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 October 2, 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 http://www.jimmunol.org/content/193/9/4602 Downloaded from 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/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 under- reporting, 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 October 2, 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 recombi- nase; 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.