Rheumatoid arthritis is associated with increased gut permeability and bacterial translocation which are reversed by inflammation control.

OBJECTIVE: to assess how rheumatoid arthritis (RA) and Disease Modifying Anti Rheumatic Drugs (DMARDs) affect gut permeability. METHODS: to explore colonic mucosa integrity, tight junction proteins ZO-1, occludin and claudin 2 were quantified by immunohistochemistry on colonic biopsies in 20 RA patients and 20 age- and sex-matched controls. Staining intensity was assessed by two blinded independent readers. To explore intestinal permeability, serum concentrations of LPS-binding protein (LBP), sCD14 and zonulin-related proteins (ZRP) were evaluated by ELISA in another cohort of 59 RA: 21 patients naive of DMARDs (17 before and after introduction of a conventional synthetic (cs) DMARDs), 38 patients with severe RA (before and after introduction of a biological (b) DMARDs), and 33 healthy controls. RESULTS: Z0-1 protein was less expressed in colon of RA patients than controls (mean score ± SEM of 1.6 ± 0.56 vs 2.0 ± 0.43; p= 0.01), while no significant difference was detected for occludin and claudin-2. RA patients had higher serum LBP and sCD14 concentrations than controls. LBP and sCD14 levels were significantly correlated with DAS28 (r = 0.61, p= 0.005 and r = 0.57, p= 0.01, respectively) while ZRP did not. bDMARD responders had significantly reduced LBP and sCD14 concentrations unlike bDMARDs non-responders and patients treated with csDMARDs. CONCLUSION: RA patients have altered colonic tight junction proteins and increased serum biomarkers of intestinal permeability. There was a correlation between serological markers of intestinal permeability and disease activity as well as bDMARD response. These results suggest a link between impaired gut integrity and systemic inflammation in RA.

Rheumatoid arthritis is associated with increased gut permeability and bacterial translocation which are reversed by inflammation control.

OBJECTIVE: to assess how rheumatoid arthritis (RA) and Disease Modifying Anti Rheumatic Drugs (DMARDs) affect gut permeability. METHODS: to explore colonic mucosa integrity, tight junction proteins ZO-1, occludin and claudin 2 were quantified by immunohistochemistry on colonic biopsies in 20 RA patients and 20 age- and sex-matched controls. Staining intensity was assessed by two blinded independent readers. To explore intestinal permeability, serum concentrations of LPS-binding protein (LBP), sCD14 and zonulin-related proteins (ZRP) were evaluated by ELISA in another cohort of 59 RA: 21 patients naive of DMARDs (17 before and after introduction of a conventional synthetic (cs) DMARDs), 38 patients with severe RA (before and after introduction of a biological (b) DMARDs), and 33 healthy controls. RESULTS: Z0-1 protein was less expressed in colon of RA patients than controls (mean score ± SEM of 1.6 ± 0.56 vs 2.0 ± 0.43; p= 0.01), while no significant difference was detected for occludin and claudin-2. RA patients had higher serum LBP and sCD14 concentrations than controls. LBP and sCD14 levels were significantly correlated with DAS28 (r = 0.61, p= 0.005 and r = 0.57, p= 0.01, respectively) while ZRP did not. bDMARD responders had significantly reduced LBP and sCD14 concentrations unlike bDMARDs non-responders and patients treated with csDMARDs. CONCLUSION: RA patients have altered colonic tight junction proteins and increased serum biomarkers of intestinal permeability. There was a correlation between serological markers of intestinal permeability and disease activity as well as bDMARD response. These results suggest a link between impaired gut integrity and systemic inflammation in RA.

Glutamine promotes the generation of B10+ cells via the mTOR/GSK3 pathway.

Alterations in cell metabolism can shift the differentiation of immune cells toward a regulatory or inflammatory phenotype, thus, opening up new therapeutic opportunities for immune-related diseases. Indeed, growing knowledge on T- cell metabolism has revealed differences in the metabolic programs of suppressive Tregs as compared to inflammatory Th1 and Th17 cells. In addition to Tregs, IL-10-producing regulatory B cells are crucial for maintaining tolerance, inhibiting inflammation, and autoimmunity. Yet, the metabolic networks regulating diverse B-lymphocyte responses are not well known. Here, we show that glutaminase blockade decreased downstream mTOR activation and attenuated IL-10 secretion. Direct suppression of mTOR activity by rapamycin selectively impaired IL-10 production by B cells whereas secretion was restored upon Glycogen synthase kinase 3 (GSK3) inhibition. Mechanistically, we found mTORC1 activation leads to GSK3 inhibition, identifying a key signalling pathway regulating IL-10 secretion by B lymphocytes. Thus, our results identify glutaminolysis and the mTOR/GSK3 signalling axis, as critical regulators of the generation of IL-10 producing B cells with regulatory functions.

CXCR5/CXCL13 pathway, a key driver for migration of regulatory B10 cells, is defective in patients with rheumatoid arthritis.

OBJECTIVES: Chemokines (CKs) are key players of immune-cell homing and differentiation. CK receptors (CKRs) can be used to define T-cell functional subsets. We aimed to characterize the CKR profile of the regulatory B-cell subset B10+ cells and investigate the CKs involved in their migration and differentiation in healthy donors and patients with RA. METHODS: RNA sequencing and cytometry were used to compare CKR expression between B10+ and B10neg cells. Migration of B10+ and B10neg cells and IL-10 secretion of B cells in response to recombinant CKs or synovial fluid (SF) were assessed. RESULTS: CXCR5 was expressed at a higher level on the B10+ cell surface as compared with other B cells (referred to as B10neg cells). In line with this, its ligand CXCL13 preferentially attracted B10+ cells over B10neg cells. Interestingly, synovial fluid from RA patients contained high levels of CXCL13 and induced strong and preferential migration of B10+ cells. Besides its role in attracting B10+ cells, CXCL13 also promoted IL-10 secretion by B cells. In RA patients, the level of CXCR5 on B-cell surface was reduced. The preferential migration of RA B10+ cells toward CXCL13-rich SF was lost and CXCL13 stimulation triggered less IL-10 secretion than in healthy donors. CONCLUSION: Our results identify that the CXCR5/CXCL13 axis is essential for B10+ cell biology but is defective in RA. Restoring the preferential migration of B10+ within the affected joints to better control inflammation may be part of the therapeutic approach for RA.

IL-10 Producing B Cells Ability to Induce Regulatory T Cells Is Maintained in Rheumatoid Arthritis.

Despite growing evidence highlighting the relevance of increasing IL-10-producing B cells (B10+cells) in autoimmune diseases, their functions in patients are still unknown. The aim of this study was to evaluate the functions of CpG-induced B10+ cells isolated from healthy controls (HC) and rheumatoid arthritis (RA) patients, on naïve T cell differentiation. We demonstrated that CpG-induced B10+ cells from HC drove naïve T cell differentiation toward regulatory T cells (Treg cells) and IL-10-producing T cells (Tr1) through IL-10 secretion and cellular contacts. B10+ cells from HC did not decrease T helper 1 (Th1) nor and tumor necrosis factor α producing T cell (TNFα+ T cell) differentiation. We showed that in RA, B10+ cells could also induce Treg cells and Tr1 from naïve T cells. Contrary to HC, B10+ cells from RA patients increased naïve T cell conversion into Th1. Interestingly, PD-L2, a programmed death-1 (PD-1) ligand that inhibits PD-L1 and promotes Th1 differentiation, was overexpressed on RA B10+ cells compared to HC B10+ cells. Together, our findings showed that CpG-induced B10+ cells may be used to increase Treg cells in patients with RA. However, CpG may not be the most adequate stimuli as CpG-induced B10+ cells also increased inflammatory T cells in those patients.

PhosphoLipid transfer protein (PLTP) exerts a direct pro-inflammatory effect on rheumatoid arthritis (RA) fibroblasts-like-synoviocytes (FLS) independently of its lipid transfer activity.

Rheumatoid arthritis (RA) is a chronic inflammatory rheumatic disease with modification of lipids profile and an increased risk of cardiovascular events related to inflammation. Plasma phospholipid transfer protein (PLTP) exerts a lipid transfer activity through its active form. PLTP can also bind to receptors such as ATP-binding cassette transporter A1 (ABCA1). In addition to its role in lipoprotein metabolism and atherosclerosis, the latest advances came in support of a complex role of PLTP in the regulation of the inflammatory response, both with pro-inflammatory or anti-inflammatory properties. The aim of the present study was to decipher the role of PLTP in joint inflammation and to assess its relevance in the context of RA. PLTP expression was examined by western-blot and by immunochemistry. ABCA1 expression was analyzed by flow cytometry. Lipid transfer activity of PLTP and pro-inflammatory cytokines were measured in sera and synovial fluid (SF) from RA patients and controls (healthy subjects or osteoarthritis patients [OA]). FLS were treated with both lipid-transfer active form and inactive form of recombinant human PLTP. IL-8, IL-6, VEGF and MMP3 produced by FLS were assessed by ELISA, and proliferation by measuring 3H-Thymidine incorporation. RA synovial tissues showed higher PLTP staining than OA and PLTP protein levels were also significantly higher in RA-FLS. In addition, RA, unlike OA patients, displayed elevated levels of PLTP activity in SF, which correlated with pro-inflammatory cytokines. Both lipid-transfer active and inactive forms of PLTP significantly increased the production of cytokines and proliferation of FLS. ABCA1 was expressed on RAFLS and PLTP activated STAT3 pathway. To conclude, PLTP is highly expressed in the joints of RA patients and may directly trigger inflammation and FLS proliferation, independently of its lipid transfer activity. These results suggest a pro-inflammatory role for PLTP in RA.

Sodium excretion is higher in patients with rheumatoid arthritis than in matched controls.

OBJECTIVE: It was shown that sodium can promote auto-immunity through the activation of the Th17 pathway. We aimed to compare sodium intake in patients with rheumatoid arthritis (RA) vs. matched controls. METHODS: This case-control study included 24 patients with RA at diagnosis and 24 controls matched by age, gender and body mass index. Sodium intake was evaluated by 24-hr urinary sodium excretion. RESULTS: Sodium excretion was greater for patients with early RA (2,849±1,350 vs. 2,182±751.7mg/day, p = 0.039) than controls. This difference remained significant after adjustment for smoking and the use of anti-hypertensive and nonsteroidal anti-inflammatory drugs (p = 0.043). Patients with radiographic erosion at the time of diagnosis had a higher sodium excretion than those without (p = 0.028). CONCLUSION: Patients with early RA showed increased sodium excretion which may have contributed to autoimmunity.

Phosphatidylserine Outer Layer Translocation Is Implicated in IL-10 Secretion by Human Regulatory B Cells.

B cells can have a regulatory role, mainly mediated by interleukin 10 (IL-10). IL-10 producing B cells (B10 cells) cells remain to be better characterized. Annexin V binds phosphatidylserine (PS), which is externalized during apoptosis. Previous works suggested that B10 cells are apoptotic cells since they bind Annexin V. Others showed that Annexin V binding could also be expressed on viable B cells. We aimed to explore if PS exposure can be a marker of B10 cells and if PS exposure has a functional role on B cell IL-10 production in healthy subjects. We found that B10 cells were significantly more often Annexin V+ than IL-10 non-producing B cells. After CpG activation, Annexin V+ B cells differentiated more often into B10 cells than Annexin Vneg B cells. Cell death and early apoptosis were similar between Annexin V+ and Annexin Vneg B cells. PS blockage, using biotinylated AnV and glyburide, decreased B10 cell differentiation. This study showed that B10 cells have an increased PS exposure independently of any apoptotic state. B cells exposing PS differentiate more into B10 cells whereas PS blockage inhibits B10 cells generation. These results strongly suggest a link between PS exposure and B10 cells.

A proliferation inducing ligand (APRIL) promotes IL-10 production and regulatory functions of human B cells.

B cells may have a negative regulatory role, mainly mediated by interleukin 10 (IL-10). We recently showed that regulatory B-cell functions are impaired in patients with rheumatoid arthritis (RA) and that mice transgenic for a proliferation-inducing ligand (APRIL) are protected against collagen-induced arthritis. We aimed to explore the effect of APRIL on human B-cell IL-10 production, in healthy subjects and in patients with RA. The IL-10 production of B-cell was greater with APRIL than with BLyS or control medium, in a dose dependent manner. TACI expression was greater in IL-10 producing B cells (B10) than non-IL-10-producing B cells whereas BAFF-R expression was lower. TNF-α and IFN-γ secretion of T-cells were decreased by APRIL-stimulated B cells. APRIL stimulated STAT3 and STAT3 inhibition decreased B10 cells. APRIL also promoted B10 cells in RA patients. In conclusion, APRIL but not BLyS promotes IL-10 production by CpG-activated B cells and enhances the regulatory role of B cells on T cells. B10 cells in RA patients are responsive to APRIL, which suggests a possible therapeutic application of APRIL to expand B10 cells. This could also explain the difference of clinical efficacy observed between belimumab and atacicept in RA.

Osteoprotegerin and tumor necrosis factor-related apoptosis-inducing ligand as prognostic factors in rheumatoid arthritis: results from the ESPOIR cohort.

INTRODUCTION: We previously reported that low ratio of osteoprotegerin (OPG) to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was associated with Disease Activity Score in 28 joints (DAS28) remission at 6 months in patients with early rheumatoid arthritis (RA). Here, we aimed to evaluate the value of baseline OPG/TRAIL ratio in predicting clinical and radiological outcomes in patients with early RA in the ESPOIR cohort. METHODS: OPG and TRAIL serum concentrations were assessed in the ESPOIR cohort patients. Patients with definite RA were included in this study. Patients were excluded if they had high erosion score at baseline (>90(th) percentile) or received biological therapy during the first 2 years of follow-up. Data were analyzed by univariate analysis and multivariate logistic regression to predict 1-year DAS28 remission and 2-year radiographic disease progression. RESULTS: On univariate analysis of 399 patients, OPG/TRAIL ratio at baseline was significantly lower in patients with than without remission at 1 year (p = 0.015). On multivariate logistic regression including age, gender, body mass index and DAS28, low OPG/TRAIL ratio was independently associated with remission at 1 year (odds ratio 1.68 [95 % confidence interval 1.01-2.79]). On univariate analysis, high OPG/TRAIL ratio at baseline was associated with rapid progression of erosion at 2 years (p = 0.041), and on multivariate logistic regression including age, anti-citrullinated protein antibody positivity and C-reactive protein level, OPG/TRAIL ratio independently predicted rapid progression of erosion at 2 years. CONCLUSIONS: OPG/TRAIL ratio at baseline was an independent predictor of 1-year remission and 2-year rapid progression of erosion for patients with early rheumatoid arthritis. Thus, OPG/TRAIL ratio could be included in matrix prediction scores to predict rapid radiographic progression. Further confirmation in an independent cohort is warranted.

High levels of natural killer cells are associated with response to tocilizumab in patients with severe rheumatoid arthritis.

OBJECTIVES: We aimed to assess the effect of tocilizumab (TCZ), an IL-6 receptor inhibitor, on B, T, NK and NKT cells in patients with RA and to study the cell type predictors of remission. We also compared NK cells in patients with RA and in controls. METHODS: RA patients included in the study met the 2010 ACR/European League Against Rheumatism (EULAR) criteria, were receiving stable doses of steroids and had not received rituximab in the previous year. Different B and T cell subsets, NK cells and NKT cells were assessed by flow cytometry along with perforin A and granzyme B to estimate NK cell cytotoxicity. RESULTS: We included 92 RA patients, including 20 requiring TCZ treatment and 15 requiring anti-TNF drugs, and 25 controls. At baseline, the proportion of CD56(dim)CD16(+)CD3(-) NK cells was inversely correlated with the 28-joint DAS (DAS28). In TCZ-treated patients, the baseline proportion of CD3(-)CD56(+) NK cells was inversely correlated with the change in DAS28 at 3 months and the proportion was 3-fold greater for patients with DAS28 remission at 3 months than other patients. Change in the proportion of CD56(bri)CD16(-) NK cells was linearly correlated with change in the DAS28 at 3 months. The baseline proportion of NK cells did not predict change in disease activity at 3 months with anti-TNF therapy. The perforin content in NK cells increased with TCZ treatment. CONCLUSION: This study supports NK cell involvement in RA and in the TCZ mechanism of action. NK cells at baseline could be a predictive factor of TCZ response if results are confirmed.

Targeting the Fas/FasL system in Rheumatoid Arthritis therapy: Promising or risky?

Rheumatoid Arthritis (RA) is a chronic inflammatory disease affecting synovial joints. Tumor necrosis factor (TNF) α is a key component of RA pathogenesis and blocking this cytokine is the most common strategy to treat the disease. Though TNFα blockers are very efficient, one third of the RA patients are unresponsive or present side effects. Therefore, the development of novel therapeutic approaches is required. RA pathogenesis is characterized by the hyperplasia of the synovium, closely associated to the pseudo-tumoral expansion of fibroblast-like synoviocytes (FLS), which invade and destroy the joint structure. Hence, depletion of RA FLS has been proposed as an alternative therapeutic strategy. The TNF family member Fas ligand (FasL) was reported to trigger apoptosis in FLS of arthritic joints by binding to its receptor Fas and therefore suggested as a promising candidate for targeting the hyperplastic synovial tissue. However, this cytokine is pleiotropic and recent data from the literature indicate that Fas activation might have a disease-promoting role in RA by promoting cell proliferation. Therefore, a FasL-based therapy for RA requires careful evaluation before being applied. In this review we aim to overview what is known about the apoptotic and non-apoptotic effects of Fas/FasL system and discuss its relevance in RA.

Distinct effects of soluble and membrane-bound fas ligand on fibroblast-like synoviocytes from rheumatoid arthritis patients.

OBJECTIVE: Injection of agonistic anti-Fas antibody has been shown to decrease disease symptoms in mouse models of arthritis. Additionally, membrane-bound FasL (mFasL) has been shown to induce cell death in fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients. However, levels of soluble FasL (sFasL) are increased in the joints of RA patients and have been associated with disease severity, indicating that mFasL and sFasL play opposing roles in RA. The purpose of this study was to analyze the effects of FasL on RA FLS responses. METHODS: The responses of FLS from RA and osteoarthritis (OA) patients to soluble and oligomeric FasL, the latter mimicking mFasL, were analyzed by fluorescence-activated cell sorting and proliferation assays, using 3 different FasL variants. The signaling pathways that trigger FasL responses were characterized by Western blotting. RESULTS: We found that mFasL and sFasL have distinct roles in RA FLS. Crosslinked FasL preferentially induced apoptosis, whereas sFasL stimulated proliferation. Moreover, sFasL activated several signaling pathways in RA FLS, such as ERK-1/2, phosphatidylinositol 3-kinase, caspase 8, and JNK, with a prominent role of JNK, since only the blockade of this pathway rendered FLS more susceptible to FasL-induced apoptosis. Crosslinked FasL induced apoptosis in FLS from OA patients, but sFasL failed to stimulate their proliferation. CONCLUSION: Our findings suggest that sFasL is a disease promoter in RA, a finding consistent with previous reports describing a tumor-promoting role of FasL. Therefore, blocking of sFasL could be a therapeutic strategy for RA.

Regulatory B10 cells are decreased in patients with rheumatoid arthritis and are inversely correlated with disease activity.

OBJECTIVE: Regulatory interleukin-10 (IL-10)-producing B cells (B10 cells) have been shown to prevent and cure collagen-induced arthritis in mice. In humans, very little is known about B10 cells in rheumatoid arthritis (RA). Several B cell subsets, such as CD24(high) CD38(high) , CD24(high) CD27+, and CD5+ B cells, were suggested to be precursors of B10 cells. We aimed to analyze these B cell subsets and B10 cells in RA patients and healthy controls. METHODS: B10 cells were generated from peripheral blood mononuclear cells stimulated for 24 hours with CpG and for 4 hours with phorbol 12-myristate 13-acetate/ionomycin/brefeldin A. Intracellular B cell IL-10 was assessed by flow cytometry. Thirty-one controls and 99 RA patients were included. RESULTS: After multiple adjustments, levels of CD24(high) CD38(high) , CD24(high) CD27+, and CD5+ B cells were found to be similar in RA patients and controls. Levels of B10 cells were lower in RA patients than in controls, especially in patients with RA of ≤5 years' duration. Levels of B10 cells correlated inversely with the Disease Activity Score in 28 joints. This was more pronounced in patients with RA of ≤5 years' duration, in whom B10 cells also correlated inversely with C-reactive protein levels. Moreover, B10 cells correlated inversely with rheumatoid factor levels. CD24(high) CD38(high) and CD24(high) CD27+ B cells induced more Treg cells than did CD24(low) B cells in controls but not in RA patients. CONCLUSION: The ability of B cells to produce IL-10 was altered in RA, and this impairment influenced disease activity, biologic inflammation, and autoantibody levels, especially in patients with RA of ≤5 years' duration. This strongly suggests a role of B10 cells in RA initiation.

The two directions of TNF-related apoptosis-inducing ligand in rheumatoid arthritis.

The TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) superfamily that has been recognized for its specific pro-apoptotic effect on cancer cells and has been therefore proposed as a treatment in cancer. Studies on animal models have shown that TRAIL could also have a beneficial effect in rheumatoid arthritis (RA). This includes reports suggesting that TRAIL could be used to control the synovial hyperplasia and hyperactivation of immune cells observed in RA, but recent reports suggest a disease promoting role of TRAIL in RA. Indeed, adverse effects and mechanism of resistance could counteract beneficial effect of TRAIL. This review focuses on the role of TRAIL in immune regulation, synovial hyperplasia and joint remodeling in RA. We will also discuss the potential use of TRAIL in RA treatment.

Mechanisms and clinical relevance of TRAIL-triggered responses in the synovial fibroblasts of patients with rheumatoid arthritis.

OBJECTIVE: Results of studies in mice suggest a protective role for TRAIL in arthritis. The aim of this study was to investigate the role of TRAIL in patients with rheumatoid arthritis (RA). METHODS: In the present study, we compared RA fibroblast-like synoviocytes (FLS) that were resistant or sensitive to TRAIL-induced apoptosis and the expression of TRAIL receptors in these cells, and also investigated the clinical features of the patients from whom the FLS were derived. Furthermore, we evaluated the levels of TRAIL and its soluble decoy receptor osteoprotegerin (OPG) in patients with RA, patients with osteoarthritis (OA), and patients with spondylarthritis (SpA). RESULTS: Sensitivity to TRAIL-induced apoptosis varied in FLS from different patients, and the severity of disease in patients with RA was inversely correlated with the susceptibility of their FLS to TRAIL-induced apoptosis. TRAIL-sensitive cells expressed significantly lower levels of TRAILR-1, and silencing of TRAILR-1 increased TRAIL-induced apoptosis in RA FLS. TRAIL levels were elevated in the arthritic joints of patients with established RA, and TRAIL levels in the synovial fluid of these patients were elevated compared with levels in the synovial fluid of patients with OA or SpA. At baseline, a low OPG-to-TRAIL ratio in the sera of patients with early RA was associated with a better evolution of disease activity, but high serum levels of TRAIL at followup were associated with joint damage. CONCLUSION: These findings suggest that TRAIL has a dual role in RA, and that the resistance of RA FLS to TRAIL-induced apoptosis is associated with a disease-promoting activity of TRAIL in RA.

Apoptosis is not the major death mechanism induced by celecoxib on rheumatoid arthritis synovial fibroblasts.

Synovial hyperplasia in rheumatoid arthritis (RA) has been associated with apoptosis deficiency of RA fibroblast-like synoviocytes (FLSs). Celecoxib is a non-steroidal anti-inflammatory drug that has been demonstrated to induce apoptosis in some cellular systems. We have therefore examined the dose- and time-dependent effects of celecoxib on RA FLS viability. Treatment of RA FLSs with celecoxib for 24 hours reduced their viability in a dose-dependent manner. Analysis of celecoxib-treated RA FLSs for their content of apoptotic and necrotic cells by Annexin V staining and TO-PRO-3 uptake displayed only few apoptotic cells. Caspase 3, a key mediator of apoptosis, was not activated in celecoxib-treated RA FLSs, and the presence of specific caspase 3 or pan-caspase inhibitors did not affect celecoxib-induced cell death. Moreover, we could not detect other signs of apoptosis, such as cleavage of poly(ADP-ribose) polymerase, caspase 8 or 9, or DNA fragmentation. We therefore conclude that apoptosis is not the major death pathway in celecoxib-treated RA FLSs.