Alantolactone alleviates collagen-induced arthritis and inhibits Th17 cell differentiation through modulation of STAT3 signalling

Context Alantolactone, the bioactive component in Inula helenium L. (Asteraceae), exhibits multiple biological effects.Objective We aimed to determine the anti-inflammatory effect of alantolactone in a collagen-induced arthritis (CIA) mouse model and its immunomodulatory effects on Th17 differentiation.Materials and methods A CIA mouse model was established with DBA/1 mice randomly divided into four groups (n = 6): healthy, vehicle and two alantolactone-treated groups (25 or 50 mg/kg), followed by oral administration of alantolactone to mice for 21 consecutive days after arthritis onset. The severity of CIA was evaluated by an arthritic scoring system and histopathological examination. Levels of cytokines and anti-CII antibodies as well as percentages of splenic Th17 and Th17 differentiation with or without alantolactone treatments (0.62, 1.2 or 2.5 μM) were detected with ELISA and flow cytometry, respectively. Western blot analysis was used to evaluate intracellular signalling in alantolactone-treated spleen cells.Results In CIA mice, alantolactone at 50 mg/kg attenuated RA symptoms, including high arthritis scores, infiltrating inflammatory cells, synovial hyperplasia, bone erosion and levels of the proinflammatory cytokines TNF-α, IL-6 and IL-17A, but not IL-10 in paw tissues. Alantolactone also reduced the number of splenic Th17 cells and the capability of naïve CD4⁺ T cells to differentiate into the Th17 subset by downregulating STAT3/RORγt signalling by as early as 24 h of treatment.Discussion and conclusions Alantolactone possesses an anti-inflammatory effect that suppresses murine CIA by inhibiting Th17 cell differentiation, suggesting alantolactone is an adjunctive therapeutic candidate to treat rheumatoid arthritis.     

Differential expression of GAS5 in rapamycin‐induced reversion of glucocorticoid resistance

This study evaluates the association between the long noncoding RNA GAS5 levels and the anti‐proliferative effect of the glucocorticoid (GC) methylprednisolone (MP) alone and in combination with rapamycin in peripheral blood mononuclear cells (PBMCs) obtained from healthy donors. The effect of MP, rapamycin, and MP plus rapamycin was determined in 17 healthy donors by labelling metabolically active cells with [methyl‐3H] thymidine and the expression levels of GAS5 gene were evaluated by real‐time RT‐PCR TaqMan analysis. We confirmed a role for GAS5 in modulating GC response: poor responders presented higher levels of GAS5 in comparison with good responders. Interestingly, when PBMCs were treated with the combination of rapamycin plus MP, the high levels of GAS5 observed for each drug in the MP poor responders group decreased in comparison with rapamycin (P value = 0.0134) or MP alone (P value = 0.0193). GAS5 is involved in GC resistance and co‐treatment of rapamycin with GCs restores GC effectiveness in poor responders through the downregulation of the long noncoding RNA. GAS5 could be considered a biomarker to personalize therapy and a novel therapeutic target useful for the development of new pharmacological approaches to restore GC sensitivity.     

Mangiferin corrects the imbalance of Th17/Treg cells in mice with TNBS-induced colitis

In the previous study, 80% ethanol extract of the rhizome mixture of Anemarrhena asphodeloides and Coptidis chinensis (AC) and its main constituent mangiferin improved TNBS-induced colitis in mice by inhibiting macrophage activation related to the innate immunity. In the preliminary study, we found that AC could inhibit Th17 cell differentiation in mice with TNBS-induced colitis. Therefore, we investigated whether AC and it main constituent mangiferin are capable of inhibiting inflammation by regulating T cell differentiation related to the adaptive immunity in vitro and in vivo. AC and mangiferin potently suppressed colon shortening and myeloperoxidase activity in mice with TNBS-induced colitis. They also suppressed TNBS-induced Th17 cell differentiation and IL-17 expression, but increased TNBS-suppressed Treg cell differentiation and IL-10 expression. Moreover, AC and mangiferin strongly inhibited the expression of TNF-α and IL-17, as well as the activation of NF-κB. Furthermore, mangiferin potently inhibited the differentiation of splenocytes into Th7 cells and increased the differentiation into Treg cells in vitro. Mangiferin also inhibited RORγt and IL-17 expression and STAT3 activation in splenocytes and induced Foxp3 and IL-10 expression and STAT5 activation. Based on these findings, mangiferin may ameliorate colitis by the restoration of disturbed Th17/Treg cells and inhibition of macrophage activation.     

Therapeutic effects of DZ2002, a reversible SAHH inhibitor,on lupus-prone NZBxNZW F1 mice via interference with TLR-mediated APC response

Aim： To examine the therapeutic effects and underlying mechanisms of DZ2002, a reversible S-adenosyI-L-homocysteine hydrolase （SAHH） inhibitor, on lupus-prone female NZBxNZW F1 （NZB/W F1） mice. Methods： Female NZB/W F1 mice were treated orally with DZ2002 （0.5 mg.kgl-d-1） for 11 weeks, and the proteinuria level and body weight were monitored. After the mice ware euthanized, serum biochemical parameters and renal damage were determined. Splenocytes of NZB/W F1 mice were isolated for ex vivo study. Toll-like receptor （TLR）-stimulated human peripheral blood mononuclear cells （PBMCs） or murine bone marrow-derived dendritic cells （BMDCs） were used for in vitro study. Results： Treatment of the mice with DZ2002 significantly attenuated the progression of glomerulonephritis and improved the overall health. The improvement was accompanied by decreased levels of nephritogenic anti-dsDNA IgG2a and IgG3 antibodies, serum IL-17, IL-23p19 and TGF-13. In ex vivo studies, treatment of the mice with DZ2002 suppressed the development of pathogenic Th17 cells, significantly decreased IL-17, TGF-13, IL-6, and IL-23p19 production and impeded activation of the STAT3 protein and JNK/NF-KB signaling in splenocytes. DZ2002 （500 pmol/L） significantly suppressed TLR agonists-stimulated up-regulation in IL-6, IL-12p40, TNF-a, and IgG and IgM secretion as well as in HLA-DR and CD40 expression of dendritic cells among human PBMCs in vitro. DZ2002 （100 pmol/L） also significantly suppressed TLR agonists-stimulated up-regulation in IL-6 and IL-23p19 production in murine BMDCs, and prevented Thl7 differentiation and suppressed IL-17 secretion by the T cells in a BMDC-T cell co-culture system. Conclusion： DZ2002 effectively ameliorates lupus syndrome in NZB/W F1 mice by regulating TLR signaling-mediated antigen presenting cell （APC） responses.     

Allergen induced Th17 response in the peripheral blood mononuclear cells (PBMCs) of patients with nasal polyposis

BackgroundNasal polyposis (NP) is a chronic inflammatory disease of the nasal cavity and sinuses. Th17 cells have been considered to play roles in allergic airway diseases and various chronic inflammatory disorders.Aim of the studyThis study aimed to investigate the population and function of peripheral Th17 cells in response to house dust mite extracts (HDM) allergen in NP patients, and evaluate the possible correlation between Th17 cells and atopy, to explore the role of atopy in the pathogenesis of NP.MethodsPeripheral blood mononuclear cells (PBMCs) obtained from atopic NP patients, non-atopic NP patients, and controls were stimulated by phytohemagglutinin (PHA) or HDM plus PHA. The resulting frequency of Th17 cells was detected by flow cytometry and the expression of RORc was measured by real-time PCR. Then the concentrations of IL-17A, INF-γ, IL-4 and IL-5 in the supernatants were assayed by specific ELISAs.ResultsThe population and function of Th17 cells in allergen stimulated PBMCs were significantly higher in atopic NP patients. In addition, in atopic group, HDM + PHA stimulation induced significant increase of Th17 population and IL-17A production versus those in PHA stimulated ones. However, the frequency of Th17 cells was not correlated with Th1, Th2 cytokine productions.ConclusionTh17 immunity is involved in the systemic immune responses to allergen in atopic NP and atopy may aggravate NP by stimulating the increase of Th17 population and IL-17A production. The mechanism of Th17 cells response to allergen may be regulated differently from the regulation of Th1 and Th2 immunity in NP.     

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.     

MicroRNA-10a-3p mediates Th17/Treg cell balance and improves renal injury by inhibiting REG3A in lupus nephritis

BackgroundThe therapeutic approaches guided toward microRNAs (miRNAs) have been extensively explored in lupus nephritis (LN), but the precise position of miR-10a-3p posted in disease is not translated thoroughly. Therein, this work pivoting on miR-10a-3p was launched with the involvement of regenerating islet-derived 3 α (REG3A).MethodsPeripheral blood samples from LN patients and healthy controls (n = 132) were collected. miR-10a-3p and REG3A expression in peripheral blood mononuclear cells were tested. Mice were injected with miR-10a-3p agomir, miR-10a-3p antagomir and/or REG3A low expression vector for presentation of their roles in renal function, T helper cell 17 (Th17)/regulatory cell (Treg) balance, renal pathological damage, JAK2/STAT3 pathway activation and renal injury in LN. The relation between miR-10a-3p and REG3A was tested.ResultsMiR-10a-3p was down-regulated while REG3A was up-regulated in LN. Restoring miR-10a-3p or silencing REG3A decreased Th17/Treg ratio in CD4⁺ T cells, inhibited JAK2/STAT3 pathway activation, ameliorated renal function, improved renal pathological damage and alleviated renal injury in LN. REG3A depletion negated the effects of down-regulated miR-10a-3p on LN. MiR-10a-3p targeted REG3A.ConclusionThe work elucidates that miR-10a-3p restoration decreases Th17/Treg ratio and attenuates renal injury in LN via inhibiting REG3A and the activation of JAK2/STAT3 pathway, which renews the therapeutic reference for LN management.     

2′-fucosyllactose inhibits imiquimod-induced psoriasis in mice by regulating Th17 cell response via the STAT3 signaling pathway

Psoriasis is a chronic immune-mediated inflammatory cutaneous disorder with Th17 cells and Th17-related cytokines playing an important role in its development. 2′-FL (2′-fucosyllactose), which makes up about 30% of all HMOs (human milk oligosaccharides) in blood type secretor positive maternal milk, plays an essential role in supporting aspects of immune development and regulation. To explore the immunomodulatory effect of 2′-FL in psoriasis, we employed the imiquimod (IMQ)-induced psoriasis-like mouse model. Our data showed that mice administered with 2′-FL exhibited attenuated skin damage and inflammation, characterized by significantly decreased erythema and thickness and reduced recruitment of pro-inflammatory cytokines, when compared to control mice. The alleviated skin inflammation in 2′-FL treated mice was associated with a reduced proportion of Th17 cells and decreased production of Th17-related cytokines. Furthermore, we have demonstrated that 2′-FL reduced the phosphorylation of STAT3 in the skin tissue from mice with IMQ stimulation, which could account for the decreasing recruitment of Th17 cells. In vitro studies showed that 2′-FL inhibited differentiation of Th17 cells, phosphorylation of STAT3, and RORγt mRNA levels in T cells under Th17 polarization. Our results indicate that 2′-FL ameliorates IMQ-induced psoriasis by inhibiting Th17 cell immune response and Th17-related cytokine secretion via modulation of the STAT3 signaling pathway.     

Thymol as a reciprocal regulator of T cell differentiation: Promotion of regulatory T cells and suppression of Th1/Th17 cells

Regulatory T cells (Tregs) are critical for maintaining immune response and enhancing their differentiation has therapeutic implications for autoimmune diseases. In this study, we investigated the effects of thymol a well-known monoterpene from Thyme on differentiation and function of Tregs. In vitro generation of Tregs from purified naïve CD4⁺CD25⁻ T cells in the presence of thymol was carried out. Suppressor activity of generated Tregs was examined by changes in the proliferation of CFSE-labeled conventional T cells. Thymol promotes differentiation of naïve CD4⁺CD25⁻ T cells to CD4⁺CD25⁺Foxp3⁺ Tregs [66.9–71.8% vs. control (47%)] and increased intensity of Foxp3 expression on Tregs (p < 0.01). In functional assay, an increased immune suppression by thymol-induced Tregs (≈2.5 times of untreated Tregs) was detected. For in vivo study, thymol was intraperitoneally administered to ovalbumin (Ova)-immunized mice. Flow cytometry assessment of spleens from thymol-treated Ova-immunized mice showed increased number of CD4⁺ Foxp3⁺ Tregs (>8%, p < 0.01(and decreased levels of CD4⁺T-bet⁺ Th1 and CD4⁺RORγt⁺ Th17 cells resulted in significant decreased Th1/Treg and Th17/Treg ratios. In ex vivo Ova challenge of splenocytes from thymol-treated Ova-immunized mice, similarly higher levels of CD4⁺ Foxp3⁺ Tregs, and also elevated TGF-β expression in CD4⁺Foxp3⁺ population (48.1% vs. 18.9% in untreated Ova-immunized group) and reduced IFN-γ-producing CD4⁺T-bet⁺ T cells and IL-17-producing CD4⁺RORγt⁺ T cells were detected. This led to marked decreased ratios of IFNγ/TGF-β and IL-17/TGF-β expressions. In conclusion, this study revealed thymol as a compound with enhancing effects on Treg differentiation and function, which may have potential benefits in treatment of immune-mediated diseases with Th1/Th17 over-activation.     

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.     

Ursolic acid ameliorates autoimmune arthritis via suppression of Th17 and B cell differentiation

Aim:

Ursolic acid (UA) is a pentacyclic triterpenoid found in most plant species, which has been shown anti-inflammatory and anti-oxidative activities. In this study, we examined the effects of UA on collagen-induced arthritis (CIA) in mice, and to identify the mechanisms underlying the effects.

Methods:

CIA was induced in mice. Two weeks later, the mice were treated with UA (150 mg/kg, ip, 3 times per week) for 4 weeks. The expression of cytokines and oxidative stress markers in joint tissues was measured with immunohistochemistry. The numbers of CD4⁺IL-17⁺, CD4⁺CD25⁺Foxp3⁺ and pSTAT3 cells in spleens were determined using confocal immunostaining or flowcytometric analyses. Serum antibody levels and B cell-associated marker mRNAs were analyzed with ELISAs and qRT-PCR, respectively. CD4⁺ T cells and CD19⁺ B cells were purified from mice spleens for in vitro studies.

Results:

UA treatment significantly reduced the incidence and severity of CIA-induced arthritis, accompanied by decreased expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-21 and IL-17) and oxidative stress markers (nitrotyrosine and iNOS) in arthritic joints. In CIA mice, UA treatment significantly decreased the number of Th17 cells, while increased the number of Treg cells in the spleens, which was consistent with decreased expression of pSTAT3, along with IL-17 and RORγt in the splenocytes. In addition, UA treatment significantly reduced the serum CII-specific IgG levels in CIA mice. The inhibitory effects of UA on Th17 cells were confirmed in an in vitro model of Th17 differentiation. Furthermore, UA dose-dependently suppressed the expression of B cell-associated markers Bcl-6, Blimp1 and AID mRNAs in purified CD19⁺ B cells pretreated with IL-21 or LPS in vitro.

Conclusion:

UA treatment significantly ameliorates CIA in mice via suppression of Th17 and differentiation. By targeting pathogenic Th17 cells and autoantibody production, UA may be useful for the treatment of autoimmune arthritis and other Th17-related diseases.     

Rapamycin inhibits differentiation of Th17 cells and promotes generation of FoxP3+ T regulatory cells

Reciprocal differentiation of immunosuppressive CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) and proinflammatory IL-17-producing cells (Th17) from naïve CD4 cells is contingent upon the cytokine environment. Using MACS-purified CD4 cells, we found that rapamycin and cyclosporine A (CsA) potently inhibited the TGFβ and IL-6-induced generation of IL-17-producing cells. Intriguingly, rapamycin promoted, while CsA markedly inhibited, TGFβ-mediated generation of Tregs. The aforementioned effects of rapamycin and CsA were also observed for Flow-sorted CD4⁺CD25⁻ T cells, indicating that the effect of these two immunosuppressive agents was based on their action on de novo generation of Tregs and Th17 cells from naïve CD4 cells. Our observation suggests a distinct mode of immunosuppressive action and tolerance induction by rapamycin and CsA. The capacity of rapamycin to generate immunosuppressive Tregs and to suppress differentiation of pathogenic Th17 cells furthers our understanding of the basis for the therapeutic immunosuppressive effects of rapamycin in patients with autoimmune diseases and allo-transplantation reactions.