Cutting Edge: c-Maf Is Required for Regulatory T Cells To Adopt ROR?t+ and Follicular Phenotypes.
ABSTRACT: Regulatory T cells (Tregs) adopt specialized phenotypes defined by coexpression of lineage-defining transcription factors, such as ROR?t, Bcl-6, or PPAR?, alongside Foxp3. These Treg subsets have unique tissue distributions and diverse roles in maintaining organismal homeostasis. However, despite extensive functional characterization, the factors driving Treg specialization are largely unknown. In this article, we show that c-Maf is a critical transcription factor regulating this process in mice, essential for generation of both ROR?t+ Tregs and T follicular regulatory cells, but not for adipose-resident Tregs. c-Maf appears to function primarily in Treg specialization, because IL-10 production, expression of other effector molecules, and general immune homeostasis are not c-Maf dependent. As in other T cells, c-Maf is induced in Tregs by IL-6 and TGF-?, suggesting that a combination of inflammatory and tolerogenic signals promote c-Maf expression. Therefore, c-Maf is a novel regulator of Treg specialization, which may integrate disparate signals to facilitate environmental adaptation.
Project description:ROR?t-expressing Tregs form a specialized subset of intestinal CD4+ Foxp3+ cells which is essential to maintain gut homeostasis and tolerance to commensal microbiota. Recently, c-Maf emerged as a critical factor in the regulation of ROR?t expression in Tregs. However, aside from c-Maf signaling, the signaling pathways involved in the differentiation of ROR?t+ Tregs and their possible interplay with c-Maf in this process are largely unknown. We show that ROR?t+ Treg development is controled by positive as well as negative signals. Along with c-Maf signaling, signals derived from a complex microbiota, as well as IL-6/STAT3- and TGF-?-derived signals act in favor of ROR?t+ Treg development. Ectopic expression of c-Maf did not rescue ROR?t expression in STAT3-deficient Tregs, indicating the presence of additional effectors downstream of STAT3. Moreover, we show that an inflammatory IFN-?/STAT1 signaling pathway acts as a negative regulator of ROR?t+ Treg differentiation in a c-Maf independent fashion. These data thus argue for a complex integrative signaling network that finely tunes ROR?t expression in Tregs. The finding that type 1 inflammation impedes ROR?t+ Treg development even in the presence of an active IL-6/STAT3 pathway further suggests a dominant negative effect of STAT1 over STAT3 in this process.
Project description:Both microbial and host genetic factors contribute to the pathogenesis of autoimmune diseases. There is accumulating evidence that microbial species that potentiate chronic inflammation, as in inflammatory bowel disease, often also colonize healthy individuals. These microorganisms, including the Helicobacter species, can induce pathogenic T cells and are collectively referred to as pathobionts. However, how such T cells are constrained in healthy individuals is not yet understood. Here we report that host tolerance to a potentially pathogenic bacterium, Helicobacter hepaticus, is mediated by the induction of ROR?t+FOXP3+ regulatory T (iTreg) cells that selectively restrain pro-inflammatory T helper 17 (TH17) cells and whose function is dependent on the transcription factor c-MAF. Whereas colonization of wild-type mice by H. hepaticus promoted differentiation of ROR?t-expressing microorganism-specific iTreg cells in the large intestine, in disease-susceptible IL-10-deficient mice, there was instead expansion of colitogenic TH17 cells. Inactivation of c-MAF in the Treg cell compartment impaired differentiation and function, including IL-10 production, of bacteria-specific iTreg cells, and resulted in the accumulation of H. hepaticus-specific inflammatory TH17 cells and spontaneous colitis. By contrast, ROR?t inactivation in Treg cells had only a minor effect on the bacteria-specific Treg and TH17 cell balance, and did not result in inflammation. Our results suggest that pathobiont-dependent inflammatory bowel disease is driven by microbiota-reactive T cells that have escaped this c-MAF-dependent mechanism of iTreg-TH17 homeostasis.
Project description:Stat3 signaling is essential for the induction of ROR?t and subsequent Th17 cell differentiation. However, the downstream targets of Stat3 for ROR?t expression remain largely unknown. We show here that a novel isoform of Sox5, named Sox5t, is induced in Th17 cells in a Stat3-dependent manner. In vivo, T cell-specific Sox5-deficient mice exhibit impaired Th17 cell differentiation and are resistant to experimental autoimmune encephalomyelitis and delayed-type hypersensitivity. Retrovirus-mediated induction of Sox5 together with c-Maf induces Th17 cell differentiation even in Stat3-deficient CD4(+) T cells but not in ROR?t-deficient CD4(+) T cells, indicating that Sox5 and c-Maf induce Th17 cell differentiation as downstream effectors of Stat3 and as upstream inducers of ROR?t. Moreover, Sox5 physically associates with c-Maf via the HMG domain of Sox5 and DNA-binding domain of c-Maf, and Sox5 together with c-Maf directly activates the promoter of ROR?t in CD4(+) T cells. Collectively, our results suggest that Sox5 and c-Maf cooperatively induce Th17 cell differentiation via the induction of ROR?t as downstream targets of Stat3.
Project description:Atopic dermatitis is an allergic inflammatory skin disease characterized by the production of the type 2 cytokines in the skin by type 2 innate lymphoid cells (ILC2s) and T helper 2 (TH2) cells, and tissue eosinophilia. Using two distinct mouse models of atopic dermatitis, we show that expression of retinoid-related orphan receptor ? (ROR?) in skin-resident T regulatory cells (Tregs) is important for restraining allergic skin inflammation. In both models, targeted deletion of ROR? in mouse Tregs led to exaggerated eosinophilia driven by interleukin-5 (IL-5) production by ILC2s and TH2 cells. Expression of ROR? in skin-resident Tregs suppressed IL-4 expression and enhanced expression of death receptor 3 (DR3), which is the receptor for tumor necrosis factor (TNF) family cytokine, TNF ligand-related molecule 1 (TL1A), which promotes Treg functions. DR3 is expressed on both ILC2s and skin-resident Tregs Upon deletion of ROR? in skin-resident Tregs, we found that Tregs were no longer able to sequester TL1A, resulting in enhanced ILC2 activation. We also documented higher expression of ROR? in skin-resident Tregs than in peripheral blood circulating Tregs in humans, suggesting that ROR? and the TL1A-DR3 circuit could be therapeutically targeted in atopic dermatitis.
Project description:Although Th17 responses may contribute to the pathogenesis of glomerulonephritis, whether the key transcription factor in Th17 cell development, ROR?t, also promotes glomerulonephritis is unknown. Here, we induced crescentic glomerulonephritis in wild-type and ROR?t-deficient (ROR?t(-/-)) mice. ROR?t(-/-) mice were protected from disease, with reduced histologic and functional injury and decreased leukocyte infiltration. Because ROR?t(-/-) mice lack lymph nodes, which may influence the development of nephritis, we performed cell-transfer studies. We reconstituted Rag1(-/-) mice, which lack adaptive immunity but otherwise have normal architecture of the lymphatic system, with splenocytes from naïve wild-type or ROR?t(-/-) mice. Mice receiving wild-type splenocytes exhibited high mortality from renal failure after induction of nephritis whereas mice receiving ROR?t(-/-) cells were protected. To determine the effect of ROR?t deficiency specifically in T helper cells, we isolated naïve CD4(+) T cells from wild-type and ROR?t(-/-) mice and transferred them into Rag1(-/-) animals. Recipients of wild-type CD4(+) T cells developed severe glomerulonephritis whereas recipients of ROR?t(-/-) cells developed less severe disease. To exclude effects of altered regulatory T cell (Treg) development caused by ROR?t deficiency, we transferred naïve CD4(+) T cells depleted of Tregs into Rag1(-/-) mice. Recipients of wild-type, Treg-depleted, CD4(+) T cells developed severe glomerulonephritis whereas recipients of ROR?t(-/-), Treg-depleted CD4(+) T cells did not. Taken together, this study demonstrates that ROR?t promotes the development of crescentic glomerulonephritis by directing nephritogenic Th17 responses.
Project description:We identify c-Maf as an essential factor regulating adoption of RORgamma t+ and T follicular regulatory phenotypes in effector Tregs. Differential expression analysis demonstrates reduced signatures of both ROR gamma t+ Tregs and T follicular regulatory cells following regulatory T cell-specific c-Maf deletion. Overall design: Differential expression analysis of Peyer's patch effector Tregs (CD4+FoxP3+CD62L-) from 13-week old Maf+/+FoxP3-YFP-cre or Mafflox/floxFoxP3-YFP-cre mice, performed in duplicate.
Project description:ROR?t+ group 3 innate lymphoid cells (ILC3s) maintain intestinal homeostasis through secretion of type 3 cytokines such as interleukin (IL)-17 and IL-22. However, CCR6- ILC3s additionally co-express T-bet allowing for the acquisition of type 1 effector functions. While T-bet controls the type 1 programming of ILC3s, the molecular mechanisms governing T-bet are undefined. Here, we identify c-Maf as a crucial negative regulator of murine T-bet+ CCR6- ILC3s. Phenotypic and transcriptomic profiling of c-Maf-deficient CCR6- ILC3s revealed a hyper type 1 differentiation status, characterized by overexpression of ILC1/NK cell-related genes and downregulation of type 3 signature genes. On the molecular level, c-Maf directly restrained T-bet expression. Conversely, c-Maf expression was dependent on T-bet and regulated by IL-1?, IL-18 and Notch signals. Thus, we define c-Maf as a crucial cell-intrinsic brake in the type 1 effector acquisition which forms a negative feedback loop with T-bet to preserve the identity of CCR6- ILC3s.
Project description:Foxp3+ regulatory T cells (Tregs) represent a major fraction of skin resident T cells. Although normally protective, Tregs have been shown to produce pro-inflammatory cytokines in human diseases, including psoriasis. A significant hurdle in the Treg field has been the identification, or development, of model systems to study this Treg plasticity. To overcome this gap, we analyzed skin resident Tregs in a mouse model of IL-23 mediated psoriasiform dermatitis. Our results demonstrate that IL-23 drove the accumulation of Tregs; including a subpopulation that co-expressed ROR?t and produced IL-17A. Genesis of this population was attenuated by a ROR?t inverse agonist compound and clinically relevant therapeutics. In vitro, IL-23 drove the generation of CD4+Foxp3+ROR?t+IL-17A+ cells from Treg cells. Collectively, our data shows that IL-23 drives Treg plasticity by inducing a population of CD4+Foxp3+ROR?t+IL-17A+ cells that could play a role in the disease pathogenesis. Through this work, we define an in vitro system and a pre-clinical in vivo mouse model that can be used to further study Treg homeostasis and plasticity in the context of psoriasis.
Project description:ROR?+ and Helios+ Treg cells in the colon are phenotypically and functionally distinct, but their origins and relationships are poorly understood. In monocolonized and normal mice, single-cell RNA-seq revealed sharing of TCR clonotypes between these Treg cell populations, potentially denoting a common progenitor. In a polyclonal Treg cell replacement system, naive conventional CD4+ (Tconv) cells, but not pre-existing tTregs, could differentiate into ROR?+ pTregs upon interaction with gut microbiota. A smaller proportion of Tconv cells converted into Helios+ pTreg cells, but these dominated when the Tconv cells originated from preweaning mice. T cells from infant mice were predominantly immature, insensitive to ROR?-inducing bacterial cues and to IL6, and showed evidence of higher TCR-transmitted signals, which are also characteristics of recent thymic emigrants (RTEs). Correspondingly, transfer of adult RTEs or Nur77high Tconv cells mainly yielded Helios+ pTreg cells, recapitulating the infant/adult difference. Thus, CD4+ Tconv cells can differentiate into both ROR?+ and Helios+ pTreg cells, providing a physiological adaptation of colonic Treg cells as a function of the age of the cell or of the individual.
Project description:Cells expressing both the regulatory T cell (Treg)-inducing transcription factor Foxp3 and the Th17 transcription factor ROR?t have been identified (biTregs). It is unclear whether ROR?t(+)Foxp3(+) biTregs belong to the Th17-specific Treg17 cells, represent intermediates during Treg/Th17 transdifferentiation, or constitute a distinct cell lineage. Because the role of biTregs in inflammatory renal disease is also unknown, we studied these cells in the nephrotoxic nephritis (NTN) model of acute crescentic GN. Induction of NTN resulted in rapid renal and systemic expansion of biTregs. Notably, analyses of the biTreg expression profile revealed production of both anti-inflammatory (IL-10, IL-35) and proinflammatory (IL-17) cytokines. Additionally, biTregs expressed a signature of surface molecules and transcription factors distinct from those of Th17 cells and conventional Tregs (cTregs), and biTregs were identified in Treg17-deficient mice. Finally, fate reporter and cell transfer studies confirmed that biTregs are not Treg/Th17 transdifferentiating cells. Therapeutic transfer of biTregs suppressed the development of nephritis to an extent similar to that observed with transferred cTregs, but in vitro studies indicated different mechanisms of immunosuppression for biTregs and cTregs. Intriguingely, as predicted from their cytokine profile, endogenous biTregs displayed additional proinflammatory functions in NTN that were abrogated by cell-specific deletion of ROR?t. In summary, we provide evidence that ROR?t(+)Foxp3(+) biTregs are a novel and independent bifunctional regulatory T cell lineage distinct from cTregs, Treg17 cells, and Th17 cells. Furthermore, biTregs appear to contribute to crescentic GN and hence may be novel therapeutic targets.