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ObjectivesRecent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK inducible isoform. We hypothesise that HK2 contributes to the synovial lining hypertrophy and plays a critical role in bone and cartilage damage.MethodsHK1 and HK2 expression were determined in RA and osteoarthritis (OA) synovial tissue by immunohistochemistry. RA FLS were transfected with either HK1 or HK2 siRNA, or infected with either adenovirus (ad)-GFP, ad-HK1 or ad-HK2. FLS migration and invasion were assessed. To study the role of HK2 in vivo, 108 particles of ad-HK2 or ad-GFP were injected into the knee of wild-type mice. K/BxN serum transfer arthritis was induced in HK2F/F mice harbouring Col1a1-Cre (HK2Col1), to delete HK2 in non-haematopoietic cells.ResultsHK2 is particular of RA histopathology (9/9 RA; 1/8 OA) and colocalises with FLS markers. Silencing HK2 in RA FLS resulted in a less invasive and migratory phenotype. Consistently, overexpression of HK2 resulted in an increased ability to migrate and invade. It also increased extracellular lactate production. Intra-articular injection of ad-HK2 in normal knees dramatically increased synovial lining thickness, FLS activation and proliferation. HK2 was highly expressed in the synovial lining after K/BxN serum transfer arthritis. HK2Col1 mice significantly showed decreased arthritis severity, bone and cartilage damage.ConclusionHK2 is specifically expressed in RA synovial lining and regulates FLS aggressive functions. HK2 might be an attractive selective metabolic target safer than global glycolysis for RA treatment.

Ferroptosis is a nonapoptotic cell death process that requires cellular iron and the accumulation of lipid peroxides. In progressive rheumatoid arthritis (RA), synovial fibroblasts proliferate abnormally in the presence of reactive oxygen species (ROS) and elevated lipid oxidation. Here we show, using a collagen-induced arthritis (CIA) mouse model, that imidazole ketone erastin (IKE), a ferroptosis inducer, decreases fibroblast numbers in the synovium. Data from single-cell RNA sequencing further identify two groups of fibroblasts that have distinct susceptibility to IKE-induced ferroptosis, with the ferroptosis-resistant fibroblasts associated with an increased TNF-related transcriptome. Mechanistically, TNF signaling promotes cystine uptake and biosynthesis of glutathione (GSH) to protect fibroblasts from ferroptosis. Lastly, low dose IKE together with etanercept, a TNF antagonist, induce ferroptosis in fibroblasts and attenuate arthritis progression in the CIA model. Our results thus imply that the combination of TNF inhibitors and ferroptosis inducers may serve as a potential candidate for RA therapy. Expansion of synovial fibroblast is associated with rheumatoid arthritis (RA) progression, but how this expansion is regulated is still not clear. Here the authors use a mouse RA model, single cell RNA sequencing and in vitro analyses to show that inducing ferroptosis and suppressing TNF signaling reduce fibroblast numbers and ameliorate experimental arthritis.

MicroRNA (miRNA) species (miR) regulate mRNA translation and are implicated as mediators of disease pathology via coordinated regulation of molecular effector pathways. Unraveling miR disease-related activities will facilitate future therapeutic interventions. miR-155 recently has been identified with critical immune regulatory functions. Although detected in articular tissues, the functional role of miR-155 in inflammatory arthritis has not been defined. We report here that miR-155 is up-regulated in synovial membrane and synovial fluid (SF) macrophages from patients with rheumatoid arthritis (RA). The increased expression of miR-155 in SF CD14⁺ cells was associated with lower expression of the miR-155 target, Src homology 2-containing inositol phosphatase-1 (SHIP-1), an inhibitor of inflammation. Similarly, SHIP-1 expression was decreased in CD68⁺ cells in the synovial lining layer in RA patients as compared with osteoarthritis patients. Overexpression of miR-155 in PB CD14⁺ cells led to down-regulation of SHIP-1 and an increase in the production of proinflammatory cytokines. Conversely, inhibition of miR-155 in RA synovial CD14⁺ cells reduced TNF-α production. Finally, miR-155-deficient mice are resistant to collagen-induced arthritis, with profound suppression of antigen-specific Th17 cell and autoantibody responses and markedly reduced articular inflammation. Our data therefore identify a role of miR-155 in clinical and experimental arthritis and suggest that miR-155 may be an intriguing therapeutic target.

Number of IL-9-expressing ILC2s are elevated in patients with inflammatory arthritis during remission, and these cells are critical in mice for the resolution of inflammatory arthritis via regulatory T cell induction. Delivery of DNA minicircles encoding IL-9 into inflamed joints ameliorates mouse experimental arthritis, suggesting possible therapeutic applications. Inflammatory diseases such as arthritis are chronic conditions that fail to resolve spontaneously. While the cytokine and cellular pathways triggering arthritis are well defined, those responsible for the resolution of inflammation are incompletely characterized. Here we identified interleukin (IL)-9-producing type 2 innate lymphoid cells (ILC2s) as the mediators of a molecular and cellular pathway that orchestrates the resolution of chronic inflammation. In mice, the absence of IL-9 impaired ILC2 proliferation and activation of regulatory T (T reg ) cells, and resulted in chronic arthritis with excessive cartilage destruction and bone loss. In contrast, treatment with IL-9 promoted ILC2-dependent T reg activation and effectively induced resolution of inflammation and protection of bone. Patients with rheumatoid arthritis in remission exhibited high numbers of IL-9 + ILC2s in joints and the circulation. Hence, fostering IL-9-mediated ILC2 activation may offer a novel therapeutic approach inducing resolution of inflammation rather than suppression of inflammatory responses.

ObjectivesT helper 9 (Th9) cells are recognised for their characteristic expression of the transcription factor PU.1 and production of interleukin-9 (IL-9), which has been implicated in various autoimmune diseases. However, its precise relationship with rheumatoid arthritis (RA) pathogenesis needs to be further clarified.MethodsThe expression levels of PU.1 and IL-9 in patients with RA were determined by ELISA, western blotting (WB) and immunohistochemical staining. PU.1-T cell-conditional knockout (KO) mice, IL-9 KO and IL-9R KO mice were used to establish collagen antibody-induced arthritis (CAIA), respectively. The inhibitor of PU.1 and IL-9 blocking antibody was used in collagen-induced arthritis (CIA). In an in vitro study, the effects of IL-9 were investigated using siRNAs and IL-9 recombinant proteins. Finally, the underlying mechanisms were further investigated by luciferase reporter analysis, WB and Chip-qPCR.ResultsThe upregulation of IL-9 expression in patients with RA exhibited a positive correlation with clinical markers. Using CAIA and CIA model, we demonstrated that interventions targeting PU.1 and IL-9 substantially mitigated the inflammatory phenotype. Furthermore, in vitro assays provided the proinflammatory role of IL-9, particularly in the hyperactivation of macrophages and fibroblast-like synoviocytes. Mechanistically, we uncovered that PU.1 and IL-9 form a positive feedback loop in RA: (1) PU.1 directly binds to the IL-9 promoter, activating its transcription and (2) Th9-derived IL-9 induces PU.1 via the IL-9R-JAK1/STAT3 pathway.ConclusionsThese results support that the PU.1-IL-9 axis forms a positive loop in Th9 dysregulation of RA. Targeting this signalling axis presents a potential target approach for treating RA.

Osteoarthritis is one of the leading causes of chronic pain, but almost nothing is known about the mechanisms and molecules that mediate osteoarthritis-associated joint pain. Consequently, treatment options remain inadequate and joint replacement is often inevitable. Here, we use a surgical mouse model that captures the long-term progression of knee osteoarthritis to longitudinally assess pain-related behaviors and concomitant changes in the innervating dorsal root ganglia (DRG). We demonstrate that monocyte chemoattractant protein (MCP)-1 (CCL2) and its high-affinity receptor, chemokine (C-C motif) receptor 2 (CCR2), are central to the development of pain associated with knee osteoarthritis. After destabilization of the medial meniscus, mice developed early-onset secondary mechanical allodynia that was maintained for 16 wk. MCP-1 and CCR2 mRNA, protein, and signaling activity were temporarily up-regulated in the innervating DRG at 8 wk after surgery. This result correlated with the presentation of movement-provoked pain behaviors, which were maintained up to 16 wk. Mice that lack Ccr2 also developed mechanical allodynia, but this started to resolve from 8 wk onwards. Despite severe allodynia and structural knee joint damage equal to wild-type mice, Ccr2 -null mice did not develop movement-provoked pain behaviors at 8 wk. In wild-type mice, macrophages infiltrated the DRG by 8 wk and this was maintained through 16 wk after surgery. In contrast, macrophage infiltration was not observed in Ccr2 -null mice. These observations suggest a key role for the MCP-1/CCR2 pathway in establishing osteoarthritis pain.

Background Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and joint stiffness, finally leading to tissue destruction. Connective tissue growth factor (CTGF) is a critical factor in RA progression, which promotes fibroblast-like synoviocyte (FLS) proliferation, pannus formation, and the damage of cartilage as well as bone. Resolvin D1 (RvD1) can promote inflammation resolution in acute inflammatory diseases, and recently, effects of RvD1 on chronic inflammatory diseases also attracted attention. This study aimed to examine the effect of RvD1 on pannus formation in RA and the underlying mechanism. Methods Serum levels of RvD1 and CTGF were determined in RA patients and healthy persons by UPLC-MS/MS and ELISA respectively. The levels of CTGF and inflammatory factors were assessed by qRT-PCR and ELISA. MicroRNA expression profile was determined by miRNA microarray. The effects of CTGF, RvD1, and miR-146a-5p on angiogenesis were evaluated with tube formation and chick chorioallantoic membrane (CAM) assays. Collagen-induced arthritis (CIA) mice were constructed to detect the effects of RvD1 and miR146a-5p on RA. STAT3 activation was determined by Western blotting. Results RvD1 levels decreased while CTGF levels increased in RA patients’ serum, and an inverse correlation of the concentrations of RvD1 and CTGF in the serum of RA patients was synchronously observed. In CIA mice, RvD1 suppressed angiopoiesis and decreased the expression of CTGF. Simultaneously, RvD1 significantly decreased CTGF and pro-inflammation cytokines levels in RA FLS. Furthermore, CTGF suppressed angiopoiesis and RvD1 inhibited the proliferation and migration of RA FLS and angiopoiesis. MiRNA microarray and qRT-PCR results showed that RvD1 upregulated miRNA-146a-5p. The transfection experiments demonstrated that miRNA-146a-5p could decrease inflammatory factors and CTGF levels. Moreover, miRNA-146a-5p decreased the proliferation of FLS and angiogenesis in vivo. MiRNA-146a-5p also suppressed angiogenesis and downregulated the expression of CTGF in CIA mice. Finally, Western blot results revealed that miRNA-146a-5p inhibited the activation of STAT3. Conclusion RvD1 is prone to alleviate RA progression through the upregulation of miRNA-146a-5p to suppress the expression of CTGF and inflammatory mediators, thereby decreasing pannus formation and cartilage damage.

In rheumatoid arthritis (RA), insufficient apoptosis of macrophages and excessive generation of pro-inflammatory cytokines are intimately connected, accelerating the development of disease. Here, a fluorinated polyamidoamine dendrimer (FP) is used to deliver miR-23b to reduce inflammation by triggering the apoptosis of as well as inhibiting the inflammatory response in macrophages. Following the intravenous injection of FP/miR-23b nanoparticles in experimental RA models, the nanoparticles show therapeutic efficacy with inhibition of inflammatory response, reduced bone and cartilage erosion, suppression of synoviocyte infiltration and the recovery of mobility. Moreover, the nanoparticles accumulate in the inflamed joint and are non-specifically captured by synoviocytes, leading to the restoration of miR-23b expression in the synovium. The miR-23b nanoparticles target Tab2 , Tab3 and Ikka to regulate the activation of NF-κB pathway in the hyperplastic synovium, thereby promoting anti-inflammatory and anti-proliferative responses. Additionally, the intravenous administration of FP/miR-23b nanoparticles do not induce obvious systemic toxicity. Overall, our work demonstrates that the combination of apoptosis induction and inflammatory inhibition could be a promising approach in the treatment of RA and possibly other autoimmune diseases. Delivery of anti-inflammatory microRNA (miRNA) could be beneficial for inflammatory diseases such as rheumatoid arthritis (RA). Here the authors show that a fluorinated polyamidoamine dendrimer nanoparticle delivers miR-23b to affected RA joints and reduces inflammation, joint damage and synovial cell influx.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia, pannus formation, and cartilage and bone destruction. Nuclear receptor subfamily 1 group D member 1 (NR1D1) functions as a transcriptional repressor and plays a vital role in inflammatory reactions. However, whether NR1D1 is involved in synovial inflammation and joint destruction during the pathogenesis of RA is unknown. In this study, we found that NR1D1 expression was increased in synovial tissues from patients with RA and decreased in RA Fibroblast-like synoviocytes (FLSs) stimulated with IL-1β in vitro. We showed that NR1D1 activation decreased the expression of proinflammatory cytokines and matrix metalloproteinases (MMPs), while NR1D1 silencing exerted the opposite effect. Furthermore, NR1D1 activation reduced reactive oxygen species (ROS) generation and increased the production of nuclear transcription factor E2-related factor 2 (Nrf2)-associated enzymes. Mitogen-activated protein kinase (MAPK) and nuclear factor κB (NF-κB) pathways were blocked by the NR1D1 agonist SR9009 but activated by NR1D1 silencing. NR1D1 activation also inhibited M1 macrophage polarization and suppressed osteoclastogenesis and osteoclast-related genes expression. Treatment with NR1D1 agonist SR9009 in collagen-induced arthritis (CIA) mouse significantly suppressed the hyperplasia of synovial, infiltration of inflammatory cell and destruction of cartilage and bone. Our findings demonstrate an important role for NR1D1 in RA and suggest its therapeutic potential.

ObjectivesTo investigate the mechanism by which intestinal epithelial cell (IEC) death induces arthritis.MethodsIEC death was assessed by staining for necroptosis and apoptosis markers and fluorescence in situ hybridisation at different time points during collagen-induced arthritis (CIA). During the development of CIA, messenger RNA (mRNA) sequencing was performed, followed by Gene Ontology enrichment analysis of differentially expressed genes. Mice deficient for hypoxia-inducible factor 1α (Hif1a) in IECs (Hif1a ∆IEC) were generated and induced for arthritis. mRNA sequencing, chromatin immunoprecipitated (ChIP) DNA sequencing and ChIP-qualitative PCR were performed on IECs from Hif1a ∆IEC mice and littermate controls. Effects of HIF1α stabilisation by inhibition of prolyl hydroxylase domain-containing enzymes and treatment with the inhibitor of receptor-interacting protein kinase-3 (RIPK3) were tested in intestinal organoids and in CIA.ResultsIEC underwent apoptotic and necroptotic cell death at the onset of arthritis, leading to impaired gut barrier function. HIF1α was identified as one of the most upregulated genes in IECs during the onset of arthritis. Deletion of Hif1a in IEC enhanced IEC necroptosis, triggered intestinal inflammation and exacerbated arthritis. HIF1α was found to be a key transcriptional repressor for the necroptosis-inducing factor RIPK3. Enhanced RIPK3 expression, indicating necroptosis, was also found in the intestinal epithelium of patients with new-onset rheumatoid arthritis. Therapeutic stabilisation of HIF1α as well as small-molecule-based RIPK3 inhibition rescued intestinal necroptosis in vitro and in vivo and suppressed the development of arthritis.ConclusionOur results identify IEC necroptosis as a critical link between the gut and the development of arthritis.