IRAK4 inhibition: a promising strategy for treating RA joint inflammation and bone erosion

Flares of joint inflammation and resistance to currently available biologic therapeutics in rheumatoid arthritis (RA) patients could reflect activation of innate immune mechanisms. Herein, we show that a TLR7 GU-rich endogenous ligand, miR-Let7b, potentiates synovitis by amplifying RA monocyte and fibroblast (FLS) trafficking. miR-Let7b ligation to TLR7 in macrophages (MΦs) and FLSs expanded the synovial inflammatory response. Moreover, secretion of M1 monokines triggered by miR-Let7b enhanced Th1/Th17 cell differentiation. We showed that IRAK4 inhibitor (i) therapy attenuated RA disease activity by blocking TLR7-induced M1 MΦ or FLS activation, as well as monokine-modulated Th1/Th17 cell polarization. IRAK4i therapy also disrupted RA osteoclastogenesis, which was amplified by miR-Let7b ligation to joint myeloid TLR7. Hence, the effectiveness of IRAK4i was compared with that of a TNF inhibitor (i) or anti-IL-6R treatment in collagen-induced arthritis (CIA) and miR-Let7b-mediated arthritis. We found that TNF or IL-6R blocking therapies mitigated CIA by reducing the infiltration of joint F480⁺iNOS⁺ MΦs, the expression of certain monokines, and Th1 cell differentiation. Unexpectedly, these biologic therapies were unable to alleviate miR-Let7b-induced arthritis. The superior efficacy of IRAK4i over anti-TNF or anti-IL-6R therapy in miR-Let7b-induced arthritis or CIA was due to the ability of IRAK4i therapy to restrain the migration of joint F480⁺iNOS⁺ MΦs, vimentin⁺ fibroblasts, and CD3⁺ T cells, in addition to negating the expression of a wide range of monokines, including IL-12, MIP2, and IRF5 and Th1/Th17 lymphokines. In conclusion, IRAK4i therapy may provide a promising strategy for RA therapy by disconnecting critical links between inflammatory joint cells.     

T-Cell Immune Imbalance in Rheumatoid Arthritis Is Associated with Alterations in NK Cells and NK-Like T Cells Expressing CD38

BackgroundCD38+ NK (CD3− CD16+ CD38+ CD56+) cells were increased in rheumatoid arthritis (RA), which suppressed Treg cell differentiation. This study explored how CD38+ NK cells regulated CD4+ T-cell differentiation into Treg cells in RA.MethodsProportions of CD38+ NK cells and their counterpart CD38+ NK-like T (CD3+ CD16+ CD38+ CD56+) cells were measured in RA and rats with collagen-induced arthritis (CIA). CD38+ NK cells and CD38+ NK-like T cells were cocultured with CD4+ T cells, respectively.ResultsA significantly increased proportion of CD38+ NK cells and a decreased proportion of CD38+ NK-like T cells were detected in RA and CIA blood and synovial fluids. When CD4+ T cells were cocultured with CD38+ NK cells, mammalian target of rapamycin (mTOR) signaling was activated, and Th1/Th2 and Th17/Treg ratios were increased. When CD38+ NK cells were pretreated with anti-CD38 antibody, Treg cell proportion was increased, and Th1/Th2 and Th17/Treg ratios were decreased. CD38+ NK-like T cells showed the opposite results. CD38+ NK cells and CD38+ NK-like-T cells activated differential gene expressions and pathways in CD4+ T cells and initiated Th1 and Th2 cell differentiation by differential gene nodes.ConclusionsThis study suggest that the high CD38+ NK cell proportion and low CD38+ NK-like T cell proportion in RA suppress Treg cell differentiation by stimulating mTOR signaling in CD4+ T cells, which consequentially disturbs the immune tolerance.     

Dual effect of IL‐7/IL‐7R signalling on the osteoimmunological system: a potential therapeutic target for rheumatoid arthritis

The IL‐7/IL‐7R pathway plays a vital role in the immune system, especially in the inflammatory response. Monocytes/macrophages (osteoclast precursors) have been recently recognized as important participants in the osteoclastogenesis of rheumatoid arthritis (RA) patients. Here, we aimed to investigate the therapeutic potential of IL‐7/IL‐7R pathway in RA and to determine whether it could restrain osteoclastogenic functions and therefore ameliorate RA. Firstly, collagen‐induced arthritis (CIA) mice were administered with IL‐7Rα‐target antibodies to assess their therapeutic effect on arthritis. We found that blockade of the IL‐7/IL‐7R pathway protected CIA mice from bone destruction in addition to inducing inflammatory remission, by altering the RANKL/RANK/OPG ratio and consequently decreasing osteoclast formation. To explore the effect and mechanism of this pathway, bone marrow cells were induced to osteoclasts and treated with IL‐7, a STAT5 inhibitor or supernatants from T cells. The results showed that the IL‐7/IL‐7R pathway played a direct inhibitory role in osteoclast differentiation via STAT5 signalling pathway in a RANKL‐induced manner. We applied flow cytometry to analyse the effect of IL‐7 on T‐cell RANKL expression and found that IL‐7/IL‐7R pathway had an indirect role in the osteoclast differentiation process by enhancing the RANKL expression on T cells. In conclusion, the IL‐7/IL‐7R pathway exhibited a dual effect on osteoclastogenesis of CIA mice by interacting with osteoimmunology processes and could be a novel therapeutic target for autoimmune diseases such as RA.     

Metallothionein‐1 suppresses rheumatoid arthritis pathogenesis by shifting the Th17/Treg balance

It is now well accepted that an imbalance between the Th17 and regulatory T‐cell responses is closely associated with the development of rheumatoid arthritis (RA). However, the precise regulatory mechanism for the differentiation of Th17 and Treg in RA is not well characterized. The present study showed that metallothionein‐1 (MT‐1), which is a low molecular weight protein that is involved in the detoxification of heavy metals and scavenging of free radicals, was upregulated in RA. Furthermore, the synovial inflammation and pathologic symptoms in collagen‐induced arthritis and collagen antibody‐induced arthritis mice were significantly suppressed when MT‐1 was expressed intraarticularly. Further investigation revealed that MT‐1 inhibited the differentiation of Th17 cells but enhanced that of Treg cells. Furthermore, it markedly decreased both STAT3 and RAR‐related orphan receptor gamma t (RORγt) expression in vitro and in vivo. Collectively, our studies demonstrated that MT‐1 might manifest as a protein involved in immunosuppression of RA pathogenesis by shifting Th17/Treg balance and may prove to be a potential therapeutic target for RA autoimmune diseases.     

IL-10-producing regulatory B10 cells ameliorate collagen-induced arthritis via suppressing Th17 cell generation

IL-10-producing CD1d(hi)CD5(+) B cells, also known as B10 cells, have been shown to possess a regulatory function in the inhibition of immune responses, but whether and how B10 cells suppress the development of autoimmune arthritis remain largely unclear. In this study, we detected significantly decreased numbers of IL-10-producing B cells, but increased IL-17-producing CD4(+) T (Th17) cells in both spleen and draining lymph nodes of mice during the acute stage of collagen-induced arthritis (CIA) when compared with adjuvant-treated control mice. On adoptive transfer of in vitro expanded B10 cells, collagen-immunized mice showed a marked delay of arthritis onset with reduced severity of both clinical symptoms and joint damage, accompanied by a substantial reduction in the number of Th17 cells. To determine whether B10 cells directly inhibit the generation of Th17 cells in culture, naive CD4(+) T cells labeled with carboxyfluorescein succinimidyl ester (CFSE) were co-cultured with B10 cells. These B10 cells suppressed Th17 cell differentiation via the reduction of STAT3 phosphorylation and retinoid-related orphan receptor γt (RORγt) expression. Moreover, Th17 cells showed significantly decreased proliferation when co-cultured with B10 cells. Although adoptive transfer of Th17 cells triggered the development of collagen-induced arthritis in IL-17(-/-)DBA/1J mice, co-transfer of B10 cells with Th17 cells profoundly delayed the onset of arthritis. Thus, our findings suggest a novel regulatory role of B10 cells in arthritic progression via the suppression of Th17 cell generation.     

Early activation of invariant natural killer T cells in a rheumatoid arthritis model and application to disease treatment

Invariant NKT (iNKT) cells are a distinctive subtype of CD1d‐restricted T cells involved in regulating autoimmunity and capable of producing various T helper type 1 (Th1), Th2 and Th17 cytokines. Activation of iNKT cells by their exogenous ligand α‐galactosylceramide (α‐GalCer) exerts therapeutic effects in autoimmune diseases such as rheumatoid arthritis (RA). However, the pathophysiological role of iNKT cells in RA, in the absence of exogenous stimulation, is incompletely understood. We investigated the potential pathophysiological effects of iNKT cells in mice with collagen‐induced arthritis (CIA), a model of RA. We found that iNKT cells underwent activation only in the early phases of the disease (6 days post‐induction). In the liver, but not the spleen or lymph nodes, this early activation led to the release of interleukins ‐4, ‐17A and ‐10 and of interferon‐γ; and an increased CD69 expression. Importantly, clinical and histological signs of arthritis were improved by the functional blockade of iNKT cells by a monoclonal antibody to CD1d at the early phase of the disease. This improvement was associated on day 6 post‐induction with decreased expression of co‐stimulatory molecules (CD80, CD86, CD40) on splenic dendritic cells and macrophages, whereas regulatory T‐cell suppressive effects and proportions were not modified. Taken in concert, these findings suggest that iNKT cells are activated early in the course of CIA and contribute to the pathogenesis of arthritis. Therefore, iNKT‐cell activation may be a valid treatment target in RA.     

Treatment of collagen‐induced arthritis by Natura‐α via regulation of Th‐1/Th‐17 responses

Cytokines and CD4⁺ Th cells play a crucial role in the pathogenesis of rheumatoid arthritis. Among the Th populations, Th‐1 and Th‐17 have been described as pathogenic in collagen‐induced arthritis (CIA) whereas Th‐2 and Treg were found to have protective effects. The objective of this study was to examine the affect of Natura‐α, a newly developed cytokine regulator, on CIA and on Th cell development. Natura‐α treatment was administered before or during arthritis induction. Anti‐type II collagen antibodies and cytokine expression were evaluated by ELISA. Emergence of CD4⁺CD25⁺Foxp3⁺ T cells was assessed by flow cytometry. Th‐17 differentiation of naive CD4 T cells was assessed in cultures with anti‐CD3 and anti‐CD28. We showed that Natura‐α both prevented and treated CIA. We further demonstrated that in vivo treatment with Natura‐α inhibited IL‐17 production and anti‐type II collagen IgG development. We showed in vitro, using an APC‐free system, that Natura‐α acted directly on differentiating T cells and inhibiting the formation of Th‐1 and Th‐17 cells but did not affect Th‐2 cells. Since Natura‐α inhibits a large spectrum of important pathogenic factors in CIA, it may provide a new and powerful approach to the treatment of rheumatoid arthritis and other inflammatory diseases.     

Increased synovial expression of IL-27 by IL-17 in rheumatoid arthritis

Objectives and design

Interleukin (IL) 17 plays an important role in synovial inflammation and bone destruction in rheumatoid arthritis (RA), while IL-27 exerts a regulatory role in T cell-mediated immune responses. Our aim was to study the influence of IL-17 on IL-27 production in RA.

Materials and methods

Following injection of IL-17 in the knee joint of CIA mice, synovium was examined for IL-17 and IL-27 expression by western blot, real-time PCR, and immunohistochemistry. IL-17 and IL-27 levels were measured by ELISA in mouse bone marrow-derived dendritic cells (BM-DCs) and in synovial fluid (SF) macrophages from RA patients.

Results

IL-17 exacerbated disease progression in CIA mice. Histological analysis showed increased pannus formation associated with cartilage and bone erosion following injection with IL-17. The expression of IL-27 was increased in CIA mice. The expression of IL-17 and IL-27 was increased more in IL-17-injected CIA mice than in control mice. The majority of cells expressing IL-27 were co-localized with synovial macrophages. Increased expression of IL-27 by application of recombinant IL-17 was confirmed in CIA BM-DCs and in SF macrophages from RA patients.

Conclusion

IL-17 enhanced expression of IL-27 in synovial macrophages from RA patients and CIA mice, indicating an interaction between IL-17 and IL-27 as an autoregulatory mechanism.     

Combination therapy with metformin and coenzyme Q10 in murine experimental autoimmune arthritis

Metformin (Met) and coenzyme Q10 (CoQ10) are reported to have therapeutic functions in several inflammatory diseases. These drugs have shown anti-inflammatory effects and have been utilized in mouse models of rheumatoid arthritis (RA). However, there is no evidence of the additive effect of Met and CoQ10 in RA. Although Met and CoQ10 may be involved in the improvement of mitochondrial dysfunction, limited information is available regarding whether this effect can improve mitochondrial dysfunction in RA in particular. In this study, we sought to determine whether Met and CoQ10 attenuate the severity of collagen-induced arthritis (CIA) and show an additive effect in a mouse model. The combination of Met and CoQ10 improved CIA, reducing joint inflammation, Th17 differentiation and IgG production. In contrast, the combination of Met and CoQ10 induced Treg differentiation. Osteoclastogenesis was reduced by the combination of Met and CoQ10. The protein expression of interleukin-1β, interleukin-6 and tumor necrosis factor-alpha in mice splenocytes exposed to lipopolysaccharide decreased after drug combination therapy. We also found that the expression of JC-1 and COX IV were enhanced by treatment with the combination of Met and CoQ10. Moreover, the combination of Met and CoQ10 promoted mitochondrial O₂ consumption. These findings suggest that the combination of Met and CoQ10 reduced CIA severity, improving mitochondrial dysfunction compared to Met or CoQ10 alone. These results present a novel, significant preventive targets in RA and may enhance our understanding of its pathogenesis.     

Atorvastatin inhibits IL-17A,TNF, IL-6, and IL-10 in PBMC cultures from patients with severe rheumatoid arthritis

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease characterized by joint damage, and it may present with comorbidities at the systemic level. The Th1/Th2/Th17 CD4⁺ lymphocyte imbalance produces inflammatory cytokines, which begin to act, injuring joint tissue. Atorvastatin is a cholesterol- lowering drug with a range of biological effects including anti-inflammatory potential. Patients with rheumatoid arthritis who used statins exhibited clinical improvement. However, the mechanism is not fully understood. Therefore, we aimed to evaluate the RA immunomodulatory activity of atorvastatin.MethodsPeripheral blood mononuclear cells (PBMCs) of RA patients and healthy donors were exposed to atorvastatin in different concentrations following a cytotoxicity assay. Th1, Th2, and Th17 cytokines profiles were evaluated in the culture supernatant by cytometric bead array (CBA). Data were analyzed using the Wilcoxon test, and differences were considered significant when p < 0.05.ResultsAtorvastatin showed no toxicity at the tested doses in RA PBMC cultures, and at 10μM, it showed the most significant results, reducing IL-17A (p = 0.002), TNF (p = 0.002), and IL-6 (p = 0.008) supernatant levels. The outcomes also revealed that only patients with more severe disease activity and those sensitive to corticoid treatments were responders to atorvastatin in vitro.ConclusionThese findings suggest the potential immunomodulatory action of atorvastatin as a mechanism in rheumatoid arthritis treatment.     

IL-17 increases cadherin-11 expression in a model of autoimmune experimental arthritis and in rheumatoid arthritis

IL-17 plays important roles in synovial inflammation and bone destruction in the mouse model of autoimmune arthritis and in rheumatoid arthritis (RA). Cadherin-11 determines the behavior of synovial cells in their proinflammatory and destructive tissue response in inflammatory arthritis, and promotes the invasive behavior of fibroblast-like synoviocytes (FLS). The purpose of this study was to examine the effect of IL-17 on the expression of cadherin-11 in autoimmune experimental arthritis and in RA synovium. The severity of synovial inflammation and bone destruction were examined in IL-17-injected knee joints of mice with collagen-induced arthritis (CIA). Cadherin-11 expression was examined in the synovium of mice with CIA, of IL-1 receptor antagonist (IL-1Ra)-deficient mice and of patients with RA and osteoarthritis (OA). Cadherin-11 expression was also examined in the synovium of IL-17 injected knee joints from CIA mice and in IL-17-stimulated FLS of CIA mice and RA patients. IL-17 aggravated synovial inflammation and bone destruction in CIA. By immunohistochemistry, cadherin-11 expression was increased in the synovium of mice with CIA and IL-1Ra-deficient mice and in patients with RA. Synovial cadherin-11 expression in IL-17-injected knee joints, measured by real-time RT-PCR, Western blot and immunohistochemistry, was increased in CIA. Cadherin-11 expression was significantly increased by IL-17 in cultured FLS of CIA mice and RA patients, and these increases were blocked by NF-κB inhibitors. IL-17 increased the expression of cadherin-11 in vivo and in vitro, which implies that an IL-17-induced increase of cadherin-11 is involved in IL-17-induced aggravation of joint destruction and inflammation.     

Galectin-9 suppresses the generation of Th17, promotes the induction of regulatory T cells, and regulates experimental autoimmune arthritis

The effects of galectin-9 on a mouse collagen-induced arthritis (CIA) model were assessed to clarify whether galectin-9 suppresses CIA by regulating T cell immune responses. Galectin-9 suppressed CIA in a dose-dependent manner, and such suppression was observed even when treatment was started on 7 days after the booster, indicating its preventive and therapeutic effects. Galectin-9 induced the decreased levels of pro-inflammatory cytokines, IL-17, IL-12, and IFNgamma in the joint. Galectin-9 induced the decreased number of CD4(+) TIM-3(+) T cells in peripheral blood. Galectin-9-deficient mice became susceptible to CIA may be by increased number of CD4(+) TIM-3(+) T cells and decreased number of Treg cells. We further found that galectin-9 induces differentiation of naive T cells to Treg cells, and it suppresses differentiation to Th17 cells in vitro. The present results suggested that galectin-9 ameliorates CIA by suppressing the generation of Th17, promoting the induction of regulatory T cells.     

Myeloid-Derived Suppressor Cells Protect Mouse Models from Autoimmune Arthritis via Controlling Inflammatory Response

Myeloid-derived suppressor cells (MDSCs) have been reported to participate in immune suppression and autoimmune disorders. However, its role in autoimmune arthritis remains to be determined. We explored whether adoptive transfer of MDSCs in vivo would block joint inflammation and histological damage using collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA) models. CD11b⁺ Gr-1⁺ MDSCs were isolated from the single cells from the spleens of CIA mice on day 41 or AIA mice on day 35. MDSCs (2?×?10⁶) were then transferred to AIA and CIA mice via tail vein before arthritis establishment at indicated time points. Phosphate buffered saline (PBS) was injected as control. Arthritis was evaluated by severity score and histology. The levels of TNF-α, IL-6, IL-17 and IL-10 in the serum and joints were detected by enzyme-linked immunosorbent assay (ELISA). The number of Th17 cells and macrophages in draining lymph nodes and joint tissues was assessed by flow cytometric analysis. Adoptive transfer of MDSCs significantly reduced the clinical score of arthritis, alleviated joint inflammation and histological damage both in AIA and CIA models compared with PBS-treated control groups. The levels of TNF-α, IL-6, IL-17, and IL-10 in the serum and joints were down-regulated by transfer of MDSCs. In addition, adoptive transfer of MDSCs significantly reduced the number of Th17 cells and macrophages in draining lymph nodes and joint tissues. Altogether, we demonstrate that adoptive transfer of MDSCs prevented autoimmune arthritis in mouse models of RA through inhibiting Th17 cells and macrophages. These new findings provide insights into the inhibitory functions of MDSCs and MDSCs may be used as a cell-based biotherapy in RA.     

微渠多孔羟基磷灰石支架诱导骨髓间充质干细胞成骨分化的miRNA表达谱分析

文题释义： 羟基磷灰石：是目前最为理想的生物活性材料,具有生物相容性、骨传导性和骨诱导性,植入人体后对组织无刺激和排斥作用,能与骨形成很强的化学结合,用作骨缺损的充填材料,能为新骨的形成提供支架,发挥骨传导作用,是理想的硬组织替代材料。 MicroRNA(miRNA)：是一类内生的、长度为20-24个核苷酸的单链小分子RNA,由具有发夹结构的70-90个碱基大小的单链RNA前体经过Dicer酶加工后生成,其可以通过几个miRNAs的组合在转录后水平精细调控基因的表达。 背景：多孔羟基磷灰石支架具有良好的体内外成骨效能,但其所涉及的miRNAs复杂调控机制相关研究较少。 目的：探讨多孔羟基磷灰石支架材料介导大鼠骨髓间充质干细胞成骨矿化过程中相关miRNA表达谱的变化。 方法：体外分离、培养和鉴定大鼠骨髓间充质干细胞,将骨髓间充质干细胞与多孔羟基磷灰石支架共培养为实验组,骨髓间充质干细胞单独培养为空白对照组,分别进行成骨诱导7 d,运用miRNA高通量测序技术分析两组骨髓间充质干细胞成骨矿化过程中相关miRNA表达谱的变化并进行GO分析,筛选出两组中表达差异明显的miRNA分子并进行qRT-PCR验证。 结果与结论：①与空白对照组比较,成骨诱导7 d时实验组BMP2、ALP、Runx2 mRNA表达上调,其中BMP2上调明显(P < 0.05);②microRNA高通量测序结果显示miR-210-3p、miR-146a-5p等13个miRNAs明显上调;let-7c-3p、let-3615等17个miRNAs明显下调;③GO分析上调的miRNA靶基因主要参与生物学调节、细胞基因表达、基因表达调节等,包括NF-κB、Toll样受体9、细胞间黏附、白细胞介素1调节、血管生成、Hippo等信号通路;④实时荧光定量qPCR验证结果显示miRNA-210在实验组上调15倍,miR-146a-5p在实验组上调10倍(P < 0.05);⑤结果表明,新型微渠多孔羟基磷灰石支架可以通过上调骨髓间充质干细胞miRNA-210-3p和miR-146a表达,促进骨髓间充质干细胞的成骨分化。 ORCID: 0000-0002-8722-1548(郑佳俊) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程