Inhibited Th17 Differentiation Research Articles

Protein arginine methyltransferase 5 promotes cholesterol biosynthesis-mediated Th17 responses and autoimmunity.

Protein arginine methyltransferase 5 (PRMT5) catalyzes symmetric dimethylation (SDM) of arginine, a posttranslational modification involved in oncogenesis and embryonic development. However, the role and mechanisms by which PRMT5 modulates Th cell polarization and autoimmune disease have not yet been elucidated. Here, we found that PRMT5 promoted SREBP1 SDM and the induction of cholesterol biosynthetic pathway enzymes that produce retinoid-related orphan receptor (ROR) agonists that activate RORγt. Specific loss of PRMT5 in the CD4+ Th cell compartment suppressed Th17 differentiation and protected mice from developing experimental autoimmune encephalomyelitis (EAE). We also found that PRMT5 controlled thymic and peripheral homeostasis in the CD4+ Th cell life cycle and invariant NK (iNK) T cell development and CD8+ T cell maintenance. This work demonstrates that PRMT5 expression in recently activated T cells is necessary for the cholesterol biosynthesis metabolic gene expression program that generates RORγt agonistic activity and promotes Th17 differentiation and EAE. These results point to Th PRMT5 and its downstream cholesterol biosynthesis pathway as promising therapeutic targets in Th17-mediated diseases.

Elevated levels of soluble Endothelial protein C receptor in rheumatoid arthritis and block the therapeutic effect of protein C in collagen-induced arthritis

BackgroundEndothelial protein C receptor (EPCR) is a membranous protein that can be combined with a variety of ligands and plays important roles in anticoagulant and anti-inflammation. Recent reports have shown that surface EPCR expression on T cells is negatively associated with Th17 differentiation and is co-expressed with other immunosuppressive molecules, such as The programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). Hence, we hypothesized that EPCR may play a critical role in rheumatoid arthritis (RA) disease progression that is mediated by Th17 differentiation. In order to explore the role of EPCR on RA disease pathogenesis, we detected membranous EPCR (mEPCR) expression in CD4+ T cells and soluble EPCR (sEPCR) expression in the sera of RA patients. MethodsThe proportion of CD4+/EPCR+ T cells in the peripheral blood of RA patients was detected by flow cytometry, and the expression of sEPCR in the sera of RA patients was detected by enzyme-linked immunosorbent assay (ELISA). For in vitro experiments, protein C (PC) and EPCR recombinant proteins were used to block peripheral blood mononuclear cell (PBMC) activation and to detect Th17 differentiation. For in vivo experiments in DBA/1 mice with collagen-induced arthritis (CIA), we administered PC and EPCR recombinant proteins, monitored disease progression, and evaluated the role of EPCR in disease progression. ResultsThe proportion of CD4+/EPCR+ T cells in the peripheral blood of RA patients was lower than that of osteoarthritis (OA) patients, while the expression level of sEPCR in the sera of RA patients was concomitantly higher than that in OA patients. Subsequent analysis revealed that sEPCR expression was positively correlated with rheumatoid factors (RF) and other inflammatory indicators in RA patients. Further studies confirmed that sEPCR administration alleviated the progression of collagen-induced arthritis and partially blocked the therapeutic effect of PC in CIA mice. ConclusionSoluble EPCR is associated with RA disease progression and induces disease remission in CIA mice by inhibiting Th17 differentiation.

A directly GP130-targeting small molecule ameliorates collagen-induced arthritis (CIA) by inhibiting IL-6/GP130 signalling and Th17 differentiation.

Rheumatoid arthritis is a chronic inflammatory disease associated with joint inflammation and destruction driven by T helper 17 (Th17) cells. Interleukin-6 (IL-6) is secreted by many cell types, including macrophages and synovial fibroblasts. It induces the differentiation and function of Th17 cells that can increase lymphocytic infiltration in the joint. LMT-28 can suppress IL-6 signalling through direct binding to glycoprotein-130 and alleviate inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. The purpose of this study was to assess whether LMT-28 could potently inhibit Th17 differentiation and to determine the mechanism involved in the attenuating effect of LMT-28 on rheumatoid arthritis through the IL-6 signalling pathway. LMT-28 reduced the arthritis score and showed protective effects against bone and cartilage destruction in collagen-induced arthritis (CIA) mice. In mice with CIA, LMT-28 markedly decreased serum levels of IL-6, TNF and IL-1β compared to vehicle control. Moreover, LMT-28 attenuated Th17 cell activation in lymph nodes of CIA mice. We demonstrated that LMT-28 suppressed differentiation of Th17 in mouse splenocytes and human peripheral blood mononuclear cells (PBMCs). Additionally, LMT-28 inhibited phosphorylation of GP130, STAT3 and ERK induced by Hyper-IL-6 in human fibroblast-like synoviocytes (FLS). Collectively, these results suggest that LMT-28 can inhibit differentiated/activated-Th17 cells in rheumatoid arthritis by blocking activation of the STAT3 pathway. LMT-28 can attenuate rheumatoid arthritis by inhibiting differentiation/activation of Th17 cells and suppressing the proliferation and signalling activation of the IL-6/solubleIL-6 receptor complex stimulated FLS.

LncRNA GAS5 inhibits Th17 differentiation and alleviates immune thrombocytopenia via promoting the ubiquitination of STAT3

BackgroundThe increased differentiation of T helper 17 cells (Th17) accelerates the development of immune thrombocytopenia (ITP), which is a common autoimmune disease with limited therapeutic methods. Recent studies have revealed that long non-coding RNAs (lncRNAs) play a critical role in autoimmune diseases, thus this study aims to investigate the effect of lncRNA GAS5 on the differentiation of Th17 cells in ITP. MethodsThe expression of GAS5 in peripheral blood mononuclear cells (PBMCs) of ITP patients and spleen tissues of ITP mice was measured by qRT-PCR. The percentage of Th17 cells in CD4+ cells was measured by flow cytometry. The combination between GAS5 and STAT3 was confirmed by RNA pull-down assay and RNA Binding Protein Immunoprecipitation (RIP). The ubiquitination of STAT3 was detected by ubiquitination assay and the interaction between STAT3 and TRAF6 was measured by Co-Immunoprecipitation (Co-IP). Finally, the effect of GAS5 on Th17 differentiation was investigated in vitro and in vivo using lentivirus (lenti)-GAS5. ResultsGAS5 expression was downregulated both in PBMCs of ITP patients and spleen tissues of ITP mice. Overexpression of GAS5 suppressed Th17 differentiation while had no effect on Treg differentiation in naïve CD4+ cells. RNA pull-down and RNA immunoprecipitation assays confirmed the interaction between GAS5 and STAT3. Further studies showed GAS5 accelerated the degradation of STAT3 via promoting TRAF6-mediated ubiquitination. Overexpressing GAS5 suppressed Th17 differentiation in vitro and alleviated ITP in vivo via reducing STAT3. ConclusionLncRNA GAS5 inhibited Th17 differentiation through promoting the TRAF6-mediated ubiquitination of STAT3, thus relieving ITP.

A Critical Regulation of Th17 Cell Responses and Autoimmune Neuro-Inflammation by Ginsenoside Rg3.

Among diverse helper T-cell subsets, Th17 cells appear to be pathogenic in diverse autoimmune diseases, and thus, targeting Th17 cells could be beneficial for the treatment of the diseases in humans. Ginsenoside Rg3 is one of the most potent components in Korean Red Ginseng (KRG; Panax ginseng Meyer) in ameliorating inflammatory responses. However, the role of Rg3 in Th17 cells and Th17-mediated autoimmunity is unclear. We found that Rg3 significantly inhibited the differentiation of Th17 cells from naïve precursors in a dendritic cell (DC)–T co-culture system. While Rg3 minimally affected the secretion of IL-6, TNFα, and IL-12p40 from DCs, it significantly hampered the expression of IL-17A and RORγt in T cells in a T-cell-intrinsic manner. Moreover, Rg3 alleviated the onset and severity of experimental autoimmune encephalomyelitis (EAE), induced by transferring myelin oligodendrocyte glycoprotein (MOG)-reactive T cells. Our findings demonstrate that Rg3 inhibited Th17 differentiation and Th17-mediated neuro-inflammation, suggesting Rg3 as a potential candidate for resolving Th17-related autoimmune diseases.

Eriodictyol suppresses Th17 differentiation and the pathogenesis of experimental autoimmune encephalomyelitis.

T helper 17 (Th17) cells that express interleukin-17 (IL-17) play a key role in various inflammatory diseases, such as multiple sclerosis (MS), and its animal model experimental autoimmune encephalomyelitis (EAE). The retinoic acid receptor-related orphan receptors γt (RORγt) have an indispensable effect on the differentiation of this cell type, and are thus considered a valuable target in the treatment of Th17-related disorders. In this study, we found that eriodictyol (EDT), a natural flavonoid abundant in citrus fruits and peanuts, was located directly in the binding pocket of RORγt, and induced a conformational change that resulted in the effective suppression of the receptor's activity, thus offering insight into the transcriptional inhibition of RORγt-dependent genes. Consistent with this, EDT dose-dependently (5-10 μM) blocked murine Th17 differentiation, and markedly reduced IL-17A secretion in vitro. Furthermore, this compound has been found to have novel properties for directly inhibiting Th1 cell development and promoting Treg cell differentiation at high doses (≥10 μM). EDT administration significantly decreased the clinical severity in the EAE model, with inhibited demyelination and reduced inflammatory responses in the periphery and in the central nervous system (CNS). In the adoptive transfer model, EDT also remarkably suppressed the Th17 cell infiltration and pathogenicity. Collectively, our data demonstrated that EDT, as an agent for the pharmacological inhibition of RORγt, has great potential for immunomodulation, and for use in the treatment of Th17-mediated autoimmune disease.

Combination of gp130-targeting and TNF-targeting small molecules in alleviating arthritis through the down-regulation of Th17 differentiation and osteoclastogenesis

Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease that is characterized by T helper 17 (Th17) cell- and osteoclast-induced joint destruction and inflammation. In RA, several cytokines (interleukin (IL)-1, 6,17, and tumor necrosis factor (TNF)) are involved in almost all aspects of articular inflammation and destruction. This study aimed to evaluate the combinatorial effect of TNF and IL-6 inhibitors on the differentiation and activation of Th17 cells and osteoclasts in the context of RA, and to identify the RA-related mechanisms through IL-6 signaling. Tetrahydropapaverine (THP) showed direct binding to TNF in screening-ELISA, and SPR and TNF-neutralization assays. In a previous study, the therapeutic effect of gp130-targeting LMT-28 was confirmed in RA. Combinatorial treatment with LMT-28 and THP reduced the arthritis index and showed protective effects against bone and cartilage destruction in CIA mice. The secretion levels of TNF, IL-6, and IL-1β significantly decreased upon combinatorial treatment with LMT-28 and THP. Further, the LMT-28 and THP combination suppressed the differentiation and activation of Th17 cells in mouse splenocytes and human PBMCs. In human RA-FLS, the LMT-28 and THP combination inhibited cell proliferation and downregulated IL-6 and/or TNF-mediated signaling relative to that observed upon independent treatment with LMT-28 or THP. Furthermore, the combination of LMT-28 and THP significantly inhibited the differentiation of mouse bone marrow monocytes (BMMs) into osteoclasts. In conclusion, the LMT-28 and THP combination can attenuate RA through the inhibition of Th17 differentiation and osteoclastogenesis, and suppression of IL-6 or TNF-induced signaling pathways. This combinatorial therapy could be used as a new strategy for the treatment of RA.

Sauchinone ameliorates intestinal inflammation and promotes Th17 cell production of IL-10 via Blimp-1

Inflammatory bowel disease (IBD) is characterized by chronic, unpredictable relapsing and inflammatory disease of the gastrointestinal tract. Daily diet patterns have long been one of the most important hotspots for IBD therapeutic strategies. Sauchinone (SAU), a key bioactive lignin isolated from the roots of the herb Saururus chinensis, has been known to play an anti-inflammatory role in several diseases. However, its effect on IBD has not yet been investigated. In the current study, we established 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice and treated them with SAU. Flow cytometric analysis was performed to determine the phenotype of T cells in the lamina propria. qRT-PCR and ELISA were performed to measure cytokine transcript and protein levels, respectively. We found that SAU ameliorated TNBS-induced mouse colitis and inflammatory responses in mucosal tissues and peripheral blood CD4+ T cells from IBD patients. SAU significantly suppressed Th17 differentiation but facilitated IL-10 production, and SAU-treated Th17 cells exhibited inhibitory functions in vitro and in vivo. Mechanistically, we demonstrated that SAU induced Blimp-1 expression (encoded by Prdm1) in Th17 cells, and SAU failed to increase IL-10 production in Prdm1-knockout Th17 cells. Our data reveal an uncharacterized mechanism through which SAU regulates intestinal inflammation and Th17 differentiation.

LncRNA H19 over-expression inhibited Th17 cell differentiation to relieve endometriosis through miR-342-3p/IER3 pathway

ObjectiveTo investigate the mechanism of LncRNA H19 in Th17 cell differentiation and endometrial stromal cells (ESCs) proliferation in endometriosis (EMS).MethodsLncRNA H19, miR-342-3p and IER3 expressions were detected by qRT-PCR and western blot. The percentage of Th17 cells/CD4+ T cells was detected by flow cytometry. IL-17 level was measured by ELISA. The interaction of miR-342-3p and IER3 was confirmed by Luciferase reporter assay.ResultsLncRNA H19 and IER3 expressions were down-regulated in mononuclear cells from peritoneal fluid (PFMCs) of patients with EMS or under Th17 differentiation conditions, whereas miR-342-3p expression was up-regulated and the percentage of Th17 cells was increased in PFMCs of patients with EMS or under Th17 differentiation conditions. Over-expression of LncRNA H19 decreased IL-17 level and the percentage of Th17 cells/CD4+ T cells. Besides, we confirmed that miR-342-3p could target to IER3 and negatively regulate IER3 expression. LncRNA H19 over-expression suppressed Th17 differentiation and ESC proliferation through regulating miR-342-3p/IER3. In vivo experiments showed LncRNA H19 over-expression suppressed the growth of Th17 cell differentiation-induced endometriosis-like lesions.ConclusionLncRNA H19 was down-regulated in PFMC of patients with EMS or under Th17 polarizing conditions, and LncRNA H19 over-expression suppressed Th17 cell differentiation and ESCs proliferation through miR-342-3p/IER3 pathway.

Retinoic Acid Receptor-Related Receptor Alpha Ameliorates Autoimmune Arthritis via Inhibiting of Th17 Cells and Osteoclastogenesis.

Rheumatoid arthritis (RA) is a chronic inflammatory polyarthritis characterized by progressive joint destruction. IL-17-producing CD4+ T (Th17) cells play pivotal roles in RA development and progression. Retinoic acid receptor-related orphan receptor alpha (RORα) is a negative regulator of inflammatory responses, whereas RORγt, another member of the ROR family, is a Th17 lineage-specific transcription factor. Here, we investigated the immunoregulatory potential of RORα in collagen-induced arthritis (CIA) mice, an experimental model of RA. Cholesterol sulfate (CS) or SR1078, a ligand of RORα, inhibited RORγt expression and Th17 differentiation in vitro. In addition, fortification of RORα in T cells inhibited the expression levels of glycolysis-associated genes. We found that RORα overexpression in CIA mice attenuated the clinical and histological severities of inflammatory arthritis. The anti-arthritic effect of RORα was associated with suppressed Th17 differentiation and attenuated mTOR-STAT3 signaling in T cells. Furthermore, altered RORα activity could directly affect osteoclastogenesis implicated in progressive bone destruction in human RA. Our findings defined a critical role of RORα in the pathogenesis of RA. These data suggest that RORα may have novel therapeutic uses in the treatment of RA.

Daurinol Attenuates Autoimmune Arthritis via Stabilization of Nrp1-PTEN-Foxp3 Signaling in Regulatory T Cells.

Optimizing Treg function and improving Treg stability are attractive treatment strategies for treating autoimmune rheumatoid arthritis (RA). However, the limited number of circulating Tregs and questions about the functional stability of in vitro-expanded Tregs are potential limitations of Treg-based cell therapy. The aim of this study was to analyze the regulatory effect of daurinol, a catalytic inhibitor of topoisomerase IIα, on Th cell differentiation and to evaluate their therapeutic potential in a preclinical experimental model of RA. We investigated the effect of daurinol on T cell differentiation by flow cytometry. Foxp3 stability and methylation were analyzed by suppression assays and bisulfite pyrosequencing. Daurinol was treated in the collagen-induced arthritis (CIA) model, and the effects in vivo were determined. We found that daurinol can promote Treg differentiation and reciprocally inhibit Th17 differentiation. This Treg-inducing property of daurinol was associated with decreased activity of Akt–mTOR and reciprocally increased activity of neuropilin-1 (Nrp1)–PTEN. Daurinol treatment inhibited aerobic glycolysis in Th17 conditions, indicating the metabolic changes by daurinol. We found that the daurinol increase the Treg stability was achieved by Foxp3 hypomethylation. In vivo daurinol treatment in CIA mice reduced the clinical arthritis severity and histological inflammation. The Treg population frequency increased and the Th17 cells decreased in the spleens of arthritis mice treated with daurinol. These results showed the anti-arthritic and immunoregulating properties of daurinol is achieved by increased differentiation and stabilization of Tregs. Our study provides first evidence for daurinol as a treatment for RA.

Let-7f-5p suppresses Th17 differentiation via targeting STAT3 in multiple sclerosis.

T helper 17 (Th17) cells are regarded as key factors in the pathogenesis of multiple sclerosis (MS). Although the involvement of certain microRNAs (miRNAs) in the development of MS has been reported, their roles in Th17 cell differentiation and MS pathogenesis remain elusive. In this study, we identified that let-7f-5p expression is significantly downregulated in CD4+ T cells from MS patients and during the process of Th17 differentiation. The overexpression of let-7f-5p suppressed Th17 differentiation, whereas the knockdown of let-7f-5p expression enhanced this progress. We then explored the molecular mechanism through which let-7f-5p suppressed Th17 differentiation and identified signal transducer and activator of transcription 3 (STAT3), a pivotal transcription factor of Th17 cells, as a direct target of let-7f-5p. In contrast to the downregulated expression of let-7f-5p, STAT3 and p-STAT3 protein levels were dramatically upregulated and inversely correlated with let-7f-5p in peripheral blood CD4+ T cells from MS patients. In conclusion, let-7f-5p functions as a potential inhibitor of Th17 differentiation in the pathogenesis of MS by targeting STAT3 and may serve as a new therapeutic target.

Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation

Asthma is a heterogeneous chronic airway inflammation in which Th2 and Th17 cells are key players in its pathogenesis. We have reported that RhoA of Rho GTPases orchestrated glycolysis for Th2 cell differentiation and allergic airway inflammation by the use of a conditional RhoA-deficient mouse line. However, the role of RhoA in Th17 cells remains to be elucidated. In this study, we investigated the effects of RhoA deficiency on Th17 cells in the context of ex vivo cell culture systems and an in vivo house dust mites (HDM)-induced allergic airway inflammation. We found that RhoA deficiency inhibited Th17 differentiation and effector cytokine secretion, which was associated with the downregulations of Stat3 and Rorγt, key Th17 transcription factors. Furthermore, loss of RhoA markedly suppressed Th17 and neutrophil-involved airway inflammation induced by HDM in mice. The infiltrating inflammatory cells in the lungs and bronchoalveolar lavage (BAL) fluids were dramatically reduced in conditional RhoA-deficient mice. Th17 as well as Th2 effector cytokines were suppressed in the airways at both protein and mRNA levels. Interestingly, Y16, a specific RhoA inhibitor, was able to recapitulate the most phenotypes of RhoA genetic deletion in Th17 differentiation and allergic airway inflammation. Our data demonstrate that RhoA is a key regulator of Th17 cell differentiation and function. RhoA might serve as a potential novel therapeutic target for asthma and other inflammatory disorders.

The IL-33/ST2 pathway shapes the regulatory T cell phenotype to promote intestinal cancer

The composition of immune infiltrates strongly affects the prognosis of patients with colorectal cancer (CRC). Interleukin (IL)-33 and regulatory T cells (Tregs) in the tumor microenvironment have been separately implicated in CRC; however their contribution to intestinal carcinogenesis is still controversial. Here, we reveal that IL-33 signaling promotes CRC by changing the phenotype of Tregs. In mice with CRC, tumor-infiltrating Tregs preferentially upregulate IL-33 receptor (ST2), and IL-33/ST2 signaling positively correlates with tumor number and size. Transcriptomic and flow cytometry analyses demonstrate that ST2 expression induces a more activated and migratory phenotype in FOXP3+ Tregs, which favors their accumulation in the tumor environment. Consequently, genetic ablation of St2 reduces Treg infiltration and concomitantly enhances the frequencies of effector CD8+ T cells, thereby restraining CRC. Mechanistically, IL-33 curtails IL-17 production by FOXP3+ Tregs and inhibits Th17 differentiation. In humans, numbers of activated ST2-expressing Tregs are increased in blood and tumor lesions of CRC patients, suggesting a similar mode of regulation. Together, these data indicate a central role of IL-33/ST2 signaling in shaping an immunosuppressive environment during intestinal tumorigenesis. Blockade of this pathway may provide a strategy to modulate the composition of CRC immune infiltrates.

Thrombospondin-1/CD47 Interaction Regulates Th17 and Treg Differentiation in Psoriasis.

Accumulating evidence on the role of Thrombospondin-1 (TSP-1) in the immune response has emerged during the last years. In spite of the importance of TSP-1 not only as anti-angiogenic factor but also as an immunomodulatory molecule, studies on the role of TSP-1 in psoriasis have been neglected. TSP-1 and CD47 expression were analyzed in skin samples from psoriasis patients and control subjects using RT-PCR and immunofluorescence. Expression of these molecules was also evaluated in peripheral blood CD4+ T cells, moDCs, and circulating primary DCs. The functional role of TSP-1/CD47 signaling axis in psoriasis was assessed in Th17 and Treg differentiation assays. Additionally, small interfering RNA assays specific to TSP-1 were performed in CD4+ T cells and monocyte derived DC to specifically evaluate the function of this protein. Lesional skin of psoriasis patients expressed lower TSP-1 and CD47 mRNA levels compared to non-lesional skin or skin from controls. Immunofluorescence staining revealed decreased expression of CD47 in CD45+ dermal cells from psoriasis samples compared to control subjects. Peripheral CD4+ T cells and circulating primary DCs from psoriasis also expressed lower levels of CD47 compared to controls. Although no significant differences were detected in TSP-1 expression in CD4+ T cells and moDCs between patients and controls, TSP-1 expression in psoriasis patients inversely correlated with disease activity evaluated by the Psoriasis Area and Index Activity. Furthermore, exogenous TSP-1 inhibited Th17 differentiation and stimulated the differentiation of CD4+ T cells toward Treg cells. Furthermore, RNA interference specific for TSP-1 confirmed the role of this molecule as a negative regulator of T cell activation. Because of the impact of TSP-1/CD47 signaling axis in Th17 and Treg differentiation, a dysregulated expression of these molecules in the immune cells from psoriasis patients may favor the exacerbated inflammatory response in this disease.

BCL6 corepressor contributes to Th17 cell formation by inhibiting Th17 fate suppressors.

CD4+ T helper 17 (Th17) cells protect vertebrate hosts from extracellular pathogens at mucosal surfaces. Th17 cells form from naive precursors when signals from the T cell antigen receptor (TCR) and certain cytokine receptors induce the expression of the RORγt transcription factor, which activates a set of Th17-specific genes. Using T cell-specific loss-of-function experiments, we find that two components of the Polycomb repressive complex 1.1 (PRC1.1), BCL6 corepressor (BCOR) and KDM2B, which helps target the complex to unmethylated CpG DNA islands, are required for optimal Th17 cell formation in mice after Streptococcus pyogenes infection. Genome-wide expression and BCOR chromatin immunoprecipitation studies revealed that BCOR directly represses Lef1, Runx2, and Dusp4, whose products inhibit Th17 differentiation. Together, the results suggest that the PRC1.1 components BCOR and KDM2B work together to enhance Th17 cell formation by repressing Th17 fate suppressors.

A directly GP130-targeting small molecule ameliorates collagen-induced arthrites by inhibiting IL-6/GP130 signalling and Th17 differentiation

Abstract Rheumatoid arthritis is a chronic inflammatory disease that is characterized by joint destruction and inflammation by T helper 17 (Th17) cells. Interleukin-6 (IL-6) is secreted in cells such as macrophage or synovial fibroblast and induces the differentiation and function of Th17 cells that increase lymphocytic infiltration in the joint. LMT-28 suppresses IL-6 signaling through direct binding to glycoprotein-130, and alleviates inflammatory disease (such as rheumatoid arthritis, inflammatory bowel disease) in previous study. The purpose of this study is to assess whether LMT-28 would potently inhibit Th17 differentiation, and the mechanism of LMT-28 that attenuates rheumatoid arthritis through IL-6 signaling pathway. LMT-28 reduced the arthritis score and showed protective effects against bone and cartilage destruction in collagen induced arthritis (CIA) mice. In mice with CIA, LMT-28 markedly decreased the serum levels of IL-6, TNF, and IL-1β compared with each vehicle treated group. Moreover, LMT-28 attenuated Th17 cell activation in lymph nodes of CIA mice. We demonstrated that LMT-28 suppressed differentiation of Th17 in mouse splenocytes and human PBMCs. Additionally, LMT-28 inhibited the phosphorylation of GP130, STAT3, and ERK induced by Hyper IL-6 in human fibroblast-like synoviocytes(FLS). Collectively, these results suggested that LMT-28 inhibits differentiated/activated-Th17 cells in rheumatoid arthritis by blocking activation of the STAT3 pathway.

Stimulating Type 1 Angiotensin Receptors on T Lymphocytes Attenuates Renal Fibrosis

Most forms of chronic kidney disease culminate in renal fibrosis that heralds organ failure. In contrast to the protective effects of globally blocking type 1 angiotensin (AT1) receptors throughout the body, activating AT1 receptors directly on immune cells may serve protective functions. However, the effects of stimulating the T-cell AT1 receptor on the progression of renal fibrosis remain unknown. In this study, mice with T-cell-specific deletion of the dominant murine AT1 receptor isoform Lck-Cre Agtraflox/flox [total knockout (TKO)] and wild-type (WT) controls were subjected to the unilateral ureteral obstruction model of kidney fibrosis. Compared with WT controls, obstructed kidneys from TKO mice at day 14 had increased collagen 1 deposition. CD4+ T cells, CD11b+Ly6Chi myeloid cells, and mRNA levels of Th1 inflammatory cytokines are elevated in obstructed TKO kidneys, suggesting that augmented Th1 responses in the TKO mice may exaggerate renal fibrosis by driving proinflammatory macrophage differentiation. In turn, T-bet deficient (T-bet knockout) mice lacking Th1 responses have attenuated collagen deposition after unilateral ureteral obstruction. We conclude that activating the AT1 receptoron T cells mitigates renal fibrogenesis by inhibiting Th1 differentiation and renal accumulation of profibrotic macrophages.

Dendritic cell-targeted CD40 DNA vaccine suppresses Th17 and ameliorates progression of experimental autoimmune glomerulonephritis.

The CD40-CD40L costimulatory pathway is critical for T cell activation in autoimmune disease. We have previously found that blocking the CD40-CD40L pathway using a dendritic cell-targeted CD40 DNA (DEC-CD40) vaccine prevented the development of Heymann nephritis. In this study, we explored the effect of a DEC-CD40 vaccine in the treatment of experimental autoimmune glomerulonephritis (EAG), an animal model of human Goodpasture's disease induced by antigen α3IV-NC1. DEC-CD40 vaccine given at week 3 and week 6 after 3IV-NC1 injection reduced kidney structural and functional injury significantly in EAG. DEC-CD40 vaccination suppressed Th17 cell numbers and Th17 immune responses in kidney and spleen, but did not alter Th1 cells number and responses. Serum derived from rats with DEC-CD40 vaccination suppressed Th17 differentiation, but not Th1 differentiation in vitro. Furthermore, B cell activation, driven by Th17 cytokines, was suppressed by serum from rats vaccinated with DEC-CD40. A DNA vaccine encoding CD40 and targeting dendritic cell, ameliorates kidney injury in both early and late stages in EAG rats, indicating DEC-CD40 vaccination has a therapeutic role in EAG. Its effect is associated with the reduction of Th17 differentiation and Th17-mediated B cell activation.

Potent Antagonists of RORγt, Cardenolides from Calotropis gigantea, Exhibit Discrepant Effects on the Differentiation of T Lymphocyte Subsets.

RORγt is the master transcription factor of IL-17 cytokine expression and Th17 lymphocyte differentiation, which are responsible for the induction of many autoimmune diseases. Recently, RORγt has become an attractive target for drug development to treat these types of diseases, and the field of RORγt antagonist research is now extremely competitive. In our current study, molecular docking was applied to demonstrate that cardenolides, including uscharin, calactin, and calotropin derived from Calotropis gigantea, probably directly bind to RORγt. Therefore, the inhibitory effect was further validated using a luciferase reporter assay. Because RORγt is the key transcriptional factor for Th17 differentiation, the effects of these compounds on Th17 differentiation were studied by flow cytometry. The results showed that uscharin, calactin, and calotropin inhibited Th17 differentiation from 100 to 500 nM. Furthermore, uscharin had a better effect than digoxin, a well-known inverse agonist of RORγt, in reducing Th17 polarization. Additionally, the effects of the cardenolides on the differentiation of other Th lineages, including Th1, Th2, and Treg, were investigated. Uscharin suppressed Th1, Th2, and Treg cell differentiation, while calactin suppressed the differentiation of Th1 cells, and calotropin did not influence the other T cell subsets, indicating that calactin suppressed Th1 and Th17 differentiation, and calotropin selectively quenched Th17 polarization. Structural analysis of the three compounds showed that the selectivity of uscharin, calactin, and calotropin on the suppression of the different subsets of T cells is correlated to the minor differences in their chemical structures. Collectively, calactin and calotropin have greater potential to be developed as lead compounds than uscharin to treat autoimmune diseases mediated by Th17 and/or Th1 cells.