Butyrate inhibit collagen-induced arthritis via Treg/IL-10/Th17 axis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder demanding the development of novel therapeutic strategy. Butyrate is a functional short-chain fatty acid produced by the anaerobic intestinal microbiota. This study aimed to investigate the attenuation of butyrate on RA. The collagen-induced arthritis (CIA) mouse model was established and butyrate was administered in drinking water along with the collagen immunization. The histopathological features, clinical score, paw swelling, as well as the production of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6 and IL-17A were measured to determine the amelioration of butyrate on arthritis. The differentiation of Treg cells and Th17 cells in the splenic cells was assessed by flow cytometry. The expression of Foxp3, IL-10, Rorγt and IL-17A were detected by RT-PCR and FACS immunostaining. Anti-IL10R antibody was used in the CIA and CD4⁺ cell cultures to mediate the effects of butyrate. Butyrate significantly inhibited expressions of IL-1β, IL-6 and IL-17A, but promoted the expression of IL-10. Butyrate also increased systematical Treg cells and reduced Th17 cells. Mechanism study revealed that butyrate directly enhanced the polarization of Treg cells but not Th17 cells. All effects of butyrate on RA were inversed by the co-administered anti-IL10R antibody. This study showed that butyrate administration inhibited arthritis in CIA mice model, suppressed the expression of inflammatory cytokines. The modulation may be mediated the differentiation of CD4 T cells towards Treg cells, which produce anti-inflammatory cytokine IL-10, and thus influenced the function of Th17 cells.     

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.     

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.     

Rosmarinic acid down-regulates endothelial protein C receptor shedding in vitro and in vivo

The endothelial protein C receptor (EPCR) plays pivotal roles in coagulation and inflammation, however, its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). According to previous studies, there are approximately 100 ng/ml sEPCR in human plasma and the levels increase in inflammatory diseases. EPCR can be shed from the cell surface, and this is mediated by tumor necrosis factor-α converting enzyme (TACE). We recently reported on the anti-inflammatory and barrier protective activities of rosmarinic acid (RA), an important component of the leaves of Perilla frutescens. However, little is known about the effects of RA on EPCR shedding. Here, we investigated this issue by monitoring the effects of RA on phorbol-12-myristate 13-acetate (PMA), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, and on cecal ligation and puncture (CLP)-mediated EPCR shedding and underlying mechanisms. Data showed that treatment with RA resulted in potent inhibition of PMA, TNF-α, IL-induced EPCR shedding by suppression of TACE expression. In addition, RA reduced PMA-stimulated phosphorylation of p38, extracellular regulated kinases (ERK) 1/2, and c-Jun N-terminal kinase (JNK). These results suggest the potential for use of RA as an anti-sEPCR shedding reagent against PMA, TNF-α, IL-1β and CLP-mediated EPCR shedding.     

CXCR3 antagonist AMG487 inhibits glucocorticoid-induced tumor necrosis factor-receptor-related protein and inflammatory mediators in CD45 expressing cells in collagen-induced arthritis mouse model

Rheumatoid arthritis (RA) is an autoimmune disease classified by uncontrolled joint inflammation leading to the destruction of both cartilage and joints. Despite progress made in RA treatment in the past decade, new drugs with high efficacy and fewer long-term adverse effects are still needed; thus, safe anti-inflammatory therapies for RA are urgently needed. Previous results demonstrated that the CXCR3 antagonist is an extremely attractive therapeutic target for the treatment of several autoimmune diseases, suggesting that it might have an inhibitory effect on RA. In this study, we investigated the effect of AMG487, a selective CXCR3 antagonist, on collagen-induced arthritis (CIA) in mice and evaluated its potential therapeutic mechanism. Following induction of CIA, mice were treated with AMG487 (5 mg/kg, intraperitoneally), to investigate their protective effects against CIA. CD4, CD25, CCR6, IL-9, NF-κB, IL-6, IL-17A, IL-21, STAT6 and Foxp3 expressing GITR⁺ and CD45⁺ cells were measured in the spleen using flow cytometry to assess anti-inflammatory effects of AMG487. The mRNA and protein expression of GITR, CCR6, IL-9, and IL-21 were measured using quantitative real-time PCR and western blot analysis in knee tissue. AMG487 significantly alleviated joint inflammation by decreasing GITR⁺CD25⁺, GITR⁺CD45⁺, GITR⁺IL-9⁺, GITR⁺NF-κB⁺ CD45⁺CD4⁺, CD45⁺CCR6⁺, CD45⁺IL-6⁺ cells, CD45⁺IL-17A⁺, and CD45⁺IL-21⁺, and increasing GITR⁺Foxp3⁺ and GITR⁺STAT6⁺ cells. There was a significant decrease in mRNA and protein expression of GITR, CD4, CCR6, IL-6, IL-9, and IL-21 in knee tissue of CIA mice. This study demonstrates that AMG487 has a potential therapeutic effect on RA and could explore novel anti-inflammatory therapies for its treatment.     

Partial depletion of natural gut flora by antibiotic aggravates collagen induced arthritis (CIA) in mice

BackgroundRheumatoid arthritis (RA) is a chronic inflammatory disease that affects about 1% of the adult population and occurs twice as frequently among women than men. At present it is accepted that pathogenesis of RA is based on inflammatory response mediated by CD4⁺ Th1 and Th17 lymphocytes. The most commonly applied model imitating RA is the collagen induced arthritis (CIA).A growing evidence shows that there is a correlation between microbial dysbiosis and human pathology which includes autoimmunity, allergic diseases, obesity, inflammatory bowel disease (IBD), metabolic syndrome.MethodsCollagen induced arthritis was used to study influence of natural gut flora on course of rheumatoid arthritis.ResultsCurrent work employing CIA model showed that partial depletion of natural gut flora with orally administered antibiotic Baytril (enrofloxacin) aggravates disease severity when compared to control mice. Observed partial depletion of both aerobic and anaerobic bacteria did not affect animal body weight. Additionally, in vitro study showed increased production of IFN-γ and IL-17A and decreased release of IL-4 by axillary lymph node cells (ALNC) isolated from mice treated with antibiotic and induced CIA when compared to positive control. Furthermore, treatment with antibiotic prior to CIA induction results in augmented production of IFN-γ, IL-17A and IL-6 by mesenteric lymph node cells (MLNC).ConclusionPresented data suggest that alteration of gut microbiota via use of enrofloxacin may play a role in modulating arthritis symptom severity in this mouse model.     

Ethyl pyruvate ameliorates inflammatory arthritis in mice

ObjectivesEthyl pyruvate (EP) is the ethyl ester of pyruvate and has antioxidative and anti-inflammatory effects. This study aimed to evaluate the therapeutic effect of EP in inflammatory arthritis and to identify the underlying mechanisms.MethodsMice with collagen-induced arthritis (CIA) were treated with the vehicle control or EP at 20 mg/kg, and clinical and histological analyses were performed on the animals. The differentiation of murine CD4 + T cells into T helper 17 (Th17) cells in the presence of EP was investigated in vitro. The effects of EP on osteoclastogenesis were determined by staining for tartrate-resistant acid phosphatase, and measuring the mRNA levels of osteoclastogenesis-related genes. The expression of high-mobility group box 1 (HMGB1) was evaluated after EP therapy using immunohistochemical staining and Western blotting.ResultsEP significantly improved the clinical and histological features of arthritis in CIA mice. EP suppressed the differentiation of CD4 + T cells into Th17 cells, and inhibited the expression of RORγt. The generation of osteoclasts and osteoclastogenic markers from murine and human monocytes was significantly reduced in the presence of EP. The expression of HMGB1 in the synovium was significantly lower in CIA mice treated with EP, compared to control CIA mice. During osteoclastogenesis, HMGB1 release from monocytes was inhibited in the presence of EP.ConclusionsEP attenuated synovial inflammation and bone destruction in the experimental arthritis model through suppression of IL-17 and HMGB-1. The data suggests that EP could be a novel therapeutic agent for the treatment of inflammatory arthritis, such as rheumatoid arthritis.     

Naltrexone (NTX) relieves inflammation in the collagen-induced- arthritis (CIA) rat models through regulating TLR4/NFκB signaling pathway

ObjectiveOur aim was to study the efficacy and mechanism by which NTX alleviate arthritis in CIA rat models in vivo.MethodsFemale Wistar rats were randomly divided into 6 groups, their weights were observed and the severity of arthritis and pathological changes were evaluated by HE staining. T lymphocyte subsets were detected by flow cytometry. The expression of cytokines was detected in peripheral serum by ELISA. Real time PCR, immunohistochemical staining and western blot analysis were utilized to detect the mRNA and protein expression of opioid receptors, TLR4, RANKL and /NF-κB in synovial tissue and the spleen.ResultsThe weight of the rats in the 10 mg/kg NTX group decreased the least, and had the least severe arthritis. CD4⁺ T cells, Th1 cells and Treg cells increased, and CD8⁺T cells, Th1 cells and Th17 cells decreased in the splenic lymphocytes. The expression of proinflammatory cytokines decreased, and the expression of anti-inflammatory cytokines increased. MOR and DOR were strongly expressed in the spleen, whereas KOR and DOR were strongly expressed in synovial tissue. The expression of TLR4, NF-κB and RANKL was reduced in the spleen and synovium in the NTX group.ConclusionsNTX relieved the severity of arthritis in the CIA rat models at a concentration of 10 mg/kg by regulating T lymphocyte subsets and the expression of cytokines. NTX affected opioid receptors to inhibit the TLR4/NF-κB signaling pathway, regulating the systemic immune response and decreasing osteoclast differentiation, thereby alleviating inflammation and the erosion of articular cartilage along with bone tissue.     

Analysis of the role of palmitoleic acid in acute anterior uveitis

PurposeTo study the role of palmitoleic acid (PA) in the pathogenesis of acute anterior uveitis (AAU).MethodsPA levels in feces from AAU patients were measured by gas chromatography coupled with a mass spectrometer (GC-MS) and compared with samples obtained from healthy individuals. Enzyme linked immunosorbent assay (ELISA) and flow cytometry (FCM) were used to assess the effect of PA on dendritic cells (DCs) and CD4⁺T cells obtained from mice, AAU patients and healthy individuals. C57BL/6 mice were fed with PA or vehicle and experimental autoimmune uveitis (EAU) was induced with a human retinal IRBP651-670 peptide. Disease severity of EAU was evaluated by clinical manifestation and histology. Differentiation of splenic Type 1 helper T cells (Th1) and Th17 cells was evaluated by FCM. Tandem mass tag (TMT)-based proteomics analysis was used to identify differentially expressed proteins following incubation of DCs with PA.ResultsThe fecal concentration of PA was increased in AAU patients as compared with healthy individuals. In vitro, PA promoted apoptosis of DCs and inhibited the secretion of TNF-α from mouse bone-marrow-derived dendritic cells (BMDCs) as well as in DCs from AAU patients and healthy individuals. It only decreased DCs surface marker expression and IL-12p70 secretion in BMDCs and healthy individuals DCs but not in AAU patient DCs. PA-treated BMDCs inhibited Th cell differentiation from mouse naïve CD4⁺T cells and IL-17 and IFN-γ secretion in co-culture supernatants. PA also inhibited the differentiation of Th cells and secretion of IFN-γ and IL-17 in CD4⁺T cells from mice, AAU patients and healthy individuals. In vivo, PA-treated EAU mice showed milder clinical and histopathological intraocular manifestations as compared with the control group. PA feeding inhibited differentiation of splenic Th17 cells, whereas Th1 cells were not affected. Up to 30 upregulated and 77 downregulated proteins were identified when comparing PA-treated DCs with controls.ConclusionAn increased expression of fecal PA was observed in AAU patients. PA was shown to have immunoregulatory effects on DCs and CD4⁺T cells and attenuated disease severity in EAU mice.     

c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 is a critical regulator for arthritis progression by meditating inflammation in mice model

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease. However, the pathogenesis of RA is not fully understood. Here, we reported that c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3 (JIP3)) was significantly important for collagen-induced arthritis (CIA) in mice. Mice with JIP3 knockout (JIP3^−/−) showed a significant decrease in arthritis index and swollen joint count in CIA mice. The histopathology of spleen and joint was markedly alleviated by JIP3 deficiency in CIA mice. Excessive macrophage activation in CIA mice was also inhibited by JIP3 deletion. CIA-induced RANKL/RANK/OPG system mRNA expression was blocked in JIP3-knockout mice. In addition, CIA-triggered cytokine secretion and TLRs/NF-κB activation was inactivated by JIP3-deficiency. In line with the inhibition of inflammation by JIP3-knockout, it also significantly suppressed JNK pathway activation induced by CIA, as evidenced by the down-regulation of p-JNK, p-c-Jun, AFT-2 and Elk-1 in joints. In vitro, RANKL-exposed RAW264.7 cells showed a significant reduction of osteoclast formation using TRAP staining. Moreover, JIP3 inhibition reduced the RANKL-caused expression of osteoclastic genes and inflammatory regulators, as well as activation of TLRs/NF-κB and JNK signaling pathways. Importantly, we found that promoting JNK activity could abrogate JIP3 knockdown-suppressed osteoclastic genes expression, inflammatory response and NF-κB activation. These findings suggested that JIP3 could significantly impede osteoclast formation and function by regulating JNK activation, illustrating a novel therapeutic strategy for managing arthritis and preventing bone destruction.     

Cholinergic dysfunction-induced insufficient activation of alpha7 nicotinic acetylcholine receptor drives the development of rheumatoid arthritis through promoting protein citrullination via the SP3/PAD4 pathway

Both cholinergic dysfunction and protein citrullination are the hallmarks of rheumatoid arthritis(RA), but the relationship between the two phenomena remains unclear. We explored whether and how cholinergic dysfunction accelerates protein citrullination and consequently drives the development of RA. Cholinergic function and protein citrullination levels in patients with RA and collageninduced arthritis(CIA) mice were collected. In both neuron-macrophage coculture system and CIA mice,the effect o...     

Bone-derived MSCs encapsulated in alginate hydrogel prevent collagen-induced arthritis in mice through the activation of adenosine A2A/2B receptors in tolerogenic dendritic cells

Tolerogenic dendritic cells (tolDCs) facilitate the suppression of autoimmune responses by differentiating regulatory T cells (Treg). The dysfunction of immunotolerance results in the development of autoimmune diseases, such as rheumatoid arthritis (RA). As multipotent progenitor cells, mesenchymal stem cells (MSCs), can regulate dendritic cells (DCs) to restore their immunosuppressive function and prevent disease development. However, the underlying mechanisms of MSCs in regulating DCs still need to be better defined. Simultaneously, the delivery system for MSCs also influences their function. Herein, MSCs are encapsulated in alginate hydrogel to improve cell survival and retention in situ, maximizing efficacy in vivo. The three-dimensional co-culture of encapsulated MSCs with DCs demonstrates that MSCs can inhibit the maturation of DCs and the secretion of pro-inflammatory cytokines. In the collagen-induced arthritis (CIA) mice model, alginate hydrogel encapsulated MSCs induce a significantly higher expression of CD39⁺CD73⁺ on MSCs. These enzymes hydrolyze ATP to adenosine and activate A_2A/2B receptors on immature DCs, further promoting the phenotypic transformation of DCs to tolDCs and regulating naïve T cells to Tregs. Therefore, encapsulated MSCs obviously alleviate the inflammatory response and prevent CIA progression. This finding clarifies the mechanism of MSCs-DCs crosstalk in eliciting the immunosuppression effect and provides insights into hydrogel-promoted stem cell therapy for autoimmune diseases.     

The therapeutic effects of daphnetin in collagen-induced arthritis involve its regulation of Th17 cells

ObjectivesDaphne odora var. marginata (D. marginata), an aiophyllus arbuscular plant, is one of the traditional Chinese medicines used to treat rheumatoid arthritis. This study investigated the therapeutic effects and mechanisms of daphnetin, an active monomer ingredient derived from D. marginata, on collagen-induced arthritis (CIA) in rats.MethodsThe effects of daphnetin on joint diseases were assessed by hematoxylin and eosin staining and radiographic and transmission electron microscopy. The protein and mRNA expression levels of T helper (Th)1/Th2/Th17-type cytokines in the spleen were determined by flow cytometry and quantitative real-time PCR.ResultsOur results showed that daphnetin significantly reduced paw swelling and was nontoxic in vivo at the tested doses. Synovial hyperplasia, joint destruction and chondrocyte degeneration in CIA rats were suppressed by daphnetin. Daphnetin treatment also reduced the levels of Th1/Th2/Th17 type cytokines in spleen lymphocytes in CIA rats. Moreover, the expression of Foxp3, which can down-regulate the activity of Th17 cells, was significantly increased in the daphnetin-treated groups.ConclusionsThese results suggest that daphnetin may have therapeutic effects in down-regulating Th17-type responses in CIA rats. The beneficial effects of daphnetin on CIA may be related to its inhibition of Th17 cell priming and activation.     

Crocin exerts anti-inflammatory and anti-arthritic effects on type II collagen-induced arthritis in rats

Context: Rheumatoid arthritis (RA) is a common systemic auto-immune disease, which is characterized by chronic and symmetry synovial inflammation. Crocin has been reported to exhibit anti-inflammatory effects in animal models.

Objective: This study investigates the anti-inflammatory and anti-arthritic effects of crocin on type II collagen-induced arthritis (CIA) in Wistar rats.

Materials and methods: The CIA rat model was established and randomly divided into five groups with or without crocin treatment (10, 20 or 40?mg/kg), which was started on day 21 after arthritis induction and persisted for 36 days. The symptoms and molecular mechanisms of CIA and crocin-treated CIA rats were compared and investigated.

Results: CIA rats presented severe RA symptoms, including high arthritis score, paw swelling, joint inflammation, bone erosion, chondrocyte death, cartilage destruction, enhanced expressions of matrix metalloproteinase (MMP) and pro-inflammatory cytokines. However, crocin could mitigate these symptoms. Crocin (40?mg/kg) exhibited the most efficient therapeutic function on CIA rats: the histological scores of joint inflammation, bone erosion, chondrocyte death, cartilage surface erosion, and bone erosion of CIA rats receiving 40?mg/kg crocin treatment were comparable to the normal rats. MMP-1, -3 and -13 protein expression levels of CIA rats with 40?mg/kg crocin treatment were decreased to levels similar to normal rats. Moreover, crocin could also inhibit the expression of TNF-α, IL-17, IL-6 and CXCL8 in serum and ankle tissues of CIA rats.

Conclusions: In summary, crocin exhibits therapeutic potential for RA, by mitigating the symptoms and inhibiting the pro-inflammatory factor expression.     

APC-EPCR-PAR1通路在类风湿关节炎中的研究进展

类风湿关节炎（RA）是一种慢性、系统性自身免疫性疾病。活化蛋白C（APC）是由其前体蛋白C（PC）转化而来的一种天然抗凝血剂,在凝血调控中起着关键的作用。APC可与内皮蛋白C受体（EPCR）结合,激活内皮细胞上的蛋白激酶受体1（PAR1）,从而启动细胞的保护和抗炎反应。现就APC-EPCR-PAR1活化通路上各靶点在RA中的研究进展进行综述,探讨APC-EPCR-PAR1通路在RA疾病中的潜在治疗价值,为RA的治疗提供更多的选择。     

Chronotherapy targeting cytokine secretion attenuates collagen-induced arthritis in mice

ObjectiveDiurnal variation of symptoms are observed in rheumatoid arthritis, especially in productions of cytokines that show peak concentrations during mid night. In contrast, cytokines of collagen-induced arthritis (CIA) mice increase in daytimes under Mid-light condition. By using chronotherapy, differences in drug efficacies according to administration time of Baricitinib, a wide ranged cytokine blocker, were examined in CIA mice.MethodsCIA mice were administered a dose of 3 mg/kg of Baricitinib once a day at zeitgeber time (ZT) 0 or ZT12 for 21 days. Arthritis scores, histopathology and factors related to joint destruction in sera were examined. Phosphorylation of STAT3 in liver, expressions of cytokines in spleen, and Interleukin (IL)-6 and tumor necrosis factor (TNF)-α in sera were measured.ResultsIn CIA mice, diurnal variations were observed both in expressions of cytokines and phosphorylation of STAT3. Arthritis scores of ZT0/12 group decreased from day3 as compared to untreated mice, and those of ZT0 group significantly decreased as compared to ZT12 group from day12. Pathological findings, immunohistochemistry of cytokines and Receptor activator of nuclear factor kappa-Β ligand (RANKL)/osteoprotegerin ratio in sera well reflected results of arthritis scores. Diurnal variation of STAT3 phosphorylation was suppressed in ZT0 group. At ZT2, expressions of IL-6/Interferon-γ/TNF/granulocyte–macrophage colony-stimulating factor in ZT0 group were significantly decreased as compared to untreated mice, though not in ZT12 group. In ZT0 group, IL-6 and TNF-α in sera were decreased for longer time than that in ZT12 group.ConclusionChronotherapy using Baricitinib targeting cytokine secretions is effective in CIA mice. Clinical applications of chronotherapy can be expected to enhance the drug efficacy.     

Costunolide,a sesquiterpene lactone,inhibits the differentiation of pro-inflammatory CD4+ T cells through the modulation of mitogen-activated protein kinases

CD4⁺ T cell activation and adequate differentiation into effector T helper (Th) cells are crucial for mediating adaptive immune responses to cope with foreign pathogens. Despite the significant role of Th cells, excessive increases in their numbers result in inflammatory and autoimmune diseases. In this study, we investigated the effects of costunolide, a plant-derived natural compound with an anti-inflammatory activity, in regulating Th cells and the underlying mechanisms. Costunolide significantly decreased cell populations of differentiated Th1, Th2, and Th17 subsets under Th subset-polarizing conditions, while exerting statistically negligible effects on Treg cell differentiation. Furthermore, costunolide inhibited the expression level of Th subset-polarizing master genes such as T-bet, GATA3, and RORγt, indicating that costunolide inhibits the differentiation of CD4⁺ T cells into Th subsets. Additionally, costunolide suppressed the proliferative activity of CD4⁺ T cells and the expression of CD69 activation marker on CD4⁺ T cells. When the molecular targets of costunolide were investigated, phosphorylation of ERK and p38 was found to be decreased under Th subset-polarizing conditions, whereas activity of JNK remained unchanged. U0126, an ERK inhibitor, and SB203580, a p38 inhibitor, decreased the expression of CD69 upon TCR stimulation and inhibited CD4⁺ T cell differentiation, indicating that both ERK and p38 are suggested to be critical molecular targets of costunolide. Taken together, these results suggest that costunolide inhibits the differentiation of CD4⁺ T cells by suppressing ERK and p38 activities and can be an effective therapeutic agent for T cell-mediated immune diseases.     

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.     

CP-25 alleviates rat collagen induced arthritis by regulating T cell subsets

Rheumatoid arthritis(RA) is a chronic,systemic and autoimmune disease. Abnormally activated T cells play a key role in the pathological mechanism of RA, and the differentiated T cell subsets also have imbalance in RA. Paeoniflorin-6-oxygen-benzenesulfonate(code CP-25) is a new active monomer derived from paeoniflorin. OBJECTIVE To investigate the therapeutic effect of CP-25 on collagen induced-arthritis(CIA) rats and regulation on T cell subsets. METHODS Rats with CIA were establisghed and administered with CP-25(50 mg·kg~(-1)) in tragastric administration, and Iguratimod(25 mg·kg~(-1)) was given as positive control drug. The therapeutic effects of CP-25 on the CIA of rats and its regulatory effects on the T cell subsets were evaluated by measuring the foot paw volume, the number of swollen joints,the histopathology of the joints and spleen and the T cell subgroup. The proliferation of T and B cells, cytokines and T lymphocyte subsets were analyzed by CCK-8, ELISA and flow cytometry. RESULTS CP-25 has therapeutic effect on CIA in rats. CP-25(50 mg·kg~(-1)) can significantly reduce the paw volume and joint swelling of CIA rats,reduce the white myelosis of spleen and synovial tissue inflammatory cell infiltration, inhibit the proliferation of T cells. The ratio of CD3~+T cells, CD3~+CD4~+T cells, CD3~+CD4~+CD25~+T cell subsets, Th1, Th2 and Th17 cells increased significantly in CIA rats. CP-25 reduce CD3~+T cels, CD3~+CD4~+T cels, CD3~+CD4~+CD25~+T cel subgroups,downregulate the ratio of Th1, Th2 and Th17 cells, and the above indicators were not statistically significant with the positive control group. CONCLUSION CP-25 has antiinflammatory effects on rats with CIA, which may be related to regulating activated T cell subsets and the differentiation of T cell subsets.