Human Rheumatoid Arthritis Patients Research Articles

TRPM7 facilitates fibroblast-like synoviocyte proliferation, metastasis and inflammation through increasing IL-6 stability via the PKCα-HuR axis in rheumatoid arthritis

Transient receptor potential melastatin 7 (TRPM7) is a cation channel that plays a role in the progression of rheumatoid arthritis (RA), yet its involvement in synovial hyperplasia and inflammation has not been determined. We previously reported that TRPM7 affects the destruction of articular cartilage in RA. Herein, we further confirmed the involvement of TRPM7 in fibroblast-like synoviocyte (FLS) proliferation, metastasis and inflammation. We observed increased TRPM7 expression in FLSs derived from human RA patients. Pharmacological inhibition of TRPM7 protected primary RA-FLSs from proliferation, metastasis and inflammation. Furthermore, we found that TRPM7 contributes to RA-FLS proliferation, metastasis and inflammation by increasing the intracellular Ca2+ concentration. Mechanistically, the PKCα-HuR axis was demonstrated to respond to Ca2+ influx, leading to TRPM7-mediated RA-FLS proliferation, metastasis and inflammation. Moreover, HuR was shown to bind to IL-6 mRNA after nuclear translocation, which could be weakened by TRPM7 channel inhibition. Additionally, adeno-associated virus 9-mediated TRPM7 silencing is highly effective at alleviating synovial hyperplasia and inflammation in adjuvant-induced arthritis rats. In conclusion, our findings unveil a novel regulatory mechanism involved in the pathogenesis of RA and suggest that targeting TRPM7 might be a potential strategy for the prevention and treatment of RA.

A new class of 7-deazaguanine agents targeting autoimmune diseases: dramatic reduction of synovial fibroblast IL-6 production from human rheumatoid arthritis patients and improved performance against murine experimental autoimmune encephalomyelitis

IL-6 production in treated human synovial fibroblasts from rheumatoid arthritis patients has been utilised to select candidates from a targeted library of queuine tRNA ribosyltransferase (QTRT) substrates for subsequent in vivo screening in murine experimental autoimmune encephalomyelitis.

AIM/CD5L ameliorates autoimmune arthritis by promoting removal of inflammatory DAMPs at the lesions

The hallmark of autoimmune arthritis is the preceding autoantibody production and the following synovial inflammation with hyperplasia and tissue destruction of the joints. The joint inflammation is mediated not only by effector lymphocytes and auto-antibodies but also chronic activation of innate immunity, particularly promoted by the danger-associated molecular patterns (DAMPs). Here we show that apoptosis inhibitor of macrophage (AIM, also called CD5L) protein regulates arthritis by promoting removal of lesional DAMPs both physiologically and therapeutically. When the autoimmune arthritis was promoted by injecting a cocktail of anti-collagen antibodies without type-II collagen immunization, AIM-deficient (AIM−/−) mice exhibited more exacerbated and sustained swelling at multiple joints with greater synovial hyperplasia and bone erosion than wild-type mice. Administration of recombinant AIM (rAIM) reduced S100A8/9, a major DAMP known to be involved in arthritis progression, and decreased various inflammatory cytokines at the lesions in antibody-injected AIM−/− mice, leading to marked prevention of arthritis symptoms. In human rheumatoid arthritis (RA) patients, AIM was more activated via dissociating from IgM-pentamer in response to DAMPs-mediated inflammation both in serum and synovial fluid than in healthy individuals or non-autoimmune osteoarthritis patients, suggesting a disease-regulatory potency of AIM also in human RA patients. Thus, our study implied a therapeutic availability of rAIM to prevent arthritis symptoms targeting DAMPs.

Chronic TNF in the Aging Microenvironment Exacerbates TET2-loss-of-Function Myeloid Expansion

Introduction: Age-associated TET2 somatic mutations impart an intrinsic hematopoietic stem cell (HSC) advantage and contribute to the phenomenon of clonal hematopoiesis of indeterminate potential (CHIP). Individuals with TET2-mutantCHIP have a higher risk of developing myeloid neoplasms and other age-related conditions, including heart, lung, liver, kidney and infectious disease, and have increased risk of all-cause mortality. Despite its role in unhealthy aging, the extrinsic mechanisms driving TET2-mutant CHIP clonal expansion remain unclear. We previously showed an environment containing TNF favours TET2-mutant HSC expansion in vitro. We therefore postulated that age-related increases in TNF also provide an advantage to HSC and progeny with TET2-mutations in vivo. Methods: All human and mouse studies met ethics approval. C57Bl/6 background wildtype (WT), TNF-α knockout ( TNF-/-), Tet2 hematopoietic knockout (Vav1-iCre-mediated; Tet2-/-) and floxed control mice ( Tet2f/f) mice were obtained from the Jackson Laboratory. Young (6-mo [n=9]) and old (18-22 mo [n=9]) WT mice, and old TNF-/- mice (n=7) were subjected to nonirradiative myeloablation with busulfan, and retro-orbital injections of 8x10 6 cells/ml T-cell-depleted bone marrow (BM), equally harvested from 4-mo-old WT CD45.1 (n=5) and Tet2-/- CD45.2 donor mice (n=5). Flow cytometry was used to confirm and monitor engraftment. Mice were harvested 8 weeks post-transplant for analysis of HSC, progenitor, monocyte and neutrophil populations, and cytokine analyses. Consenting human research participants diagnosed with rheumatoid arthritis (RA) were recruited from the Greater Hamilton Area (Ontario, Canada) from 2016-2018. Blood draws occurred prior to any immunomodulatory treatment (baseline), and at 3- and 6-months following treatment with Adalimumab (Humira®), an anti-TNF agent. CHIP status was determined with our successful 48-gene, targeted, Ion-Torrent based sequencing approach to isolated genomic DNA from PBMC. Confirmation of calls and increased sensitivity to detect clones with VAF < 0.02 employed our established single molecule molecular inversion probes (smMIP)-targeted genomic capture technique and high-depth (47,500X avg. coverage) paired-end sequencing, employing high-stringency filters for error-suppression. Statistical analyses were performed in GraphPad Prism V9.2 or R 4.1.2. Results: 1. Mixed BM chimeric mice of WT and TNF -/- genotypes reconstituted with WT CD45.1 + and Tet2 -/- CD45.2 + HSC showed that age-associated increases in TNF significantly increased by 2- to 2.5-fold the proportions of HSC in old recipient mice, with myeloid lineage skewing (2- to 2.5-fold more granulocyte-monocyte, monocyte-dendritic and common monocyte progenitor cells in BM, and dramatic expansion of Tet2-/- monocytes and neutrophils in blood). This aberrant myelomonocytic advantage was mitigated in old TNF -/- recipient mice, suggesting that TNF signalling in the BM is essential for Tet2-mutant myeloid expansion. 2. Age-associated TNF predisposed Tet2-/- HSC to the development of Ly6C high inflammatory monocytes, including increased intracellular and serum TNF levels, further exacerbating an inflammatory environment in favor of Tet2-mutantexpansion. 3. Examination of human RA patients (n=7; avg. age 57y) with serial PBMC sampling revealed one patient with CHIP driven by CBL and PPM1D variants, with a 32% reduction in clone size between 3 and 6 months of anti-TNF therapy (adalimumab). Two additional variants were detected at baseline in this RA patient, TP53 R196* (VAF = 1.34%) and ASXL1 P689L (VAF = 3.13%), but these were not detected at 3- and 6-months post-treatment even with high-depth, error suppression sequencing. Two additional patients had detectible low-level CH clones prior to anti-TNF treatment - one with a STAG2 R1033Q variant at baseline (VAF = 1.65%) and the other with a TET2 baseline E798K variant (VAF = 1.68%) - but not detected during anti-TNF treatment. Conclusions: We present novel findings that TNF in the aging BM environment has a causal role in driving TET2-mutant CHIP in vivo, and the first examination of the impact of TNF blockade on CH dynamics in humans, suggesting promise for reducing clonal burden. Larger, prospective studies are required to confirm these findings and to determine if inflammatory cytokine blockade may improve CHIP-comorbid conditions and myeloid cancer risk.

DHA Induces Cell Death through the Production of ROS and the Upregulation of CHOP in Fibroblast-like Synovial Cells from Human Rheumatoid Arthritis Patients.

Rheumatoid arthritis (RA) is an inflammatory disease marked by a massive proliferation of synovial cells in the joints. In this study, we investigated the pro-apoptotic effects of docosahexaenoic acid (DHA) in human fibroblast-like synovial cells from RA patients (RA-FLS). An in vitro study using MH7A cells showed that DHA treatment induced caspase-8-dependent apoptosis in a dose-dependent manner and reduced the TNF-α-mediated induction of MMP-9 and IL-1β. DHA also induced the phosphorylation of eIF2α, the expression of the ER stress markers ATF4 and C/EBP homologous protein (CHOP), and death receptor 5 (DR5). The knockdown of CHOP or DR5 increased cell viability and reduced apoptosis in DHA-treated cells. Furthermore, the knockdown of CHOP reduced DHA-mediated DR5 expression, while the overexpression of CHOP increased DR5 expression. We also found that DHA treatment induced the accumulation of reactive oxygen species (ROS), and pretreatment with the anti-oxidant Tiron effectively abrogated not only the expression of CHOP and DR5, but also DHA-induced apoptosis. Under this condition, cell viability was increased, while PARP-1 cleavage and caspase-8 activation were reduced. All the findings were reproduced in human primary synovial cells obtained from RA patients. These results suggest that the DHA-mediated induction of ROS and CHOP induced apoptosis through the upregulation of DR5 in RA-FLSs, and that CHOP could be used as a therapy for RA.

TRPM7 channel inhibition attenuates rheumatoid arthritis articular chondrocyte ferroptosis by suppression of the PKCα-NOX4 axis

A role for ferroptosis in articular cartilage destruction associated with rheumatoid arthritis (RA) has not been identified. We previously reported transient receptor potential melastatin 7 (TRPM7) expression was correlated with RA cartilage destruction. Herein, we further characterized a role for TRPM7 in chondrocyte ferroptosis. The expression of TRPM7 was found to be elevated in articular chondrocytes derived from adjuvant arthritis (AA) rats, human RA patients, and cultured chondrocytes treated with the ferroptosis inducer, erastin. TRPM7 knockdown or pharmacological inhibition protected primary rat articular chondrocytes and human chondrocytes (C28/I2 cells) from ferroptosis. Moreover, TRPM7 channel activity was demonstrated to contribute to chondrocyte ferroptosis by elevation of intracellular Ca2+. Mechanistically, the PKCα-NOX4 axis was found to respond to stimulation with erastin, which resulted in TRPM7-mediated chondrocyte ferroptosis. Meanwhile, PKCα was shown to directly bind to NOX4, which could be reduced by TRPM7 channel inhibition. Adeno-associated virus 9-mediated TRPM7 silencing or TRPM7 blockade with 2-APB alleviated articular cartilage destruction in AA rats and inhibited chondrocyte ferroptosis. Collectively, both genetic and pharmacological inhibitions of TRPM7 attenuated articular cartilage damage and chondrocyte ferroptosis via the PKCα-NOX4 axis, suggesting that TRPM7-mediated chondrocyte ferroptosis is a promising target for the prevention and treatment of RA.

Disease status in human and experimental arthritis, and response to TNF blockade, is associated with MHC class II invariant chain (CD74) isoform expression

Splice variants of CD74 differentially modulate the activity of cathepsin L (CTSL). As CD74 and CTSL participate in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA), we determined whether splice variants of CD74 could be biomarkers of disease activity. Gene expression was measured in mice with collagen-induced arthritis using quantitative PCR (qPCR). In vitro studies using murine macrophage/DC-lineage cells determined the relative influence of macrophage phenotype on isoform expression and the potential to produce CTSL in response to TNF. CD74 splice variants were measured in human RA synovium and RA patients’ monocytes. In arthritic mice, the expression of the p41 CD74 isoform was significantly higher in severely affected paws compared with unaffected paws or the paws of naïve mice; the p41 isoform significantly correlated with the expression of TNF in arthritic paws. Compared with M2-like macrophages, M1-like macrophages expressed increased levels of CD74 and had higher expression, secretion and activity of CTSL. RA patients that responded to TNF blockade had significantly higher expression levels of CD74 in circulating monocytes after treatment, compared with non-responders. The expression of the human CD74 isoform a was significantly higher in RA synovia, compared with osteoarthritis synovia, and was associated with CSTL enzymatic activity. This study is the first to demonstrate differential expression of the CD74 p41 isoform in an auto-immune disorder and in response to therapy. The differential expression of CD74 splice variants indicates an association, and potentially a mechanistic role, in the pathogenesis of RA.

MiR-let-7c-5p and miR-149-5p inhibit proinflammatory cytokine production in osteoarthritis and rheumatoid arthritis synovial fibroblasts.

Osteoarthritis (OA) and rheumatoid arthritis (RA) are two of the most common types of arthritis. Both are characterized by the infiltration of a number of proinflammatory cytokines into the joint microenvironment. miRNAs play critical roles in the disease processes of arthritic disorders. However, little is known about the effects of miRNAs on critical inflammatory cytokine production with OA and RA progression. Here, we found higher levels of proinflammatory cytokines including interleukin 1 beta (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) in human OA and RA synovial fibroblasts (SFs) compared with normal SFs. Searches of open-source microRNA (miRNA) software determined that miR-let-7c-5p and miR-149-5p interfere with IL-1β, IL-6 and TNF-α transcription; levels of all three proinflammatory cytokines were lower in human OA and RA patients compared with normal controls. Anti-inflammatory agents dexamethasone, celecoxib and indomethacin reduced proinflammatory cytokine production by promoting the expression of miR-let-7c-5p and miR-149-5p. Similarly, ibuprofen and methotrexate also enhanced miR-let-7c-5p and miR-149-5p expression in human SFs. The evidence suggests that increasing miR-let-7c-5p and miR-149-5p expression is a novel strategy for OA and RA.

Cy3-tilmanocept labeling of macrophages in joints of mice with antibody-induced arthritis and synovium of human patients with rheumatoid arthritis.

γ-Tilmanocept (99m Tc-tilmanocept) is a receptor-directed, radiolabeled tracer that is FDA-approved for guiding sentinel lymph node biopsy. Tilmanocept binds the C-type lectin mannose receptor (MR, CD206) on macrophages. In this study, nonradioactive, fluorescently-labeled Cy3-tilmanocept was used to detect CD206+ mononuclear cells in the cartilage of mice with antibody-induced arthritis and in the synovial fluid and tissue of human subjects with rheumatoid arthritis (RA) for comparison withosteoarthritis (OA), and healthy volunteer (HV) controls. Murine arthritis was induced by injection of monoclonal anti-cartilage antibody followed by injection of Escherichiacoli lipopolysaccharide. Post-arthritis development (7-11 days), the mice were injected intravenously with Cy3-tilmanocept followed by in vivo and ex vivo epifluorescence imaging. Two-photon imaging, immunofluorescence, and immunohistochemistry were used to identify articular and synovial macrophages (CD206, F4/80, and Cy3-tilmanocept binding) in murine tissues. Cy3-tilmanocept epifluorescence was present in arthritic knees and elbows of murine tissues; no radiographic changes were noted in the skeletons. However, inflammatory arthritic changes were apparent by histopathology and immunohistochemistry (F4/80), immunofluorescence (CD206) and Cy3-tilmanocept binding. In human RA synovial fluid, Cy3-tilmanocept staining correlated with CD206+ /CD16+ cells; negligible labeling was observed in OA samples. Cy3-tilmanocept colocalized with CD206 and staining was significantly higher in RA synovial tissue compared to OA or HV. Our results demonstrate that imaging with Cy3-tilmanocept can detect in vivo inflammatory, CD206+ macrophages in an early arthritis animal model and in human RA patients. These data establish a novel tool for preclinical research of early arthritis and have implications for early RA detection and monitoring of therapeutic efficacy in humans.

The Joint-Brain Axis: Insights From Rheumatoid Arthritis on the Crosstalk Between Chronic Peripheral Inflammation and the Brain.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by erosive polyarthritis. Beyond joint pathology, RA is associated with neuropsychiatric comorbidity including depression, anxiety, and an increased risk to develop neurodegenerative diseases in later life. Studies investigating the central nervous system (CNS) in preclinical models of RA have leveraged the understanding of the intimate crosstalk between peripheral and central immune responses. This mini review summarizes the current knowledge of CNS comorbidity in RA patients and known underlying cellular mechanisms. We focus on the differential regulation of CNS myeloid and glial cells in different mouse models of RA reflecting different patterns of peripheral immune activation. Moreover, we address CNS responses to anti-inflammatory treatment in human RA patients and mice. Finally, to illustrate the bidirectional communication between the CNS and chronic peripheral inflammation, we present the current knowledge about the impact of the CNS on arthritis. A comprehensive understanding of the crosstalk between the CNS and chronic peripheral inflammation will help to identify RA patients at risk of developing CNS comorbidity, setting the path for future therapeutic approaches in both RA and neuropsychiatric diseases.

Chebulinic acid is a safe and effective antiangiogenic agent in collagen-induced arthritis in mice

BackgroundAlthough vascular endothelial growth factor-A (VEGF)-induced angiogenesis has been reported to play an important role in the pathogenesis of rheumatoid arthritis (RA), serious side effects, mainly grade 2–3 hypertension, which is commonly observed with currently available anti-VEGF agents, can be detrimental for RA patients due to hypertension and associated cardiovascular complications seen in these patients. Thus, identification of anti-VEGF molecules that do not increase blood pressure could be useful for the treatment of RA. Chebulinic acid (CI), a water-soluble small-molecule tannin, can inhibit the actions of VEGF, and a report suggested that CI might not increase blood pressure due to its compensatory effects on the cardiovascular system. Therefore, the effects of CI on blood pressure in mice and the progression of the disease in a murine collagen-induced arthritis (CIA) model were investigated.MethodsCIA was induced in DBA/1J mice with type II collagen. The effects of CI in these animals were then evaluated by determination of clinical, histopathological, and immunohistochemical parameters. The effects of CI on VEGF-induced proangiogenic genes and signaling pathways were examined in vitro and in vivo.ResultsSignificant CD31 and VEGF expressions were detected in the synovial tissues of mice with CIA, similar to their expressions observed in human RA patients. However, treatment with CI significantly inhibited paw swelling, decreased the mean articular index and joint pathology scores in these animals through inhibition of VEGF-induced proangiogenic gene expressions and signaling pathways that regulate angiogenesis. Unlike currently used antiangiogenic agents, CI at a dose that inhibits VEGF actions did not increase blood pressure in mice.ConclusionCI can act as a safe and potent anti-VEGF antiangiogenic agent for the treatment of types of inflammatory arthritis, such as RA.

Histone deacetylase 1 (HDAC1): A key player of T cell-mediated arthritis

Rheumatoid Arthritis (RA) represents a chronic T cell-mediated inflammatory autoimmune disease. Studies have shown that epigenetic mechanisms contribute to the pathogenesis of RA. Histone deacetylases (HDACs) represent one important group of epigenetic regulators. However, the role of individual HDAC members for the pathogenesis of arthritis is still unknown. In this study we demonstrate that mice with a T cell-specific deletion of HDAC1 (HDAC1-cKO) are resistant to the development of Collagen-induced arthritis (CIA), whereas the antibody response to collagen type II was undisturbed, indicating an unaltered T cell-mediated B cell activation. The inflammatory cytokines IL-17 and IL-6 were significantly decreased in sera of HDAC1-cKO mice. IL-6 treated HDAC1-deficient CD4+ T cells showed an impaired upregulation of CCR6. Selective inhibition of class I HDACs with the HDAC inhibitor MS-275 under Th17-skewing conditions inhibited the upregulation of chemokine receptor 6 (CCR6) in mouse and human CD4+ T cells. Accordingly, analysis of human RNA-sequencing (RNA-seq) data and histological analysis of synovial tissue samples from human RA patients revealed the existence of CD4+CCR6+ cells with enhanced HDAC1 expression. Our data indicate a key role for HDAC1 for the pathogenesis of CIA and suggest that HDAC1 and other class I HDACs might be promising targets of selective HDAC inhibitors (HDACi) for the treatment of RA.

Exploiting CD22 To Selectively Tolerize Autoantibody Producing B-Cells in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease that primarily affects the synovial joints and can lead to bone erosion and cartilage damage. One hallmark of RA is anticitrullinated protein autoantibodies (ACPA) and memory citrulline-specific B-cells, which have been implicated in RA pathogenesis. While depletion of B-cells with Rituximab improves clinical responses in RA patients, this treatment strategy leaves patients susceptible to infections. Here we use of Siglec-engaging Tolerance-inducing Antigenic Liposomes (STALs) to selectively target the citrulline-specific B-cells. ACPA production from purified human RA patients' B-cells in vitro was achieved through a set of stimulation conditions, which includes the following: BAFF, anti-CD40, IL-21, and LPS. In vivo generation of citrulline specific B-cells and ACPA production was accomplished by antigenic liposomes consisting of monophosphoryl lipid A (MPLA) and a cyclic citrullinated peptide (CCP) administered to SJL/J mice. We show that STALs that codisplay a high affinity CD22 glycan ligand and synthetic citrullinated antigen (CCP STALs) can prevent ACPA production from RA patients' memory B-cells in vitro. These CCP STALs were also effective in inducing tolerance to citrullinated antigens in SJL/J mice. The results demonstrate that tolerization of the B-cells responsible for ACPA can be achieved by exploiting the inhibitory receptor CD22 with high-affinity glycan ligands. Such a treatment strategy could be beneficial in the treatment of RA.

Effects of Morinda citrifolia on Rheumatoid Arthritis in SKG Mice.

Morinda citrifolia L., known as noni, originated from Indonesia exhibits various pharmacological activities including anti-inflammatory properties. However, the validity of noni fruit juice as a treatment for rheumatoid arthritis (RA), an autoimmune disorder, has not been confirmed yet. Therefore, the main purpose of this research was to evaluate the efficacy of noni fruit juice (INFJ) made in Indonesia using SKG mice as an animal model of RA, which shows the resembling characteristics of human RA patients. Furthermore, the safety of INFJ was examined by repeated dose experiments in mice. INFJ was mixed with water at 50% and administered to SKG mice sensitized with mannan, free access for 4 weeks. Arthritis scores of fore- and hind-leg joints were measured and the joints were histopathologically examined. The sub-acute and sub-chronic toxicities of INFJ were evaluated using BALB/c mice. The arthritic scores were significantly lower from the 7 d after sensitization in the INFJ group than the control group. Histopathological examinations of the joints revealed inhibition of severity of RA. In both toxicity studies, INFJ did not show any toxicities. INFJ exhibited anti-arthritic activity in arthritic and histopathological examinations of the joints in SKG mice. Present study was the first report where noni juice may be effective against RA. The dose of noni juice showing efficacy against RA was confirmed safe from repeated dose studies in mice.

B Cell Tolerance to Deiminated Histones in BALB/c, C57BL/6, and Autoimmune-Prone Mouse Strains.

Deimination, a posttranslational modification of arginine to citrulline carried out by peptidylarginine deiminases, may compromise tolerance of self-antigens. Patients with connective tissue autoimmunity, particularly rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or Felty’s syndrome, present with autoantibodies to deiminated histones (dH), which thus form a category of antibodies to citrullinated protein antigens (ACPA). In general, ACPA are a sensitive diagnostic for RA and may form in response to the release of nuclear chromatin (DNA plus dH) from granulocytes, usually referred to as neutrophil extracellular traps. The aim of this study was to examine spontaneously autoimmune mice for autoantibodies and T cell responses to dH. We compared IgG binding to deiminated and non-deiminated histones (nH) by ELISA and Western blotting in spontaneously autoimmune strains of (NZB × NZW) F1 and NZM2410 together with their derivative congenic strains, C57BL/6.Sle1 and C57BL/6.Sle1.Sle3, which display profound autoreactivity against nuclear self-antigens. The splenocyte proliferation against the two antigens was determined in the spontaneously autoimmune (NZB × NZW) F1 strain from which other autoimmune strains used in the study were derived. Immunizations with dH and nH were attempted in BALB/c mice to assess their splenocyte response. Splenocytes from BALB/c mice and from autoimmune mice at the time of conversion to autoimmunity proliferated strongly in response to dH, yet serum IgG from autoimmune (NZB × NZW) F1, NZM2410, and C57BL/6.Sle1.Sle3 mice displayed a remarkable bias against binding to dH. At the time of seroconversion, the antibodies already exhibited preference for nH, and only nH were recovered from circulating immune complexes. Analysis of histone deimination showed constitutive deimination in thymic extracts from C57BL/6 and C57BL/6.Sle1.Sle2.Sle3 triply congenic mice and in spleens of autoimmune triply congenic mice. Our study demonstrates that tolerance mechanisms against dH are intact in BALB/c and C57BL/6 mice and continue to be effective in mice with overt autoimmunity to nH. We conclude that, in contrast to human RA and SLE patients, where we frequently observe autoantibodies against dH, autoimmune mice maintain strong tolerance mechanisms to prevent the development of autoantibodies to dH.

Altered composition of gut microbiota in rheumatoid arthritis patients.

Manifestation of rheumatoid arthritis (RA) can be attributed to both genetic and environmental factors. Some researchers have been focusing on intestinal microbiota which is thought to be one of the environmental factors that may enhance the development of RA. The advancement of culture-independent, high throughput microbial DNA sequencing had enabled us to understand the interplay between intestinal microbiota and host immune systems. In this study, we have reviewed the previous findings in animal and human studies with respect to the role of intestinal microbiota in RA. Mouse models of arthritis have demonstrated that gut microbiota plays a critical role in the disease development. K/BxN and IL-1 receptor-antagonist knock-out mice did not develop disease in germ free condition, however, colonization of particular intestinal bacteria was sufficient to induce arthritis. Moreover, the dysbiosis was observed in the human RA patients from United States, China and Finland. Thus, we believe that endeavors to improve the dysbiosis would serve as a novel therapeutic or preventive strategy in RA patients.

SAT0192 In Vivo Detection of Cortical Micro Channels in Metacarpophalangeal Joints with Xtreme-Ct - A New Instrument to Visualize Communication between the Bone Marrow and the Joint Space

BackgroundSynovitis and osteitis are distinct pathologic findings in patients with rheumatoid arthritis (RA). Their frequent simultaneous occurrence led to the theory of increased communication between the bone marrow and synovial...

Adjuvant arthritis is associated with changes in the glycosylation of serum IgG1 and IgG2b.

The increased amounts of agalactosyl IgG (N-linked oligosaccharides terminating with N-acetylglucosamine (GlcNAc) in the serum of patients with rheumatoid arthritis (RA) and other chronic inflammatory diseases have suggested that agalactosyl IgG may be involved in the pathogenesis of RA. We have now evaluated the incidence of agalactosyl IgG in the Lewis rat during the course of adjuvant arthritis (AA). The modification in glycosylation of IgG was measured by means of polyclonal and monoclonal anti GlcNAc antibodies as well as by the lectin concanavalin A (Con A). The results show that Lewis rats undergo a change in serum IgG glycosylation during the course of AA. As in human RA patients, rats with AA lack terminal galactose on IgG heavy chain oligosaccharides, and the terminal GlcNAc or mannose residues are thus exposed. The degree of agalactosyl IgG was positively correlated with the incidence of disease, peaked 20 days after disease induction, and the IgG gradually reverted to the fully glycosylated form thereafter. The post-arthritic glycosylation profile was very similar to that characteristic of the naive animal. Purified IgG was shown to contain two IgG subclasses, IgG1 and IgG2b, which underwent changes in glycosylation. Western blot analysis revealed that IgG1 expressed a higher degree of terminal mannose, whereas IgG2b expressed a higher degree of terminal GlcNAc. These findings raise the question of the possible involvement of agalactosyl IgG in immune complex-mediated inflammation.

Circulating immune complexes contain citrullinated fibrinogen in rheumatoid arthritis

IntroductionThere is increasing evidence that autoantibodies and immune complexes (ICs) contribute to synovitis in rheumatoid arthritis (RA), yet the autoantigens incorporated in ICs in RA remain incompletely characterised.MethodsWe used the C1q protein to capture ICs from plasma derived from human RA and control patients. Antibodies specific for immunoglobulin were used to detect ICs, and fibrinogen antibodies were used to detect fibrinogen-containing ICs. RA and control plasma were separated by liquid chromatography, and fractions then characterised by ELISA, immunoblotting and mass spectrometry. Immunohistochemical staining was performed on rheumatoid synovial tissue.ResultsC1q-immunoassays demonstrated increased levels of IgG (p = 0.01) and IgM (p = 0.0002) ICs in plasma derived from RA patients possessing anti-cyclic citrullinated peptide (CCP+) autoantibodies as compared with healthy controls. About one-half of the anti-CCP+ RA possessed circulating ICs containing fibrinogen (p = 0.0004). Fractionation of whole RA plasma revealed citrullinated fibrinogen in the high molecular weight fractions that contained ICs. Positive correlations were observed between fibrinogen-containing ICs and anti-citrullinated fibrinogen autoantibodies, anti-CCP antibody, rheumatoid factor and certain clinical characteristics. Immunohistochemical staining demonstrated co-localisation of fibrinogen, immunoglobulin and complement component C3 in RA pannus tissue. Mass spectrometry analysis of immune complexes immunoprecipitated from RA pannus tissue lysates demonstrated the presence of citrullinated fibrinogen.ConclusionCirculating ICs containing citrullinated fibrinogen are present in one-half of anti-CCP+ RA patients, and these ICs co-localise with C3 in the rheumatoid synovium suggesting that they contribute to synovitis in a subset of RA patients.

Suppression of Collagen-Induced Arthritis by Oral Administration of theCitrusFlavonoid Hesperidin

The preventive and therapeutic effects of the Citrus flavonoid hesperidin (HES) on the development of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA), were investigated. Mice were administered orally HES three times a week starting from either the onset (day 21) of secondary immunization or on day 31, when the CIA development had reached a plateau. In both cases, treatment with HES resulted in a significant suppression of clinical scores and improvement of histological features. These results suggest that oral administration of HES could be effective for treating human RA patients.