Length Defective regulation of autoreactive IL-6-producing transitional B lymphocytes is associated with disease in patients with systemic sclerosis

Background: Systemic Sclerosis (SSc) has the highest case-specific mortality of any rheumatic disease and has no effective therapy. A clear manifestation of SSc is the presence of auto-antibodies. However, the origin of autoantibody-producing B lymphocytes and mechanisms of their activation, auto-antibody production and role remain unclear. Objective: To identify mechanisms that contribute to pathogenic B cell generation, involvement in SSc and assess the altered distribution and function of B cells in patients. Methods: Multi-colour flow cytometry was used to determine B cell subset distribution, cytokine production and tolerance induction in SSc patients and healthy controls. Cytokine production following stimulation of the cells ex vivo was carried out by multiplex analysis. Results: The study reveals a range of defects in B lymphocyte tolerance and cytokine production in SSc. Evidence is provided for altered distribution of transitional B cell subsets, increased production of IL-6 and IL-8 and defective tolerance induction in SSc B cells. In addition, the study reveals that B cells in SSc have a reduced ability to produce IL-10 when stimulated through innate immune pathways. In contrast to healthy individuals, tolerance checkpoints in SSc patients fail to suppress the emergence of B cells that produce autoantibodies with specificity to the Scl-70 antigen, a specificity strongly associated with SSc. These defects are paralleled by altered intracellular signalling and apoptosis following B cell receptor (BCR) engagement. A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. Conclusion: The study provides new insights into mechanisms underlying defective B lymphocyte responses in patients with SSc and their contribution to pathology. INTRODUCTION Systemic sclerosis (SSc) is a severe inflammatory disease characterized by excessive extracellular matrix (ECM) deposition in the skin and visceral organs (1). It has complex pathogenesis with two major hallmarks: autoimmunity and inflammation leading to widespread damage to blood vessels and progressive interstitial and perivascular fibrosis (2). A key feature of autoimmunity in SSc is high levels of auto-antibodies (auto-Abs) to nuclear proteins including topoisomerase I enzyme, centromere and RNA polymerase and endothelial cell and platelet derived growth factor receptor (1, 3). Evidence for pathogenic roles of these auto-Abs comes from studies revealing the association between their specificity and which tissues and organs are involved. Importantly also, these auto-Abs appear prior to disease onset. Furthermore, B lymphocytes accumulate at sites of disease, around small vessels in the skin (4) and in alveolar interstitium in patients with lung involvement (5). In addition to producing auto-Abs, B lymphocytes contribute to fibrosis through producing interleukin 6 (IL-6) (6). The role played by B lymphocytes in SSc is supported by studies of disease pathogenesis in mouse models (7-9) and the success of treating patients with rituximab, a chimeric monoclonal antibody against human CD20 (10, 11). In the tight-skin mouse model of SSc that is associated with constitutive CD19-mediated signaling (7, 12), inhibition of CD19 expression abrogates auto-Ab production, IL-6 production by B cells and ameliorate skin fibrosis (7, 8). Similar effects were noted when CD19 expression was suppressed in the bleomycin-treated mouse model of SSc (9). Furthermore, SSc patients with interstitial lung A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. and diffuse disease who are positive for anti-Scl70 auto-Abs benefit significantly from treatment with rituximab (10). Thus, no patient receiving rituximab exhibited lung function deterioration, whereas 5 out of 6 placebo-treated patients had a worsening of their disease. Additionally, skin thickening, collagen deposition in the skin and health quality improved in rituximab-treated patients but not in placebo-treated patients (10-14). Furthermore, rituximab reduced the level of plasma IL-6, activity index, depleted skin B lymphocytes and reduced dermal myofibroblasts and hyalinised collagen in the skin (15). The cause of defective B cell responses in SSc remains unclear. However, defective tolerance and deregulation of autoreactive B cell responses are potential causes of B cell-mediated pathology. High affinity self-reactive B cells are normally deleted in the bone marrow but some that recognize self with low affinity or those that do not encounter self-antigens in the bone marrow escape censure and migrate to the periphery as transitional B cells. Migrant “transitional” B cells go through series of tolerance checkpoints and maturational phases to become mature B cells. This pathway was initially described in mice as immature B cells transiting to the spleen to mature into to B cell receptor(BCR) responsive cells to antigen engagement (16). Murine transitional B cells are distinguishable from mature cells based on the level of CD23, CD21 and the developmental marker CD24 expression (17). In humans, recent studies from a number of laboratories including ours have revealed that transitional human B cells (CD24CD38) do not constitute a single population but can be divided into 4 subsets (18). These subsets have subtle differences in maturational and tolerance status and capacity to produce IL-10 and IL-6 (18, 19). The current study was carried out to explore if defects in the maturation of, or tolerance induction in B cells at the transitional stage could relate to the emergence of high affinity pathogenic B cells in patients. Transitional B cells were studied for their ability to produce IL-6 as its production is a key mechanism by which B cells could promote fibrosis (6-8). In addition, the potential of transitional B cells in SSc to A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. produce IL-10 was studied to assess their ability to be involved in immune regulation (19, 20). We used a multi-colour flow cytometry and a secretome profiling approach to characterize the distribution and functional characteristics of B cells in SSc to identify potential pathways through which pathogenic B cells could emerge and contribute to pathogenesis. METHODS Patients All patients fulfilled the 2013 EULAR/ACR criteria for SSc (21). Disease subsets were defined as patients with limited cutaneous SSc (lcSSc) when skin thickening was present distal to elbows and knees and as diffuse cutaneous SSc (dsSSc) when skin thickening affected both distal and proximal areas. Blood samples from 88 patients with SSc attending the clinics were obtained for the purpose of this study after their informed consents. Patients with interstitial lung disease were diagnosed based on characteristic changes visualised by high-resolution computed tomography (CT). Pulmonary arterial hypertension was diagnosed by right heart catheterization with mean pulmonary artery pressure of ≥25 mm Hg and normal pulmonary capillary wedge pressure. Scleroderma renal crisis (SRC) was defined as newonset systemic hypertension >150/85 mm Hg and a documented decrease in estimated glomerular filtration rate of ≥30% or confirmed features on renal biopsy (22). Blood samples from 17 healthy controls (HCs) (mean age 40.2±11.3 years; 25-60 years) were also included for comparisons. Some of the tested samples from both patients and controls were studied on multiple occasions. The study was approved by the London-Hampstead National Research Ethics Service Committee (REC reference: 6398) and was conducted in compliance with the Helsinki Declaration of 2013. A cc ep te d A rt ic le This article is protected by copyright. All rights reserved. B lymphocyte enrichment B lymphocytes were enriched from whole blood by negative selection using the EasySep Human B Cell Enrichment Kit (StemCell Technologies, Grenoble, France). All experiments presented in this study were carried out using freshly-enriched B lymphocytes from blood samples taken on the same day. The protocol of B cell enrichment involves using a cocktail of monoclonal antibodies (mAbs) with dual specificity for non-B cell components of blood mononuclear cells (MNCs) and red blood cells (RBCs). The cocktail aggregates all non-B cells with RBCs and these are then separated on Ficoll-Paque. The protocol is fast and purity of enriched B cells is >95%. Cell staining and flow cytometry Enriched B cells were stained with combinations of fluorochrome-conjugated antibodies (18). mAbs were purchased from the suppliers indicated as follows: APC-eFlour780-conjugated anti-human CD10 (Clone SN5c), PE-Cy5.5-anti-human CD19 (Clone: HIB19), PE-Cy7-antihuman CD32 (Clone 6C4), FITC-anti-human IgD (Clone: IA6-2), eFlour 450-anti-human CD21 (Clone: HB5) and PE-anti-human CD24 (Clone: eBioSN3) mAbs were all from eBioscience. Brilliant Violet 605-anti-human IgM (Clone: MHM-88), brilliant Violet 785anti-human CD19 (Clone: HIB19) and brilliant Violet 711-anti-human CD27 (Clone: O323) were from BioLegend. PerCP-Cy5.5-anti-CD38 mAb (Clone: HIT2) was from BD Biosciences. For intracellular cytokines, stained cells were fixed, permeabilized and stained with either APC-anti-human IL-6 mAb (Clone: MQ2-13A5), APC-anti-human IL-10 mAb (Clone: JES3-9D7) (both from BD Bioscience) and were assessed by LSR-Fortessa FACS machine and FacsDiva software.