Generation of ROR?t+ Antigen-Specific T Regulatory 17 Cells from Foxp3+ Precursors in Autoimmunity.
ABSTRACT: Th17 cells are potent mediators in autoimmune diseases, and ROR?t is required for their development. Recent studies have shown that ROR?t+ Treg cells in the gut regulate intestinal inflammation by inhibiting effector T cell function. In the current study, we report that ROR?t+ Treg cells were also found in lymph nodes following immunization. Not only distinct from intestinal ROR?t+ Treg cells in their transcriptomes, peripheral ROR?t+ Treg cells were derived from Foxp3+ thymic Treg cells in an antigen-specific manner. Development of these ROR?t+ Treg cells, coined T regulatory 17 (Tr17) cells, depended on IL-6/Stat3 signaling. Tr17 cells showed suppressive activity against antigen-specific effector T cells in vitro. In addition, Tr17 cells efficiently inhibited myelin-specific Th17-cell-mediated CNS auto-inflammation in a passive EAE model. Collectively, our study demonstrates that Tr17 cells are effector Treg cells that potentially restrict autoimmunity.
Project description:Foxp3+ regulatory T cells (Treg) are essential modulators of immune responses, but the molecular mechanisms underlying their function are not fully understood. Here we show that the transcription factor Blimp-1 is a crucial regulator of the Foxp3+ROR?t+ Treg subset. The intrinsic expression of Blimp-1 in these cells is required to prevent production of Th17-associated cytokines. Direct binding of Blimp-1 to the Il17 locus in Treg is associated with inhibitory histone modifications but unaltered binding of ROR?t. In the absence of Blimp-1, the Il17 locus is activated, with increased occupancy of the co-activator p300 and abundant binding of the transcriptional regulator IRF4, which is required, along with ROR?t, for IL-17 expression in the absence of Blimp-1. We also show that despite their sustained expression of Foxp3, Blimp-1-/- ROR?t+IL-17-producing Treg lose suppressor function and can promote intestinal inflammation, indicating that repression of Th17-associated cytokines by Blimp-1 is a crucial requirement for ROR?t+ Treg function.
Project description:The ability of CD4+ T cells to change their phenotype and to specialize into different functional subsets may enhance the risk of autoimmune diseases. Here we investigate how a pleiotropic cytokine interleukin (IL)-15 may modify the functional commitment of CD4+ T cells expressing the lineage-associated transcription factors: forkhead box P3 (Foxp3; Treg) and ROR?t (Th17) in the context of inflammatory bowel disease (IBD). We demonstrate in mice that impaired delivery of IL-15 to CD4+ T cells in the colon downmodulates Foxp3 expression (diminishing STAT5 phosphorylation) and enhances ROR?t expression (by upregulating the expression of Runx1). In consequence, CD4+ T cells deprived of IL-15 rapidly trigger IBD characterized by enhanced production of pro-inflammatory cytokines (interferon-?, IL-6) and accumulation of Th1/Th17 cells. Overall, our findings indicate a potentially beneficial role of IL-15 in IBD by fine-tuning the balance between Treg and Th17 cells and controlling intestinal inflammation.
Project description:Th17 cells are potent mediators in autoimmune diseases and RORγt is required for their development. Recent studies have shown that RORγt+ Treg cells in the gut regulate intestinal inflammation by inhibiting effector T cell function. In the current study, we report that RORγt+ Treg cells were also found in lymph nodes following immunization. Not only distinct from intestinal RORγt+ Treg in their transcriptomes, peripheral RORγt+ Treg cells were derived from Foxp3+ thymic Treg cells, in an antigen-specific manner. Development of these RORγt+ Treg cells, coined as T regulatory 17 (Tr17) cells, depended on IL-6/Stat3 signaling. Tr17 cells showed suppressive activity against antigen-specific effector T cells in vitro. In addition, Tr17 cells efficiently inhibited myelin-specific Th17 cell-mediated CNS auto-inflammation in a passive EAE model. Collectively, our study demonstrates Tr17 cells as effector Treg cells that potentially restrict autoimmunity. Overall design: Examination of mRNA profiles of RORγt+ Tr17 cells in draining lymph nodes of immunized mice by deep sequencing, using SOLiD v4
Project description:Microbiota are thought to influence the development and progression of inflammatory bowel disease (IBD), but determining generalizable effects of microbiota on IBD etiology requires larger-scale functional analyses. We colonized germ-free mice with intestinal microbiotas from 30 healthy and IBD donors and determined the homeostatic intestinal T cell response to each microbiota. Compared to microbiotas from healthy donors, transfer of IBD microbiotas into germ-free mice increased numbers of intestinal Th17 cells and Th2 cells and decreased numbers of ROR?t+ Treg cells. Colonization with IBD microbiotas exacerbated disease in a model where colitis is induced upon transfer of naive T cells into Rag1-/- mice. The proportions of Th17 and ROR?t+ Treg cells induced by each microbiota were predictive of human disease status and accounted for disease severity in the Rag1-/- colitis model. Thus, an impact on intestinal Th17 and ROR?t+ Treg cell compartments emerges as a unifying feature of IBD microbiotas, suggesting a general mechanism for microbial contribution to IBD pathogenesis.
Project description:Infections with Giardia are among the most common causes of food and water-borne diarrheal disease worldwide. Here, we investigated Th17, Treg and IgA responses, and alterations in gut microbiota in two mouse lines with varying susceptibility to Giardia muris infection. Infected BALB/c mice shed significantly more cysts compared with C57BL/6 mice. Impaired control of infection in BALB/c mice was associated with lower Th17 activity and lower IgA levels compared with C57BL/6 mice. The limited metabolic activity, proliferation and cytokine production of Th17 cells in BALB/c mice was associated with higher proportions of intestinal Foxp3+ROR?t+ regulatory T cells and BALB/c mice developed increased ROR?t+ Treg:Th17 ratios in response to G. muris infection. Furthermore, G. muris colonization led to a significantly reduced evenness in the gut microbial communities of BALB/c mice. Our data indicate that differential susceptibility to Giardia infections may be related to ROR?t+ Treg controlling Th17 activity and that changes in the microbiota composition upon Giardia infection partially depend on the host background.
Project description:<b>Background and Aims:</b> Despite the negative results of blocking IL-17 in Crohn's disease (CD) patients, selective modulation of Th17-dependent responses warrants further study. Inhibition of retinoic acid-related orphan receptor gamma (ROR?t), the master regulator of the Th17 signature, is currently being explored in inflammatory diseases. Our aim was to determine the effect of a novel oral ROR?t antagonist (BI119) in human CD and on an experimental model of intestinal inflammation. <b>Methods:</b> 51 CD patients and 11 healthy subjects were included. The effects of BI119 were tested on microbial-stimulated peripheral blood mononuclear cells (PBMCs), intestinal crypts and biopsies from CD patients. The ability of BI119 to prevent colitis <i>in vivo</i> was assessed in the CD4<sup>+</sup>CD45RB<sup>high</sup> T cell transfer model. <b>Results:</b> In bacterial antigen-stimulated PBMCs from CD patients, BI119 inhibits Th17-related genes and proteins, while upregulating Treg and preserving Th1 and Th2 signatures. Intestinal crypts cultured with supernatants from BI119-treated commensal-specific CD4<sup>+</sup> T cells showed decreased expression of <i>CXCL1, CXCL8</i> and <i>CCL20</i>. BI119 significantly reduced <i>IL17</i> and <i>IL26</i> transcription in colonic and ileal CD biopsies and did not affect <i>IL22</i>. BI119 has a more profound effect in ileal CD with additional significant downregulation of <i>IL23R, CSF2, CXCL1, CXCL8</i>, and <i>S100A8</i>, and upregulation of <i>DEFA5</i>. BI119 significantly prevented development of clinical, macroscopic and molecular markers of colitis in the T-cell transfer model. <b>Conclusions:</b> BI119 modulated CD-relevant Th17 signatures, including downregulation of <i>IL23R</i> while preserving mucosa-associated IL-22 responses, and abrogated experimental colitis. Our results provide support to the use of ROR?t antagonists as a novel therapy to CD treatment.
Project description:RAR-related orphan receptor-?t (ROR-?t) directs differentiation of proinflammatory T helper 17 (TH17) cells and is a potential therapeutic target in chronic autoimmune and inflammatory diseases. However, ROR-?t-dependent group 3 innate lymphoid cells ILC3s provide essential immunity and tissue protection in the intestine, suggesting that targeting ROR-?t could also result in impaired host defense after infection or enhanced tissue damage. Here, we demonstrate that transient chemical inhibition of ROR-?t in mice selectively reduces cytokine production from TH17 but not ILCs in the context of intestinal infection with Citrobacter rodentium, resulting in preserved innate immunity. Temporal deletion of Rorc (encoding ROR-?t) in mature ILCs also did not impair cytokine response in the steady state or during infection. Finally, pharmacologic inhibition of ROR-?t provided therapeutic benefit in mouse models of intestinal inflammation and reduced the frequency of TH17 cells but not ILCs isolated from primary intestinal samples of individuals with inflammatory bowel disease (IBD). Collectively, these results reveal differential requirements for ROR-?t in the maintenance of TH17 cell and ILC3 responses and suggest that transient inhibition of ROR-?t is a safe and effective therapeutic approach during intestinal inflammation.
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:<h4>Objective</h4>Cardiovascular diseases (CVDs) are the leading cause of mortality in Western countries. Atherosclerosis is a multi-step inflammatory disease characterized at early stages by accumulation of cholesterol in the arterial wall followed by recruitment of immune cells. We sought to determine if pharmacological suppression of ROR?/? activity is beneficial in treatment of atherosclerosis.<h4>Methods</h4>To identify the role of ROR? and ROR? in atherosclerosis, we used the LDL-R(-/-) mouse model of atherosclerosis placed on a high cholesterol diet treated with SR1001, a ROR?/? inverse agonist, for four weeks.<h4>Results</h4>Our results demonstrate that treatment with the ROR inverse agonist substantially decreases plaque formation in vivo. The mechanism of the anti-atherogenic activity of the inhibition of ROR?/? activity appeared to be due to targeting two distinct pathways. SR1001 treatment reduced plasma low density lipoprotein (LDL) level without affecting high density lipoprotein (HDL) via increasing intestinal cholesterol excretion. Treatment with SR1001 also induced an anti-atherogenic immune profile that was characterized by a reduction in Th17 cells and an increase in Treg and Th2 cells. Our data suggest that ROR? and ROR? play a critical role in atherosclerosis development by regulating at least two major pathways important in the pathology of this disease: cholesterol flux and inflammation.<h4>Conclusion</h4>Our data suggest that pharmacological targeting of ROR?/? may be an effective method for treatment of atherosclerosis offering a distinct mechanism of action relative to statins.
Project description:T cell functional differentiation is mediated by lineage-specific transcription factors. T helper 17 (Th17) has been recently identified as a distinct Th lineage mediating tissue inflammation. Retinoic acid receptor-related orphan receptor gamma (ROR gamma) was shown to regulate Th17 differentiation; ROR gamma deficiency, however, did not completely abolish Th17 cytokine expression. Here, we report Th17 cells highly expressed another related nuclear receptor, ROR alpha, induced by transforming growth factor-beta and interleukin-6 (IL-6), which is dependent on signal transducer and activator of transcription 3. Overexpression of ROR alpha promoted Th17 differentiation, possibly through the conserved noncoding sequence 2 in Il17-Il17f locus. ROR alpha deficiency resulted in reduced IL-17 expression in vitro and in vivo. Furthermore, ROR alpha and ROR gamma coexpression synergistically led to greater Th17 differentiation. Double deficiencies in ROR alpha and ROR gamma globally impaired Th17 generation and completely protected mice against experimental autoimmune encephalomyelitis. Therefore, Th17 differentiation is directed by two lineage-specific nuclear receptors, ROR alpha and ROR gamma.