Synovial Lymphocytes Research Articles

IL-1β stimulated human umbilical cord mesenchymal stem cells ameliorate rheumatoid arthritis via inducing apoptosis of fibroblast-like synoviocytes

Rheumatoid arthritis (RA) is characterized by synovial proliferation and lymphocyte accumulation leading to progressive damage of the periarticular bone and the articular cartilage. The hyperplasia of the synovial intima lining mainly consists of fibroblast-like synoviocytes-rheumatoid arthritis (HFLS-RA) which exhibit apoptosis-resistance, hyper-proliferation, and high invasiveness. The therapeutic efficacy of mesenchymal stem cells (MSCs) treatment in RA has been shown to be due to its immuno-regulatory ability. However, the exact factors and mechanisms involved in MSCs treatment in RA remain unclear. In this study, TRAIL receptor-Death receptor 4 (DR4), DR5, and LFA-1 ligand-intercellular adhesion molecule-1 (ICAM-1) were upregulated in IL-1β-stimulated HFLS-RA. We demonstrated that the total cell number of IL-1β-stimulated hUCMSCs adhering to IL-1β-stimulated HFLA-RA increased via LFA-1/ICAM-1 interaction. Direct co-culture of IL-1β-stimulated hUCMSCs with IL-1β-stimulated HFLS-RA increased the apoptosis of HFLS-RA. RA symptoms in the CIA mouse model improved after administration of IL-1β-stimulated hUCMSCs. In conclusion, IL-1β-stimulated hUCMSCs adhering to HFLS-RA occurred via LFA-1/ICAM-1 interaction, apoptosis of HFLS-RA was induced via TRAIL/DR4, DR5 contact, and RA symptoms and inflammation were significantly improved in a CIA mouse model. The results of this study suggest that IL-1β-stimulated hUCMSCs have therapeutic potential in RA treatment.

T Helper 17–Like Regulatory T Cells in Equine Synovial Fluid Are Associated With Disease Severity of Naturally Occurring Posttraumatic Osteoarthritis

Background: Infiltration of cluster of differentiation (CD) 3+ (CD3+) T cells into the synovium and synovial fluid occurs in most patients with posttraumatic osteoarthritis. During disease progression, proinflammatory T helper 17 cells and anti-inflammatory regulatory T cells infiltrate the joint in response to inflammation. This study aimed to characterize the dynamics of regulatory T and T helper 17 cell populations in synovial fluid from equine clinical patients with posttraumatic osteoarthritis to determine whether phenotype and function are associated with potential immunotherapeutic targets. Hypothesis: An imbalance of the ratio of regulatory T cells and T helper 17 cells would be associated with disease progression in posttraumatic osteoarthritis, suggesting opportunities for immunomodulatory therapy. Study Design: Descriptive laboratory study. Methods: Synovial fluid was aspirated from the joints of equine clinical patients undergoing arthroscopic surgery for posttraumatic osteoarthritis resulting from intra-articular fragmentation. Joints were classified as having mild or moderate posttraumatic osteoarthritis. Synovial fluid was also obtained from nonoperated horses with normal cartilage. Peripheral blood was obtained from horses with normal cartilage and those with mild and moderate posttraumatic osteoarthritis. Synovial fluid and peripheral blood cells were analyzed by flow cytometry, and native synovial fluid was analyzed by enzyme-linked immunosorbent assay. Results: CD3+ T cells represented 81% of lymphocytes in synovial fluid, which increased in animals with moderate posttraumatic osteoarthritis to 88.3% (P = .02). CD14+ macrophages were doubled in those with moderate posttraumatic osteoarthritis compared with mild posttraumatic osteoarthritis and controls (P < .001). Less than 5% of CD3+ T cells found within the joint were forkhead box P3 protein+ (Foxp3+) regulatory T cells, but a 4- to 8-times higher percentage of nonoperated and mild posttraumatic osteoarthritis joint regulatory T cells secreted interleukin (IL)–10 than peripheral blood Tregs (P < .005). T regulatory-1 cells that secreted IL-10 but did not express Foxp3 accounted for approximately 5% of CD3+ T cells in all joints. T helper 17 cells and Th17-like regulatory T cells were increased in those with moderate posttraumatic osteoarthritis (P < .0001) compared with mild and nonoperated patients. IL-10, IL-17A, IL-6, chemokine (C-C motif) ligand (CCL) 2 (CCL2), and CCL5 concentrations detected by enzyme-linked immunosorbent assay in synovial fluid were not different between groups. Conclusions: An imbalance of the ratio of regulatory T cells and T helper 17 cells and an increase in T helper 17 cell–like regulatory T cells in synovial fluid from joints with more severe disease provide novel insights into immunological mechanisms that are associated with posttraumatic osteoarthritis progression and pathogenesis. Clinical Relevance: Early and targeted use of immunotherapeutics in the mitigation of posttraumatic osteoarthritis may improve patient clinical outcomes.

Roles of CD3, CD4 and CD8 in synovial lymphocytes of rheumatoid arthritis.

In this study, theimmunohistochemical EnVision method was applied to detect CD3, CD4 and CD8 in synovial tissues of40 patients with rheumatoid arthritis (RA) and 10 patients with osteoarthritis (OA). In 92.5% (37/40) RA cases, lymphocytes were focally aggregated, and even germinal centers appeared, forming lymphoid follicle-like structures. Theexpression ofCD3, CD4, and CD8 were high in synovial tissue ofRA group, but low in OA group. Thenumber ofCD3, CD4+, and CD8+ lymphocytes in OA group were significantly lower than that in RA group (p<0.05); CD4+lymphocytes in RA accounted for themajority, and mostly were focally distributed. Thenumber ofCD8+lymphocytes in thesynovial tissue were small, and were mostly scattered. Thenumber ofCD4+lymphocytes were significantly higher than CD8+lymphocytes (p<0.05). Compared with theOA group, thenumber ofCD4+T and CD8+T lymphocytes in RA group were higher, and theratio ofCD4/CD8 was higher in RA group (p < 0.05). In conclusion, theCD3, CD4 and CD8 with high level may promote theoccurrence and development ofRA. Theratio ofCD4+/CD8+ may be used as areference index for thediagnosis and prognosis ofRA.

New Proteins Contributing to Immune Cell Infiltration and Pannus Formation of Synovial Membrane from Arthritis Diseases.

An inflamed synovial membrane plays a major role in joint destruction and is characterized by immune cells infiltration and fibroblast proliferation. This proteomic study considers the inflammatory process at the molecular level by analyzing synovial biopsies presenting a histological inflammatory continuum throughout different arthritis joint diseases. Knee synovial biopsies were obtained from osteoarthritis (OA; n = 9), chronic pyrophosphate arthropathy (CPPA; n = 7) or rheumatoid arthritis (RA; n = 8) patients. The histological inflammatory score was determined using a semi-quantitative scale based on synovial hyperplasia, lymphocytes, plasmocytes, neutrophils and macrophages infiltration. Proteomic analysis was performed by liquid chromatography-mass spectrometry (LC-MS/MS). Differentially expressed proteins were confirmed by immunohistochemistry. Out of the 1871 proteins identified and quantified by LC-MS/MS, 10 proteins (LAP3, MANF, LCP1, CTSZ, PTPRC, DNAJB11, EML4, SCARA5, EIF3K, C1orf123) were differentially expressed in the synovial membrane of at least one of the three disease groups (RA, OA and CPPA). Significant increased expression of the seven first proteins was detected in RA and correlated to the histological inflammatory score. Proteomics is therefore a powerful tool that provides a molecular pattern to the classical histology usually applied for synovitis characterization. Except for LCP1, CTSZ and PTPRC, all proteins have never been described in human synovitis.

Targeting of Janus Kinases Limits Pro-Inflammatory but Also Immunosuppressive Circuits in the Crosstalk between Synovial Fibroblasts and Lymphocytes.

Crosstalk between synovial fibroblasts (SF) and immune cells plays a central role in the development of rheumatoid arthritis (RA). Janus kinase inhibitors (JAKi) have proven efficacy in the treatment of RA, although clinical responses are heterogeneous. Currently, little is known regarding how JAKi affect pro- and anti-inflammatory circuits in the bidirectional interplay between SF and immune cells. Here, we examined the effects of tofacitinib, baricitinib and upadacitinib on crosstalk between SF and T or B lymphocytes in vitro and compared them with those of biologic disease modifying anti-rheumatic drugs (bDMARDs). JAKi dose-dependently suppressed cytokine secretion of T helper (Th) cells and decreased interleukin (IL)-6 and matrix metalloproteinase (MMP)3 secretion of SF stimulated by Th cells. Importantly, JAK inhibition attenuated the enhanced memory response of chronically stimulated SF. Vice versa, JAKi reduced the indoleamine-2,3-dioxygenase (IDO)1-mediated suppression of T cell-proliferation by SF. Remarkably, certain bDMARDs were as efficient as JAKi in suppressing the IL-6 and MMP3 secretion of SF stimulated by Th (adalimumab, secukinumab) or B cells (canakinumab) and combining bDMARDs with JAKi had synergistic effects. In conclusion, JAKi limit pro-inflammatory circuits in the crosstalk between SF and lymphocytes; however, they also weaken the immunosuppressive functions of SF. Both effects were dose-dependent and may contribute to heterogeneity in clinical response to treatment.

The Diagnostic Value of Synovial Fluid Lymphocytes in Gout Patients.

Objective This study is aimed at investigating the diagnostic value of synovial fluid cell counts in gout patients. Methods A total of 185 gout, 64 rheumatoid arthritis (RA), 26 axial spondyloarthritis (axSpA), and 24 osteoarthritis (OA) patients were included in the study. According to serum uric acid (sUA) levels on attack, gout patients were divided into normal sUA gout patients and high sUA gout patients. The laboratory data were recorded. ROC curves were generated to evaluate the diagnostic value of the variables for gout patients and normal sUA gout patients compared with RA, axSpA, and OA patients. Results The synovial fluid white blood cell (WBC), peripheral blood mononuclear cell (PBMC), monocyte, polymorphonuclear (PMN), and neutrophil counts in gout patients were higher than those in OA patients (P < 0.05). The synovial fluid PBMC and lymphocyte counts in gout patients were lower than those in RA and axSpA patients (P < 0.05). ROC curve results showed that the AUC values of lymphocytes and sUA for gout patients were 0.728 and 0.881, respectively, which were higher than those of other variables. The optimal cutoff value of lymphocytes for gout was 1.362, with a Youden index of 0.439, a sensitivity of 83.3%, and a specificity of 60.6%. The AUC values of lymphocytes, sUA, and CRP for normal sUA gout patients were 0.694, 0.643, and 0.700, respectively, which were higher than those of other variables. The optimal cutoff value of lymphocytes for normal sUA gout patients was 1.362, with a Youden index of 0.422, a sensitivity of 81.6%, and a specificity of 60.6%. Conclusions The synovial fluid cell counts of gout patients were different from those of RA, axSpA, and OA patients. Synovial fluid lymphocytes had a higher diagnostic value for gout.

Immunosuppressive regulatory T cells are unable to suppress T helper 17 cells in post-traumatic osteoarthritis

Immunosuppressive regulatory T cells are unable to suppress T helper 17 cells in post-traumatic osteoarthritis

Cell-specific epigenetic drivers of pathogenesis in rheumatoid arthritis.

Rheumatoid arthritisis a complex, inflammatory autoimmune disease, which is characterized by pain, swelling and joint damage driven by the altered behavior of a number of different cell types such as synovial fibroblastsmacrophages and lymphocytes. The mechanism underlying pathogenesis is unclear but increasing evidence points to altered epigenetic regulation within these cell typeswhich promotes the activated destructive behavior that underlies disease pathogenesis. This review summarizes the key epigenetic modifications in the most important cells types in rheumatoid arthritis, which are associated with disease activity. We also discuss emerging avenues of research focusing on readers of epigenetic markers which may serve to be potential therapeutic targets.

The relationship between synovial inflammation, structural pathology, and pain in post-traumatic osteoarthritis: differential effect of stem cell and hyaluronan treatment

BackgroundSynovitis is implicated in the severity and progression of pain and structural pathology of osteoarthritis (OA). Increases in inflammatory or immune cell subpopulations including macrophages and lymphocytes have been reported in OA synovium, but how the particular subpopulations influence symptomatic or structural OA disease progression is unclear. Two therapies, hyaluronan (HA) and mesenchymal stem cells (MSCs), have demonstrated efficacy in some clinical settings: HA acting as device to improve joint function and provide pain relief, while MSCs may have immunomodulatory and disease-modifying effects. We used these agents to investigate whether changes in pain sensitization or structural damage were linked to modulation of the synovial inflammatory response in post-traumatic OA.MethodsSkeletally mature C57BL6 male mice underwent medial-meniscal destabilisation (DMM) surgery followed by intra-articular injection of saline, a hyaluronan hexadecylamide derivative (Hymovis), bone marrow-derived stem cells (MSCs), or MSC + Hymovis. We quantified the progression of OA-related cartilage, subchondral bone and synovial histopathology, and associated pain sensitization (tactile allodynia). Synovial lymphocytes, monocyte/macrophages and their subpopulations were quantified by fluorescent-activated cell sorting (FACS), and the expression of key inflammatory mediators and catabolic enzyme genes quantified by real-time polymerase chain reaction (PCR).ResultsMSC but not Hymovis significantly reduced late-stage (12-week post-DMM) cartilage proteoglycan loss and structural damage. Allodynia was initially reduced by both treatments but significantly better at 8 and 12 weeks by Hymovis. Chondroprotection by MSCs was not associated with specific changes in synovial inflammatory cell populations but rather regulation of post-injury synovial Adamts4, Adamts5, Mmp3, and Mmp9 expression. Reduced acute post-injury allodynia with all treatments coincided with decreased synovial macrophage and T cell numbers, while longer-term effect on pain sensitization with Hymovis was associated with increased M2c macrophages.ConclusionsThis therapeutic study in mice demonstrated a poor correlation between cartilage, bone or synovium (histo)pathology, and pain sensitization. Changes in the specific synovial inflammatory cell subpopulations may be associated with chronic OA pain sensitization, and a novel target for symptomatic treatment.

Histopathological patterns seen around failed metal-on-metal hip replacements: Cluster and latent class analysis of patterns of failure.

We aimed to establish latent subtypes of histopathological patterns in failed metal-on-metal hip replacements. Tissue samples of the synovia from the neocapsule were retrieved from 284 revised ASR (Articular Surface Replacement) hip replacements and analyzed histologically. Hierarchical cluster analysis and polytomous latent class analysis were performed to establish the underlying structure and relationships of the histological observations and to find similar cohorts of cases. Clustering analyses suggested four distinct subtypes that could be readily and reasonably labeled and mapped against a recent consensus statement. The first two subtypes showed synovial necrosis, lymphocyte sheets, and abundant or thin histiocyte sheets. In addition, the first subtype showed abundant germinal centers and no metal particles either extracellularly or intracellularly. Metal particles were, however, seen in the second subtype. Hence, the first subtype was labeled "immunologic Type IV neosynovitis" and the second subtype "abrasion-induced inflammatory lymphocytic Type I neosynovitis." The third and fourth subtypes showed no perivascular and diffuse lymphocytes, but a higher number of metal particles intracellularly and extracellularly. The third subtype had synovial necrosis along with granulomas and was labeled "abrasion-induced necrotic Type I neosynovitis," whereas the fourth subtype had readily intact synovial lining, and this subtype was labeled "abrasion-induced foreign body Type I neosynovitis." Histopathological findings in failed MoM hips are not just one wide entity. These hips evince four different histological patterns that also differ at the macroscopic level. Moreover, the often stated "ALVAL-type reaction" seems to be dualistic in nature, which is a novel finding.

Mesenchymal stem cell therapy modulates synovial macrophages in a murine model of osteoarthritis

Purpose: Inflammation is one of the major hallmarks of osteoarthritis (OA), with participation of synovial macrophages and infiltrated lymphocytes. Mesenchymal stem cells (MSCs) are known to modulate the response of all cells of the immune system, with a central role for apoptosis becoming evident in many disease models. Intra-articular (IA) injections of MSCs are known to protect from cartilage degeneration and modulate local inflammation, although the lack of engraftment suggests that they exert their action via paracrine mechanisms. It is also hypothesised that locally-delivered MSCs undergo apoptosis or necrosis.The aim of this study was to examine the mechanism of action of IA-delivered MSCs in an animal model of OA. Macrophages were investigated as recipients of the MSC action through ex vivo cultures. Also, the role of apoptosis was assessed with an in vitro model of cell death. Methods: Collagenase-induced OA (CIOA) was performed on C57BL/6 mice, with a saline-treated group (SHAM) used as a control. 2×10ˆ5 GFP+ MSCs were IA-injected in the animals’ knees after one week. Three days later, knee joints were digested into a single-cell suspension and MSCs retrieved by cell sorting, based on cell fluorescence intensity, and expanded for two passages in standard culture conditions. Capacity to form colonies in vitro was assessed by CFU-F assay and expression of surface markers characteristic of MSCs was measured by flow cytometry. Conditioned medium (CM) of retrieved cells was tested on syngeneic bone marrow-derived macrophages (BMDMs) activated with lipopolysaccharides (LPS) for 4 hours. Expression of major histocompatibility complex class II (MHC-II) and co-stimulatory molecule CD86 were measured by flow cytometry, while secretion of cytokines interleukin (IL)-10 and tumor necrosis factor (TNF)-α was quantified by enzyme-linked immunosorbent assay (ELISA). Apoptosis of MSCs was induced in vitro with staurosporine (STS) and confirmed by pre-treatment with the pan-caspase inhibitor Q-VD-OPh, while necrosis was induced by heat treatment at 90°C. Cell death was evaluated by Annexin V/Sytox Blue staining, analysed by flow cytometry. CM were collected and tested on BMDMs as previously described. Results: 1.63% of injected cells were retrieved and proliferated in culture. Compared to naïve (CTRL) MSCs, retrieved cells maintained morphology, proliferation rates, clonogeneicity and surface marker expression profile. CM of CIOA-retrieved MSCs modulated activation of macrophages, significantly downregulating expression of MHC-II and CD86 and upregulating secretion of IL-10, compared to both SHAM-retrieved MSCs and CTRL MSCs. TNF-α was downregulated in a similar fashion, but not significantly. STS induced apoptosis, which was completely prevented by pre-treatment with Q-VD-OPh, thus confirming initiation of caspase pathway. Necrosis was also confirmed by Annexin V/Sytox staining. Apoptotic but not necrotic MSC-CM prevented activation of BMDMs, with suppressed secretion of TNF-α and significantly increased production of IL-10. Conclusions: MSCs can be administered and retrieved from murine knees by cell sorting. Retrieval yield was low, consistently with previous in vivo cell tracking studies, confirming that most therapeutic action is performed via a hit-and-run mechanism. MSCs were licensed from the OA joint to produce an immunosuppressive milieu that modulated macrophages ex vivo, inducing an M2-like (anti-inflammatory) switch. In vitro, apoptotic MSCs showed an increased immunomodulatory potential in a way that reflected the mechanism observed in OA-challenged MSCs. This set of data suggests that the OA synovial environment licensed the MSCs to secrete immunomodulatory factors, but also that apoptosis is a contributing factor to the effects of cell therapy in OA. As expected, macrophages have a crucial role in propagating the MSC-mediated immunomodulation, by acquiring an M2-like phenotype, with inhibition of stimulatory and co-stimulatory molecules and further release of anti-inflammatory cytokines. These findings are key to the understanding of OA and the development of future cell therapies, which could be focused on apoptosis rather than integration in the degenerated tissues.

Reduction-Responsive Polypeptide Nanogel for Intracellular Drug Delivery in Relieving Collagen-Induced Arthritis.

Rheumatoid arthritis (RA) induces the destruction of cartilage and bone. Methotrexate (MTX) functions as an effective first-line drug to relieve RA in the clinic. However, patients treated with MTX often suffer from severe side effects mainly due to its off-target effects. Therefore, selective delivery of MTX to the affected joints may achieve upregulated efficacy and safety. The affected joints of RA feature hypoxic microenvironment and increased level of glutathione (GSH), resulting from synovial proliferation, lymphocyte infiltration, and neovascularization. In this study, a disulfide-cross-linked nanogel (NG) of methoxy poly(ethylene glycol)-poly(L-phenylalanine-co-L-cystine) (mPEG-P(LP-co-LC)) was synthesized as an intracellular delivery system of MTX. The loading nanogel NG/MTX exhibited apparent reduction-responsiveness and GSH-triggered release behavior of MTX. It also showed efficient internalization and high cytotoxicity toward activated macrophages. Moreover, NG/MTX possessed selective biodistribution in the inflammatory joints of collagen-induced arthritis mouse model. The clinical and histological scores of the mice after NG/MTX treatment were lower than those of the other groups, and the progress of collagen-induced arthritis was overall relieved. To conclude, the controlled delivery of MTX by smart polymer nanoparticles to the RA-affected joints may be a promising approach in the clinical therapy of RA.

Methods for high-dimensional analysis of cells dissociated from cryopreserved synovial tissue

BackgroundDetailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples.MethodsMultiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq.ResultsUpon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified.ConclusionsWe have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers.

Targeted delivery of hyaluronic acid-coated solid lipid nanoparticles for rheumatoid arthritis therapy

Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disease. Long-term, high-dose glucocorticoid therapy can be used to treat the disease, but the fact that the drug distributes systemically can give rise to severe adverse effects. Here we develop a targeted system for treating RA in which the glucocorticoid prednisolone (PD) is encapsulated within solid lipid nanoparticles (SLNs) coated with hyaluronic acid (HA), giving rise to HA-SLNs/PD. HA binds to hyaluronic receptor CD44, which is over-expressed on the surface of synovial lymphocytes, macrophages and fibroblasts in inflamed joints in RA. As predicted, HA-SLNs/PD particles accumulated in affected joint tissue after intravenous injection into mice with collagen-induced arthritis (CIA), and HA-SLNs/PD persisted longer in circulation and preserved bone and cartilage better than free drug or drug encapsulated in SLNs without HA. HA-SLNs/PD reduced joint swelling, bone erosion and levels of inflammatory cytokines in serum. These results suggest that encapsulating glucocorticoids such as PD in HA-coated SLNs may render them safe and effective for treating inflammatory disorders.

Interleukin-20 is triggered by TLR ligands and associates with disease activity in patients with rheumatoid arthritis

BackgroundInterleukin (IL)-20 is a pro-inflammatory cytokine that may be implicated in the pathogenesis of rheumatoid arthritis (RA). This study aimed to determine the association between IL-20 and disease activity in patients with RA. MethodsThe levels of serum and synovial fluid IL-20 were measured in patients with RA and OA. The disease activity was assessed based on the Disease Activity Score of 28 joints (DAS28). The expression of IL-20 in synovial tissue samples from patients with RA and OA were determined by immunohistochemistry. Immunofluorescence staining was used to co-localize IL-20 with selected cells. The secretion of IL-20 was analysed in human peripheral blood mononuclear cells (PBMCs) of patients with RA. ResultsSynovial fluid and synovial tissue IL-20 were significantly increased in patients with RA compared with patients with OA. The expression of IL-20 in RA synovial tissue was particularly associated with macrophages and neutrophil granulocytes, but also with synovial fibroblasts and lymphocytes. The IL-20 levels in synovial fluid correlated with DAS28 (r=0.434; p=0.015) and were significantly elevated in anti-CCP positive RA compared with anti-CCP negative RA (122.3±104.1pg/ml and 45.9±35.8pg/ml; p=0.008). IL-20 production from PBMCs was induced by Poly I:C and LPS but not with pro-inflammatory cytokines, such as TNF-α or IL-1. ConclusionOur data showed that IL-20 is independently associated with RA disease activity and may be triggered by TLR ligands at local sites of inflammation.

Mesenchymal Stem Cell-Derived Exosomes: Immunomodulatory Evaluation in an Antigen-Induced Synovitis Porcine Model.

Synovitis is an inflammatory process associated with pain, disability, and discomfort, which is usually treated with anti-inflammatory drugs or biological agents. Mesenchymal stem cells (MSCs) have been also successfully used in the treatment of inflammatory-related diseases such as synovitis or arthritis. In the last years, the exosomes derived from MSCs have become a promising tool for the treatment of inflammatory-related diseases and their therapeutic effect is thought to be mediated (at least in part) by their immunomodulatory potential. In this work, we aimed to evaluate the anti-inflammatory effect of these exosomes in an antigen-induced synovitis animal model. To our knowledge, this is the first report where exosomes derived from MSCs have been evaluated in an animal model of synovitis. Our results demonstrated a decrease of synovial lymphocytes together with a downregulation of TNF-α transcripts in those exosome-treated joints. These results support the immunomodulatory effect of these exosomes and point out that they may represent a promising therapeutic option for the treatment of synovitis.

Clinical features and pathological joint changes in dogs with erosive immune-mediated polyarthritis: 13 cases (2004-2012).

OBJECTIVE To evaluate the clinical features and pathological joint changes in dogs with erosive immune-mediated polyarthritis (IMPA). DESIGN Retrospective case series. ANIMALS 13 dogs with erosive IMPA and 66 dogs with nonerosive IMPA. PROCEDURES The medical record database of a veterinary teaching hospital was reviewed to identify dogs with IMPA that were examined between October 2004 and December 2012. For each IMPA-affected dog, information extracted from the medical record included signalment, diagnostic test results, radiographic findings, and treatments administered. Dogs were classified as having erosive IMPA if review of radiographs revealed the presence of bone lysis in multiple joints, and descriptive data were generated for those dogs. All available direct smears of synovial fluid samples underwent cytologic evaluation. The synovial fluid total nucleated cell count and WBC differential count were estimated and compared between dogs with erosive IMPA and dogs with nonerosive IMPA. RESULTS 13 of 79 (16%) dogs had erosive IMPA. Dogs with erosive IMPA had a mean ± SD age of 7.1 ± 2.4 years and body weight of 8.3 ± 3.4 kg (18.3 ± 7.5 lb). All 13 dogs had erosive lesions in their carpal joints. The estimated median synovial fluid lymphocyte count for dogs with erosive IMPA was significantly greater than that for dogs with nonerosive IMPA. All dogs received immunosuppressive therapy with leflunomide (n = 9), prednisone (3), or prednisone-azathioprine (1). CONCLUSIONS AND CLINICAL RELEVANCE Results indicated erosive IMPA most commonly affected the carpal joints of middle-aged small-breed dogs. Further genetic analyses and analysis of lymphocyte-subsets are warranted for dogs with erosive IMPA.

Intra-articular injections of mesenchymal stem cells (MSCs) as a treatment for hemophilic arthropathy

ABSTRACTIntraarticular (IA) injections of mesenchymal stem cells (MSCs) in idiopathic osteoarthritis (OA) have shown encouraging results in the literature. Hemophilic arthropathy (HA) represents an enormous societal burden. The similarity between OA and HA is very limited. HA resembles much more to the rheumatoid arthritis because a presence of thick synovial membrane and large lymphocytes infiltration. However, in its final stages, HA resembles OA and that is when IA injections of MSCs are commonly used. In this article, we review the concept of IA injections of MSCs as a treatment for HA, using the literature on OA as an example. Regarding HA, only two experimental studies have been reported to date. Such studies stated that IA injections of MSCs appear to be efficacious for decreasing pain in patients with OA and HA. The use of IA injections of MSCs is thus a promising therapeutic modality for treating HA.

Design, synthesis and preliminary evaluation of the anti-inflammatory of the specific selective targeting druggable enzymome cyclooxygenase-2 (COX-2) small molecule

Context: Development of a reliable and selective anti-inflammatory agent of cyclooxygenase-2 (COX-2), induced or up-regulated by inflammatory/injury stimulus such as IL-1β, TNF-α and LPS in the various types of organs, tissues and cells, with low side effects is a long-standing medicinal chemistry problem with significant social implications.Objective: To target druggable enzymome COX-2 by exploiting NSAIDs and genipin (GEP) in anti-inflammatory infection.Materials and methods: The compound aspirin GEP ester (AGE) was designed by computer-assisted screening, synthesized in the esterification of the acylate derivative and the methylate derivative with Et3N, and evaluated with 20, 40 and 60 mg/kg from days 18 to 24 after immunization in collagen-induced arthritis (CIA) rats by the sequential enzymatic experiments, western-blot analysis and pathological observation methods.Results: AGE exhibited higher binding affinity with COX-1 and displayed the lowest estimated free energy with COX-2 than other ligands built by hanging NSAIDs with GEP, and was characterized by 1H NMR, 13C NMR and HRMS. AGE was competed against COX-2 with molecule-dependent potencies and selectivity (IC50: 0.36 mM; selectivity index: 275) in the sequential enzymatic experiments and decreased the expression of COX-2 in peripheral blood lymphocytes of CIA rats. AGE (40 and 60 mg/kg) could significantly relieve the secondary hind paw swelling and arthritis index, along with observing AGE attenuated histopathological changes of fibroblast like synovial tissue (FLST) and mesenteric lymph node lymphocytes (MLNL) in CIA rats.Discussion and conclusion: AGE pharmacophore reported herein may be an effective strategy to develop a novel anti-inflammatory agent and potential inhibitor of COX-2.

Human CD21low CD86pos B Cells Are Potent Antigen Presenting Cells, Which Are Expanded in Inflammatory Conditions

Accumulating evidence from animal models demonstrates that antigen presentation by B cells plays a crucial role during the natural immune response as well as in several important diseases. While resting B cells are poor antigen-presenting cells they can acquire strong immunostimulatory properties after activation via receptors such as CD40. Studies in animal models suggest that ex vivo generated CD40-actived B cells could serve a promising platform of cellular cancer immunotherapy. However, our understanding of the function of antigen presentation by B cells in human remains limited. This is partly due to the lack of well-defined cell surface markers that can be used to identify distinct immunostimulatory B cell subsets. We hypothesized that antigen-presenting B cells in humans would be characterized by a similar phenotype than in vitro generated CD40-activated B cells. Using a transcriptomic approach combined with flow cytometric phenotypic screening, we were able to show that CD40-activated human B cells can be distinguished from resting B cells by high expression of costimulatory receptors and low expression of BCR-associated coreceptors such as CD21 and FcγRIIB. Here, we report that CD21low CD86pos B cells represent a distinct B lymphocyte subpopulation with potent immunostimulatory capacity. This antigen-presenting B-cell subset possesses phenotypic and functional features of strong antigen-presenting cells, i.e. they express high levels of costimulatory molecules and inflammatory adhesion molecules and low levels of ITIM-bearing inhibitory receptors. In vitro, purified human CD21low CD86pos B cells induced stronger T cell proliferation than the other three subsets, which are defined by the markers CD21 and CD86. Since Epstein Barr Virus (EBV) transforms human B cells with the help of the viral protein LMP1, which mimics CD40-mediated activation of B cells, we first studied patients with replicative EBV infection. We found that CD21low CD86pos B cells were expanded in severely immunosuppressed patients with EBV-reactivation. Analysis of CD21low CD86pos B lymphocytes in blood samples of healthy volunteers and patients with rheumatoid arthritis revealed that this subpopulation was increased in inflammatory conditions. One week following vaccination there was an increase in the frequency of this B-cell subset. Furthermore, compared to healthy controls CD21low CD86pos B cells were significantly elevated in the blood of patients with rheumatoid arthritis (RA). Interestingly, the CD21low CD86pos B-cell subset made up the majority of the B lymphocytes in synovial fluid of RA patients with inflammatory knee effusion. In summary, we demonstrate that CD21low CD86pos B cells are a novel antigen-presenting B-cell subset, which is expanded in inflammatory conditions. Our findings suggest that this subset might represent a promising therapeutic target for the treatment of inflammatory diseases, such as rheumatoid arthritis, were antigen presentation by B cells plays a pathogenetic role. DisclosuresHallek:Mundipharma: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Celgene: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Roche: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Boehringher Ingelheim: Honoraria, Other: Speakers Bureau and/or Advisory Boards; Pharmacyclics: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; AbbVie: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Gilead: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding; Janssen: Honoraria, Other: Speakers Bureau and/or Advisory Boards, Research Funding.