Nikola Lepse

Bacterial DNA motifs trigger ANCA production in ANCA-associated vasculitis in remission

OBJECTIVES CpG motifs, which are highly prevalent in bacterial DNA, have been shown to trigger the production of ANCA in vitro by B lymphocytes from patients with active ANCA-associated vasculitis (AAV). Staphylococcus aureus is associated with relapses in AAV, and CpG motifs from staphylococcal DNA may trigger ANCA production in AAV patients in remission. We investigated the presence of ANCA-producing B lymphocytes during quiescent disease and tested the capacity of these cells to produce ANCA in response to CpG. METHODS Expression of Toll-like receptor 9 (TLR9) by B lymphocytes from AAV patients and controls was assessed. Peripheral blood mononuclear cells were isolated from 23 PR3-ANCA and 15 MPO-ANCA patients (33 quiescent, 5 active disease) and 14 healthy controls, and cultured for 12 days in the presence of cytosine-phosphate-guanine oligodeoxynucleotide (CpG-ODN) and IL-2. B-lymphocyte activation, differentiation, immunoglobulin production and in vitro ANCA production were studied. RESULTS TLR9 expression by B lymphocytes was comparable in AAV patients and controls. B lymphocytes were activated and differentiated towards a plasma cell phenotype in response to CpG-ODN and IL-2. ANCA were produced in vitro by 13 out of 23 PR3-ANCA patients and 3 out of 15 MPO-ANCA patients. CONCLUSIONS We conclude that ANCA-producing B lymphocytes can be present in the peripheral blood of AAV patients during remission. These autoreactive B lymphocytes are triggered by CpG-ODN and IL-2 to produce ANCA in vitro. CpG motifs may trigger the production of ANCA in vivo, contributing to the development of relapses in AAV.

Immune regulatory mechanisms in ANCA-associated vasculitides

A group of primary vasculitides is associated with the presence of anti-neutrophil cytoplasmic autoantibodies (ANCA). In these diseases, the contribution of ANCA to disease pathogenesis has been studied extensively. Also, in patients with ANCA-associated vasculitides, the T lymphocyte compartment is dysregulated, as aberrant distribution and function of T cell subsets has been shown. In this review we will discuss the putative role of T lymphocytes in inflammation and granuloma formation, as well as the involvement of B cell compartments in the pathophysiology of ANCA-associated vasculitis.

Altered B cell balance, but unaffected B cell capacity to limit monocyte activation in anti-neutrophil cytoplasmic antibody-associated vasculitis in remission

OBJECTIVE Regulatory B cells (Bregs) constitute a subset of B cells with immunomodulatory properties. Numerical and functional alterations in the Breg compartment have been associated with autoimmunity. The aim of this study was to assess the frequency and function of Bregs in patients with ANCA-associated vasculitis (AAV). METHODS B cell subsets were determined in the peripheral blood of 48 AAV patients (12 active, 36 in remission) and 41 healthy controls (HCs) by flow cytometry. Bregs were defined within the CD19(+) population as CD24(hi)CD38(hi) or CD24(hi)CD27(+) cells. The percentage of IL-10-positive B cells in circulation was analysed by flow cytometry. Sorted CD19(+) B cells were co-cultured with monocytes to evaluate their capacity to inhibit monocyte TNF-α production upon lipopolysaccharide stimulation. RESULTS The frequency of circulating CD19(+)CD24(hi)CD38(hi) cells was not different in AAV patients in remission compared with HCs, but was decreased in patients with active disease [mean in HCs 5.5% (s.d. 1.6) vs active 3.8% (s.d. 2.8), P = 0.0104]. Furthermore, the percentage of CD19(+)CD24(hi)CD27(+) cells was significantly decreased in both remission and active patients when compared with HCs [HCs 15.0% (s.d. 9.3) vs remission 6.6% (s.d. 4.4) (P < 0.0001) vs active 6.4% (s.d. 6.2) (P = 0.0006)]. The frequency of IL-10-positive B cells was comparable between patients and HCs. B cells from AAV patients suppressed monocyte TNF-α production to a similar extent to cells from HCs. CONCLUSION Based on immunophenotypic classification, Bregs are numerically diminished in AAV patients. However, B cell function in terms of IL-10 production and their capacity to suppress monocyte activation is not compromised in AAV patients in remission.

Increased frequency of circulating IL-21 producing Th-cells in patients with granulomatosis with polyangiitis (GPA)

IntroductionThe present study aimed to explore a possible role for IL-21 producing Th-cells in the immunopathogenesis of granulomatosis with polyangiitis (GPA).MethodsPeripheral blood from 42 GPA patients in remission and 29 age-matched healthy controls (HCs) were stimulated in vitro, and the frequencies of IL-21 producing Th-cells were determined by flow cytometry. Since Th17-cells produce a low level of IL-21, IL-17 was also included in the analysis. Given that IL-21 is a hallmark cytokine for T follicular helper cells (TFH), we next evaluated the expression of their key transcription factor BCL-6 by RT-PCR and flow cytometry. To investigate the effect of IL-21 on autoantibody-production, PBMCs from GPA patients were stimulated in vitro with BAFF/IL-21 and total IgG and ANCA levels were measured in supernatants. In addition, the expression of IL-21-receptor on B-cells was analyzed.ResultsPercentages of IL-21 producing Th-cells were significantly elevated in GPA-patients compared to HCs, and were restricted to ANCA-positive patients. The expression of BCL-6 was significantly higher in ANCA-positive GPA-patients, as compared with ANCA-negative patients and HCs. IL-21 enhanced the production of IgG and ANCA in vitro in stimulated PBMCs from GPA patients. No difference was found in the expression of the IL-21-receptor on B-cells between ANCA-negative patients, ANCA-positive patients, and HCs.ConclusionThe increased frequency of circulating IL-21 producing Th-cells in ANCA-positive GPA patients and the stimulating capacity of IL-21 on ANCA-production suggest a role for these cells in the immunopathogenesis of GPA. Blockade of IL-21 could constitute a new therapeutic strategy for GPA.

Toll-like receptor 9 activation enhances B cell activating factor and interleukin-21 induced anti-proteinase 3 autoantibody production in vitro

OBJECTIVES Granulomatosis with polyangiitis (GPA) is a relapsing small-vessel vasculitis characterized by circulating ANCA against PR3. The mechanisms that trigger PR3-ANCA production are unknown. The aim of this study was to determine whether endogenous factors [B cell activating factor (BAFF) and IL-21] and exogenous factors [oligodeoxynucleotides containing CpG motifs (CpG-ODN)] synergize in stimulating PR3-ANCA production in GPA patients. METHODS Peripheral blood mononuclear cells from GPA patients and healthy controls (HCs) were cultured in the presence of BAFF and IL-21, with or without CpG-ODN, for 12 days. PR3-ANCA production in culture supernatants was quantified by Phadia EliA. Phenotypic characterization and the influence of CpG-ODN treatment on IL-21 receptor (IL-21R), transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) and BAFF receptor (BAFF-R) expression on B cells was analysed by flow cytometry. RESULTS Stimulation with BAFF and IL-21 significantly increased ANCA production in patient samples, which could be augmented further by addition of CpG-ODN. Stimulation with CpG-ODN increased the percentage of IL-21R(+) and TACI(+) B cells but did not affect BAFF-R expression. GPA patients had an increased percentage of circulating IL-21R(+) and a decreased percentage of TACI(+) circulating memory B cells when compared with HCs. Additionally, patients had decreased expression of BAFF-R on B cells, which was inversely correlated with BAFF concentrations in plasma. CONCLUSION Our data demonstrate that endogenous and exogenous factors can synergize to promote PR3-ANCA production. Mechanistically, CpG-ODN up-regulated IL-21R and TACI expression on B cells, possibly sensitizing these cells for IL-21- and BAFF-mediated signals. Agents inhibiting Toll-like receptor 9, BAFF and IL-21 signalling pathways may serve as potential therapeutics for intervention in GPA patients.

Interleukin-21, B cell activating factor and unmethylated CPG oligodeoxynucleotides synergize in promoting anti-proteinase 3 autoantibody production in vitro

Background/Purpose: MicroRNAs (miRs) are a novel class of posttranscriptional regulators. A single miR can have profound effects on cell activation due to its ability to modulate multiple pathways at once. We have previously shown that miR-155 is upregulated in rheumatoid arthritis (RA) synovial macrophages and promotes the development of autoimmunity and joint inflammation. Pre-clinical arthritis may be associated with lung changes e.g. bronchial wall thickening, thus the aim of this study was to investigate the contribution of miR-155 regulated pathways to lung homeostasis. Methods: Normal human lung tissue was tested by in situ hybridisation with miR-155 and control probes. To model the fibrotic response, WT and miR-155 / mice were given bleomycin (0.06 unit/mouse) intranasally. Intervention included intraperitoneal injections of the Liver X Receptor (LXR) agonist (GW3965 daily; 40 mg/kg). End-points included bronchial lavage (BAL) cytology, lung tissue histology, evaluation of the expression of inflammatory and fibrotic genes by qPCR and concentrations of soluble mediators in serum and BAL fluid by multiplex assays. The validation of miR-155 binding to LXR, and the LXR response element in collagen gene promoters were performed with reporter assays. Results: In situ hybridisation showed an abundant expression of miR-155 in the normal human lung suggesting that this miR may contribute to normal lung homeostasis. miR-155 / mice developed more severe bleomycininduced lung fibrosis compared to WT mice, as seen by increased collagen 1a/3a mRNA expression and protein deposition in the lungs, as well as accumulation of macrophages and lymphocytes in BAL. Gene expression analysis of lung extracts revealed an increase in the M2 pro-fibrotic macrophage markers Arginase 2, IL-13R and Ym1. In addition, the levels of pro-fibrotic cytokines such as VEGF and bFGF were significantly higher in BAL and serum of miR-155 / mice. Primary lung fibroblast lines derived from miR-155 / mice showed higher proliferation rates and motility compared to WT cells in wound healing assays. Computational analysis followed by functional luciferase assays revealed that the transcription activator LXR alpha is a direct target of miR-155 in the lungs. Expression of LXR alpha was significantly upregulated in the lungs of naive miR-155 / mice and was further increased in mice given bleomycin compared to similarly treated WT controls. Injection of the LXR agonist to WT mice increased LXR expression and mirrored the same phenotypic response to bleomycin as the miR-155 deficient mice; shown by increased collagen deposition and M2 macrophage and fibroblast activation. Promoter analysis revealed that LXRs could directly induce collagen production by binding to col1a and col3a promoters. / Conclusion: miR-155 appears important for lung homeostasis, likely by fine tuning levels of LXR thereby protecting from excessive remodelling. Given this and the emerging contribution of miR-155 to development of autoimmunity, this miR may act as a master-switch determining the duration of inflammation and the initiation of remodelling, as well as the balance between the immune and auto-immune responses.Background/Purpose: High mobility group box 1 (HMGB1) is a non-histone DNA binding protein that is passively released by dying cells or actively secreted by immunocompetent cells and the receptor for advanced glycation end-products (RAGE) is one of its receptors. Higher levels of HMGB1 have been found in patients with granulomatosis with polyangiitis (GPA) with active disease whereas higher HMGB1 and lower soluble (sRAGE) levels have been found in patients with acute atherosclerotic events suggesting sRAGE acts as a decoy receptor. This study aims to evaluate HMGB1 levels in relation to subclinical carotid atherosclerosis in GPA, and the impact of therapy on HMGB1 levels. Methods: A cross-sectional study was performed on 23 GPA patients during a quiescent phase of the disease in comparison to 20 matched controls. All study participants underwent carotid ultrasound to assess atherosclerotic plaques and intima-media thickness (IMT) and were tested for traditional risk factors for atherosclerosis, serum HMGB1 levels (ELISA-Shino Test, Kanagawa, Japan), and sRAGE levels (ELISA RD P = 0.978), HDLcholesterol (1.41 ± 0.37 vs. 1.51±0.33 mmol/L; P = 0.359), LDLcholesterol (3.01±0.79 vs. 3.29±0.82 mmol/L; P = 0.267), and a similar frequency of smoking (8.7% vs. 5.0%; P = 0.635), family history of premature coronary artery disease (CAD) (39.1% vs. 40.0%; P = 0.954), and obesity (4.3% vs. 10.0%; P = 0.446). Hypertension was only found in GPA patients (39.1% vs. 0.0%; P = 0.002) while no study participants had diabetes. Overt cardiovascular disease was found only in 13.0% of GPA patients. Statins were prescribed for 21.7% of GPA patients and 5.0% of controls (P = 0.127). Among GPA patients, prednisolone was being used by 34.8% with a median daily dose of 5.0mg (2.5-15.0) and azathioprine by 34.8%. Only two GPA patients used statins and prednisolone concomitantly. Carotid plaques were found in 30.4% of GPA patients and in 15.0% of controls (P = 0.203) and the overall IMT was similar in GPA patients and in controls (0.833±0.256 vs. 0.765±0.133mm; P = 0.861). Median serum HMGB1 levels were similar between GPA patients and controls [2.13ng/mL (1.11-7.22) vs. 2.42ng/mL (0.38-6.75); P = 0.827] as well as mean sRAGE levels (1256.1±559.6 vs. 1483.3±399.8pg/mL; P = 0.155). No correlations were found between HMGB1 and sRAGE ( = 0.068; P = 0.681) and between HMGB1 and maximum IMT in carotid arteries ( = -0.067; P = 0.720). GPA patients on prednisolone (1.77±0.76 vs. 3.53±2.06ng/ mL; P = 0.017) and statins (1.39±0.28 vs. 3.34±1.94ng/mL; P = 0.001) presented significantly lower serum HMGB1 levels whereas no difference in mean HMGB1 levels was found regarding azathioprine use (2.89±2.28 vs. 2.93±1.75; P = 0.970). Conclusion: No association was found between subclinical atherosclerosis in carotid arteries and HMGB1 levels in GPA patients. Furthermore, the use of either prednisone or statins was associated with lower HMGB1 levels in GPA patients. These findings suggest that the anti-inflammatory properties of statins include effects on serum HMGB1 levels in GPA.