Elisa Corsiero

Neutrophil-derived microvesicles enter cartilage and protect the joint in inflammatory arthritis

Neutrophils generate chrondroprotective AnxA1-containing microvesicles that enter immune cell–impenetrable cartilage. Microparticles provide protection Neutrophils play an active role in protecting cartilage from damage by dispatching microvesicles (MVs) to do their bidding in this tissue they otherwise can’t access. Headland and colleagues found MVs present in the synovial fluid of patients with rheumatoid arthritis—an autoimmune disease that degrades cartilage in the joints. Cartilage is normally thought of as impenetrable to cells, so neutrophils send MVs, which easily enter the tissue and prevent damage induced by disease through a complex mechanism that involves the proresolving protein annexin A1 and its receptor. In two different mouse models of rheumatoid arthritis, MVs delivered locally entered the cartilage, prevented the loss of proteoglycans, and maintained cartilage integrity. This study suggests that immune cells can provide protection against tissue degradation in inflammatory arthritis and that the MVs may be manipulated to deliver therapeutics to diseased joints. Microvesicles (MVs) are emerging as a new mechanism of intercellular communication by transferring cellular lipid and protein components to target cells, yet their function in disease is only now being explored. We found that neutrophil-derived MVs were increased in concentration in synovial fluid from rheumatoid arthritis patients compared to paired plasma. Synovial MVs overexpressed the proresolving, anti-inflammatory protein annexin A1 (AnxA1). Mice deficient in TMEM16F, a lipid scramblase required for microvesiculation, exhibited exacerbated cartilage damage when subjected to inflammatory arthritis. To determine the function of MVs in inflammatory arthritis, toward the possibility of MV-based therapeutics, we examined the role of immune cell–derived MVs in rodent models and in human primary chondrocytes. In vitro, exogenous neutrophil-derived AnxA1+ MVs activated anabolic gene expression in chondrocytes, leading to extracellular matrix accumulation and cartilage protection through the reduction in stress-adaptive homeostatic mediators interleukin-8 and prostaglandin E2. In vivo, intra-articular injection of AnxA1+ MV lessened cartilage degradation caused by inflammatory arthritis. Arthritic mice receiving adoptive transfer of whole neutrophils displayed abundant MVs within cartilage matrix and revealed that MVs, but not neutrophils themselves, can penetrate cartilage. Mechanistic studies support a model whereby MV-associated AnxA1 interacts with its receptor FPR2 (formyl peptide receptor 2)/ALX, increasing transforming growth factor–β production by chondrocytes, ultimately leading to cartilage protection. We envisage that MVs, either directly or loaded with therapeutics, can be harnessed as a unique therapeutic strategy for protection in diseases associated with cartilage degeneration.

Role of lymphoid chemokines in the development of functional ectopic lymphoid structures in rheumatic autoimmune diseases

A sizeable subset of patients with the two most common organ-specific rheumatic autoimmune diseases, rheumatoid arthritis (RA) and Sjögrens syndrome (SS) develop ectopic lymphoid structures (ELS) in the synovial tissue and salivary glands, respectively. These structures are characterized by perivascular (RA) and periductal (SS) clusters of T and B lymphocytes, differentiation of high endothelial venules and networks of stromal follicular dendritic cells (FDC). Accumulated evidence from other and our group demonstrated that the formation and maintenance of ELS in these chronic inflammatory conditions is critically dependent on the ectopic expression of lymphotoxins (LT) and lymphoid chemokines CXCL13, CCL19, CCL21 and CXCL12. In this review we discuss recent advances highlighting the cellular and molecular mechanisms, which regulate the formation of ELS in RA and SS, with particular emphasis on the role of lymphoid chemokines. In particular, we shall focus on the evidence that in the inflammatory microenvironment of the RA synovium and SS salivary glands, several cell types, including resident epithelial, stromal and endothelial cells as well as different subsets of infiltrating immune cells, have been shown to be capable of producing lymphoid chemokines. Finally, we summarize accumulating data supporting the conclusion that ELS in RA and SS represent functional niches for B cells to undergo affinity maturation, clonal selection and differentiation into plasma cells autoreactive against disease-specific antigens, thus contributing to humoral autoimmunity over and above that of secondary lymphoid organs.

Single cell cloning and recombinant monoclonal antibodies generation from RA synovial B cells reveal frequent targeting of citrullinated histones of NETs.

Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated antigens with generation of anti-citrullinated peptide/proteins antibodies (ACPA). Currently, the nature and source of citrullinated antigens driving the humoral autoimmune response within synovial ectopic lymphoid structures (ELS) is a crucial unknown aspect of RA pathogenesis. Here we characterised the autoreactive B-cell response of lesional B cells isolated from ELS+RA synovium. Methods Single synovial tissue CD19+cells were Fluorescence Activated Cell Sorting (FACS)-sorted and VH/VL Ig genes cloned to generate recombinant monoclonal antibodies (rmAbs) from patients with ELS+/ACPA+RA. Results RA-rmAbs immunoreactivity analysis provided the following key findings: (1) in a chIP-based array containing 300 autoantigens and in a ‘citrullinome’ multiplex assay, a strong reactivity against citrullinated histones H2A/H2B (citH2A/H2B) was observed in ∼40% of RA-rmAbs, followed by cit-fibrinogen and cit-vimentin; (2) anti-citH2A/H2B-reactive RA-rmAbs (but not anti-citH2A/H2B negative) selectively recognised neutrophil extracellular traps (NETs) from peripheral blood and/or RA joint neutrophils; (3) anti-citH2A/citH2B and anti-NET immunobinding was dependent on affinity maturation and was completely abrogated following reversion of hypermutated IgVH/VL genes to germline sequences; (4) ELS+ (not ELS−) RA synovial tissues engrafted into Severe Combined ImmunoDeficiency (SCID) mice released human anti-citH2A/citH2B and anti-NET antibodies in association with the intra-graft expression of CXCL13 and lymphotoxin (LT)-β, two master regulators of ELS. Conclusion We provided novel evidence that B cells differentiated within synovial ELS in the RA joints frequent target deiminated proteins which could be generated during NETosis of RA synovial neutrophils including histones. Thus, NETs could represent a source of citrullinated antigens fuelling the ACPA autoimmune response within the RA synovium.

NETosis as Source of Autoantigens in Rheumatoid Arthritis

In neutrophils (but also in eosinophils and in mast cells), different inflammatory stimuli induce histone deimination, chromatin decondensation, and NET formation. These web-like structures that trap and kill microbes contain DNA, cationic granule proteins, and antimicrobial peptides, but the most abundant proteins are core histones. Histones contained in NETs have been deiminated, and arginines are converted in citrullines. While deimination is a physiological process amplified in inflammatory conditions, only individuals carrying genetic predisposition to develop rheumatoid arthritis (RA) make antibodies to deiminated proteins. These antibodies, collectively identified as anti-citrullinated proteins/peptides antibodies (ACPA), react with different deiminated proteins and display partially overlapping specificities. In this paper, we will summarize current evidence supporting the role of NETosis as critical mechanism in the breach of tolerance to self-antigens and in supporting expansion and differentiation of autoreactive cells. In fact, several lines of evidence connect NETosis with RA: RA unstimulated synovial fluid neutrophils display enhanced NETosis; sera from RA patients with Felty’s syndrome bind deiminated H3 and NETs; a high number of RA sera bind deiminated H4 contained in NETs; human monoclonal antibodies generated from RA synovial B cells decorate NETs and bind deiminated histones. In RA, NETs represent on one side an important source of autoantigens bearing posttranslational modifications and fueling the production of ACPA. On the other side, NETs deliver signals that maintain an inflammatory milieu and contribute to the expansion and differentiation of ACPA-producing B cells.

Ectopic Lymphoid Structures: Powerhouse of Autoimmunity.

Ectopic lymphoid structures (ELS) often develop at sites of inflammation in target tissues of autoimmune diseases, such as rheumatoid arthritis, Sjögren’s syndrome, multiple sclerosis, myasthenia gravis, and systemic lupus erythematosus. ELS are characterized by the formation of organized T/B cells aggregates, which can acquire follicular dendritic cells network supporting an ectopic germinal center response. In this review, we shall summarize the mechanisms that regulate the formation of ELS in tertiary lymphoid organs, with particular emphasis on the role of lymphoid chemokines in both formation and maintenance of ELS, the role of emerging positive and negative regulators of ELS development and function, including T follicular helper cells and IL-27, respectively. Finally, we shall discuss the main functions of ELS in supporting the affinity maturation, clonal selection, and differentiation of autoreactive B cells contributing to the maintenance and perpetuation of humoral autoimmunity.

Accumulation of Self-Reactive Naïve and Memory B Cell Reveals Sequential Defects in B Cell Tolerance Checkpoints in Sjögren’s Syndrome

Sjögren’s syndrome (SS) is an autoimmune disease characterised by breach of self-tolerance towards nuclear antigens resulting in high affinity circulating autoantibodies. Although peripheral B cell disturbances have been described in SS, with predominance of naïve and reduction of memory B cells, the stage at which errors in B cell tolerance checkpoints accumulate in SS is unknown. Here we determined the frequency of self- and poly-reactive B cells in the circulating naïve and memory compartment of SS patients. Single CD27−IgD+ naïve, CD27+IgD+ memory unswitched and CD27+IgD− memory switched B cells were sorted by FACS from the peripheral blood of 7 SS patients. To detect the frequency of polyreactive and autoreactive clones, paired Ig VH and VL genes were amplified, cloned and expressed as recombinant monoclonal antibodies (rmAbs) displaying identical specificity of the original B cells. IgVH and VL gene usage and immunoreactivity of SS rmAbs were compared with those obtained from healthy donors (HD). From a total of 353 VH and 293 VL individual sequences, we obtained 114 rmAbs from circulating naïve (n = 66) and memory (n = 48) B cells of SS patients. Analysis of the Ig V gene repertoire did not show significant differences in SS vs. HD B cells. In SS patients, circulating naïve B cells (with germline VH and VL genes) displayed a significant accumulation of clones autoreactive against Hep-2 cells compared to HD (43.1% vs. 25%). Moreover, we demonstrated a progressive increase in the frequency of circulating anti-nuclear naïve (9.3%), memory unswitched (22.2%) and memory switched (27.3%) B cells in SS patients. Overall, these data provide novel evidence supporting the existence of both early and late defects in B cell tolerance checkpoints in patients with SS resulting in the accumulation of autoreactive naïve and memory B cells.

Peripheral and synovial mechanisms of humoral autoimmunity in rheumatoid arthritis.

One of the hallmarks of rheumatoid arthritis (RA) is the development of humoral autoimmunity resulting in circulating autoantibodies. The clinical efficacy of B cell-depleting biologic treatments highlighted a key role for autoreactive B cell activation in the pathogenesis of RA. In this review, we discuss the key mechanisms leading to breach of B cell self-tolerance in the peripheral compartment. We also highlight the contribution of synovial ectopic lymphoid structures (ELS) in the development of functional niches of autoreactive B cells promoting humoral autoimmunity in the inflamed RA joints over and above secondary lymphoid organs (SLO).

A1.31 Monoclonal antibodies from CD19+ synovial B cells of RA patients with tertiary lymphoid structures display a strong immunoreactivity towards citrullinated histones from neutrophils NETs

Background and Objectives Rheumatoid arthritis (RA) is characterised by breach of self-tolerance towards citrullinated proteins. Moreover, RA patients are characterised by increased neutrophils in their synovial fluid, in particular at early stage of the disease. Recent evidence suggests a critical role of neutrophils in sustaining the inflammatory response in the RA joint. Around 40% of patients display synovial tertiary lymphoid structures (TLS) with functional B cell follicles supporting a germinal-centre response and local autoantibody production. However, the nature of the main (auto)antigenic reactivity of synovial B cells is unknown. Here we characterised the autoreactive B cell response of lesional B cells isolated from TLS + RA synovium. Materials and Methods Single CD19+ B cells were FACS sorted from synovial cell suspension of 4 TLS + RA patients. RNA was used to amplify Ig VH and VL genes and PCR products were cloned and expressed as recombinant monoclonal antibodies displaying identical specificity of the original B cells. Recombinant antibodies were then tested 1) to determine the frequency of polyreactive clones and 2) to define their immunoreactivity towards native and citrullinated antigens using a synovial antigen microarray platform. Results We obtained 139 individual VH sequences of which 33% were IgM, 40% IgG, 27% IgA and 175 VL sequences and we generated a total of 66 complete (H + L chains) recombinant monoclonal antibodies. Analysis of the VH gene somatic mutation rate showed evidence of antigen selection and intra-synovial clonal diversification. No skewed distribution of the VH and VL gene usage was observed. Around 30% of synovial monoclonal antibodies were reactive towards citrullinated histones in the antigen microarray, in particular citH2A and citH2B. This reactivity was confirmed by citH2A and citH2B ELISA. Moreover, when the synovial monoclonal antibodies were tested on neutrophil extracellular traps (NETs), which contained citrullinated histones, reactivity towards NETs proteins but not neutrophil nuclear antigens was observed. Conclusions Here we provided novel evidence that highly mutated, locally differentiated B cells within RA synovial germinal centre-like structures display a strong immunoreactive bias towards citrullinated histones which likely derived from neutrophil NETs that continuously form in the RA synovial fluid. This suggests that citrullinated histones are the main antigens driving in situ B cell activation and differentiation sustaining the humoral autoimmune response within the RA joints.

Generation of Recombinant Monoclonal Antibodies from Single B Cells Isolated from Synovial Tissue of Rheumatoid Arthritis Patients

Ectopic lymphoid structure (ELS) can form in the target tissues of patients with chronic inflammatory autoimmune diseases such as rheumatoid arthritis (RA) and Sjögrens syndrome (SS). Although it is still not clear why ELS form only in a subset of patients, it is well known that these structures can acquire features of ectopic germinal centers and contribute actively to the production of autoantibodies. Here, we describe a method to generate recombinant monoclonal antibodies from single ELS+ synovial tissue B cells obtained from RA patients. This chapter gives a detailed description of the method beginning from the mononuclear cell preparation from RA synovial tissue, single-cell sort of B cells by flow cytometry, amplification of the immunoglobulin (Ig) genes (both heavy- and light-chain genes) by PCR, and subsequent Ig gene expression vector cloning for full recombinant IgG1 monoclonal antibody (rmAb) production in vitro. The recombinant mAbs generated can be then characterized for (1) analysis of the Ig gene repertoires for clonal studies, (2) immunoreactivity profile, and (3) functional studies both in vitro and in vivo.

Characterization of a Synovial B Cell–Derived Recombinant Monoclonal Antibody Targeting Stromal Calreticulin in the Rheumatoid Joints

Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS+ RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast–like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)+ versus ACPA− RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti–cit-CRT Abs were preferentially detected in ACPA+ versus ACPA− RA sera. We identified a synovial B cell–derived RA-rmAb locally differentiated within the ELS+ RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.