2B, 2C). Among the eYFP+ ILC Generating an IL-22 fate reporter mouse fraction in the SI LP and LI LP (see gating strategy in Fig. 3A), the To generate a reporter system that would allow for the identifi- majority was NKp462CD42, but CD4+ (LTi) and NKp46+ ILCs cation of IL-22 cytokine–producing cells, as well as mapping the also were present (Fig. 3C–E). All eYFP+ ILCs expressed RORgt fate of these cells in vivo, we cloned a sequence encoding Cre and, therefore, can be assigned to the ILC3 subset (Fig. 3B). recombinase into the Il22 locus (Il22Cre). To visualize Cre activity, IL22Cre mice were crossed with reporter mice expressing eYFP under Induction of eYFP expression during inflammatory responses the control of the endogenous Rosa26 promoter (R26ReYFP). In IL-22 expression has been documented in inflammatory skin con- IL22CreR26ReYFP mice, the fluorescent reporter permanently labels ditions, such as psoriasis, and in the mouse model of psoriasiform cells that have switched on Il22 expression, regardless of the inflammation induced by the TLR7 ligand imiquimod (20, 21). present production status of this cytokine. The endogenous Il22 Therefore, we treated IL22CreR26ReYFP mice with imiquimod- locus, the gene-targeting vector, the targeted Il22 allele, including containing Aldara cream for five consecutive days and analyzed the Neo resistance gene cassette (Il22Cre-Neo), and the final tar- eYFP expression on day 5. This treatment resulted in increased geted allele (Il22Cre) after FLPe-mediated recombination are shown epidermal thickness and scaling (Fig. 4A, 4B), expansion of pri- in Fig. 1A. Fig. 1B shows the sequence of Il22 exon 1, with a linker marily gd T cells that expressed eYFP, and a considerably smaller consisting of 6 aa placed between the original Il22 ATG site and the number of eYFP+ CD4 T cells (Fig. 4C–E), consistent with reports respective start of the iCre minigene. of a predominant role for gd T cells in this model (20, 22). As We first tested reporter activity in vitro following stimulation of recently reported (17), we did not detect any CD8 T cells or ILCs FACS-selected naive CD4 T cells for 3 d under Th17, Th1, Th2, expressing eYFP in this model. The concordance of IL-22 and Th9, or iTreg conditions. IL-22 was previously identified as a Th17 eYFP expression in gd T cells was ∼30% (Fig. 4F), which is con- cytokine (4). In accordance, we detected eYFP expression in Th17 siderably higher than what was seen under in vitro culture con- cells but not in any other in vitro–differentiated CD4 T cell subset ditions. Importantly, there was a considerably larger fraction of IL-17 (Fig. 1C). This was confirmed by measuring the secreted IL-22 in producers in the eYFP+ fraction, indicating that IL-22 production the culture supernatants (Fig. 1D). IL-22 expression by Th17 cells may be a specialization of the Th17 program, rather than an in- differentiated in the presence of IL-6 and TGF-b is normally low dependent program. to nonexistent, depending on the culture medium (18), whereas We next investigated the induction of eYFP during the course of stimulation in the presence of agonists of the aryl hydrocarbon infection with C. rodentium, an effacing attaching bacterial patho- receptor (AhR) induces IL-22 expression (19). Culture under Th17 gen that causes severe inflammation of the large intestine and The Journal of Immunology 4605 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 1. Fate-reporter eYFP+ cells are induced among IL-22–producing cells. (A) Schematic presentation of the endogenous Il22 locus, the targeting vector, the targeted Il22 allele, including the Neo resistance cassette (Il22Cre-Neo), and the ﬁnal targeted allele (Il22Cre) after FLPe-mediated recombination. The iCre component is a minigene composed of a codon improved Cre, followed by an intron and polyadenylation site derived from SV40. (B) Sequence of Il22 exon 1 after targeting showing the original 6 aa after the Il22 ATG-site and the respective start of the iCre minigene. (C) FACS plots showing the expression of eYFP and intracellular cytokines or Foxp3 in Th1, Th2, Th9, or Th17 cells or iTregs after 4 d of in vitro differentiation. (D) Concentration of IL-22 in day-4 supernatants of in vitro–differentiated Th1, Th2, Th9, and Th17 cells or iTregs. Data are mean 6 SEM and are representative of three independent experiments. (E) FACS plots showing the expression of eYFP and intracellular IL-22 in naive CD4+CD44loCD252 (Figure legend continues) 4606 FATE MAPPING OF IL-22 PRODUCTION

lower IL-22 expression than that of heterozygous IL-22+/2 mice. However, at the time points these mice were analyzed (from day 28) they had all cleared the infection and the kinetics of Citrobacter clearance in IL22CreR26ReYFP followed those reported for wt mice (23) (data not shown). Kinetic analysis, between days 2 and 82 p.i., for appearance and expansion of eYFP+-expressing CD4 T cells (Fig. 5B) and other cell types showed that cellular expansion was prominent in the CD4 cell population in the LI LP, whereas eYFP+ cells in the SI LP increased only in the later stages, presumably due to overspill from the LI LP (Fig. 4C, 4D). eYFP+ ILCs or gd T cells did not expand significantly throughout the course of infection. IL-22 protein was detectable in serum from day 5 p.i. (Fig. 5E). Histo- logical analysis of colon from IL22CreR26ReYFP mice showed eYFP+ cells in structures resembling isolated lymphoid follicles (ILFs) and their expansion throughout the colon following infection. Initially, eYFP+ cells were CD3-ve, indicative of ILCs, whereas eYFP expression was extended to CD3+ cells p.i. (Fig. 6). Downloaded from Homozygous IL22CreR26ReYFP cells expand in infection but fail to control Citrobacter We next investigated the fate of eYFP+ CD4 T cells in IL22Cre R26ReYFP mice with homozygous expression of Cre. Such mice are IL-22 deficient but still allow identification of the cell type via Cre/Cre

eYFP reporting (Fig. 7B). IL22 reporter mice were unable to http://www.jimmunol.org/ clear Citrobacter, as indicated by a log higher CFU count on day 5 p.i. (the mice succumbed to the infection by day 8) (Fig. 7A). Interestingly, the number of eYFP+ CD4 T cells, but not ILCs, in LI LP and mLNs was substantially higher (Fig. 7C). This could indicate either continuous stimulation of CD4 T cells to curb bacterial expansion or that homozygous Cre expression improved the reporter intensity. To avoid complications due to the differential outgrowth of Citrobacter, we generated chimeric mice in which we

cotransferred wt B6 bone marrow with bone marrow from either by guest on September 28, 2021 IL22CreR26ReYFP mice (heterozygous for Cre, IL22wt/Cre) or Cre homozygous reporter mice (IL22Cre/Cre) into B6.Rag22/2CD45.1

+ hosts. This allowed us to monitor the extent of eYFP induction in FIGURE 2. Prevalence of eYFP cells in untreated fate reporter mouse. CD4 T cells from heterozygous or homozygous donors in mice (A) Number of eYFP+ cells in various organs of untreated fate reporter 6 with comparable bacterial burden. As shown in Fig. 7D, there was mice. Data are mean SEM and are representative of two to nine inde- + pendent experiments. (B) FACS plots showing the expression of eYFP and no difference in eYFP CD4 T cells in recipients of homozygous CD45 in various organs of untreated fate reporter mice. Data are repre- versus heterozygous Cre reporter bone marrow, and the bacterial sentative of two to nine independent experiments. (C) Relative number of burden was similar (Fig. 7E). This makes it less likely that the eYFP+ cell types in intestinal tissues, skin, and lung. Cells were gated first hyperexpansion of eYFP+ cells in homozygous reporter mice for lymphocytes (forward scatter–area versus side scatter–area) and sin- (Fig. 7B) was simply the result of a higher reporting efficiency due glets (forward scatter–area versus forward scatter–width). These cells were to the presence of two Cre alleles; rather, it suggests an ongoing + analyzed for their CD45 expression. CD45 cells were analyzed for their stimulatory drive to CD4 T cells to combat the outgrowth of + + + + + surface markers as follows: CD4 T cells: TCRb CD4 ;CD8a T cells: TCRb Citrobacter, which is thwarted because of the lack of IL-22. CD8a+; gdTcells:gdTCR+; NK cells: NK1.1+; and ILCs: lineage2 Thy1.2+ (lineage mixture: CD11c, CD11b, DX5, Gr1, Ter119, gdTCR, TCRb, CD3, Functional plasticity of cytokine expression in eYFP+ cells is CD8a, and CD19). Data shown are mean and are representative of two prominent in T cells but limited in ILCs to nine independent experiments. BM, bone marrow; pLN, peripheral To investigate the cytokine profile of eYFP+ cells, T cells (gated on lymph nodes; PP,Peyer’s patches; SI IEL, small intestinal intraepithelial lymphocytes. TCRb expression) and ILCs (TCRb-ve) were analyzed throughout the course of infection for the single expression, as well as the requires IL-22 for restoration of the intestinal barrier following coexpression of, IL-22, IL-17, and IFN-g by intracellular staining. infection (7–9). It was noticeable that IL22CreR26ReYFP mice showed First, we investigated cytokine production of FACS-purified eYFP+ 2 a more pronounced transient weight loss following infection than and eYFP CD4 T cells from Citrobacter-infected mice (day 14) did wt B6 mice and even heterozygous IL-22+/2 mice (Fig. 5A). that were restimulated with anti-CD3 in vitro. Interestingly, IL-22, This is indicative of stochastic monoallelic expression of IL-22, IL-17, and IFN-g all were predominantly produced by eYPF+ which, in combination with only one functional allele, results in CD4 T cells, indicating the substantial plasticity of these cells

T cells cultured for 4 d in Th17-differentiating conditions (TGF-b+IL-6) supplemented with FICZ, IL-1b, or IL-23 or their combination. Fold induction in the percentage of eYFP+ cells cultured for 4 d in modiﬁed Th17 conditions (as indicated on the x-axis) compared with Th17 cells cultured in the presence of TGF-b and IL-6 only (F) or in the presence of IL-6 and anti–TGF-b (G). Data in (C) and (E) are representative of three independent experiments. The Journal of Immunology 4607 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 3. eYFP+ ILC3 cells in untreated fate reporter mice. (A) Gating strategy for ﬂow cytometry analysis. Cells were gated ﬁrst for lymphocytes (forward scatter–area versus side scatter–area) and singlets (forward scatter–area versus forward scatter–width). These cells were analyzed for their CD45 expression. CD45+ cells were analyzed for their Thy1.2 and lineage marker (CD11c, CD11b, DX5, Gr1, Ter119, gdTCR, TCRb, CD3, CD8a, and CD19) expression. Thy1.2+ lineage2 cells were further analyzed for eYFP, IL-7R, c-Kit, Sca-1, and NK1.1 expression. (B) Thy1.2+ lineage2 cells (left panels) and eYFP+ Thy1.2+ lineage2 cells (middle panels) were analyzed for Rorgt and Gata3 expression. eYFP expression on gated Thy1.2+ RORgt+ lineage2 cells (right panels). (C) Thy1.2+ lineage2 cells analyzed for eYFP and NKp46 expression (left panels). NKp462 cells analyzed for (Figure legend continues) 4608 FATE MAPPING OF IL-22 PRODUCTION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Fate reporter eYFP+ cells in skin inflammation. (A) Representative images of H&E staining of skin sections from untreated (d0) and imiquimod-treated (d5) IL-22 fate reporter mice at day 5. Scale bars, 100 mm. (B) Quantification of epidermal (left panel) and scale (right panel) thickness of untreated (d0) fate reporter mice and mice at day 5 after imiquimod treatment (d5). Data are mean 6 SEM (n = 4 mice/group). (C) Number of eYFP+ gdTCR+ and eYFP+ TCRb+ cells in dorsal skin of untreated fate reporter mouse (d0) and after 5 d of imiquimod treatment (d5). Data are mean 6 SEM and are representative of five independent experiments. (D) FACS plot of eYFP+ CD45+ cells from the dorsal skin of untreated (d0) and imiquimod-treated (5 d; d5) fate reporter mice analyzed for their gdTCR and TCRb expression. (E) FACS plot of gdTCR+ and TCRb+ cells analyzed for their eYFP expression in dorsal skin of untreated (d0) and imiquimod-treated (d5) fate reporter mice. (F) FACS plot of CD45+ gdTCR+ and TCRb+ cells from the dorsal skin of imiquimod-treated fate reporter mice analyzed for their eYFP, IL-22, and IL-17A expression. Data in (C–F) are representative of five independent experiments. **p , 0.01, ****p , 0.001. during the course of infection (Fig. 8A). Fig. 8B shows the con- ciation of IL-22 with the Th17 subset on the one hand and indi- cordance of eYPF and IL-22 expression in CD45+ cells, which is cated substantial plasticity on the other hand, as previously seen higher than what was observed for the in vitro culture (Fig. 1). with Th17 cells in intestinal inflammation (24). In an attempt to eYFP+ ILCs remained predominantly IL-22+ throughout infection, identify the developmental origin and fate of eYFP-expressing with only a proportion expressing IL-17 and/or IFN-g. In contrast, CD4 T cells in Citrobacter infection, we performed single-cell the predominant cytokine expressed in eYFP+ T cells was IL-17, qPCR for a range of markers associated with different T cell alone or together with IL-22 in the early phase of infection, with subsets, including transcription factors and cytokines. a gradual increase in IFN-g expression, alone or together with the The expression changes for these markers were determined from other cytokines (Fig. 8C). This confirmed the predominant asso- the uninfected basal state to day 5 (the onset of CD4 T cell expansion)

eYFP and CD4 expression (right panels). (D) Numbers of eYFP+ and eYFP2 total ILCs and ILC3 subpopulations. (E) Relative number of eYFP+ ILC3 subpopulations in SI LP and LI LP. Data in (C) and (D) represent the mean and are representative of at least four independent experiments. The Journal of Immunology 4609 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 5. Response of fate reporter mice to infection with Citrobacter. (A) Weight curve of B6 (wt/wt), IL-22+/2 (wt/2), and IL-22 fate reporter (wt/cre) mice infected with C. rodentium (n = 6/group). Data are mean 6 SEM. (B) FACS plot of CD45+ cells from LI LP of untreated (d0) or C. rodentium–infected fate reporter mice at days 12 (d12) and 28 (d28) analyzed for their eYFP and CD4 expression. Data are representative of two to eight independent experiments. (C) Kinetics of expansion of eYFP+ CD4 T cells, CD8 T cells, ILCs, and gdT cells in LI LP and SI LP of fate reporter mice during the course of infection with C. rodentium. Data are mean 6 SEM are representative of 2–12 independent experiments. (Figure legend continues) 4610 FATE MAPPING OF IL-22 PRODUCTION

without having to rely on active production of the cytokine. We used the same approach for the generation of the Cre knock-in construct that we had used successfully for the generation of IL17Cre R26ReYFP mice. However, we noticed immediately that the pen- etrance of reporting, as well as the coexpression of eYFP and IL- 22 cytokine, was considerably lower in IL22CreR26ReYFP mice. IL-22 belongs to the IL-10 family of cytokines, and it was shown that expression of IL-10 is largely monoallelic and stochastically regulated via control of expression frequency (25). It is likely that IL-22–producing CD4 T cells, similar to IL-10–producing cells, have a low probability for activation of the IL-22 allele so that expression is predominantly monoallelic. Because our construct disables one allele by Cre insertion, IL-22 expression is reduced further, which is particularly evident in the single-cell qPCR analysis. In hindsight, it would have been better to avoid destruction of one allele by Cre insertion. Nevertheless, despite these technical shortcomings, the reporter appears faithful and under in vitro conditions reveals IL-22 pro-

duction exclusively under Th17 conditions, with important con- Downloaded from tributions by IL-1b and the AhR ligand FICZ. AhR stimulation promotes the expression of IL-1R (26), and the induction of IL-22 in Th17 conditions with IL-1b and without addition of the AhR ligand reﬂects the presence of AhR agonists in culture medium (18). IL-23, which is important for IL-22 induction in vivo (8, 27),

does not play a noticeable role in vitro under these culture con- http://www.jimmunol.org/ ditions, which are too short to promote expression of IL-23R (28). We have not been able to detect substantial eYFP expression under IL-6 stimulation, and TGF-b is essential in vitro. The in vitro con- FIGURE 6. Immunohistology of colon from fate reporter mice. Sections ditions for induction of IL-22 production remain controversial, and from the large intestine (distal) of untreated (d0) and C. rodentium– they might also be influenced by ambient TGF-b concentrations in infected fate reporter mice at day 5 (d5) and day 25 (d25) p.i. were stained with anti-GFP (green), anti-CD45 (blue), and anti-CD3 (red) (original the medium. We strongly believe that in vitro culture conditions for magnification 320). Representative results from one of at least three both IL-17 and IL-22 producers do not accurately reflect the stimuli experiments/time point are shown. that these cells obtain in vivo, where multiple cell types, including stromal cells, might contribute to optimize their polarization. by guest on September 28, 2021 In vivo, the fate reporter revealed IL-22 producers in the steady- to day 25 (when Citrobacter was cleared). They indicate a gradual state and under inflammatory conditions, consistent with previous shift toward induction of Tbet and Ifng (not determined for day 5) reports that emphasized the importance of this cytokine at barrier in the later stages while retaining the expression of Rora, with less sites of intestine, lung, and skin. The expression of eYFP was prominent expression of Rorc (not determined at day 5) and restricted to tissue-resident cell types at these locations, such as gd reduced AhR expression. The coexpression of IL-22 with eYFP T cells in skin and lung and ILC3 in the intestine, indicating their was low in the steady-state, as well as in late stages of infection, important role in homeostatic maintenance of epithelial barrier but it was more pronounced on day 5 (Fig. 8D). The magnitude of integrity. Interestingly, the resident Th17 population present in gene expression during the three time points is given in the for- the SI LP of nonimmune mice did not show eYFP expression, mat of a heat map (Fig. 8E, gray fields indicate absence of data). which instead was restricted to ILCs that were characteristic of + the ILC3 subset (29) in this location, with the caveat that low Overall, these data indicate substantial heterogeneity of eYFP + CD4 T cells, suggesting that IL-22 induction may not be strictly numbers of eYFP cells might have been missed as a result of associated with a particular CD4 T cell subset. underreporting. In line with earlier reports, IL-22–producing intestinal ILC3 control C. rodentium infection in the early phase for ∼5 d until the Discussion adaptive T cell response ensues (30). This could involve the action Generation of an IL-22 fate reporter mouse was driven by the of retinoic acid, which was shown to attenuate colon inflammation technical difficulties encountered in identifying such cells via increased IL-22 production by ILC3 (31). The induction of IL- without resorting to in vitro culture. Although Abs for intra- 22/eYFP expression in CD4 T cells was noticeable first in the colon cellular staining are available, it is difficult to predict the ki- lamina propria and, to a lesser degree, in the mLNs, suggesting that netics of IL-22 production, and expression of this cytokine the adaptive CD4 response was induced locally. Histologically, it appears to be transient and tightly controlled. For this reason, the appeared that structures resembling colonic patches and ILFs were fate reporter approach offers the advantage of allowing iden- the main sites of eYFP expression. These structures are induced tification of cells that had initiated this pathway at any time point by the lymphotoxin pathway upstream of IL-22, and both coop-

For the statistical analysis, all time points were compared with an untreated sample. (D) Relative number of eYFP+ NK cells, CD8 T cells, gdT cells, CD4 T cells, and ILCs in LI LP and SI LP of C. rodentium–infected fate reporter mice at different time points p.i. Data shown are mean and are representative of 2–12 independent experiments. (E) Serum levels of IL-22 protein in IL-22 fate reporter mice during the course of infection with C. rodentium. Data shown are mean 6 SEM and are representative of two to four independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. n.s., nonsigniﬁcant. The Journal of Immunology 4611

FIGURE 7. Homozygous and heterozygous fate reporter mice infected with C. rodentium. (A)Log10 CFU of C. rodentium in colon of fate reporter mice on day 5 p.i. Horizontal lines represent mean CFU values. (B)FACS plot of CD45+ cellsfromLILPofun- treated (d0) or C. rodentium–infected (day 5; d5) fate reporter mice analyzed for eYFP and CD4 expression. (C) Number of eYFP+ CD4 T cells (upper panels)andILCs(lower panels)inLI LP (left panels)andmLNs(right panels) of untreated (d0) or C. rodentium– infected (day 5; d5) fate reporter mice. Data in (B)and(C)aremean6 SEM

and are representative of three to eight Downloaded from independent experiments. (D)FACS plots showing eYFP expression within donor CD45.2+ CD4 T cells from LI LP of C. rodentium–infected chimeric mice at 12 d p.i. Donors: CD45.2 heterozygous (IL-22wt/Cre) or homozygous Cre/Cre (IL22 ) fate reporter mice. Recip- http://www.jimmunol.org/ ients: CD45.1 Rag22/2 mice. Data are representative of three independent experiments. (E)Log10 CFU of C. rodentium in colon of chimeric mice on day 12 p.i. Data are mean 6 SEM and are representative of three independent experiments. **p , 0.01, ***p , 0.001. cre/cre, homozygous IL-22 fate reporter mice; wt/cre, heterozygous IL-22 fate by guest on September 28, 2021 reporter mice.

erate in the organization and maintenance of colonic patches and bacteria (34), or the perpetuation of bacteria-induced colon cancer ILFs during infection with C. rodentium (32). (35). CD4 T cells from the small intestine did not seem to be involved C. rodentium was cleared around 21 d p.i. (data not shown), but in the immune response to Citrobacter, and eYFP+ cells in this elevated numbers of CD4 T cells persisted in the colon (see Fig. 6 location could only be detected at later stages when they might for day 25) up to $82 d, although most were no longer producing have spilled over from the colon. To some extent, this may be due cytokines (data not shown). Detection of cells that had initiated the to the fact that Citrobacter infection targets the distal colon (33) IL-22 program via expression of eYFP allowed the analysis of and, thus, does not directly involve the small intestinal Th17 cell single cells via qPCR (36) and confirmed the considerable hetero- population. In addition, however, it may indicate that SI LP Th17 geneity of eYFP+ CD4 T cells throughout the course of infection. cells are not participating in inflammatory immune responses. Single-cell data indicated a gradual shift in profile from a more The ILC3 response to Citrobacter infection remained largely Th17-like phenotype toward a Th1-like phenotype at later stages. focused on IL-22, with only a minor contribution of IFN-g and IL- Expression of Rora was quite consistent throughout, whereas Rorc 17 production. In contrast, the T cell response was highly plastic, expression was not prominent and decreased further at the expense with a large fraction of IL-17 producers and a gradual shift toward of Tbet on day 25; Ahr expression was reduced at the later time IFN-g, as well as dual cytokine production. Cytokine analysis of points. Runx3 was uniformly expressed in eYFP+ CD4 T cells at FACS-purified CD4 cell populations confirmed that production of day 25 p.i. and, together with Tbet, promoted the generation of IL-22, IL-17, and IFN-g was restricted to eYFP+ cells, empha- pathogenic IFN-g–producing Th17 cells (37). Overall, the emerging sizing their plasticity, as well as the fidelity, of this reporter mouse. picture from the IL-22 fate reporter suggests that, in CD4 T cells, IL-22 was detected maximally around day 2 of infection, prior to IL-22 is transiently associated with a Th17 population that is highly the main expansion of CD4 T cells, and it appeared transient plastic and prone to adopt a more Th1-like profile during the compared with IL-17, which remained dominant throughout the course of infection. The shortcomings of this reporter, resulting in infection period. It is conceivable that tight control of IL-22 se- underreporting of IL-22, make it impossible to unequivocally state cretion is necessary to avoid negative side effects associated with that there is no Th22 subset dedicated to producing this cytokine; this cytokine, such as the promotion of colonization by patho- however, the substantial plasticity of inflammatory CD4 T cells in gens (e.g., Salmonella), through suppression of related commensal this setting makes such a scenario less likely. 4612 FATE MAPPING OF IL-22 PRODUCTION Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 8. Cytokine and transcription factor expression profile of eYFP+ fate reporter mice. (A) eYFP+ and eYFP2 CD4 T cells purified from LI LP of C. rodentium–infected mice at day 14 p.i. were stimulated with anti-CD3 or medium for 18 h. Bar graphs show the amount of IL-22, IL-17A, and IFN-g produced after stimulation. (B) FACS plot of CD45+ cells from LI LP of C. rodentium–infected fate reporter mice at day 2 analyzed for eYFP and IL-22 expression. Data are representative of four independent experiments. (C) Relative numbers of eYFP+ TCRb+ and TCRb2 cytokine-expressing cells, based on intracellular cytokine staining, in LI LP of untreated (d0) fate reporter mice and of mice at different time points p.i. with C. rodentium. Data are mean and are representative of two to seven independent experiments. (D) Heat map presentation for single-cell gene expression of selected transcription factors, cytokines, or cytokine receptors, as well as eYFP, in FACS-purified eYFP+ CD4 T cells from LI LP of untreated (d0) and C. rodentium–infected fate reporter mice at day 5 (d5) and day 25 (d25) p.i. Expression levels are shown as Ct values. (E) Summary of data in (B). Heat map represents quantitative values of gene expression (Ct , 35) at different time points. Missing data are shown in gray. The Journal of Immunology 4613

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