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The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However, the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd53−/− mice showed a defect in leukocyte transmigration induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow. Comparison of adhesion molecule expression in wild-type and Cd53−/− neutrophils revealed no alteration in expression of β2 integrins, whereas L-selectin was almost completely absent from Cd53−/− neutrophils. In addition, Cd53−/− neutrophils showed defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient transendothelial migration, as well as increased α3 integrin expression. These alterations were associated with effects on inflammation, so that in Cd53−/− mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and cytoskeletal function.

International audience; OBJECTIVES:Interleukin (IL)-38 is a newly characterised cytokine that belongs to the IL-1 family. This cytokine is expressed in the rheumatoid arthritis (RA) synovial tissue and IL-38 deficient mice have exacerbated arthritis. Here, we analysed the effect of IL-38 overexpression in the joints of arthritic mice, in human macrophages and synovial fibroblasts in vitro.METHODS:Articular injections of an adeno-associated virus (AAV) 2/8 encoding IL-38 were performed in collagen-induced arthritis (CIA), K/BxN serum transfer-induced arthritis (STIA) and antigen-induced arthritis (AIA) in mice. The effect of IL-38 overexpression was evaluated through clinical scores, immunohistochemistry, microCT, Luminex and RT-qPCR analysis. THP-1 macrophages were transduced with a lentiviral vector to overexpress IL-38.RESULTS:Clinical inflammatory scores were significantly decreased after AAV IL-38 injection in joints of mice with CIA and STIA, but not AIA. This decrease was accompanied by reduced macrophage infiltration and a decreased expression of Th17 cytokines (IL-17, IL-23, IL-22) and TNFα. However, IL-38 overexpression had no effect on cartilage or bone destruction. In vitro, the THP-1 monocytic cell line expressed less IL-6, TNFα and IL-23 after IL-38 overexpression. Conditioned media from these cells, containing released IL-38, also exert an anti-inflammatory effect on human primary macrophages and synovial fibroblasts from patients with RA.CONCLUSIONS:This study shows for the first time that IL-38 overexpression attenuates the severity of experimental arthritis. IL-38 may exert its anti-inflammatory effects by decreasing the production of proinflammatory cytokines by macrophages and synovial fibroblasts. This effect can lead to the development of novel treatment strategies in arthritis.

Microfibrillar-associated protein 4 (MFAP4) is a non-structural matrix protein with cell regulatory activities and a potential as seromarker for fibrosis. We aimed to study the occurrence of MFAP4 in the synovial membrane from patients with rheumatoid arthritis (RA) vs osteoarthritis (OA). Formaldehyde-fixed synovial tissue sections, from patients with RA (N = 6) and OA (N = 6) undergoing total hip arthroplasty, were deparaffinized and immunostained with monoclonal antibodies against MFAP4. Elastin was detected using ElastiKit. MFAP4 in serum (sMFAP4) and synovial fluid was measured by an immunoassay. MFAP4 was present in synovial biopsies from both RA and OA patients, particularly prominent in deep arterioles where it colocalized with elastin. Notably however, MFAP4 was absent from the internal elastic lamina in RA arterioles irrespective of disease duration and synovitis activity, while present although with irregular staining patterns in OA specimens. sMFAP4 was increased in RA and OA serum vs healthy controls: median (interquartile range) 29.8 (25.3-39.1) and 25.5 U/L (18.1-43.3) vs 17.7 U/L (13.7-21.2), p = 0.006 and p = 0.02, respectively The concentration of synovial fluid was lower than in serum in both RA and OA. These findings may suggest that MFAP4 is involved in adaptive vessel wall remodeling associated with chronic joint disease.

Objective: Anti-citrullinated protein antibodies (ACPAs) develop many years before the clinical onset of rheumatoid arthritis (RA). This study was undertaken to address the molecular basis of the specificity and cross-reactivity of ACPAs from patients with RA. Methods: Antibodies isolated from RA patients were expressed as monoclonal chimeric antibodies with mouse Fc. These antibodies were characterized for glycosylation using mass spectrometry, and their cross-reactivity was assessed using Biacore and Luminex immunoassays. The crystal structures of the antigen-binding fragment (Fab) of the monoclonal ACPA E4 in complex with 3 different citrullinated peptides were determined using x-ray crystallography. The prevalence of autoantibodies reactive against 3 of the citrullinated peptides that also interacted with E4 was investigated by Luminex immunoassay in 2 Swedish cohorts of RA patients. Results: Analysis of the crystal structures of a monoclonal ACPA from human RA serum in complex with citrullinated peptides revealed key residues of several complementarity-determining regions that recognized the citrulline as well as the neighboring peptide backbone, but with limited contact with the side chains of the peptides. The same citrullinated peptides were recognized by high titers of serum autoantibodies in 2 large cohorts of RA patients. Conclusion: These data show, for the first time, how ACPAs derived from human RA serum recognize citrulline. The specific citrulline recognition and backbone-mediated interactions provide a structural explanation for the promiscuous recognition of citrullinated peptides by RA-specific ACPAs.

Key cellular functions including those related to energy metabolism, organization of the genetic information or production of membrane-bound and secreted proteins are compartmentalized within organelles. Various stresses such as differentiation programs, viral and bacterial infections, perturbations in protein production, mechanical constraints, changes in the environment and nutriment accessibility can impact cellular homeostasis and organelle integrity. Perturbations of these cellular compartments trigger repair and adaptation programs aimed at restoring homeostasis. These events are often associated with low-grade inflammation also termed parainflammation. While the nature and mechanisms of danger signals released by irremediably damaged cells are well understood, how transiently stressed cells trigger inflammation is still poorly understood. Emerging studies highlighted new mechanisms by which stress pathways promote inflammation. Cytosolic innate immune pathways are engaged by signals stemming from perturbed organelles such as the mitochondria, the endoplasmic reticulum (ER) or the nuclear envelope (NE). These observations indicate that these pathways function as guardians of cellular homeostasis and may contribute to disease in pathologies characterized by perturbations of cellular homoeostasis. Mitochondria-stress, ER-stress or NE-stress are emerging as proinflammatory signals that contribute to human conditions and diseases.

Cytokines and chemokines represent two important groups of proteins that control the human immune system. Dysregulation of the network in which these immunomodulators function can result in uncontrolled inflammation, leading to various diseases including rheumatoid arthritis (RA), characterized by chronic inflammation and bone erosion. Potential triggers of RA include autoantibodies, cytokines and chemokines. The tight regulation of cytokine and chemokine production, and biological activity is important. Tumor necrosis factor-α (TNF-α) is abundantly present in RA patients' serum and the arthritic synovium. This review, therefore, discusses first the role and regulation of the major proinflammatory cytokine TNF-α, in particular the regulation of TNF-α production, post-translational processing and signaling of TNF-α and its receptors. Owing to the important role of TNF-α in RA, the TNF-α-producing cells and the dynamics of its expression, the direct and indirect action of this cytokine and possible biological therapy for RA are described.

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1β and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1β, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.

Background The contribution of smoking to rheumatoid arthritis (RA) is hypothesized to be mediated through formation of anti-citrullinated protein antibodies (ACPA). In RA, however, autoantibodies such as ACPA, rheumatoid factor (RF), and anti-carbamylated protein antibodies (anti-CarP) often occur together, and it is thus unclear whether smoking is specifically associated with some autoantibodies rather than others. We therefore investigated whether smoking is only associated with ACPA or with the presence of multiple RA-related autoantibodies. Methods A population-based Japanese cohort (n = 9575) was used to investigate the association of smoking with RF and anti-cyclic citrullinated peptide antibodies (anti-CCP2) in individuals without RA. Furthermore, RA patients fulfilling the 1987 criteria from three early arthritis cohorts from the Netherlands (n = 678), the United Kingdom (n = 761), and Sweden (n = 795) were used. Data on smoking, RF, anti-CCP2, and anti-CarP were available. A total score of autoantibodies was calculated, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated by logistic regression. Results In the population-based non-RA cohort, no association was found between smoking and one autoantibody (RF or anti-CCP2), but smoking was associated with double-autoantibody positivity (OR 2.95, 95% CI 1.32–6.58). In RA patients, there was no association between smoking and the presence of one autoantibody (OR 0.99, 95% CI 0.78–1.26), but smoking was associated with double-autoantibody positivity (OR 1.32, 95% CI 1.04–1.68) and triple-autoantibody positivity (OR 2.05, 95% CI 1.53–2.73). Conclusions Smoking is associated with the concurrent presence of multiple RA-associated autoantibodies rather than just ACPA. This indicates that smoking is a risk factor for breaking tolerance to multiple autoantigens in RA. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1177-9) contains supplementary material, which is available to authorized users.

BACKGROUND: Hepatitis E virus (HEV) is the most recently discovered of the hepatotropic viruses, and is considered an emerging pathogen in developed countries with the possibility of fulminant hepatitis in immunocompromised patients. Especially in the latter elevated transaminases should be taken as a clue to consider HEV infection, as it can be treated by discontinuation of immunosuppression and/or ribavirin therapy. To our best knowledge, this is a unique case of autochthonous HEV infection with coincident reactivation of Epstein-Barr virus (EBV) infection in an immunosuppressed patient with rheumatoid arthritis (RA). CASE PRESENTATION: A 68-year-old Swiss woman with RA developed hepatitis initially diagnosed as methotrexate-induced liver injury, but later diagnosed as autochthonous HEV infection accompanied by reactivation of her latent EBV infection. She showed confounding serological results pointing to three hepatotropic viruses (HEV, Hepatitis B virus (HBV) and EBV) that could be resolved by detection of HEV and EBV viraemia. The patient recovered by temporary discontinuation of immunosuppressive therapy. CONCLUSIONS: In immunosuppressed patients with RA and signs of liver injury, HEV infection should be considered, as infection can be treated by discontinuation of immunosuppression. Although anti-HEV-IgM antibody assays can be used as first line virological tools, nucleic acid amplification tests (NAAT) for detection of HEV RNA are recommended--as in our case--if confounding serological results from other hepatotropic viruses are obtained. After discontinuation of immunosuppressive therapy, our patient recovered from both HEV infection and reactivation of latent EBV infection without sequelae.

Traditional treatments, including a variety of thermal therapies have been known since ancient times to provide relief from rheumatoid arthritis (RA) symptoms. However, a general absence of information on how heating affects molecular or immunological targets relevant to RA has limited heat treatment (HT) to the category of treatments known as “alternative therapies”. In this study, we evaluated the effectiveness of mild HT in a collagen-induced arthritis (CIA) model which has been used in many previous studies to evaluate newer pharmacological approaches for the treatment of RA, and tested whether inflammatory immune activity was altered. We also compared the effect of HT to methotrexate, a well characterized pharmacological treatment for RA. CIA mice were treated with either a single HT for several hours or daily 30 minute HT. Disease progression and macrophage infiltration were evaluated. We found that both HT regimens significantly reduced arthritis disease severity and macrophage infiltration into inflamed joints. Surprisingly, HT was as efficient as methotrexate in controlling disease progression. At the molecular level, HT suppressed TNF-α while increasing production of IL-10. We also observed an induction of HSP70 and a reduction in both NF-κB and HIF-1α in inflamed tissues. Additionally, using activated macrophages in vitro, we found that HT reduced production of pro-inflammatory cytokines, an effect which is correlated to induction of HSF-1 and HSP70 and inhibition of NF-κB and STAT activation. Our findings demonstrate a significant therapeutic benefit of HT in controlling arthritis progression in a clinically relevant mouse model, with an efficacy similar to methotrexate. Mechanistically, HT targets highly relevant anti-inflammatory pathways which strongly support its increased study for use in clinical trials for RA.

With interest we read the article by Hecht et al 1 who studied the associations of anticitrullinated peptide antibodies (ACPA) and rheumatoid factor (RF) with bone erosions in rheumatoid arthritis (RA). The background of this study was that ACPAs directed against citrullinated vimentin induce differentiation of osteoclasts, indicating that ACPA has a direct effect on bone resorption.2 No biological data are available on whether RF also affects bone cells and, given the overlap between ACPA-positivity and RF-positivity, the authors wanted to distinguish the association between RF and bone erosions from the association between ACPA and bone erosions. In a cross-sectional study, high-resolution peripheral quantitative CT (HR-pQCT) images of metacarpophalangeal (MCP) 2–4 joints of 238 patients with RA were made and groups of patients with different combinations of autoantibodies were compared. The authors observed mainly differences in erosive burden in patients with both antibodies compared with those without antibodies. In a subgroup analysis, they also reported a higher erosive burden in ACPA+/RF+ patients compared with ACPA+/RF− patients. Based on their findings the authors suggested that RF may act as an enhancer of bone loss in ACPA-positive patients. HR-pQCT is an interesting tool that admits …

Rationale: In the absence of active tuberculosis, a positive tuberculin skin test (TST) or interferon-gamma release assay (IGRA) result defines latent infection with Mycobacterium tuberculosis, although test results may vary depending on immunodeficiency. Objectives: This study compared the performance of TST and IGRAs in five different groups of immunocompromised patients, and evaluated their ability to identify those at risk for development of tuberculosis. Methods: Immunocompromised patients with HIV infection, chronic renal failure, rheumatoid arthritis, solid-organ or stem-cell transplantation, and healthy control subjects were evaluated head-to-head by the TST, QuantiFERON-TB-Gold in-tube test (ELISA), and T-SPOT. TB test (enzyme-linked immunospot) at 17 centers in 11 European countries. Development of tuberculosis was assessed during follow-up. Measurements and Main Results: Frequencies of positive test results varied from 8.7 to 15.9% in HIV infection (n = 768), 25.3 to 30.6% in chronic renal failure (n = 270), 25.0% to 37.2% in rheumatoid arthritis (n = 199), 9.0 to 20.0% in solid-organ transplant recipients (n = 197), 0% to 5.8% in stem-cell transplant recipients (n = 103), and 11.2 to 15.2% in immunocompetent control subjects (n = 211). Eleven patients (10 with HIV infection and one solid-organ transplant recipient) developed tuberculosis during a median follow-up of 1.8 (interquartile range, 0.2-3.0) years. Six of the 11 patients had a negative or indeterminate test result in all three tests at the time of screening. Tuberculosis incidence was generally low, but higher in HIV-infected individuals with a positive TST (3.25 cases per 100 person-years) than with a positive ELISA (1.31 cases per 100 person-years) or enzyme-linked immunospot result (1.78 cases per 100 person-years). No cases of tuberculosis occurred in patients who received preventive chemotherapy. Conclusions: Among immunocompromised patients evaluated in this study, progression toward tuberculosis was highest in HIV-infected individuals and was poorly predicted by TST or IGRAs.

Dendritic cells (DCs) are professional APCs that have a role in the initiation of adaptive immune responses and tolerance. Among the tolerogenic mechanisms, the expression of the enzyme IDO1 represents an effective tool to generate T regulatory cells. In humans, different DC subsets express IDO1, but less is known about the IDO1-related enzyme IDO2. In this study, we found a different pattern of expression and regulation between IDO1 and IDO2 in human circulating DCs. At the protein level, IDO1 is expressed only in circulating myeloid DCs (mDCs) and is modulated by PGE2, whereas IDO2 is expressed in both mDCs and plasmacytoid DCs and is not modulated by PGE2. In healthy subjects, IDO1 expression requires the presence of PGE2 and needs continuous transcription and translation, whereas IDO2 expression is constitutive, independent from suppressor of cytokine signaling 3 activity. Conversely, in patients suffering from inflammatory arthritis, circulating DCs express both IDO1 and IDO2. At the functional level, both mDCs and plasmacytoid DCs generate T regulatory cells through an IDO1/IDO2-dependent mechanism. We conclude that, in humans, whereas IDO1 provides an additional mechanism of tolerance induced by proinflammatory mediators, IDO2 is stably expressed in steady-state conditions and may contribute to the homeostatic tolerogenic capacity of DCs.

Cytokines and chemokines represent two important groups of proteins that control the immune system. Dysregulation of the network in which these immunomodulators function can result in uncontrolled inflammation leading to various diseases, including rheumatoid arthritis, characterized by chronic inflammation and bone erosion. Chemokine activity is regulated at multiple levels, such as post-translational modification (PTM) of chemokines and their receptors by specific enzymes including proteases and peptidylarginine deiminases. Many in vitro experiments underscore the importance of post-translational processing of human chemokines. PTMs may enhance or reduce chemokine activity or may alter the receptor specificity of chemokine ligands. However, identification of chemokine isoforms in physiological in vivo settings forms the ultimate proof that PTM of chemokines is relevant in regulating the biological activity of these molecules. This review summarizes current knowledge on the in vivo role for PTMs in the regulation of chemokine activity.

Objective: The antigenic targets of the rheumatoid arthritis (RA)-associated autoantibodies to “citrullinated” proteins are generated following citrullination by a peptidylarginine deiminase (PAD). Of the five PAD isotypes, two – PAD2 and PAD4 – are expressed in RA synovial tissue. Within this tissue, the activation of macrophages and fibroblasts mediated by T-cell contact or driven by cytokines plays a prominent part in the pathogenesis. We wanted to determine whether cytokine stimulation and contact with T cells could play a role in PAD expression by peripheral blood monocytes and fibroblastic synoviocytes. Methods: Human monocytes and T lymphocytes were derived from the blood of healthy donors. HUT-78 cells and T lymphocytes were stimulated with PHA and PMA. Human synovial fibroblasts were isolated after surgical synoviectomy. The expression of PAD was determined by real-time PCR and immunoblot. Results: In monocytes, the PADI2 and PADI4 mRNAs were transiently up-regulated by contact with stimulated HUT-78 and/or T lymphocytes. Positive modulation of the PAD2 and PAD4 proteins were also observed upon contact with stimulated HUT-78 T cells. Stimulation by IL-1β or IFN-β did not modify the PADI2 and PADI4 mRNAs, but enhanced PAD4 protein expression. No isotype of PAD was detected at the mRNA or protein level in resting or stimulated synovial fibroblasts. Conclusion: Contact between stimulated T cells and monocyte-macrophages or cytokine-activated monocyte-macrophages constitutes a highly likely source of PAD2 and PAD4, which are observed in inflamed synovial tissues. In contrast, it is most unlikely that fibroblastic synoviocytes contribute to PAD expression in rheumatoid synovial membranes.

Clinically relevant joint destruction has now become infrequent in patients with newly diagnosed rheumatoid arthritis (RA), while other disease outcomes such as functional ability and disease persistence have become more important. Recently, novel auto-antibody reactivities have been identified in RA, with the ultimate aim to better characterise patients with RA. Anticarbamylated protein (anti-CarP) antibodies were recently identified and associated with more severe radiographic progression,1 but the association with other outcomes was unexplored. With great interest we read the study by Humphreys et al ,2 reporting more severe functional disability in anti-CarP positive patients with inflammatory polyarthritis. According to the reported unadjusted β-coefficient (95% CI), patients with anti-CarP antibodies had 0.21 (0.14 to 0.29) higher scores on the Health Assessment Questionnaire (HAQ) during a median follow-up duration of 8 years. This association was statistically independent of the presence of anticitrullinated peptide antibodies (ACPA) and rheumatoid factor (RF). In general replication …

The presence of rheumatoid factors (RF) is a characteristic feature of patients with rheumatoid arthritis, but not systemic lupus erythematosus. In this study, we have explored the role of the anti-apoptotic Bcl-2 protein and the Y-linked autoimmune acceleration (Yaa) mutation in the production of IgG RF in comparison with IgG anti-DNA autoimmune responses. Analysis in C57BL/6 mice, in their F1 hybrids with lupus-prone NZW mice, and in bone marrow chimeras containing mixtures of C57BL/6 bcl-2-transgenic and BXSB non-transgenic cells demonstrated that an enforced Bcl-2 expression in B cells promoted the induction of IgG anti-DNA production in these mice, while significant IgG RF responses were observed only in mice developing high levels of gp70-anti-gp70 immune complexes and lethal glomerulonephritis. Moreover, in contrast to a synergistic interaction between the Yaa mutation and Bcl-2 overexpression on IgG anti-DNA production, the Yaa mutation failed to enhance the production of IgG RF induced in bcl-2-transgenic mice. Our results reveal that defects in the regulation of B cell apoptosis play a critical role in the production of IgG RF, and that the Yaa mutation differentially modulates RF and anti-DNA autoimmune responses, likely related to the nature of autoantigens involved in each autoimmune response.