Regulatory Innate Lymphoid Cells Control Innate Intestinal Inflammation

•ILCregs exist in mouse and human intestines•ILCregs contribute to the resolution of innate intestinal inflammation•ILCregs suppress the activation of ILC1s and ILC3s via secretion of IL-10•Autocrine TGF-β1 is required for the expansion of ILCregs during inflammation An emerging family of innate lymphoid cells (termed ILCs) has an essential role in the initiation and regulation of inflammation. However, it is still unclear how ILCs are regulated in the duration of intestinal inflammation. Here, we identify a regulatory subpopulation of ILCs (called ILCregs) that exists in the gut and harbors a unique gene identity that is distinct from that of ILCs or regulatory T cells (Tregs). During inflammatory stimulation, ILCregs can be induced in the intestine and suppress the activation of ILC1s and ILC3s via secretion of IL-10, leading to protection against innate intestinal inflammation. Moreover, TGF-β1 is induced by ILCregs during the innate intestinal inflammation, and autocrine TGF-β1 sustains the maintenance and expansion of ILCregs. Therefore, ILCregs play an inhibitory role in the innate immune response, favoring the resolution of intestinal inflammation. An emerging family of innate lymphoid cells (termed ILCs) has an essential role in the initiation and regulation of inflammation. However, it is still unclear how ILCs are regulated in the duration of intestinal inflammation. Here, we identify a regulatory subpopulation of ILCs (called ILCregs) that exists in the gut and harbors a unique gene identity that is distinct from that of ILCs or regulatory T cells (Tregs). During inflammatory stimulation, ILCregs can be induced in the intestine and suppress the activation of ILC1s and ILC3s via secretion of IL-10, leading to protection against innate intestinal inflammation. Moreover, TGF-β1 is induced by ILCregs during the innate intestinal inflammation, and autocrine TGF-β1 sustains the maintenance and expansion of ILCregs. Therefore, ILCregs play an inhibitory role in the innate immune response, favoring the resolution of intestinal inflammation. The intestine represents a major gateway for potential pathogens, which also contains dietary antigens and an extensive and diverse microbial flora that need to be tolerated. Due to these requirements, the gut constitutes the largest lymphoid organ in the body, encompassing an extensive network of secondary lymphoid organs and is home to an enormous number of lymphocytes (Izcue et al., 2009Izcue A. Coombes J.L. Powrie F. Regulatory lymphocytes and intestinal inflammation.Annu. Rev. Immunol. 2009; 27: 313-338Crossref PubMed Scopus (410) Google Scholar). Intestinal immune responses take place in the epithelium, lamina propria, and gut-associated lymphoid tissue (GALT). The gut harbors several intestine-specific subpopulations specialized in antigen presentation, antimicrobial immunity, and maintenance of tolerance. Breakdown in the regulatory pathways leads to chronic intestinal inflammation (Kaser et al., 2010Kaser A. Zeissig S. Blumberg R.S. Inflammatory bowel disease.Annu. Rev. Immunol. 2010; 28: 573-621Crossref PubMed Scopus (1482) Google Scholar). For example, dysregulation of mucosal T cell responses may cause a loss of tolerance, leading to harmful intestinal inflammation reminiscent of human inflammatory bowel disease (IBD) (Strober et al., 2002Strober W. Fuss I.J. Blumberg R.S. The immunology of mucosal models of inflammation.Annu. Rev. Immunol. 2002; 20: 495-549Crossref PubMed Scopus (1147) Google Scholar). Moreover, the regulatory T cells (Tregs) play a critical role in the maintenance of intestinal homeostasis and self-tolerance (Asseman et al., 1999Asseman C. Mauze S. Leach M.W. Coffman R.L. Powrie F. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation.J. Exp. Med. 1999; 190: 995-1004Crossref PubMed Scopus (1318) Google Scholar, Sakaguchi, 2005Sakaguchi S. Naturally arising Foxp3-expressing CD25+CD4+ regulatory T cells in immunological tolerance to self and non-self.Nat. Immunol. 2005; 6: 345-352Crossref PubMed Scopus (2316) Google Scholar). However, how the intestinal innate immune system is regulated during the intestinal inflammation remains elusive. Innate lymphoid cells (ILCs) are located in mucosal surfaces to potentiate immune responses, sustain mucosal integrity and promote lymphoid organogenesis (Diefenbach et al., 2014Diefenbach A. Colonna M. Koyasu S. Development, differentiation, and diversity of innate lymphoid cells.Immunity. 2014; 41: 354-365Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, Eberl et al., 2015Eberl G. Colonna M. Di Santo J.P. McKenzie A.N. Innate lymphoid cells. Innate lymphoid cells: a new paradigm in immunology.Science. 2015; 348: aaa6566Crossref PubMed Scopus (537) Google Scholar). Group 1 (ILC1) cells are characterized by their capacity to secrete interferon γ (IFN-γ) responding to interleukin-12 (IL-12), IL-15, and IL-18 (Gordon et al., 2012Gordon S.M. Chaix J. Rupp L.J. Wu J. Madera S. Sun J.C. Lindsten T. Reiner S.L. The transcription factors T-bet and Eomes control key checkpoints of natural killer cell maturation.Immunity. 2012; 36: 55-67Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar, Klose et al., 2014Klose C.S.N. Flach M. Möhle L. Rogell L. Hoyler T. Ebert K. Fabiunke C. Pfeifer D. Sexl V. Fonseca-Pereira D. et al.Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.Cell. 2014; 157: 340-356Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). Group 2 (ILC2) cells generate type 2 T helper (Th2) cell cytokines, such as IL-5, IL-9, and IL-13, in response to IL-25 and IL-33 stimulation (Brestoff et al., 2015Brestoff J.R. Kim B.S. Saenz S.A. Stine R.R. Monticelli L.A. Sonnenberg G.F. Thome J.J. Farber D.L. Lutfy K. Seale P. Artis D. Group 2 innate lymphoid cells promote beiging of white adipose tissue and limit obesity.Nature. 2015; 519: 242-246Crossref PubMed Scopus (659) Google Scholar, Moro et al., 2010Moro K. Yamada T. Tanabe M. Takeuchi T. Ikawa T. Kawamoto H. Furusawa J. Ohtani M. Fujii H. Koyasu S. Innate production of T(H)2 cytokines by adipose tissue-associated c-Kit(+)Sca-1(+) lymphoid cells.Nature. 2010; 463: 540-544Crossref PubMed Scopus (1506) Google Scholar). Group 3 (ILC3) cells produce IFN-γ, IL-17, and IL-22 following stimulation with IL-1β and IL-23 (Buonocore et al., 2010Buonocore S. Ahern P.P. Uhlig H.H. Ivanov I.I. Littman D.R. Maloy K.J. Powrie F. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology.Nature. 2010; 464: 1371-1375Crossref PubMed Scopus (850) Google Scholar, Klose et al., 2013Klose C.S. Kiss E.A. Schwierzeck V. Ebert K. Hoyler T. d’Hargues Y. Göppert N. Croxford A.L. Waisman A. Tanriver Y. Diefenbach A. A T-bet gradient controls the fate and function of CCR6-RORγt+ innate lymphoid cells.Nature. 2013; 494: 261-265Crossref PubMed Scopus (520) Google Scholar). Given that ILCs produce substantial effector cytokines when stimulated, they play a critical role in the regulation of type 1, type 2, and type 3 (or Th17 cell) responses, controlling host protective immunity and intestinal homeostasis (Bedoui et al., 2016Bedoui S. Gebhardt T. Gasteiger G. Kastenmüller W. Parallels and differences between innate and adaptive lymphocytes.Nat. Immunol. 2016; 17: 490-494Crossref PubMed Scopus (28) Google Scholar, Gasteiger and Rudensky, 2014Gasteiger G. Rudensky A.Y. Interactions between innate and adaptive lymphocytes.Nat. Rev. Immunol. 2014; 14: 631-639Crossref PubMed Scopus (151) Google Scholar, Shih et al., 2016Shih H.Y. Sciumè G. Mikami Y. Guo L. Sun H.W. Brooks S.R. Urban Jr., J.F. Davis F.P. Kanno Y. O’Shea J.J. Developmental acquisition of regulomes underlies innate lymphoid cell functionality.Cell. 2016; 165: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). Accumulating evidence shows that inflammatory responses can be induced and regulated independently of adaptive immunity (Sonnenberg and Artis, 2015Sonnenberg G.F. Artis D. Innate lymphoid cells in the initiation, regulation and resolution of inflammation.Nat. Med. 2015; 21: 698-708Crossref PubMed Scopus (381) Google Scholar). Rag (V[D]J recombination activation gene)-deficient mice develop colitis after treatment with anti-CD40 antibody or dextran sodium sulfate (DSS) (Buonocore et al., 2010Buonocore S. Ahern P.P. Uhlig H.H. Ivanov I.I. Littman D.R. Maloy K.J. Powrie F. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology.Nature. 2010; 464: 1371-1375Crossref PubMed Scopus (850) Google Scholar, Wirtz et al., 2007Wirtz S. Neufert C. Weigmann B. Neurath M.F. Chemically induced mouse models of intestinal inflammation.Nat. Protoc. 2007; 2: 541-546Crossref PubMed Scopus (1157) Google Scholar). Intraepithelial ILC1s undergo expansion and produce large amounts of IFN-γ in the human Crohn’s disease. Blocking of ILC1 with anti-NK1.1 antibody in mice is able to attenuate colitis (Fuchs et al., 2013Fuchs A. Vermi W. Lee J.S. Lonardi S. Gilfillan S. Newberry R.D. Cella M. Colonna M. Intraepithelial type 1 innate lymphoid cells are a unique subset of IL-12- and IL-15-responsive IFN-γ-producing cells.Immunity. 2013; 38: 769-781Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar). In addition, ILC3s are implicated in the innate intestinal inflammation via secretion of IL-17 and IL-22. IL-17-producing ILC3s are responsible for the pathogenesis of Tbx21−/−Rag2−/− ulcerative colitis mice (Garrett et al., 2007Garrett W.S. Lord G.M. Punit S. Lugo-Villarino G. Mazmanian S.K. Ito S. Glickman J.N. Glimcher L.H. Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system.Cell. 2007; 131: 33-45Abstract Full Text Full Text PDF PubMed Scopus (752) Google Scholar, Powell et al., 2012Powell N. Walker A.W. Stolarczyk E. Canavan J.B. Gökmen M.R. Marks E. Jackson I. Hashim A. Curtis M.A. Jenner R.G. et al.The transcription factor T-bet regulates intestinal inflammation mediated by interleukin-7 receptor+ innate lymphoid cells.Immunity. 2012; 37: 674-684Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar). Depletion of ILC3s by anti-Thy1 antibody abrogates innate colitis (Buonocore et al., 2010Buonocore S. Ahern P.P. Uhlig H.H. Ivanov I.I. Littman D.R. Maloy K.J. Powrie F. Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology.Nature. 2010; 464: 1371-1375Crossref PubMed Scopus (850) Google Scholar), suggesting a critical role of ILC3s in the pathogenesis of intestinal inflammation. However, it is unknown whether a regulatory subset of ILCs exists and how they function in the regulation of intestinal inflammation. Here, we identify a novel regulatory subpopulation of ILCs (called ILCregs) that exists in the gut and expands in the intestine following pathogenic stimulation. ILCregs exert an inhibitory role in the innate immune response against intestinal inflammation. Given that Treg cell-mediated suppression through secretion of their feature cytokines IL-10 and TGF-β (Izcue et al., 2009Izcue A. Coombes J.L. Powrie F. Regulatory lymphocytes and intestinal inflammation.Annu. Rev. Immunol. 2009; 27: 313-338Crossref PubMed Scopus (410) Google Scholar, Josefowicz et al., 2012Josefowicz S.Z. Lu L.F. Rudensky A.Y. Regulatory T cells: mechanisms of differentiation and function.Annu. Rev. Immunol. 2012; 30: 531-564Crossref PubMed Scopus (1968) Google Scholar), we then gated out Lin−CD45+CD127+ ILCs from multiple tissues in IL-10-GFP reporter mice and analyzed IL-10 expression (Kamanaka et al., 2006Kamanaka M. Kim S.T. Wan Y.Y. Sutterwala F.S. Lara-Tejero M. Galán J.E. Harhaj E. Flavell R.A. Expression of interleukin-10 in intestinal lymphocytes detected by an interleukin-10 reporter knockin tiger mouse.Immunity. 2006; 25: 941-952Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). We noticed that a unique subset of ILCs constitutively expressed IL-10 in the intestine (Figures 1A and S1A). These Lin−CD45+CD127+IL-10+ ILCs were mainly located in the lamina propria of the small intestine (sLP), and some of them in the small intestinal epithelium (sIE) and colon lamina propria (cLP) (Figure 1A). Lin−CD45+CD127+IL-10+ ILCs were small in size and had a high nucleus/cytoplasm ratio and scanty cytoplasm, which are all characteristics of lymphoid morphology (Figures 1B and 1C). Of note, Lin−CD45+CD127+IL-10+ ILCs were devoid of expression of CD4 and FoxP3 (Figure S1B), which are signature markers of Tregs (Sakaguchi et al., 2008Sakaguchi S. Yamaguchi T. Nomura T. Ono M. Regulatory T cells and immune tolerance.Cell. 2008; 133: 775-787Abstract Full Text Full Text PDF PubMed Scopus (3694) Google Scholar), indicating that the Lin−CD45+CD127+IL-10+ ILC subset was different from the Tregs subset. In addition, Lin−CD45+CD127+IL-10+ ILCs also expressed ILC markers, such as CD25 (IL-2Rα) and CD90 (Thy1). This population also highly expressed IL-2Rγ and Sca-1, but lacked ILC1 markers (NK1.1 and NKp46), ILC2 markers (ST2 and KLRG1), ILC3 markers (NKp46, CD4, and RORγt), or other leukocyte lineage markers (Gury-BenAri et al., 2016Gury-BenAri M. Thaiss C.A. Serafini N. Winter D.R. Giladi A. Lara-Astiaso D. Levy M. Salame T.M. Weiner A. David E. et al.The spectrum and regulatory landscape of intestinal innate lymphoid cells are shaped by the microbiome.Cell. 2016; 166: 1231-1246Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, Robinette et al., 2015Robinette M.L. Fuchs A. Cortez V.S. Lee J.S. Wang Y. Durum S.K. Gilfillan S. Colonna M. Immunological Genome ConsortiumTranscriptional programs define molecular characteristics of innate lymphoid cell classes and subsets.Nat. Immunol. 2015; 16: 306-317Crossref PubMed Scopus (483) Google Scholar) (Figures 1D and S1C). These Lin−CD45+CD127+IL-10+ ILCs represented a new IL-10-producing ILC population that we have named regulatory ILCs (ILCregs).Figure S1Identification of ILCregs in Mouse and Human Intestines, Related to Figures 1 and 2Show full caption(A) Gate strategy of ILCreg identification. Lamina propria cells from IL-10-GFP mice were isolated and blocked with anti-CD16/32 antibody followed by surface marker staining with antibodies against Lin, CD45, and CD127. Lymphocytes were gated out by FSC-A and SSC-A. Unconjugated cells were gated out by FSC-W and SSC-W. Lin−CD45+ lymphocytes were gated out for CD127 versus IL-10-GFP analysis. (Lin = CD3e,CD4,CD8,CD19,NK1.1,CD11b,CD11c,Gr1,Ter119).(B) Analysis of Tregs and ILCregs in mouse intestines after treatment with anti-CD3 monoclonal antibody. Mouse lamina propria cells were isolated and stained with the indicated antibodies. CD127+IL-10+CD4−FoxP3− population was ILCregs, and CD127+IL-10+CD4+FoxP3+ population was Tregs.(C) Flow cytometry analysis of lineage markers on ILCregs. Gray histograms depict isotype control of each antibody.(D) Immunofluorescence staining of ILCregs in mouse colons. Scale bar, 50 μm. White arrowhead indicates ILCregs.(E) Immunofluorescence staining of ILCregs in human colon. Scale bar, 50 μm. White arrowhead indicates ILCregs.(F) Flow cytometry analysis of markers on ILCregs. Gray histograms depict isotype control of each antibody.(G) Flow cytometry analysis of cytokines and transcription factors of ILCregs. Blue lines indicated positive controls. For TNFα and IFN-γ, positive controls are ILC1s cultured in 10ng/ml IL-12/IL-18 for 24 hr. For IL-5 and IL-13, positive controls are ILC2s cultured in 10ng/ml IL-33 for 24 hr. For IL-17A and IL-22, positive controls are ILC3s cultured in 10ng/ml IL-23 for 24 hr. For Eomes, Gata3 and T-bet, positive controls are Lin−CD45+CD127+ cells.(H) Analysis of ILCregs in different immunodeficient mouse strains. Cell numbers of ILCreg were calculated and shown as means ± SD (right panel). ∗∗∗p < 0.01 by One Way ANOVA.(I) Analysis of Tregs in Id2 deficient mice. Tregs in lamina propria of small intestine from Id2+/+ or Id2−/− mice were analyzed by flow cytometry. CD4+Foxp3+ gate indicates Tregs. Data are representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Gate strategy of ILCreg identification. Lamina propria cells from IL-10-GFP mice were isolated and blocked with anti-CD16/32 antibody followed by surface marker staining with antibodies against Lin, CD45, and CD127. Lymphocytes were gated out by FSC-A and SSC-A. Unconjugated cells were gated out by FSC-W and SSC-W. Lin−CD45+ lymphocytes were gated out for CD127 versus IL-10-GFP analysis. (Lin = CD3e,CD4,CD8,CD19,NK1.1,CD11b,CD11c,Gr1,Ter119). (B) Analysis of Tregs and ILCregs in mouse intestines after treatment with anti-CD3 monoclonal antibody. Mouse lamina propria cells were isolated and stained with the indicated antibodies. CD127+IL-10+CD4−FoxP3− population was ILCregs, and CD127+IL-10+CD4+FoxP3+ population was Tregs. (C) Flow cytometry analysis of lineage markers on ILCregs. Gray histograms depict isotype control of each antibody. (D) Immunofluorescence staining of ILCregs in mouse colons. Scale bar, 50 μm. White arrowhead indicates ILCregs. (E) Immunofluorescence staining of ILCregs in human colon. Scale bar, 50 μm. White arrowhead indicates ILCregs. (F) Flow cytometry analysis of markers on ILCregs. Gray histograms depict isotype control of each antibody. (G) Flow cytometry analysis of cytokines and transcription factors of ILCregs. Blue lines indicated positive controls. For TNFα and IFN-γ, positive controls are ILC1s cultured in 10ng/ml IL-12/IL-18 for 24 hr. For IL-5 and IL-13, positive controls are ILC2s cultured in 10ng/ml IL-33 for 24 hr. For IL-17A and IL-22, positive controls are ILC3s cultured in 10ng/ml IL-23 for 24 hr. For Eomes, Gata3 and T-bet, positive controls are Lin−CD45+CD127+ cells. (H) Analysis of ILCregs in different immunodeficient mouse strains. Cell numbers of ILCreg were calculated and shown as means ± SD (right panel). ∗∗∗p < 0.01 by One Way ANOVA. (I) Analysis of Tregs in Id2 deficient mice. Tregs in lamina propria of small intestine from Id2+/+ or Id2−/− mice were analyzed by flow cytometry. CD4+Foxp3+ gate indicates Tregs. Data are representative of at least three independent experiments. IL-10-producing ILCregs were further confirmed through imaging flow cytometry by using anti-IL-10 antibody (Figure 1E). Furthermore, ILCregs with co-expression of CD127 and IL-10 indeed existed in mouse small and large intestines by immunofluorescence staining (Figures 1F and S1D). These observations were further validated through immunohistochemical staining (Figure 1G). In addition, ILCregs also existed in the human intestine from biopsies (Figures 1H, 1I, and S1E). Collectively, the Lin−CD45+CD127+IL-10+ cells are a new ILC subset that exists in mouse and human intestines. We isolated ILCregs from mouse sLP and performed transcriptome microarray assays. Comparing existing transcriptome datasets of ILCs with natural killer (NK) cells (GEO: GSE37448) and Tregs (GEO: GSE68009) highlights that ILCregs do not show any significant gene profile similarity to known ILC subsets and Tregs (Figure 2A). Of note, ILCregs do express high levels of Il10 and several ILC feature markers, including Il7r (encoding CD127), Il2ra (encoding CD25), Il2rg, and Ly6a (encoding Sca-1), which is consistent with our previous observations (Figure 1D). ILCregs uniquely express transcription factors, such as Id3 and Sox4 (Figure 2A), and they also highly expressed Id2, which is required for development of ILCs (Diefenbach et al., 2014Diefenbach A. Colonna M. Koyasu S. Development, differentiation, and diversity of innate lymphoid cells.Immunity. 2014; 41: 354-365Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar, Spits and Cupedo, 2012Spits H. Cupedo T. Innate lymphoid cells: emerging insights in development, lineage relationships, and function.Annu. Rev. Immunol. 2012; 30: 647-675Crossref PubMed Scopus (545) Google Scholar). However, ILCregs lack typical transcription factors of other ILCs and Tregs, such as Rorc (encoding RORγt), Tbx21 (encoding T-bet), Gata3, and Foxp3, respectively (Serafini et al., 2015Serafini N. Vosshenrich C.A. Di Santo J.P. Transcriptional regulation of innate lymphoid cell fate.Nat. Rev. Immunol. 2015; 15: 415-428Crossref PubMed Scopus (231) Google Scholar). Intriguingly, ILCregs constitutively express Tgfbr1, Tgfbr2, Il2rb, and Il2rg (Figure 2A), suggesting they responded to TGF-β and IL-2 signaling. Principal component analysis further verified that ILCregs harbor a unique gene profile (Figure 2B). Similarly, human ILCregs show distinct gene profiles of transcription factors and cytokines compared to other ILCs (Figure 2C). Additionally, some feature cytokines, such as IL-10 and transforming growth factor β1 (TGF-β1), are also highly expressed in human ILCregs as well. High expression levels of TGF-βRI, TGF-βRII, IL-2Rβ, and Id2 (inhibitor of DNA binding 2) in ILCregs were validated by flow cytometry (Figures S1F and S1G). Moreover, Il2rg- or Id2-deficient mice abrogated ILCregs (Figure S1H), which are common characters similar to other ILC subsets (Serafini et al., 2015Serafini N. Vosshenrich C.A. Di Santo J.P. Transcriptional regulation of innate lymphoid cell fate.Nat. Rev. Immunol. 2015; 15: 415-428Crossref PubMed Scopus (231) Google Scholar). In contrast, Rag1−/− mice show a normal number of ILCregs compared to wild-type (WT) mice (Figure S1H), and ILCregs of Rag1−/− mice do not express immature T cell markers (data not shown), suggesting they develop independent of Rag. By contrast, Id2 deficiency failed to impact the development of Tregs (Figure S1I). To further determine whether ILCregs are derived from myeloid or T cells, we generated Rosa26-STOP-YFP;Lyz2-Cre;IL-10-GFP mice and Rosa26-STOP-YFP;CD4-Cre;IL-10-GFP mice. We found that ILCregs were indeed negative for YFP signals in the above two mouse strains, whereas their respective lineage positive cells virtually displayed YFP signals (Figures S2A and S2B), indicating that ILCregs are not derived from myeloid or T cells. Recently, integrin α4β7+Id2high CHILP (common helper-like innate lymphoid precursor) was identified as a common precursor for all helper-like ILCs, including LTi and ILC1-3 subsets (Klose et al., 2014Klose C.S.N. Flach M. Möhle L. Rogell L. Hoyler T. Ebert K. Fabiunke C. Pfeifer D. Sexl V. Fonseca-Pereira D. et al.Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.Cell. 2014; 157: 340-356Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar), whereas PLZF+ ILCP (common ILC precursor) is the precursor for ILC1-3s, only giving rise to ILC1s, ILC2s, and ILC3s (Ishizuka et al., 2016Ishizuka I.E. Constantinides M.G. Gudjonson H. Bendelac A. The innate lymphoid cell precursor.Annu. Rev. Immunol. 2016; 34: 299-316Crossref PubMed Scopus (51) Google Scholar). Next, we crossed Rosa26-STOP-YFP mice with different Cre mice to determine the precursor of ILCregs. We observed that only Rosa26-STOP-YFP;Id2-CreERT2 mice displayed YFP-positive ILCregs in the presence or absence of DSS treatment (Figures S2C and S2D). However, ILCregs in Rosa26-STOP-YFP;PLZF-Cre mice or Rosa26-STOP-YFP;RORγt-Cre mice were YFP negative (Figures S2C and S2D). These data suggest that ILCregs are derived from Id2-driven CHILPs, but not ILCPs or ILC3s. To further validate IL-10-producing ILCregs represented a distinct lineage as opposed to a further differentiation state of ILC1, ILC2, or ILC3 cells, we established Id2- CreERT2;RosaDTR, PLZF-Cre;RosaDTR, and RORγt-Cre;RosaDTR mice, followed by adoptive transfer assays. With diphtheria toxin (DT) treatment, Id2-CreERT2;RosaDTR mice abrogated ILCreg cells (Figure S2E), suggesting ILCregs were generated from the progenitor CHILP (Klose et al., 2014Klose C.S.N. Flach M. Möhle L. Rogell L. Hoyler T. Ebert K. Fabiunke C. Pfeifer D. Sexl V. Fonseca-Pereira D. et al.Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages.Cell. 2014; 157: 340-356Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar). However, deletion of PLZF- or RORγt-expressing cells does not affect the development of ILCregs (Figure S2E), indicating that ILCregs do not differentiate from the progenitor of LTi, ILC1, ILC2, or ILC3 cells (Artis and Spits, 2015Artis D. Spits H. The biology of innate lymphoid cells.Nature. 2015; 517: 293-301Crossref PubMed Scopus (1065) Google Scholar). To further verify the progenitor of ILCregs, we transferred CLP, αLP, CHILP, and ILCP progenitors into Rag1−/−Il2rg−/− mice for adoptive transfer assays. We noticed that CLP, αLP, and CHILP were able to produce ILCregs, whereas ILCPs could not generate ILCregs (Figure S2F). Depletion of CHILPs by DT treatment in adoptively reconstituted Rag1−/−Il2rg−/− mice abrogated generation of ILCregs (Figure S2G). Collectively, ILCregs are derived from CHILPs, but not ILCPs, which are a separate subset distinct from Tregs and other ILC lineages. Since ILCregs mainly reside in the intestine and develop independent of Rag, we next used innate intestinal inflammation models to monitor ILCreg functions. We treated Rag1−/− mice with several inflammatory stimuli, including DSS, anti-CD40 antibody, Salmonella typhimurium (S. typhimurium), and Citrobacter Rodentium (C. rodentium). We noticed that ILCregs could be induced in the intestines of Rag1−/− mice with these inflammatory stimuli (Figure 3A). Notably, ILCregs gradually expanded in the intestines during the process of inflammation (Figures 3B, 3C, and S3A), reaching a peak level at 8 days post-stimulation, which came up at peak inflammation (Figure S3B). By contrast, the number of ILC1s and ILC3s immediately increased at early stages post-stimulation and decreased soon afterward (Figure S3C), whereas the number of ILC2s did not change during the process of inflammation (Figure S3C). These data indicate that ILCregs can be induced by innate intestinal inflammation.Figure S3ILCregs Attenuate Innate Colitis, Related to Figure 3Show full caption(A) Isotype control staining for anti-IL-10 (left panel) or anti-Ki67 antibody (right panel) by flow cytometry.(B) WT mice were treated with 3% DSS in drinking water for the indicated days or 150 μg anti-CD40 antibody (i.p.) on day 0, or infected by oral gavage of 5x104 c.f.u. S. Typhimurium or 1x109 c.f.u. C. Rodentium on day 0. Colitis scores of indicated mice with different treatments were analyzed and shown as means ± SD n = 5 for each group.(C) Cell numbers of ILC1s, ILC2s and ILC3s after different stimulation as (B) were analyzed by flow cytometry and shown as means ± SD.(D) Rescue of ILCregs in Rag1−/−Il10−/− mice. ILCregs were transferred into Rag1−/−Il10−/− mice. After 2 weeks, lymphocytes from lamina propria of small intestines were analyzed by flow cytometry. Lymphocytes were pre-gated by Lin−CD45+.(E) Serum IL-10 levels in mice treated as in (D) with indicated days were analyzed by ELISA. Data were shown as means ± SD.(F) ILCregs were labeled with CellTrace (violet) and transferred into Rag1−/−Il10−/− mice for cell proliferation assays. After 7 days transferred mice were treated with 3% DSS for indicated days, CellTrace-labeled ILCregs were analyzed by flow cytometry.(G) ILCregs attenuate innate colitis induced by S.Typhimurium infection. ILCregs transferred mice were infected by oral gavage of 5x104 c.f.u. S. Typhimurium. After 8 days, colitis scores were analyzed. ∗∗p < 0.01 by One Way ANOVA. n = 5 for each group.(H) H&E staining of colon from indicated mice after S. Typhimurium infection. Scale bar, 50 μm. Data are representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Isotype control staining for anti-IL-10 (left panel) or anti-Ki67 antibody (right panel) by flow cytometry. (B) WT mice were treated with 3% DSS in drinking water for the indicated days or 150 μg anti-CD40 antibody (i.p.) on day 0, or infected by oral gavage of 5x104 c.f.u. S. Typhimurium or 1x109 c.f.u. C. Rodentium on day 0. Colitis scores of indicated mice with different treatments were analyzed and shown as means ± SD n = 5 for each group. (C) Cell numbers of ILC1s, ILC2s and ILC3s after different stimulation as (B) were analyzed by flow cytometry and shown as means ± SD. (D) Rescue of ILCregs in Rag1−/−Il10−/− mice. ILCregs were transferred into Rag1−/−Il10−/− mice. After 2 weeks, lymphocytes from lamina propria of small intestines were analyzed by flow cytometry. Lymphocytes were pre-gated by Lin−CD45+. (E) Serum IL-10 levels in mice treated as in (D) with indicated days were analyzed by ELISA. Data were shown as means ± SD. (F) ILCregs were labeled with CellTrace (violet) and transferred into Rag1−/−Il10−/− mice for cell proliferation assays. After 7 days transferred mice were treated with 3% DSS for indicated days, CellTrace-labeled ILCregs were analyzed by flow cytometry. (G) ILCregs attenuate innate colitis induced by S.Typhimurium infection. ILCregs transferred mice were infected by oral gavage of 5x104 c.f.u. S. Typhimurium. After 8 days, colitis scores were analyzed. ∗∗p < 0.01 by One Way ANOVA. n = 5 for each group. (H) H&E staining of colon from indicated mice after S. Typhimurium infection. Scale bar, 50 μm. Data are representative of at least three independent experiments. It has been reported that IL-10-deficient mice display spontaneous colitis (Josefowicz et al., 2012Josefowicz S.Z. L