Akilan Krishnamurthy

Identification of a novel chemokine-dependent molecular mechanism underlying rheumatoid arthritis-associated autoantibody-mediated bone loss

Objectives Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) appear before disease onset and are associated with bone destruction. We aimed to dissect the role of ACPAs in osteoclast (OC) activation and to identify key cellular mediators in this process. Methods Polyclonal ACPA were isolated from the synovial fluid (SF) and peripheral blood of patients with RA. Monoclonal ACPAs were isolated from single SF B-cells of patients with RA. OCs were developed from blood cell precursors with or without ACPAs. We analysed expression of citrullinated targets and peptidylarginine deiminases (PAD) enzymes by immunohistochemistry and cell supernatants by cytometric bead array. The effect of an anti-interleukin (IL)-8 neutralising antibody and a pan-PAD inhibitor was tested in the OC cultures. Monoclonal ACPAs were injected into mice and bone structure was analysed by micro-CT before and after CXCR1/2 blocking with reparixin. Results Protein citrullination by PADs is essential for OC differentiation. Polyclonal ACPAs enhance OC differentiation through a PAD-dependent IL-8-mediated autocrine loop that is completely abolished by IL-8 neutralisation. Some, but not all, human monoclonal ACPAs derived from single SF B-cells of patients with RA and exhibiting distinct epitope specificities promote OC differentiation in cell cultures. Transfer of the monoclonal ACPAs into mice induced bone loss that was completely reversed by the IL-8 antagonist reparixin. Conclusions We provide novel insights into the key role of citrullination and PAD enzymes during OC differentiation and ACPA-induced OC activation. Our findings suggest that IL8-dependent OC activation may constitute an early event in the initiation of the joint specific inflammation in ACPA-positive RA.

Autoantibodies to citrullinated proteins induce joint pain independent of inflammation via a chemokine-dependent mechanism

Objective An interesting and so far unexplained feature of chronic pain in autoimmune disease is the frequent disconnect between pain and inflammation. This is illustrated well in rheumatoid arthritis (RA) where pain in joints (arthralgia) may precede joint inflammation and persist even after successful anti-inflammatory treatment. In the present study, we have addressed the possibility that autoantibodies against citrullinated proteins (ACPA), present in RA, may be directly responsible for the induction of pain, independent of inflammation. Methods Antibodies purified from human patients with RA, healthy donors and murinised monoclonal ACPA were injected into mice. Pain-like behaviour was monitored for up to 28 days, and tissues were analysed for signs of pathology. Mouse osteoclasts were cultured and stimulated with antibodies, and supernatants analysed for release of factors. Mice were treated with CXCR1/2 (interleukin (IL) 8 receptor) antagonist reparixin. Results Mice injected with either human or murinised ACPA developed long-lasting pronounced pain-like behaviour in the absence of inflammation, while non-ACPA IgG from patients with RA or control monoclonal IgG were without pronociceptive effect. This effect was coupled to ACPA-mediated activation of osteoclasts and release of the nociceptive chemokine CXCL1 (analogue to human IL-8). ACPA-induced pain-like behaviour was reversed with reparixin. Conclusions The data suggest that CXCL1/IL-8, released from osteoclasts in an autoantibody-dependent manner, produces pain by activating sensory neurons. The identification of this new pain pathway may open new avenues for pain treatment in RA and also in other painful diseases associated with autoantibody production and/or osteoclast activation.

Dendritic cell reprogramming by endogenously produced lactic acid.

The demand for controlling T cell responses via dendritic cell (DC) vaccines initiated a quest for reliable and feasible DC modulatory strategies that would facilitate cytotoxicity against tumors or tolerance in autoimmunity. We studied endogenous mechanisms in developing monocyte-derived DCs (MoDCs) that can induce inflammatory or suppressor programs during differentiation, and we identified a powerful autocrine pathway that, in a cell concentration–dependent manner, strongly interferes with inflammatory DC differentiation. MoDCs developing at low cell culture density have superior ability to produce inflammatory cytokines, to induce Th1 polarization, and to migrate toward the lymphoid tissue chemokine CCL19. On the contrary, MoDCs originated from dense cultures produce IL-10 but no inflammatory cytokines upon activation. DCs from high-density cultures maintained more differentiation plasticity and can develop to osteoclasts. The cell concentration–dependent pathway was independent of peroxisome proliferator–activated receptor γ (PPARγ), a known endogenous regulator of MoDC differentiation. Instead, it acted through lactic acid, which accumulated in dense cultures and induced an early and long-lasting reprogramming of MoDC differentiation. Our results suggest that the lactic acid–mediated inhibitory pathway could be efficiently manipulated in developing MoDCs to influence the immunogenicity of DC vaccines.

Human plasma cell derived monoclonal antibodies to post-translationally modified proteins recognize amino acid motifs rather than specific proteins

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.

A1.16 Role of IL-8 and its receptor in anti-citrullinated protein antibody mediated osteoclastogenesis in ra

Background and objectives Circulating levels of IL-8 is related to the bone loss associated with breast cancer metastasis. Recently, we have shown that anti-citrullinated protein antibodies (ACPA) can induce osteoclastogenesis. We aimed to investigate the role of IL-8 and its receptors in mediating osteoclastogenesis in presence or absence of ACPAs Methods IL-8 levels was measured by ELISA in serum of risk RA patients and synovial fluid samples of RA patients. CD14 positive monocytes were used to generate osteoclasts with M-CSF and RANKL. Inhibition of IL-8 and its receptor were tested with IL-8 neutralising antibody or small molecule CXCR1/2 anta-agonist (Reparixin and SCH-527123) on osteoclasts (OC) in the presence or absence of ACPA. The number of the OC’s were counted in light microscope after TRAP staining. Cell counting kit 8 (CCK8) assay was performed to detect cytotoxicity. CXCR1 and CXCR2 expression was analysed using flow cytometry and immunohistochemistry during different days of osteoclasts maturation. Results Increased IL-8 levels were observed in the serum of ACPA positive at risk RA individuals and in synovial fluid of established ACPA positive RA patients. Exogenous IL-8 dose dependently increased OC numbers in presence of RANKL. IL-8 neutralising antibody inhibited the OC numbers even in the presence of ACPA. Reparixin and SCH-527123 significantly inhibited RANKL mediated osteoclastogenesis at 100 µM concentration. No cytotoxicity of the drugs were detected with CCK8 assay. With flow cytometry, we observed CXCR1 and CXCR2 expression in the OC precursors at day 0 and in presence of RANKL the expression was completely lost at day 1 and it reappeared at day 4. Immunohistochemistry staining confirmed the CXCR1/2 expression on OC precursors. Conclusions Our data provides insights in to the importance of IL-8 autocrine loop in RANKL and ACPA mediated OC development. IL-8 receptor blockade might be a novel way of targeting osteoclasts in RA.

A4.17 Anti-citrullinated proteins antibodies promotes osteoclastogenesis and bone destruction in rheumatoid arthritis

Background/Purpose Presence of ACPA is a major risk factor for bone erosion in RA and antibodies against modified citrullinated vimentin induce osteoclast (OC) formation from monocytes. We aimed to identify new molecular mechanisms responsible for ACPA-mediated bone destruction by investigating the direct effect of ACPA (obtained from the synovial fluid (SF) and peripheral blood (PB) of RA patients) on osteoclastogenesis and their influence on distinct osteoclasts precursors (monocytes/macrophages (MΦ) and immature dendritic cells (DC)). Methods ACPA and non-ACPA IgGs were isolated from SF, (n = 26) and PB (n = 38) samples of RA patients. Osteoclasts precursors were generated (DC and MΦ) from ACPA+ RA patient and healthy donors PB and then differentiated into OC in presence of RANKL and M-CSF. In parallel, cells were grown on osteoassay surfaces and bone resorption area was quantified. In-house generated monoclonal anti-citrulline antibodies cloned from SF B-cells were also tested. Cytokines were measured by cytometric bead array in culture supernatants. Mass spectrometry (MS) analysis was performed on different stages of OC maturation. Immunohistochemistry (IHC) was used to stain the OCs with biotinylated ACPA IgG and monoclonal anti-citrulline antibodies. The effect of PAD inhibition (Cl amidine) and IL-8 inhibition was tested in the cultures. Results ACPAs pools enhanced osteoclastogenesis from both DC (fold change of (FC) 1.6 ± 0.14 for OC number) and MΦ (FC 2.0 ± 0.6 for OC and 1.6 ± 0.4 for bone resorption area (erosion)) of healthy donors. Similar effect was observed when the precursor cells were derived from ACPA+ RA patients in both DC (FC 2.3 ± 0.9 OC and 2.6 ± 0.1 erosion) and MΦ (FC 1.8 ± 0.6 OC and 2.3 ± 0.7 erosion). Increased osteoclastogenesis was associated with significantly higher levels of IL-8 levels in culture supernatants measured at all stages of osteoclasts maturation from both DC (FC 2.4 ± 0.5) and MΦ (FC 2.0 ± 0.5). Interestingly SF levels of IL-8 were higher in ACPA+RA patients as compared to disease controls. IL-8 neutralisation completely abolished ACPAs effects while blocking PAD enzymes resulted in a complete inhibition of OC formation. MS analysis showed the presence of actin and vimentin citrullinated during osteoclastogenesis. Two of the tested anti-citrullinated monoclonal antibodies (D10 and B2) but not a third anti-citrulline antibody (C7) nor a negative anti-tetanus control antibody (E2) bound to osteoclasts and promoted osteoclastogenesis and bone destruction, interestingly Fab fragments of the D10 and B2 antibodies retained similar effects. Conclusion In conclusion, SF and PB derived ACPA IgGs with broad specificities enhanced osteoclastogenesis from both DC and MΦ. This effect appears to be restricted to certain ACPAs specificities, IL-8 dependent and at least partially mediated through a Fab mediated mechanism.

Rheumatoid Arthritis: Transition from Systemic Autoimmunity to Joint Inflammation and Bone Loss

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease resulting from the complex interaction between genetic and environmental factors, with at least two distinct clinical phenotypes that are differentiated by the presence or absence of anti-citrullinated protein antibodies (ACPAs), i.e., ACPA-positive RA and ACPA-negative RA (Klareskog et al. 2009). These two phenotypes differ both in terms of risk factors and disease mechanisms. Specific environment-gene interactions (such as smoking and presence of the HLA-DRB1 risk allele variants) only confer risk for developing of ACPA-positive RA but not ACPA-negative RA. ACPA-positive RA has a more severe disease course, and more frequently associates with bone destruction as compared to ACPA-negative RA. More recent findings suggest that ACPA might play an important role in the transition from systemic autoimmunity (that develops before joint inflammation) to joint disease through specific targeting of the bone and activation of osteoclasts. The current chapter will review existing evidence showing that ACPA might be generated at extra-articular mucosal sites and contribute to the initiation of chronic joint inflammation.

FRI0006 Protein citrullinations by pad enzymes promote dendritic cell transdifferentiation into osteoclast and generate targets for ra-specific antibodies

Background Immature dendritic cells (DCs) are able to trans-differentiate into osteoclasts (OCs) although the mechanisms regulating this process are little understood. We have recently described an important role for protein citrulliantion and peptidylarginine deiminase (PAD) enzyme activity in the regulation of OC development (1). Objectives We studied the molecular bases of DC-OC trans-differentiation and aimed at understanding the role of protein citrullination in this process. Methods Monocyte-derived DCs and peripheral blood CD1c+ DCs were cultured in the presence of osteoclastogenic cytokines. Polyclonal ACPAs were isolated from the serum of RA patients and applied in OC cultures. DC and OC differentiation was analyzed in vitro using gene expression analyses, flow cytometry-based methods, DC-T cell co-culture experiments, tartrate-resistant acid phosphatase stainings and osteolysis assays. Protein citrullination was monitored with the help of mass spectrometry. PAD activity was measured by ELISA. Results Different DC types showed different capacities to develop into OCs. OC-prone DCs were characterized by little immunogenicity and their development was potentiated by the increase of lactic acid, a side product of glycolytic metabolism. The more immunogenic DC types, characterized by prominent ability to migrate towards secondary lymphoid tissues and trigger T cell activation, showed a limited capacity to develop into OCs (2). The differentiation switch towards the OC lineage was associated with increased activity of the Protein Arginine Deiminase (PAD) enzymes and with higher level of protein citrullination in DCs. The PAD inhibitor Cl-Amidine efficiently interfered with OC development form DC precursors. In addition, the deposition of citrullinated proteins on the cell surface made the cells sensitive for anti-citrullinated protein autoantibodies, which could further stimulate DC-OC trans-differentiation through inducing the cytokine IL-8. Conclusions Our results indicated that DCs are heterogenic in their ability to form OCs and lineages for immunostimulatory and OC-prone DCs might separate early during DC differentiation. Plasticity towards OC differentiation might be influenced by the metabolic environment and the upregulation of PAD activity in DCs. References Krishnamurthy A et al. Ann Rheum Dis 2016. Nasi A et al. J Immunol 2013. Disclosure of Interest None declared

08.24 Monoclonal acpa antibodies recognising a common citrulline motif are mainly dependent on light chain hypermutations for antigen recognition

Background Anti-citrullinated protein antibodies (ACPA) can be detected many years before disease onset, implicating that not all ACPAs are pathogenic and that the autoantibodies undergo critical maturation steps closer to disease onset. We have studied the ACPA profile of synovial fluid and of monoclonal antibodies generated from plasma cells of the same synovial fluid to address these questions. Materials and methods IgG-secreting cells from RA synovial fluid were isolated, the variable Ig regions were amplified, and recombinant monoclonal antibodies were expressed. The antibody reactivity to citrullinated peptides were addressed both by a planar multi array of RA associated peptides, as well as on a 1 75 000 peptide micro-array of arginine/citrulline peptides from extracellular matrix proteins. The matching synovial fluid was also analysed on the peptide arrays. Chimeric monoclonal antibodies were produced based on IMGT predicted germline sequences of the ACPAs, to address the importance of hypermutations in the heavy versus the light chain. The induction of osteoclastogenesis by the ACPAs were tested in an in vitro assay. Results Sera from RA-patients display wide cross-reactivity between many citrullinated proteins and peptides. Synovial fluid replicates this pattern, and a glycine-rich consensus sequence could be deduced. The monoclonal ACPAs recognised different consensus sequences, of which some replicated the dominant polyclonal pattern. To dissect the contribution of the heavy and light chain to antigen recognition, chimeric variants were expressed where either the light or the heavy chain was exchanged with the respective germline sequence, or to an unrelated chain. Surprisingly, the ACPAs with the common consensus epitope were highly dependent on the light chain mutations for citrulline reactivity, while the heavy chain appeared primarily important for stability and conformation, independent on the acquired mutations. Even if these ACPA were highly cross-reactive, they did not cause osteoclastogenesis. The pathogenic ACPA was instead dependent on hypermutations in both heavy and light chains for citrulline reactivity, and had a more restricted citrulline peptide reactivity. Conclusions Our data lend support to the observed high cross-reactivity of ACPA between citrullinated peptides. We also find differences in citrulline-reactivity patterns and light chain contribution between pathogenic and non-pathogenic ACPA.

A2.29 Immature dendritic cells are potent osteoclasts precursors in ra and are targeted by ra-specific antibodies

Background and purpose We have recently shown that the immature dendritic cells (iDC) developing in dense cultures in presence of high lactic acid levels can efficiently differentiate into osteoclasts (OC). We aimed to investigate influence of Anti-citrullinated protein antibodies (ACPA) on iDC differentiation to OC in rheumatoid arthritis (RA). Methods CD14+ monocytes from PB of ACPA+ RA patients and healthy individuals were first used to generate iDC and MΦ and these cells were then differentiated to OC with RANKL and M-CSF. Mass spectrometry analysis was performed on different stages of differentiation of iDC and MΦ derived OC. ACPA positive and negative polyclonal IgGs were isolated from synovial fluid (SF, n = 26) and peripheral blood (PB, n = 38) samples of RA patients and the effects of these antibodies was tested in OC cultures. Cytokines were measured by CBA in cultures supernatants. Immunoflourescence was done on OCs with monoclonal ACPAs derived from single SF derived B cells. The effect of IL-8 and PAD inhibition was tested on OC differentiation. Results Principal component analysis confirmed distinct proteomics profiles in sparse or dense DC cultures and in MΦ but both DC and MΦ precursors differentiated into OC with similar protein composition. Citrullinated-actin was identified during iDC-OC maturation and citrullinated-vimentin in matured OC. With immunofluorescent staining we demonstrated the presence of citrullinated proteins on the surface of both iDC precursors and iDC-derived mature OCs. PAD2 and PAD4 showed faint staining in CD14 monocytes with increased staining intensity in both iDC precursors and more mature OCs. Polyclonal ACPAs enhanced osteoclastogenesis and bone resorption from iDC (fold increase 1.6 ± 0.2 for OC number and 2.0 ± 0.3 for bone resorption area). Similar effect was observed when the iDC were derived from ACPA+ RA patients (fold increase of 2.3 ± 0.9 for OC number and 2.6 ± 0.1 for bone resorption area). The importance of citrullination and PAD enzymes for ACPA mediated iDC transdifferentiation to OC was confirmed by a dose-dependent inhibition of OC differentiation using the PAD inhibitor Cl-Amidine. Increased osteoclastogenesis was associated with significantly higher levels of IL-8 levels in cultures supernatants (fold increase of 2.4 ± 0.5). Conclusion iDC can efficiently transdifferentiate in to OC in RA and this process is enhanced by ACPA.