Molecular studies on the autoantibody-mediated bone destruction in rheumatoid arthritis

Autoantibody-positive rheumatoid arthritis (RA), also called seropositive RA, is characterised by the presence of anti citrullinated proteins antibodies (ACPA) that can be detected in blood several years before disease onset. Joint inflammation, pain and bone destruction are major features of the disease. Classically bone destruction and pain have been considered to be late events in the disease development, resulting from long lasting and uncontrolled inflammation. However, both bone loss and pain have been reported in both seropositive individuals not yet having the disease and those seropositive individuals just being diagnosed with seropositive RA. Taken together these findings suggest that other factors than uncontrolled joint inflammation might drive the process of bone loss and pain in RA. We hypothesized that antibodies themselves and specifically ACPA might play a direct role in mediating bone loss and pain and aimed to investigate the role of ACPAs in bone metabolism and pain-like behaviour in mice. To investigate bone metabolism, we focused on studies of osteoclasts (OC), cells responsible for bone loss in vitro (by assessing formation and maturation of OC in cell cultures and estimating their capacity to degrade bone matrix in vitro) and in vivo (by micro-CT analysis of the bone density). We have demonstrated that polyclonal and monoclonal ACPAs isolated from the blood and synovial fluid of RA patients enhance the number of OC and of their bone resorptive capacity in vitro. This effect was accompanied by a significant increase of IL-8 levels in OC supernatants and abolished by neutralizing anti IL-8 antibodies. Further, ACPA injected in mice were shown to bind to CD68-positive OC precursors in bone marrow in the vicinity of the joints and to promote trabecular bone loss, which was also reversed by blocking the mice homologues of IL-8, CXCL1/2 by using reparixin. In parallel to bone destruction ACPA also induced pain-like behaviour in mice, that similar to bone loss was also abolished by CXCL1/2 blocking. Taken together these findings suggest that ACPA promote bone loss by inducing IL-8 that in turn can further amplify the bone loss process and induce pain-behaviour. As ACPA, but no other immunoglobulins (non-ACPA immunoglobulins from RA patients, non-ACPA Ig from healthy individuals) were able to promote bone loss, we investigated the role of citrullination in ACPA-mediated osteoclastogenesis. We demonstrated that citrullination by peptidyl arginine deiminases (PAD) enzymes is essential for the physiological development and maturation of OCs but no other cells (such as synovial fibroblasts). This finding might explain the ACPA preference for OCs. Further we showed that ACPA bind to targets expressed on the surface of OCs. Blocking the citrullination machinery by PAD enzyme inhibitors significantly abrogates ACPA binding to OCs and ACPA-mediated osteoclastogenesis. OCs could develop from different cell precursors and inflammatory conditions, such as joint inflammation in RA, promote the transdifferentitation of immature DC (iDC) into OCs. In order to investigate if ACPA might also play a role in this proces, we analyzed the capacity of polyclonal and monoclonal ACPA to promote the in vitro transdifferentation of iDC to OC. We showed that despite a clearly distinct protein profile as compared to classical macrophage OC precursors, iDC are able to develop into remarkably similar OCs. Plasticity towards OC differentiation correlated with PAD activity and protein citrullination expression levels in iDC cultures. Citrullinated actin and vimentin were present in iDCs and iDC-derived OCs and both proteins were deposited on the cell surface, co-localising with ACPAs binding to the cells. ACPAs enhanced OC differentiation from both monocyte-derived iDCs and from circulating CD1c+ DCs. Blocking either PAD activity or ACPA-induced IL-8 secretion completely abolished the stimulatory effects of citrulline-targeting antibodies on DC-OC transdifferentiation. We further explored the mechanisms involved in the plasticity of iDC and their capacity to develop into OCs showing that cell culture densities and lactate concentrations are essential mediators. DCs originating from dense cultures developed in the presence of high lactic acid doses, have high PAD activity and increased efficiency to convert into OC and erode bone. In contrast, DCs from sparse cultures have low PAD activity with decreased OC potential. In conclusion, the current thesis describes novel mechanisms by which RA-associated antibodies target OC to induce bone loss and pain. Our studies provide insights into the mechanisms by which systemic autoimmunity might target the joints and suggest potential novel ways to prevent this

Autoreactivity to malondialdehyde-modifications in rheumatoid arthritis is linked to disease activity and synovial pathogenesis

Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA are associated with inflammatory conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications. The mAbs recognized MDA-adducts in a variety of proteins. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. These anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses

Current view on the pathogenic role of anti-citrullinated protein antibodies in rheumatoid arthritis

Epidemiological findings suggest a potential role for anti-citrullinated protein antibodies (ACPAs) in rheumatoid arthritis (RA) pathogenesis. ACPA-positive RA is associated with unique genetical and environmental risk factors, in contrast to seronegative RA. ACPA-positive healthy individuals are at risk of developing RA and can develop joint pain and bone loss already before disease onset. ACPA injection triggered bone loss and pain-like behaviour in mice and, in the presence of additional arthritis inducers, exacerbated joint inflammation. In cell culture experiments, ACPAs could bind to and modulate a variety of cellular targets, such as macrophages, osteoclasts, synovial fibroblasts, neutrophil granulocytes, mast cells, dendritic cells and platelets, further underlying a potential role for these autoantibodies in triggering pathogenic pathways and providing clues for their mechanisms of action. Patient-derived ACPA clones have been characterised by unique cellular effects and multiple ways to act on the target cells. ACPAs might directly induce stimulatory signals by ligating key citrullinated cell surface molecules or, alternatively, act as immune complexes on Fc receptors and potentially other molecules that recognise carbohydrate moieties. On the contrary to experimentally manufactured ACPA clones, patient-derived ACPAs are highly promiscuous and cross-reactive, suggesting a simultaneous binding to a range of functionally relevant and irrelevant targets. Moreover, several ACPA clones recognise carbamylated or acetylated targets as well. These features complicate the identification and description of ACPA-induced pathogenic mechanisms. In the current review, we summarise recent data on the functional properties of patient-derived ACPAs and present mechanistic models on how these antibodies might contribute to RA pathogenesis

Anti-Citrullinated Protein Antibody Reactivity towards Neutrophil-Derived Antigens: Clonal Diversity and Inter-Individual Variation

Background: Why the adaptive immune system turns against citrullinated antigens in rheumatoid arthritis (RA) and whether anti-citrullinated protein antibodies (ACPAs) contribute to pathogenesis are questions that have triggered intense research, but still are not fully answered. Neutrophils may be crucial in this context, both as sources of citrullinated antigens and also as targets of ACPAs. To better understand how ACPAs and neutrophils contribute to RA, we studied the reactivity of a broad spectrum of RA patient-derived ACPA clones to activated or resting neutrophils, and we also compared neutrophil binding using polyclonal ACPAs from different patients. Methods: Neutrophils were activated by Ca2+ ionophore, PMA, nigericin, zymosan or IL-8, and ACPA binding was studied using flow cytometry and confocal microscopy. The roles of PAD2 and PAD4 were studied using PAD-deficient mice or the PAD4 inhibitor BMS-P5. Results: ACPAs broadly targeted NET-like structures, but did not bind to intact cells or influence NETosis. We observed high clonal diversity in ACPA binding to neutrophil-derived antigens. PAD2 was dispensable, but most ACPA clones required PAD4 for neutrophil binding. Using ACPA preparations from different patients, we observed high patient-to-patient variability in targeting neutrophil-derived antigens and similarly in another cellular effect of ACPAs, the stimulation of osteoclast differentiation. Conclusions: Neutrophils can be important sources of citrullinated antigens under conditions that lead to PAD4 activation, NETosis and the extrusion of intracellular material. A substantial clonal diversity in targeting neutrophils and a high variability among individuals in neutrophil binding and osteoclast stimulation suggest that ACPAs may influence RA-related symptoms with high patient-to-patient variability

Anticitrullinated protein antibodies facilitate migration of synovial tissue-derived fibroblasts

OBJECTIVES Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology. METHODS Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting. RESULTS Challenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis. CONCLUSION We propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration

Recognition of Amino Acid Motifs, Rather Than Specific Proteins, by Human Plasma Cell-Derived Monoclonal Antibodies to Posttranslationally Modified Proteins in Rheumatoid Arthritis

Objective: Antibodies against posttranslationally modified proteins are a hallmark of rheumatoid arthritis (RA), but the emergence and pathogenicity of these autoantibodies are still incompletely understood. The aim of this study was to analyze the antigen specificities and mutation patterns of monoclonal antibodies (mAb) derived from RA synovial plasma cells and address the question of antigen cross-reactivity. Methods: IgG-secreting cells were isolated from RA synovial fluid, and the variable regions of the immunoglobulins were sequenced (n = 182) and expressed in full-length mAb (n = 93) and also as germline-reverted versions. The patterns of reactivity with 53,019 citrullinated peptides and 49,211 carbamylated peptides and the potential of the mAb to promote osteoclastogenesis were investigated. Results: Four unrelated anti-citrullinated protein autoantibodies (ACPAs), of which one was clonally expanded, were identified and found to be highly somatically mutated in the synovial fluid of a patient with RA. The ACPAs recognized >3,000 unique peptides modified by either citrullination or carbamylation. This highly multireactive autoantibody feature was replicated for Ig sequences derived from B cells from the peripheral blood of other RA patients. The plasma cell-derived mAb were found to target distinct amino acid motifs and partially overlapping protein targets. They also conveyed different effector functions as revealed in an osteoclast activation assay. Conclusion: These findings suggest that the high level of cross-reactivity among RA autoreactive B cells is the result of different antigen encounters, possibly at different sites and at different time points. This is consistent with the notion that RA is initiated in one context, such as in the mucosal organs, and thereafter targets other sites, such as the joints