Proteomic Profiling of Tears in Blau Syndrome Patients in Identification of Potential Disease Biomarkers.

Blau syndrome (BS) is a rare autoinflammatory granulomatosis characterized by granulomatous arthritis, uveitis, and dermatitis. Ocular complications are particularly severe in BS, significantly contributing to morbidity. This study aims to identify potential biomarkers for BS ocular degeneration through proteomic profiling of tear samples from affected patients. Seven subjects from the same family, including four carriers of the BS-associated NOD2 mutation (p.E383K), were recruited alongside healthy controls. Tear samples were collected using Schirmer strips and analyzed via mass spectrometry. A total of 387 proteins were identified, with significant differences in protein expression between BS patients, healthy familial subjects, and healthy controls. Key findings include the overexpression of alpha-2-macroglobulin (A2M) and immunoglobulin heavy constant gamma 4 (IGHG4) in BS patients. Bioinformatic analysis revealed that differentially expressed proteins are involved in acute-phase response, extracellular exosome formation, and protein binding. Notably, neutrophils' azurophilic granule components, as azurocidin (AZU1), myeloperoxidases (MPO), and defensins (DEFA3), were highly expressed in the most severely affected subject, suggesting a potential role of neutrophils in BS ocular severity. These proteins might be promising biomarkers for ocular involvement in BS, facilitating early detection and tailored treatment strategies.

Gallic acid ameliorates synovial inflammation and fibrosis by regulating the intestinal flora and its metabolites.

Gallic acid (GA) has been found by a large number of studies to have pharmacological effects such as antioxidant and anti-inflammatory properties. However, the underlying therapeutic mechanisms are not fully understood.. Studies have shown that altering the intestinal flora affects host metabolism and effectively mediates the development of synovitis. The aim of this study was to explore the pharmacological effects of GA in the treatment of synovial inflammation and anti-synovial fibrosis in knee osteoarthritis (KOA) and the underlying mechanisms by macrogenomics combined with off-target metabolomics. We established a synovitis model via in vivo and in vitro experiments to observe the effect of GA intervention on synovitis. Moreover, we collected serum and feces from rats and analyzed the changes in intestinal flora by macro-genome sequencing and the changes in metabolites in the serum by untargeted metabolomics. We found that GA reduced the levels of IL-1ß, IL-6, and TNF-α, and decreased the protein expression levels of α-SMA, TGF-ß, and Collagen I in synovial tissues and cells, and the composition and function of the intestinal flora were similarly altered. Combined with macrogenomic pathway enrichment analysis and metabolic pathway enrichment analysis, these findings revealed that GA impacts Bacteroidia and Muribaculaceae abundance, and via the following metabolic pathways: sphingolipid metabolism, glycerophospholipid metabolism, and arginine biology.to ameliorate synovial inflammation and fibrosis in KOA. The therapeutic effect of GA on KOA synovitis and fibrosis is partly attributed to the alleviation of metabolic disorder and the rebalancing of the intestinal flora. These results provides a rationale for the therapeutic application of GA in the treatment of synovitis.

Disease-Modifying Effects of Lenvatinib, a Multiple Receptor Tyrosine Kinase Inhibitor, on Posttraumatic Osteoarthritis of the Knee.

Angiogenesis and vascular endothelial growth factor (VEGF) are involved in osteoarthritis (OA). We previously reported the inhibitory effect of bevacizumab in a rabbit model of OA. In the current study, we investigated the effects of lenvatinib, an angiogenesis inhibitor targeting the VEGF and fibroblast growth factor receptors, on synovitis, osteophyte formation, and cartilage degeneration in a rabbit OA model. Posttraumatic OA was induced by anterior cruciate ligament transection (ACLT) on one knee of each rabbit. Rabbits were placed into four groups according to the following lenvatinib doses: untreated control (n = 12), L0.3: 0.3 mg/kg/day (n = 15), L1.0: 1.0 mg/kg/day (n = 14), and L3.0: 3.0 mg/kg/day (n = 13) groups. We evaluated limb pain using the weight distribution ratio measured with an incapacitance tester, macroscopic osteophyte formation, and femoral condyle synovium and cartilage histology. For cartilage evaluation, the following distal sites of the femur were evaluated separately: femoral-tibial (FT), femoral-patellar (FP), and femoral corner (between FP and FT). The weight distribution ratio at 12 weeks after surgery was higher in the L0.3 and L1.0 groups than in the control group. Osteophyte formation and synovitis scores were significantly lower in the L0.3, L1.0, and L3.0 groups than in the control group. The Osteoarthritis Research Society International scores of the FT, corner, and FP sites in the L0.3 group were lower than in the control group. The cartilage thickness ratio at the FT and corner sites was significantly lower in the L0.3 group than in the control group. Krenn's grading system of cartilage synovitis showed that all lenvatinib-administered groups had significantly lower scores than the control group. MMP3 expression level in cartilage tissue was significantly lower in the L3.0 group compared with the other three groups. ADAMTS5 expression was lower in the L3.0 group compared with the control and L0.3 groups. Oral administration of lenvatinib inhibited synovitis, osteophyte formation, and cartilage degeneration and reduced pain in a rabbit ACLT model. Lenvatinib is an oral VEGF inhibitor that is easier to administer than other VEGF inhibitors and may have potential as a treatment of posttraumatic OA.

Mid1 promotes synovitis in rheumatoid arthritis via ubiquitin-dependent post-translational modification.

INTRODUCTION: Current anti-rheumatic drugs are primarily modulating immune cell activation, yet their effectiveness remained suboptimal. Therefore, novel therapeutics targeting alternative mechanisms, such as synovial activation, is urgently needed. OBJECTIVES: To explore the role of Midline-1 (Mid1) in synovial activation. METHODS: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice were used to establish a subcutaneous xenograft model. Wild-type C57BL/6, Mid1-/-, Dpp4-/-, and Mid1-/-Dpp4-/- mice were used to establish a collagen-induced arthritis model. Cell viability, cell cycle, qPCR and western blotting analysis were used to detect MH7A proliferation, dipeptidyl peptidase-4 (DPP4) and Mid1 levels. Co-immunoprecipitation and proteomic analysis identified the candidate protein of Mid1 substrates. Ubiquitination assays were used to determine DPP4 ubiquitination status. RESULTS: An increase in Mid1, an E3 ubiquitin ligase, was observed in human RA synovial tissue by GEO dataset analysis, and this elevation was confirmed in a collagen-induced mouse arthritis model. Notably, deletion of Mid1 in a collagen-induced arthritis model completely protected mice from developing arthritis. Subsequent overexpression and knockdown experiments on MH7A, a human synoviocyte cell line, unveiled a previously unrecognized role of Mid1 in synoviocyte proliferation and migration, the key aspects of synovial activation. Co-immunoprecipitation and proteomic analysis identified DPP4 as the most significant candidate of Mid1 substrates. Mechanistically, Mid1 promoted synoviocyte proliferation and migration by inducing ubiquitin-mediated proteasomal degradation of DPP4. DPP4 deficiency led to increased proliferation, migration, and inflammatory cytokine production in MH7A, while reconstitution of DPP4 significantly abolished Mid1-induced augmentation of cell proliferation and activation. Additionally, double knockout model showed that DPP4 deficiency abolished the protective effect of Mid1 defect on arthritis. CONCLUSION: Overall, our findings suggest that the ubiquitination of DPP4 by Mid1 promotes synovial cell proliferation and invasion, exacerbating synovitis in RA. These results reveal a novel mechanism that controls synovial activation, positioning Mid1 as a promising target for therapeutic intervention in RA.

Correlation of meniscus tear type with synovial inflammation and the therapeutic potential of docosapentaenoic acid.

BACKGROUND: Synovitis, characterized by inflammation of the synovial membrane, is commonly induced by meniscus tears. However, significant differences in inflammatory responses and the key inflammatory mediators of synovium induced by different types of meniscal tears remain unclear. METHODS: Magnetic resonance imaging (MRI) was employed to identify the type of meniscus tear, and the quantification of synovial inflammation was assessed through H&E staining assay. Transcription and expression levels of IL-1ß and IL-6 were evaluated using bioinformatics, ELISA, RT-qPCR, and IHC of CD68 staining assays. The therapeutic potential of Docosapentaenoic Acid (DPA) was determined through network pharmacology, ELISA, and RT-qPCR assays. The safety of DPA was assessed using colony formation and EdU staining assays. RESULTS: The results indicate that both IL-1ß and IL-6 play pivotal roles in synovitis pathogenesis, with distinct expression levels across various subtypes. Among tested meniscus tears, oblique tear and bucket handle tear induced the most severe inflammation, followed by radial tear and longitudinal tear, while horizontal tear resulted in the least inflammation. Furthermore, in synovial inflammation induced by specific meniscus tears, the anterior medial tissues exhibited significantly higher local inflammation than the anterior lateral and suprapatellar regions, highlighting the clinical relevance and practical guidance of anterior medial tissues' inflammatory levels. Additionally, we identified the essential omega-3 fatty acid DPA as a potential therapeutic agent for synovitis, demonstrating efficacy in blocking the transcription and expression of IL-1ß and IL-6 with minimal side effects. CONCLUSION: These findings provide valuable insights into the nuanced nature of synovial inflammation induced by various meniscal tear classifications and contribute to the development of new adjunctive therapeutic agents in the management of synovitis.

Syndecans, Exostosins and Sulfotransferases as Potential Synovial Inflammation Moderators in Patients with Hip Osteoarthritis.

The gradual deterioration of articular cartilage was thought to be the central event in osteoarthritis (OA), but recent studies demonstrated the importance of low-grade synovitis in the progression of OA. The Syndecan (SDC) family of membrane proteoglycans is known to be involved in the regulation of inflammation, but there is limited evidence considering the role of syndecans in OA synovitis. Our study aimed to investigate the hip OA synovial membrane expression patterns of SDC1, SDC2 and SDC4, as well as exostosins and sulfotransferases (enzymes involved in the polymerisation and modification of syndecans' heparan sulphate chains). Synovial membrane samples of patients with OA (24) were divided into two groups according to their Krenn synovitis score severity. The immunohistochemical expressions of SDC1, SDC2, SDC4, EXT1, EXT2, NDST1 and NDST2 in synovial intima and subintima were then analysed and compared with the control group (patients with femoral neck fracture). According to our study, the immunoexpression of SDC1, NDST1 and EXT2 is significantly increased in the intimal cells of OA synovial membrane in patients with lower histological synovitis scores and SDC4 in patients with higher synovitis scores, in comparison with non-OA controls. The difference in the expression of SDC2 among the OA and non-OA groups was insignificant. SDC1, SDC4, NDST1 and EXT2 seem to be involved as inflammation moderators in low-grade OA synovitis and, therefore, should be further investigated as potential markers of disease progression and therapeutic goals.

Deconvolution of synovial myeloid cell subsets across pathotypes and role of COL3A1+ macrophages in rheumatoid arthritis remission.

Background: Monocyte/macrophage (Mo/Mp) is a critical cell population involved in immune modulation of rheumatoid synovitis (RA) across different pathotypes. This study aims to investigate the contribution of Mo/Mp clusters to RA activity, and the biological function of particular subtypes in RA remission. Methods: We integrated single-cell RNA sequencing datasets from 4 published and 1 in-house studies using Liger selected by comparison. We estimated the abundance of Mo/Mp subtypes in bulk RNA-seq data from the 81 patients of the Pathobiology of Early Arthritis Cohort (PEAC) using deconvolution analysis. Correlations between Mo/Mp subtypes and RA clinical metrics were assessed. A particular cell type was identified using multicolor immunofluorescence and flow cytometry in vivo and successfully induced from a cell line in vitro. Potential immune modulation function of it was performed using immunohistochemical staining, adhesion assay, and RT-qPCR. Results: We identified 8 Mo/Mp clusters. As a particular subtype among them, COL3A1+ Mp (CD68+, COL3A1+, ACTA2-) enriched in myeloid pathotype and negatively correlated with RA severity metrics in all pathotypes. Flow cytometry and multicolor immunofluorescence evidenced the enrichment and M2-like phenotype of COL3A1+ Mp in the myeloid pathotype. Further assays suggested that COL3A1+ Mp potentially attenuates RA severity via expressing anti-inflammatory cytokines, enhancing Mp adhesion, and forming a physical barrier at the synovial lining. Conclusion: This study reported unexplored associations between different pathologies and myeloid cell subtypes. We also identified a fibroblast-and-M2-like cluster named COL3A1+ Mp, which potentially contributes to synovial immune homeostasis. Targeting the development of COL3A1+ Mp may hold promise for inducing RA remission.

Increased expression of omentin-1 is associated with synovitis and bone destruction in rheumatoid arthritis.

OBJECTIVES: The aberrant expression of omentin-1 had been reported in type 2 diabetes and cardiovascular disease. Here, we investigated the expression and role of omentin-1 in rheumatoid arthritis (RA). METHODS: The expression of omentin-1 in RA and in the normal population was detected by ELISA and immunohistochemistry, and collagen-induced arthritis (CIA) mice were used to detect the role of omentin-1 in RA. RESULTS: We found that the expression of omentin-1 was elevated in serum of RA patients compared with healthy controls (p=0.004), and in the RA disease activity group compared with the disease remission group (p<0.001). In addition, the level of omentin-1 in RA patients was positively correlated with CRP (r=0.427, p=0.002), ESR (r=0.454, p<0.001) and DAS28 (r=0.496, p<0.001; r=0.661, p<0.001, respectively). Multivariable analysis showed that omentin-1 alone was associated with disease activity state (OR=1.018, p=0.004). Immunohistochemical results showed that omentin-1 was increased in the synovium of RA and CIA mice. Omentin-1 injection resulted in an earlier onset of arthritis, an aggravated arthritic progression, more severe synovial hyperplasia and bone erosion in CIA mice. Moreover, omentin-1 treatment markedly enhanced IL-6, TNF-α, MMP-3, MMP-13 and RANKL in the joint tissue of CIA mice. CONCLUSIONS: Our results suggested that omentin-1 was up-regulated in RA and can exacerbate synovitis and joint destruction which may provide new insight into the pathogenesis of RA.

Nanomedicines Promote Cartilage Regeneration in Osteoarthritis by Synergistically Enhancing Chondrogenesis of Mesenchymal Stem Cells and Regulating Inflammatory Environment.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive erosion of the articular cartilage and inflammation. Mesenchymal stem cells' (MSCs) transplantation in OA treatment is emerging, but its clinical application is still limited by the low efficiency in oriented differentiation. In our study, to improve the therapeutic efficiencies of MSCs in OA treatment by carbonic anhydrase IX (CA9) siRNA (siCA9)-based inflammation regulation and Kartogenin (KGN)-based chondrogenic differentiation, the combination strategy of MSCs and the nanomedicine codelivering KGN and siCA9 (AHK-CaP/siCA9 NPs) was used. In vitro results demonstrated that these NPs could improve the inflammatory microenvironment through repolarization of M1 macrophages to the M2 phenotype by downregulating the expression levels of CA9 mRNA. Meanwhile, these NPs could also enhance the chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) by upregulating the pro-chondrogenic TGF-ß1, ACAN, and Col2α1 mRNA levels. Moreover, in an advanced OA mouse model, compared with BMSCs alone group, the lower synovitis score and OARSI score were found in the group of BMSCs plus AHK-CaP/siCA9 NPs, suggesting that this combination approach could effectively inhibit synovitis and promote cartilage regeneration in OA progression. Therefore, the synchronization of regulating the inflammatory microenvironment through macrophage reprogramming (CA9 gene silencing) and promoting MSCs oriented differentiation through a chondrogenic agent (KGN) may be a potential strategy to maximize the therapeutic efficiency of MSCs for OA treatment.

Metabolic changes in fibroblast-like synoviocytes in rheumatoid arthritis: state of the art review.

Fibroblast-like synoviocytes (FLS) are important components of the synovial membrane. They can contribute to joint damage through crosstalk with inflammatory cells and direct actions on tissue damage pathways in rheumatoid arthritis (RA). Recent evidence suggests that, compared with FLS in normal synovial tissue, FLS in RA synovial tissue exhibits significant differences in metabolism. Recent metabolomic studies have demonstrated that metabolic changes, including those in glucose, lipid, and amino acid metabolism, exist before synovitis onset. These changes may be a result of increased biosynthesis and energy requirements during the early phases of the disease. Activated T cells and some cytokines contribute to the conversion of FLS into cells with metabolic abnormalities and pro-inflammatory phenotypes. This conversion may be one of the potential mechanisms behind altered FLS metabolism. Targeting metabolism can inhibit FLS proliferation, providing relief to patients with RA. In this review, we aimed to summarize the evidence of metabolic changes in FLS in RA, analyze the mechanisms of these metabolic alterations, and assess their effect on RA phenotype. Finally, we aimed to summarize the advances and challenges faced in targeting FLS metabolism as a promising therapeutic strategy for RA in the future.

Macrophage Activation in Synovitis and Osteoarthritis of Temporomandibular Joint and Its Relationship with the Progression of Synovitis and Bone Remodeling.

This study investigates the regulatory mechanisms of synovial macrophages and their polarization in the progression of temporomandibular joint osteoarthritis (TMJOA). Macrophage depletion models were established by intra-articular injection of clodronate liposomes and unloaded liposomes. TMJOA was induced by intra-articular injection of 50 µL Complete Freund's Adjuvant and the surgery of disc perforation. The contralateral joint was used as the control group. The expression of F4/80, CD86, and CD206 in the synovium was detected by immunofluorescence staining analysis. Hematoxylin and eosin staining and TMJOA synovial score were detected to show the synovial changes in rat joints after TMJOA induction and macrophage depletion. Changes in rat cartilage after TMJOA induction and macrophage depletion were shown by safranin fast green staining. The bone-related parameters of rats' joints were evaluated by micro-computed tomography analysis. The TMJOA model induced by Complete Freund's Adjuvant injection and disc perforation aggravated synovial hyperplasia and showed a significant up-regulation of expression of F4/80-, CD86-, and CD206-positive cells. F4/80, CD86, and CD206 staining levels were significantly decreased in macrophage depletion rats, whereas the synovitis score further increased and cartilage and subchondral bone destruction was slightly aggravated. Macrophages were crucially involved in the progression of TMJOA, and macrophage depletion in TMJOA synoviocytes promoted synovitis and cartilage destruction.

κ-Opioid receptor activation attenuates osteoarthritis synovitis by regulating macrophage polarization through the NF-κB pathway.

Osteoarthritis (OA) is a prevalent and chronic joint disease that affects the aging population, causing pain and disability. Macrophages in synovium are important mediators of synovial inflammatory activity and pathological joint pain. Previous studies have demonstrated the significant involvement of κ-opioid receptor (KOR) in the regulation of pain and inflammation. Our study reveals a significant reduction in synovial KOR expression among patients and mice with OA. Here, we find that KOR activation effectively inhibits the expressions of the LPS-induced-inflammatory cytokines TNF-α and IL-6 by inhibiting macrophage M1 phenotype. Mechanistically, KOR activation effectively suppresses the proinflammatory factor secretion of macrophages by inhibiting the translocation of NF-κB into the nucleus. Our animal experiments reveal that activation of KOR effectively alleviates knee pain and prevents synovitis progression in OA mice. Consistently, KOR administration suppresses the expressions of M1 macrophage markers and the NF-κB pathway in the synovium of the knee. Collectively, our study suggests that targeting KOR may be a viable strategy for treating OA by inhibiting synovitis and improving joint pain in affected patients.

Moxibustion intervention improves synovitis by down-regulating NLRP3/Caspase-1/IL-1ß signaling of synovial tissue in rats with adjuvant arthritis. / 基于NLRP3/Caspase-1/IL-1ßä¿¡å·é€šè·¯æŽ¢è®¨è‰¾ç¸æ”¹å–„ä½å‰‚æ€§å ³èŠ‚ç‚Žå¤§é¼ æ»‘è†œç‚Žçš„æœºåˆ¶.

OBJECTIVES: To observe the effect of moxibustion on activities of NOD-like receptor family protein 3 (NLRP3)/cysteine aspartic acid specific protease-1 (Caspase-1)/interleukin-1ß (IL-1ß) signaling pathway in rats with adjuvant arthritis (AA), so as to explore its mechanisms underlying improvement of rheumatoid arthritis (RA). Me-thods Thirty male Wistar rats were randomly divided into normal control, AA model and moxibustion groups, with 10 rats in each group. The AA model was replicated by raising in wind, cold and damp environment combined with complete Freund's adjuvant injection. In the moxibustion group, moxibustion was applied to bilateral "Shenshu" (BL23) and "Zusanli"(ST36) for 20 min each time, once daily for 21 days. Changes of joint swelling degree (JSD) and arthritis index (AI) in each group were observed. The ultrastructural changes of synovial cells in each group were observed by transmission electron microscopy. The protein expression levels of NLRP3, apoptosis-associated speck-like protein (ASC), Caspase-1, tumor necrosis factor-α (TNF-α) and IL-1ß in the synovial tissues of the knee joint were measured by Western blot. RESULTS: Compared with the normal control group, JSD, AI and the protein expressions of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß in the synovial tissues were significantly increased (P<0.01) in the model group. In comparison with the model group, JSD, AI and the protein expression levels of NLRP3, ASC, Caspase-1, TNF-α and IL-1ß were significantly decreased (P<0.01) in the moxibustion group. Results of transmission electron microscope showed an irregular and vague nuclear membrane of synovial cells, and unclear mitochondrial membrane boundary with sparse, swelling crests in the model group, which was relatively milder in the damage degree in the moxibustion group. CONCLUSIONS: Moxibustion can relieve the inflammatory response in the synovial membrane of AA rats, which may be related to its function in down-regulating synovial NLRP3/Caspase-1/IL-1ß inflammatory signaling.

Microscopic and transcriptomic changes in porcine synovium one year following disruption of the anterior cruciate ligament.

OBJECTIVE: There is no disease-modifying treatment for posttraumatic osteoarthritis (PTOA). This may be partly due to an incomplete understanding of synovitis, which has been causally linked to PTOA progression. The microscopic and transcriptomic changes in synovium seen in early- to mid-stage PTOA were evaluated to better characterize this knowledge gap. METHODS: Seventy-two Yucatan minipigs underwent transection of the anterior cruciate ligament (ACL). Subjects were randomized to no further intervention, ligament reconstruction, or ligament repair, followed by microscopic synovium evaluation and RNA-sequencing at 1, 4, and 52 weeks. Six additional subjects received no ligament transection and served as 1- and 4-week controls and 12 contralateral knees served as 52-week controls. RESULTS: Synovial lining thickness, stromal cellularity, and overall microscopic synovitis reached their highest levels in the first few weeks following injury. Inflammatory infiltration continued to increase over the course of a year. Leaving the ACL transected, reconstructing the ligament, or repairing the ligament did not modulate synovitis development at 1, 4, or 52 weeks. Differential gene expression analysis of PTOA-affected synovium compared to control synovium revealed increased cell proliferation, angiogenesis, collagen breakdown, and diminished lipid metabolism at 1 and 4 weeks, and increased axonogenesis and focal adhesion with reduced immune activation at 52 weeks. CONCLUSIONS: Synovitis was present one year after ACL injury and was not alleviated by surgical intervention. Gene expression in early synovitis was characterized by cell proliferation, angiogenesis, proteolysis, and reduced lipolysis, which was followed by nerve growth and cellular adhesion with less immune activation at 52 weeks.

Role of mitochondria-bound HK2 in rheumatoid arthritis fibroblast-like synoviocytes.

Background: Glucose metabolism, specifically, hexokinase 2 (HK2), has a critical role in rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) phenotype. HK2 localizes not only in the cytosol but also in the mitochondria, where it protects mitochondria against stress. We hypothesize that mitochondria-bound HK2 is a key regulator of RA FLS phenotype. Methods: HK2 localization was evaluated by confocal microscopy after FLS stimulation. RA FLSs were infected with Green fluorescent protein (GFP), full-length (FL)-HK2, or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (Ad). RA FLS was also incubated with methyl jasmonate (MJ; 2.5 mM), tofacitinib (1 µM), or methotrexate (1 µM). RA FLS was tested for migration and invasion and gene expression. Gene associations with HK2 expression were identified by examining single-cell RNA sequencing (scRNA-seq) data from murine models of arthritis. Mice were injected with K/BxN serum and given MJ. Ad-FLHK2 or Ad-HK2ΔN was injected into the knee of wild-type mice. Results: Cobalt chloride (CoCl2) and platelet-derived growth factor (PDGF) stimulation induced HK2 mitochondrial translocation. Overexpression of the HK2 mutant and MJ incubation reversed the invasive and migrative phenotype induced by FL-HK2 after PDGF stimulation, and MJ also decreased the expression of C-X-C Motif Chemokine Ligand 1 (CXCL1) and Collagen Type I Alpha 1 Chain (COL1A1). Of interest, tofacitinib but not methotrexate had an effect on HK2 dissociation from the mitochondria. In murine models, MJ treatment significantly decreased arthritis severity, whereas HK2FL was able to induce synovial hypertrophy as opposed to HK2ΔN. Conclusion: Our results suggest that mitochondrial HK2 regulates the aggressive phenotype of RA FLS. New therapeutic approaches to dissociate HK2 from mitochondria offer a safer approach than global glycolysis inhibition.

Effects of synovial macrophages in osteoarthritis.

Osteoarthritis (OA) is a common degenerative disease in mammals. However, its pathogenesis remains unclear. Studies indicate that OA is not only an aging process that but also an inflammation-related disease. Synovitis is closely related to the progression of OA, and synovial macrophages are crucial participants in synovitis. Instead of being a homogeneous population, macrophages are polarized into M1 or M2 subtypes in OA synovial tissues. Polarization is highly associated with OA severity. However, the M1/M2 ratio cannot be the only factor in OA prognosis because intermediate stages of macrophages also exist. To better understand the mechanism of this heterogeneous disease, OA subtypes of synovial macrophages classified by gene expression were examined. Synovial macrophages do not act alone; they interact with surrounding cells such as synovial fibroblasts, osteoclasts, chondrocytes, lymphocytes and even adipose cells through a paracrine approach to exacerbate OA. Treatments targeting synovial macrophages and their polarization are effective in relieving pain and protecting cartilage during OA development. In this review, we describe how synovial macrophages and their different polarization states influence the progression of OA. We summarize the current knowledge of the interactions between macrophages and other joint cells and examine the current research on new medications targeting synovial macrophages.

CD10-Bound Human Mesenchymal Stem/Stromal Cell-Derived Small Extracellular Vesicles Possess Immunomodulatory Cargo and Maintain Cartilage Homeostasis under Inflammatory Conditions.

The onset and progression of human inflammatory joint diseases are strongly associated with the activation of resident synovium/infrapatellar fat pad (IFP) pro-inflammatory and pain-transmitting signaling. We recently reported that intra-articularly injected IFP-derived mesenchymal stem/stromal cells (IFP-MSC) acquire a potent immunomodulatory phenotype and actively degrade substance P (SP) via neutral endopeptidase CD10 (neprilysin). Our hypothesis is that IFP-MSC robust immunomodulatory therapeutic effects are largely exerted via their CD10-bound small extracellular vesicles (IFP-MSC sEVs) by attenuating synoviocyte pro-inflammatory activation and articular cartilage degradation. Herein, IFP-MSC sEVs were isolated from CD10High- and CD10Low-expressing IFP-MSC cultures and their sEV miRNA cargo was assessed using multiplex methods. Functionally, we interrogated the effect of CD10High and CD10Low sEVs on stimulated by inflammatory/fibrotic cues synoviocyte monocultures and cocultures with IFP-MSC-derived chondropellets. Finally, CD10High sEVs were tested in vivo for their therapeutic capacity in an animal model of acute synovitis/fat pad fibrosis. Our results showed that CD10High and CD10Low sEVs possess distinct miRNA profiles. Reactome analysis of miRNAs highly present in sEVs showed their involvement in the regulation of six gene groups, particularly those involving the immune system. Stimulated synoviocytes exposed to IFP-MSC sEVs demonstrated significantly reduced proliferation and altered inflammation-related molecular profiles compared to control stimulated synoviocytes. Importantly, CD10High sEV treatment of stimulated chondropellets/synoviocyte cocultures indicated significant chondroprotective effects. Therapeutically, CD10High sEV treatment resulted in robust chondroprotective effects by retaining articular cartilage structure/composition and PRG4 (lubricin)-expressing cartilage cells in the animal model of acute synovitis/IFP fibrosis. Our study suggests that CD10High sEVs possess immunomodulatory miRNA attributes with strong chondroprotective/anabolic effects for articular cartilage in vivo. The results could serve as a foundation for sEV-based therapeutics for the resolution of detrimental aspects of immune-mediated inflammatory joint changes associated with conditions such as osteoarthritis (OA).

Targeting macrophagic PIM-1 alleviates osteoarthritis by inhibiting NLRP3 inflammasome activation via suppressing mitochondrial ROS/Cl- efflux signaling pathway.

BACKGROUND: Osteoarthritis (OA), in which macrophage-driven synovitis is considered closely related to cartilage destruction and could occur at any stage, is an inflammatory arthritis. However, there are no effective targets to cure the progression of OA. The NOD-, LRR-,and pyrin domain-containing protein 3 (NLRP3) inflammasome in synovial macrophages participates in the pathological inflammatory process and treatment strategies targeting it are considered to be an effective approach for OA. PIM-1 kinase, as a downstream effector of many cytokine signaling pathways, plays a pro-inflammatory role in inflammatory disease. METHODS: In this study, we evaluated the expression of the PIM-1 and the infiltration of synovial macrophages in the human OA synovium. The effects and mechanism of PIM-1 were investigated in mice and human macrophages stimulated by lipopolysaccharide (LPS) and different agonists such as nigericin, ATP, Monosodium urate (MSU), and Aluminum salt (Alum). The protective effects on chondrocytes were assessed by a modified co-culture system induced by macrophage condition medium (CM). The therapeutic effect in vivo was confirmed by the medial meniscus (DMM)-induced OA in mice. RESULTS: The expression of PIM-1 was increased in the human OA synovium which was accompanied by the infiltration of synovial macrophages. In vitro experiments, suppression of PIM-1 by SMI-4a, a specific inhibitor, rapidly inhibited the NLRP3 inflammasome activation in mice and human macrophages and gasdermin-D (GSDME)-mediated pyroptosis. Furthermore, PIM-1 inhibition specifically blocked the apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization in the assembly stage. Mechanistically, PIM-1 inhibition alleviated the mitochondrial reactive oxygen species (ROS)/chloride intracellular channel proteins (CLICs)-dependent Cl- efflux signaling pathway, which eventually resulted in the blockade of the ASC oligomerization and NLRP3 inflammasome activation. Furthermore, PIM-1 suppression showed chondroprotective effects in the modified co-culture system. Finally, SMI-4a significantly suppressed the expression of PIM-1 in the synovium and reduced the synovitis scores and the Osteoarthritis Research Society International (OARSI) score in the DMM-induced OA model. CONCLUSIONS: Therefore, PIM-1 represented a new class of promising targets as a treatment of OA to target these mechanisms in macrophages and widened the road to therapeutic strategies for OA.

Innate Immunity and Sex: Distinct Inflammatory Profiles Associated with Murine Pain in Acute Synovitis.

Joint pain severity in arthritic diseases differs between sexes and is often more pronounced in women. This disparity is thought to stem from biological mechanisms, particularly innate immunity, yet the understanding of sex-specific differences in arthritic pain remains incomplete. This study aims to investigate these disparities using an innate immunity-driven inflammation model induced by intra-articular injections of Streptococcus Cell Wall fragments to mimic both acute and pre-sensitized joint conditions. Nociceptive behavior was evaluated via gait analysis and static weight-bearing, and inflammation was evaluated via joint histology and the synovial gene expression involved in immune response. Although acute inflammation and pain severity were comparable between sexes, distinct associations between synovial inflammatory gene expression and static nociceptive behavior emerged. These associations delineated sex-specific relationships with pain, highlighting differential gene interactions (Il6 versus Cybb on day 1 and Cyba/Gas6 versus Nos2 on day 8) between sexes. In conclusion, our study found that, despite similar pain severity between sexes, the association of inflammatory synovial genes revealed sex-specific differences in the molecular inflammatory mechanisms underlying pain. These findings suggest a path towards more personalized treatment strategies for pain management in arthritis and other inflammatory joint diseases.

B-cell capacity for expansion and differentiation into plasma cells are altered in osteoarthritis.

OBJECTIVE: Autoantibody (autoAbs) production in osteoarthritis (OA), coupled with evidence of disturbed B-cell homoeostasis, suggest a potential role for B-cells in OA. B-cells can differentiate with T-cell help (T-dep) or using alternative Toll like recptor (TLR) co-stimulation (TLR-dep). We analysed the capacity for differentiation of B-cells in OA versus age-matched healthy controls (HCs) and compared the capacity of OA synovitis-derived stromal cells to provide support for plasma cell (PC) maturation. METHODS: B-cells were isolated from OA and HC. Standardised in vitro models of B-cell differentiation were used comparing T-dep (CD40 (cluster of differentiation-40/BCR (B-cell receptor)-ligation) versus TLR-dep (TLR7/BCR-activation). Differentiation marker expression was analysed by flow-cytometry; antibody secretion (immunnoglobulins IgM/IgA/IgG) by ELISA (enzyme-linked immunosorbent assay), gene expression by qPCR (quantitative polymerase chain reaction). RESULTS: Compared to HC, circulating OA B-cells showed an overall more mature phenotype. The gene expression profile of synovial OA B-cells resembled that of PCs. Circulating B-cells differentiated under both TLR-dep and T-dep, however OA B-cells executed differentiation faster in terms of change in surface marker and secreted more antibody at Day 6, while resulting in similar PC numbers at Day 13, with an altered phenotype at Day 13 in OA. The main difference was reduced early B-cells expansion in OA (notably in TLR-dep) and reduced cell death. Stromal cells support from OA-synovitis allowed better PC survival compared to bone marrow, with an additional population of cells and higher Ig-secretion. CONCLUSION: Our findings suggest that OA B-cells present an altered capacity for proliferation and differentiation while remaining able to produce antibodies, notably in synovium. These findings may partly contribute to autoAbs development as recently observed in OA synovial fluids.