Differentiation Of Pathogenic Th17 Cells Research Articles

TPN10475 Constrains Effector T Lymphocytes Activation and Attenuates Experimental Autoimmune Encephalomyelitis Pathogenesis by Facilitating TGF-β Signal Transduction.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) mediated by immune cells, in which auto-reactive CD4+ T cells have been implicated as a major driver in the pathogenesis of the disease. In this study, we aimed to investigate whether the artemisinin derivative TPN10475 could alleviate experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of MS and its possible mechanisms. TPN10475 effectively resisted the reduction of TGF-β signal transduction induced by TCR stimulation, suppressed the activation and function of effector CD4+ T cells in vitro, and restricted the differentiation of pathogenic Th1 and Th17 cells. It was also found to negatively regulate the inflammatory response in EAE by reducing the peripheral activation drive of auto-reactive helper T lymphocytes, inhibiting the migration of inflammatory cells into the CNS to attenuate EAE. The above results suggested that the upregulation of TGF-β signal transduction may provide new ideas for the study of MS pathogenesis and have positive implications for the development of drugs for the treatment of autoimmune diseases.

BTN2A2-Ig protein inhibits the differentiation of pathogenic Th17 cells and attenuates EAE in mice

Pathogenic Th17 cells play a key role in the pathogenesis of many autoimmune diseases. Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) is the commonly used animal model for human MS and is characterized by autoreactive CD4+T cells attacking autoantigens in the CNS and causing myelin sheath damage. Although the recombinant BTN2A2-IgG2aFc (BTN2A2-Ig) fusion protein has been shown to inhibit T cell functions in vitro, it's unclear whether BTN2A2-Ig affects pathogenic Th17 cells and EAE development. We show here that BTN2A2-Ig protein attenuates established EAE, as compared with control Ig protein treatment. This is associated with reduced activation and proliferation of T cells in BTN2A2-Ig-treated EAE mice. Furthermore, BTN2A2-Ig protein inhibits the differentiation of CD4 naïve T cells into pathogenic Th17 cells and reduces the expression levels of Th1/Th17 cytokines and the Th1/Th17 pathway related genes and proteins but increases the expression levels of Th2-related genes and proteins. Our studies not only provide new insights into the mechanisms by which BTN2A2-Ig affects T cells, but also have the potential to provide a new strategy to treat MS and other autoimmune diseases.

Growth hormone releasing hormone signaling promotes Th17 cell differentiation and autoimmune inflammation

Dysregulation of Th17 cell differentiation and pathogenicity contributes to multiple autoimmune and inflammatory diseases. Previously growth hormone releasing hormone receptor (GHRH-R) deficient mice have been reported to be less susceptible to the induction of experimental autoimmune encephalomyelitis. Here, we show GHRH-R is an important regulator of Th17 cell differentiation in Th17 cell-mediated ocular and neural inflammation. We find that GHRH-R is not expressed in naïve CD4+ T cells, while its expression is induced throughout Th17 cell differentiation in vitro. Mechanistically, GHRH-R activates the JAK-STAT3 pathway, increases the phosphorylation of STAT3, enhances both non-pathogenic and pathogenic Th17 cell differentiation and promotes the gene expression signatures of pathogenic Th17 cells. Enhancing this signaling by GHRH agonist promotes, while inhibiting this signaling by GHRH antagonist or GHRH-R deficiency reduces, Th17 cell differentiation in vitro and Th17 cell-mediated ocular and neural inflammation in vivo. Thus, GHRH-R signaling functions as a critical factor that regulates Th17 cell differentiation and Th17 cell-mediated autoimmune ocular and neural inflammation.

Towards molecular mechanisms for pathogenic Th17 cell differentiation in ocular autoimmunity

Abstract CD4+ T helper (Th) cells play critical roles in coordinating proper immune responses by differentiating into effector subtypes specialized against different types of pathogens, while dysregulated Th cells can drive autoimmune disease by triggering inappropriate immune responses. Pathogenic Th17 (pTh17) cells, which combine behaviors of classic Th1 and Th17 effector cells by producing both interferon-gamma (IFNg) and interleukin-17A (IL17A), have been implicated in autoimmune diseases. Our goal is to map regulatory networks and identify novel regulators underlying pTh17 differentiation and activation using the murine model experimental autoimmune uveitis (EAU), in which ocular autoimmunity is driven by pTh17 cells. To track pTh17 differentiation, we bred a double reporter mouse with IL17A-cre/tdTomato-flox tracer and IFNg-YFP. We sorted both Tomato+YFP+ and Tomato+YFP- CD4+ T cells from eyes and spleens after EAU induction for single-cell RNA-seq. Clustering of single-cell transcriptomes revealed distinct transcriptomes between eyes and spleen. In the eye, we identified clusters similar to pTh17 and non-pathogenic (np)Th17 populations defined in a myelin autoimmunity model. The pTh17 cluster contained both double positive and Tomato single positive cells, suggesting a heterogeneous pathogenic population. In contrast, the npTh17 cluster only contained single positives. Differential gene expression analysis showed that pathogenic and non-pathogenic clusters utilize distinct sets of transcription factors. We are further characterizing epigenetic differences between pTh17 and npTh17 to better understand how distinct regulatory networks shape pTh17 differentiation. Supported by the NEI Intramural Research Program fund (ZIAEY000569-01)

Fecal microbiota transplantation and replenishment of short-chain fatty acids protect against chronic cerebral hypoperfusion-induced colonic dysfunction by regulating gut microbiota, differentiation of Th17 cells, and mitochondrial energy metabolism

BackgroundLittle is known about the association between gut microbiota and intestinal injury under a state of chronic cerebral hypoperfusion (CCH). Here, the effects of gut microbiota and short-chain fatty acids (SCFAs), as important metabolic products, on intestinal function and potential mechanisms after CCH were investigated.MethodsRats were subjected to bilateral common carotid artery occlusion (BCCAo) to induce CCH. The gut microbiota and metabolites of SCFAs were assessed by 16S rRNA sequencing and targeted metabolomics, respectively. Transcriptomic analysis of colon tissues was also conducted. Subsequently, potential molecular pathways and differentially expressed genes were verified by western blot, immunoprecipitation, and immunofluorescence analyses. Furthermore, the integrity of the colonic barrier was evaluated by hematoxylin and eosin and mucin 2 staining and expression levels of tight junction proteins. Besides, colonic inflammation was further assessed by flow cytometry and expression levels of inflammatory cytokines. In addition, colonic mitochondrial dysfunction was analyzed via membrane potential, reactive oxygen species, electron transport chain (ETC) activities, adenosine triphosphate content, and mitochondrial ultrastructure.ResultsCCH modified gut microbial composition and microbial metabolism of SCFAs, which may be associated with inhibition of mitochondrial ETC activities and oxidative phosphorylation, leading to dysregulation of mitochondrial energy metabolism. Furthermore, CCH induced differentiation of pathogenic Th17 cells, promoted the formation of complexes of interferon regulatory factor 4 and signal transducer and activator of transcription 3 (STAT3), and increased the phosphorylation of STAT3. This was associated with an impairment of colonic barrier function and chronic colonic inflammation. In contrast, FMT and SCFA replenishment ameliorated CCH-induced gut microbial dysbiosis by increasing the intestinal content of Ruminococcus_sp_N15_MGS_57 and modulating microbial metabolism of SCFAs by increasing acetic acid contents associated with an improvment of the balance between Tregs and Th17 cells, mitochondrial ETC activities, and oxidative phosphorylation to prevent colonic inflammation and dysregulation of mitochondrial energy metabolism.ConclusionThese findings indicate that FMT and SCFA replenishment present a promising therapeutic strategy against colonic dysfunction under a state of chronic cerebral ischemia.

IL-3 regulates the differentiation of pathogenic Th17 cells.

IL-17-producing Th17 cells play an important role in pathogenesis of rheumatoid arthritis (RA). Aberrant immune activation due to an imbalance between Th17 and regulatory T (Treg) cells is associated with the development of RA and other autoimmune diseases. Targeting pathogenic Th17 cells and their associated molecules is emerging as a promising strategy to treat and reverse RA. Here, we demonstrate that IL-3 inhibits the differentiation of Th17 cells and promotes the development of Treg cells in IL-2-dependent manner. In IL-2 KO mice, we observed that IL-3 has no effect on differentiation of both Th17 and Treg cells. In addition, IL-3 decreases pathogenic IL-17A+ TNF-α+ , IL-17A+ IFN-γ+ and IL-23R+ Th17 cells, secretion of GM-CSF and IFN-γ, and osteoclastogenesis when presented in the culture together with Th17 polarizing cytokines. Mechanistically, IL-3 regulates the development of Th17 cells through the inhibition of STAT3 phosphorylation. IL-3 treatment significantly decreases the pathogenic Th17 cell responses and arthritic scores in the mouse model of RA. Importantly, IL-3 inhibits the differentiation of human Th17 cells. Thus, our results suggest a novel therapeutic role of IL-3 in the regulation of Th17 cell-mediated pathophysiology of RA.

650 Target the activin receptor 1c on CD4+ T cells to achieve anti-tumor therapeutic effects

BackgroundActivins, members of the transforming growth factor-ß (TGF-ß) superfamily, were isolated and identified in endocrine system, and have been widely studied in endocrine-related cancers,1 2 but not substantially in the context of immune system and endocrine-unrelated cancers.3–5 It has been reported that upon binding to the receptors, activins cause the intracellular recruitment and phosphorylation of smad proteins, which mediate the expression of Foxp3.6–9 Therefore, we hypothesized that the blockade of the interaction of activins and their receptors will inhibit the activins-mediated Foxp3 induction in CD4+ T cells, thus modify the immune suppressive tumor microenvironment and achieve the goal of cancer immunotherapy.MethodsELISA (enzyme-linked immunosorbent assay) has been performed to determine the plasma level of Activin A in tumor-bearing mice and cancer patients. In vitro iTreg (induced regulatory T cells) differentiation has been done to naïve CD4+ cells isolated from wild type mice in the presence or absence of Activin A, and the percentage of Foxp3+ cells was demonstrated by flow cytometric analysis. qRT-PCR analysis has been conducted to determine the mRNA level of activin receptor isotypes in the immune subpopulations sorted from Foxp3-YFP mice. In the end, in vivo subcutaneous transplanted tumor studies have been done to evaluate the anti-tumor therapeutic effects of activin-receptor 1c blockade.ResultsWe show here that tumor-bearing mice had elevated Activin A levels, which correlated directly with tumor burden. Likewise, cancer patients had elevated plasma Activin A compared to healthy controls. Importantly, our in vitro studies suggested that Activin A promoted differentiation of conventional CD4+ cells into Foxp3-expressing induced Tregs, especially when TGF-ß was limited. Database and qRT-PCR analysis of sorted major immune cell subsets in mice revealed that activin receptor 1C (Acvr1c) was uniquely expressed on Tregs and was highly upregulated during iTreg differentiation. Mice deficient in Acvr1c were more resistant to cancer progression compared to wild type mice. This phenotype correlated with reduced expression of the FoxP3 transcription factor in CD4+ cells. Similar phenomena were observed when we treated the mice with anti-Acvr1c antibody after tumor inoculation. This anti-tumor therapeutic effect was more significant when anti-Acvr1c antibody was administrated in combination with anti-PD-1 antibody.ConclusionsBlocking Activin A signaling through its receptor 1c is a promising and disease-specific strategy for preventing the accumulation of immunosuppressive iTregs in cancer. Hence it represents a potential candidate for cancer immunotherapy.AcknowledgementsThis research is supported by the Bloomberg-Kimmel Institute (Immunometabolism Program & Immune Modulation Program), the Melanoma Research Alliance, the NIH (RO1AI099300, RO1AI089830, and R01AI137046), and The DoD (PC130767).ReferencesRisbridger GP, Schmitt JF, Robertson DM. Activins and inhibins in endocrine and other tumors. Endocr Rev 2001;22(6):836–858.Cui X, et al. Perspectives of small molecule inhibitors of activin receptor-like kinase in anti-tumor treatment and stem cell differentiation (Review). Mol Med Rep 2019;19(6):5053–5062.Michael IP, et al. ALK7 signaling manifests a homeostatic tissue barrier that is abrogated during tumorigenesis and metastasis. Dev Cell 2019;49(3):409–424.Wu B, et al. The TGF-ß superfamily cytokine Activin-A is induced during autoimmune neuroinflammation and drives pathogenic Th17 cell differentiation. Immunity 2021;54(2):308–323.Antsiferova M, et al. Activin promotes skin carcinogenesis by attraction and reprogramming of macrophages. MBO Mol Med 2017;9(1):27–45.Tsuchida K, et al. Activin isoforms signal through type I receptor serine/threonine kinase ALK7. Mol Cell Endocrinol 2004;220(1–2):59–65.Khalil AM, et al. Differential binding activity of TGF-ß family proteins to select TGF-ß receptors. J Pharmacol Exp Ther 2016;358(3):423–430.Huber S, et al. Activin a promotes the TGF-beta-induced conversion of CD4+CD25- T cells into Foxp3+ induced regulatory T cells. J Immunol 2009;182(8):4633–4640.Iizuka-Koga M, et al. Induction and maintenance of regulatory T cells by transcription factors and epigenetic modifications. J Autoimmun 2017;83:113–121.Ethics ApprovalAll animal experiments were performed under protocols approved by the Johns Hopkins University Institutional Animal Care and Use Committee (IACUC).

ZEB1 promotes pathogenic Th1 and Th17 cell differentiation in multiple sclerosis.

SUMMARYInappropriate CD4+ T helper (Th) differentiation can compromise host immunity or promote autoimmune disease. To identify disease-relevant regulators of T cell fate, we examined mutations that modify risk for multiple sclerosis (MS), a canonical organ-specific autoimmune disease. This analysis identified a role for Zinc finger E-box-binding homeobox (ZEB1). Deletion of ZEB1 protects against experimental autoimmune encephalitis (EAE), a mouse model of multiple sclerosis (MS). Mechanistically, ZEB1 in CD4+ T cells is required for pathogenic Th1 and Th17 differentiation. Genomic analyses of paired human and mouse expression data elucidated an unexpected role for ZEB1 in JAK-STAT signaling. ZEB1 inhibits miR-101-3p that represses JAK2 expression, STAT3/STAT4 phosphorylation, and subsequent expression of interleukin-17 (IL-17) and interferon gamma (IFN-γ). Underscoring its clinical relevance, ZEB1 and JAK2 downregulation decreases pathogenic cytokines expression in T cells from MS patients. Moreover, a Food and Drug Administration (FDA)-approved JAK2 inhibitor is effective in EAE. Collectively, these findings identify a conserved, potentially targetable mechanism regulating disease-relevant inflammation.

SKI Expression Suppresses Pathogenic Th17 Cell Response and Mitigates Experimental Autoimmune Encephalomyelitis.

Pathogenic Th17 cells are critically involved in many autoimmune diseases, while non-pathogenic Th17 cells are more immune regulatory. Understanding the mechanisms of the induction and maintenance of pathogenic Th17 cells will benefit the development of therapeutic treatments of related diseases. We have shown that the transforming growth factor-β (TGFβ) induced SKI degradation and dissociation from Smad4 complex is a prerequisite for TGFβ-induced Th17 cell differentiation. However, it is unclear whether and how SKI regulates pathogenic Th17 differentiation, which does not require TGFβ cytokine. Here we showed that SKI expression was downregulated during pathogenic Th17 cell differentiation and the ectopic expression of SKI abrogated the differentiation of pathogenic Th17 cells. Functionally, using a knock-in mouse model, we found ectopic SKI expression specifically in T cells prevented myelin oligodendrocyte glycoprotein peptide (MOG33–55) induced experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. We further revealed that induced SKI expression in already differentiated pathogenic Th17 cells reduced the maintenance of Th17 program and ameliorated EAE in an adoptive T cell transfer model. Therefore, our study provides valuable insights of targeting SKI to modulate pathogenic Th17 cell function and treat Th17-related diseases.

Calcium Signaling in T Cells and Chronic Inflammatory Disorders of the Oral Cavity

Acute immune responses to microbial insults in the oral cavity often progress to chronic inflammatory diseases such as periodontitis and apical periodontitis. Chronic oral inflammation causes destruction of the periodontium, potentially leading to loss of the dentition. Previous investigations have demonstrated that the composition of oral immune cells, rather than the overall extent of cellular infiltration, determines the pathological development of chronic inflammation. The role of T lymphocyte populations, including Th1, Th2, Th17, and Treg cells, has been extensively described. Studies now propose pathogenic Th17 cells as a distinct subset, uniquely classifiable from traditional Th17 populations. In situ differentiation of pathogenic Th17 cells has been verified as a source of destructive inflammation, which critically drives pathogenesis in chronic inflammatory diseases such as diabetes, rheumatoid arthritis, and inflammatory bowel disease. Pathogenic Th17 cells resemble a Th1 penotype and produce not only interleukin 17 (IL-17) but also γ-interferon (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The proinflammatory cytokine-specific mechanisms known to induce IL-17 expression in Th17 cells are well characterized; however, differentiation mechanisms that lead to pathogenic Th17 cells are less understood. Recently, Ca2+ signaling through Ca2+ release-activated Ca2+ channels (CRAC) in T cells has been uncovered as a major signaling axis involved in the regulation of T-cell-mediated chronic inflammation. In particular, pathogenic Th17 cell–mediated immunological diseases appear to be effectively targeted via such Ca2+ signaling pathways. Pathogenic plasticity of Th17 cells has been extensively illustrated in autoimmune and chronic inflammatory diseases. Although their specific causal relationship to oral infection-induced chronic inflammatory diseases is not fully established, pathogenic Th17 cells may be involved in the underlining mechanism. This review highlights the current understanding of T-cell phenotype regulation, calcium signaling pathways in this event, and the potential role of pathogenic Th17 cells in chronic inflammatory disorders of the oral cavity.

The TGF-β superfamily cytokine Activin-A is induced during autoimmune neuroinflammation and drives pathogenic Th17 cell differentiation

Th17 cells are known to exert pathogenic and non-pathogenic functions. Although the cytokine transforming growth factor β1 (TGF-β1) is instrumental for Th17 cell differentiation, it is dispensable for generation of pathogenic Th17 cells. Here, we examined the Tcell-intrinsic role of Activin-A, a TGF-β superfamily member closely related to TGF-β1, in pathogenic Th17 cell differentiation. Activin-A expression was increased in individuals with relapsing-remitting multiple sclerosis and in mice with experimental autoimmune encephalomyelitis. Stimulation with interleukin-6 and Activin-A induced a molecular program that mirrored that of pathogenic Th17 cells and was inhibited by blocking Activin-A signaling. Genetic disruption of Activin-A and its receptor ALK4 in Tcells impaired pathogenic Th17 cell differentiation invitro and invivo. Mechanistically, extracellular-signal-regulated kinase (ERK) phosphorylation, which was essential for pathogenic Th17 cell differentiation, was suppressed by TGF-β1-ALK5 but not Activin-A-ALK4 signaling. Thus, Activin-A drives pathogenic Th17 cell differentiation, implicating the Activin-A-ALK4-ERK axis as a therapeutic target for Th17 cell-related diseases.

Chronic Toxoplasma gondii Infection Alleviates Experimental Autoimmune Encephalomyelitis by the Immune Regulation Inducing Reduction in IL-17A/Th17 Via Upregulation of SOCS3

Experimental autoimmune encephalomyelitis (EAE) is a mouse model of multiple sclerosis (MS), a demyelinating autoimmune disease caused by the infiltration of a harmful autoreactive Th1 and Th17 cells. To mitigate MS, which is impossible to cure with medication only, immunomodulatory interventions that prevent Th17 cell activation are ideal. The objective of the present study was to analyze the effect of Toxoplasma gondii infection on the onset of EAE. Our results found that Toxoplasma gondii infection in the brain increases SOCS3 expression and decreases the phosphorylation of STAT3, resulting in reducing IL-17A and IL-23, which suppress the differentiation and expansion of pathogenic Th17 cells, an important factor in MS development. These immune responses resulted in a reduction in the clinical scoring of EAE induced by myelin oligodendrocyte glycoprotein 35–55 immunization. In the EAE group with T. gondii infection (Tg + EAE group), Th17-related immune responses that exacerbate the onset of EAE were reduced compared to those in the EAE group. This study suggests that the alleviation of EAE after T. gondii infection is regulated in a SOCS3/STAT3/IL-17A/blood–brain barrier integrity-dependent manner. Although parasite infection would not be permitted for MS treatment, this study using T. gondii infection identified potential targets that contribute to disease attenuation.

TonEBP in dendritic cells mediates pro-inflammatory maturation and Th1/Th17 responses

Dendritic cells (DCs) are potent antigen-presenting cells that link the innate and adaptive immune responses; as such they play pivotal roles in initiation and progression of rheumatoid arthritis (RA). Here, we report that the tonicity-responsive enhancer-binding protein (TonEBP or NFAT5), a Rel family protein involved in the pathogenesis of autoimmune disease and inflammation, is required for maturation and function of DCs. Myeloid cell-specific TonEBP deletion reduces disease severity in a murine model of collagen-induced arthritis; it also inhibits maturation of DCs and differentiation of pathogenic Th1 and Th17 cells in vivo. Upon stimulation by TLR4, TonEBP promotes surface expression of major histocompatibility complex class II and co-stimulatory molecules via p38 mitogen-activated protein kinase. This is followed by DC-mediated differentiation of pro-inflammatory Th1 and Th17 cells. Taken together, these findings provide mechanistic basis for the pathogenic role of TonEBP in RA and possibly other autoimmune diseases.

IFN-γ-induced nitric oxide leads to inhibition of Th17 differentiation via induction of iNOS and nitration of ROR-γ T

Abstract Elucidating the molecular mechanisms involved in pleotropic cytokine signaling can lead to novel treatment approaches for autoimmune diseases that target proinflammatory functions while maintaining immune-regulatory functions. To that end, we sought to further investigate the immune-regulatory roles of the pleotropic cytokine IFN-γ. Previous studies demonstrated that IFN-γ prevents the differentiation of pathogenic Th17 cells. In addition, nitric oxide generated via inducible nitric oxide synthase (iNOS) prevents differentiation of Th17 cells by nitration of ROR-γT. However, it remains unresolved whether IFN-γ prevents Th17 differentiation directly via inducing nitric oxide (NO) and nitration of ROR-γT. In line with previous observations, we found an increase in the frequency of autoreactive Th17 cells and a decrease in iNOS expression and NO in the absence of IFN-γ in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Additionally, the increase in Th17 cells in the absence of IFN-γ was reversible both in vitro and in vivo with treatment with a NO donor. This indicates a role for IFN-γ in inhibiting the differentiation of Th17 cells via iNOS-derived NO. We are currently elucidating the underlying mechanisms of NO mediated suppression and the molecular link between ROR-γt-nitration and decreased Th17 differentiation by flow cytometry, single-cell western blotting of key transcription factors, and in vivo adoptive transfer studies. Our studies may lead to a better understanding of the role of IFN-g and NO/iNOS axis in Th17 cell differentiation and autoimmune diseases.

SUMO-specific protease 2 restrains the differentiation of Th17 cells

Abstract Accumulating evidence suggests that posttranslational modifications (PTMs) play a critical role in the regulation of cellular functions and disease progression. Several reports indicated that SUMOylation, a type of PTMs, contributes to diseases through modulation of cell signaling and functions. The process of SUMOylation is dynamic and can be reversibly removed by the sentrin-specific protease (SENP) family. Smad4 has been shown to suppress the differentiation of pathogenic Th17 cells and can be deSUMOylated by SENP2. Here, we would like to study whether SUMOylation may affect T cell development and differentiation, and T cell-mediated diseases. To study the roles of Senp2 in T cell lineage, we generated Senp2f/f×Lck-cre (CKO) mice. We found that there were more Th1 and Th17 cells in CKO mice in the steady state, as compared with those in littermate controls (wild type, WT) mice. Besides, Senp2-deficiency in T cells also promotes the progression of dextran sodium sulfate (DSS)-induced colitis in mice. The levels of IFNg and IL-17 produced by in vitro differentiated Th1 and Th17 cells, respectively, were higher when Senp2 is deleted. The enzymatic activity of Senp2 is required for the inhibition of Th17 differentiation; as compared with the effects of lentiviral vector expressing wild type Senp2, transduction of differentiating CKO Th17 cells with a lentiviral vector expressing a catalytic domain mutant Senp2 failed to suppress the frequency of Th17 cells. We are examining if SUMOylation affects the function of Smad4 in Th17 cells. Results from this study may provide insights into the new therapeutic strategy for the control of Th17-mediated diseases.

Pathogenic Th17 cell differentiation is negatively regulated by let-7 microRNAs in a mouse model of multiple sclerosis

Abstract Autoimmune disorders such as multiple sclerosis (MS) are caused by proinflammatory events mediated by pathogenic Th17 cells. In MS, these cells arise in response to autoantigen recognition and exposure to the cytokines IL-1β and IL-23, migrate to the central nervous system (CNS) by following gradients of CCR2- and CCR5-cognate chemokines, and secrete GM-CSF. GM-CSF is essential for disease development, as it promotes the activation, differentiation, and recruitment of peripheral inflammatory myeloid cells to the CNS that directly demyelinate neurons and damage axons. Th17 cell pathogenicity in MS has been correlated with microRNA (miRNA) dysregulation, which leads to aberrant post-transcriptional regulation of gene expression and enhanced autoreactive phenotype. We found that the lethal-7 (let-7) miRNA family is abundantly expressed in naive CD4+ T cells, but gets dramatically downregulated over time following antigen encounter, indicating that let-7 may control the differentiation of pathogenic Th17 cells. To investigate a potential regulatory role for let-7 in Th17 cell autoreactivity, we used experimental autoimmune encephalomyelitis (EAE), the animal model of MS. Specifically, we demonstrated that let-7 confers protection from EAE by negatively regulating the proliferation, IL-1β/IL-23-dependent differentiation, and CCR2/CCR5-dependent migration of pathogenic Th17 cells to the CNS. Conversely, absence of let-7 led to enhanced Th17 cell autoreactivity and aggravated disease. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for the treatment of MS-related autoimmune diseases.

Chloroquine modulates inflammatory autoimmune responses through Nurr1 in autoimmune diseases

Abstract For over a half-century the anti-malarial drug chloroquine (CQ) has been used as a therapeutic agent, alone or in combination, to treat autoimmune diseases. However, neither the underlying mechanism(s) of action nor their molecular target(s) are well defined. The orphan nuclear receptor Nurr1 (also known as NR4A2) is an essential transcription factor affecting the development and maintenance of midbrain dopaminergic neurons. In this study, using in vitro T cell differentiation models, we demonstrate that CQ activates TREG cell differentiation and induces Foxp3 gene expression in a Nurr1-dependent manner. Remarkably, CQ appears to induce Nurr1 function by two distinct mechanisms: firstly, by direct binding to Nurr1’s ligand-binding domain and promoting its transcriptional activity and secondly by upregulation of Nurr1 expression through the CREB signaling pathway. In contrast, CQ suppressed gene expression and differentiation of pathogenic TH17 cells. Importantly, using a valid animal model of inflammatory bowel disease (IBD), we demonstrated that CQ promotes Foxp3 expression and differentiation of TREG cells in a Nurr1-dependent manner, leading to significant improvement of IBD-related symptoms. Taken together, these data suggest that CQ ameliorates autoimmune diseases via regulating Nurr1 function/expression and that Nurr1 is a promising target for developing effective therapeutics of human inflammatory autoimmune diseases.

Differentiation of Pathogenic Th17 Cells Is Negatively Regulated by Let-7 MicroRNAs in a Mouse Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a disabling demyelinating autoimmune disorder of the central nervous system (CNS) which is driven by IL-23- and IL-1β-induced autoreactive Th17 cells that traffic to the CNS and secrete proinflammatory cytokines. Th17 pathogenicity in MS has been correlated with the dysregulation of microRNA (miRNA) expression, and specific miRNAs have been shown to promote the pathogenic Th17 phenotype. In the present study, we demonstrate, using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), that let-7 miRNAs confer protection against EAE by negatively regulating the proliferation, differentiation and chemokine-mediated migration of pathogenic Th17 cells to the CNS. Specifically, we found that let-7 miRNAs may directly target the cytokine receptors Il1r1 and Il23r, as well as the chemokine receptors Ccr2 and Ccr5. Therefore, our results identify a novel regulatory role for let-7 miRNAs in pathogenic Th17 differentiation during EAE development, suggesting a promising therapeutic application for disease treatment.

Melatonin ameliorates necrotizing enterocolitis by preventing Th17/Treg imbalance through activation of the AMPK/SIRT1 pathway.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease affecting premature infants. Mounting evidence supports the therapeutic effect of melatonin on NEC, although the underlying mechanisms remain unclear.Methods: NEC was induced in 10-day-old C57BL/6 pups via hypoxia and gavage feeding of formula containing enteric bacteria, and then, mice received melatonin, melatonin + recombinant IL-17, melatonin + anti-CD25 monoclonal antibody, melatonin + Ex-527, or melatonin + Compound C treatment. Control mice were left with their dams to breastfeed and vehicle-treated NEC pups were used as controls for treatment. Ileal tissues were collected from mice and analyzed by histopathology, immunoblotting, and flow cytometry. FITC-labeled dextran was administered to all surviving pups to evaluate gut barrier function by fluorometry. We used molecular biology and cell culture approaches to study the related mechanisms in CD4+ T cells from umbilical cord blood.Results: We demonstrated that melatonin treatment ameliorates disease in an NEC mouse model in a manner dependent on improved intestinal Th17/Treg balance. We also showed that melatonin blocks the differentiation of pathogenic Th17 cells and augments the generation of protective Treg cells in vitro. We further demonstrated that the Th17/Treg balance is influenced by melatonin through activation of AMPK in the intestine, in turn promoting SIRT1 activation and stabilization.Conclusions: These results demonstrate that melatonin-induced activation of AMPK/SIRT1 signaling regulates the balance between Th17 and Treg cells and that therapeutic strategies targeting the Th17/Treg balance via the AMPK/SIRT1 pathway might be beneficial for the treatment of NEC.

Serum Amyloid A Proteins Induce Pathogenic Th17 Cells and Promote Inflammatory Disease

Lymphoid cells that produce interleukin (IL)-17 cytokines protect barrier tissues from pathogenic microbes but are also prominent effectors of inflammation and autoimmune disease. T helper 17 (Th17) cells, defined by RORγt-dependent production of IL-17A and IL-17F, exert homeostatic functions in the gut upon microbiota-directed differentiation from naive CD4+ Tcells. In the non-pathogenic setting, their cytokine production is regulated by serum amyloid A proteins (SAA1 and SAA2) secreted by adjacent intestinal epithelial cells. However, Th17 cell behaviors vary markedly according to their environment. Here, we show that SAAs additionally direct a pathogenic pro-inflammatory Th17 cell differentiation program, acting directly on Tcells in collaboration with STAT3-activating cytokines. Using loss- and gain-of-function mouse models, we show that SAA1, SAA2, and SAA3 have distinct systemic and local functions in promoting Th17-mediated inflammatory diseases. These studies suggest that Tcell signaling pathways modulated by the SAAs may be attractive targets for anti-inflammatory therapies.