Serum microRNA screening and functional studies reveal miR-483-5p as a potential driver of fibrosis in systemic sclerosis

Abstract Objective MicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease. Methods The levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (n = 22), systemic lupus erythematosus (SLE) (n = 33) and primary Sjogren's syndrome (pSS) (n = 23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells. Results 30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes. Conclusions Our findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc

Additive immunosuppressive effect of leflunomide and hydroxychloroquine supports rationale for combination therapy for Sjögren’s syndrome

Objective: Effective treatment for primary Sjögren’s syndrome (pSS) is not available. pSS immunopathology involves a variety of immune-cells and dysregulated pathways; targeting several pathways instead of only one could therefore be effective. Treatment with leflunomide (LEF) and hydroxychloroquine (HCQ) might be successful given their unique immunosuppressive properties. We aimed to study the in vitro effects of LEF, HCQ and their combination on T- and B-cell proliferation, cytokine and immunoglobulin production by activated PBMCs. Methods: PBMCs of six healthy individuals and nine pSS patients were stimulated with superantigen and TLR9 agonist to mimic the hallmark features. LEF, HCQ and their combinations were tested at clinically observed concentrations and proliferation, cytokine and immunoglobulin production were measured. Results: TCR/TLR9 activation of PBMCs induced strong proliferation of T and B-cells and production of CXCL13, IFN-α, IFN-γ, IgG and IgM. LEF dose-dependently inhibited all measured parameters, where HCQ potently and dose-dependently decreased B cell proliferation, CXCL13, IFN-α, IgG and IgM production. At different concentration combinations, HCQ and LEF inhibited several immune hallmark features more potently than each single compound. Conclusion: A combination of LEF and HCQ at clinically applicable concentrations additively inhibits immune activation, supporting a potential implementation of this drug combination in pSS treatment

Additive immunosuppressive effect of leflunomide and hydroxychloroquine supports rationale for combination therapy for Sjögren’s syndrome

Objective: Effective treatment for primary Sjögren’s syndrome (pSS) is not available. pSS immunopathology involves a variety of immune-cells and dysregulated pathways; targeting several pathways instead of only one could therefore be effective. Treatment with leflunomide (LEF) and hydroxychloroquine (HCQ) might be successful given their unique immunosuppressive properties. We aimed to study the in vitro effects of LEF, HCQ and their combination on T- and B-cell proliferation, cytokine and immunoglobulin production by activated PBMCs. Methods: PBMCs of six healthy individuals and nine pSS patients were stimulated with superantigen and TLR9 agonist to mimic the hallmark features. LEF, HCQ and their combinations were tested at clinically observed concentrations and proliferation, cytokine and immunoglobulin production were measured. Results: TCR/TLR9 activation of PBMCs induced strong proliferation of T and B-cells and production of CXCL13, IFN-α, IFN-γ, IgG and IgM. LEF dose-dependently inhibited all measured parameters, where HCQ potently and dose-dependently decreased B cell proliferation, CXCL13, IFN-α, IgG and IgM production. At different concentration combinations, HCQ and LEF inhibited several immune hallmark features more potently than each single compound. Conclusion: A combination of LEF and HCQ at clinically applicable concentrations additively inhibits immune activation, supporting a potential implementation of this drug combination in pSS treatment

CXCL4 exposure potentiates TLR-driven polarization of human monocyte-derived dendritic cells and increases stimulation of T cells

Chemokines have been shown to play immune-modulatory functions unrelated to steering cell migration. CXCL4 is a chemokine abundantly produced by activated platelets and immune cells. Increased levels of circulating CXCL4 are associated with immunemediated conditions, including systemic sclerosis. Considering the central role of dendritic cells (DCs) in immune activation, in this article we addressed the effect of CXCL4 on the phenotype and function of monocyte-derived DCs (moDCs). To this end, we compared innate and adaptive immune responses of moDCs with those that were differentiated in the presence of CXCL4. Already prior to TLR- or Ag-specific stimulation, CXCL4-moDCs displayed a more matured phenotype. We found that CXCL4 exposure can sensitize moDCs for TLR-ligand responsiveness, as illustrated by a dramatic upregulation of CD83, CD86, and MHC class I in response to TLR3 and TLR7/8-agonists. Also, we observed a markedly increased secretion of IL-12 and TNF-α by CXCL4- moDCs exclusively upon stimulation with polyinosinic-polycytidylic acid, R848, and CL075 ligands. Next, we analyzed the effect of CXCL4 in modulating DC-mediated T cell activation. CXCL4-moDCs strongly potentiated proliferation of autologous CD4+ T cells and CD8+ T cells and production of IFN-γ and IL-4, in an Ag-independent manner. Although the internalization of Ag was comparable to that of moDCs, Ag processing by CXCL4-moDCs was impaired. Yet, these cells were more potent at stimulating Ag-specific CD8+ T cell responses. Together our data support that increased levels of circulating CXCL4 may contribute to immune dysregulation through the modulation of DC differentiation

Increased expression of Fas on group 2 and 3 innate lymphoid cells is associated with an interferon signature in systemic lupus erythematosus and Sjögren's syndrome

The role of innate lymphoid cells (ILCs) in the pathophysiology of rheumatic diseases is emerging. Evidence from animal studies implicate type I IFN, produced by plasmacytoid dendritic cells, to be involved in regulating the survival of group 2 and group 3 ILCs (ILC2s and ILC3s) via the upregulation of Fas (CD95) expression. For the first time, we explored the frequency and phenotype of circulating ILCs in SLE and primary Sjögren's syndrome (pSS) in relationship to the IFN signature. Methods: Frequencies and phenotypes of ILC subsets and plasmacytoid dendritic cells were assessed by flow cytometry in peripheral blood of patients with SLE (n = 20), pSS (n = 20) and healthy controls (n = 17). Patients were stratified by the presence or absence of an IFN signature as assessed by RT-qPCR on circulating mononuclear cells. Results: ILC1 frequencies were increased in peripheral blood of patients with SLE as compared with healthy controls and correlate with disease activity in pSS patients. Overall, the frequencies of ILC2s or ILC3s did not differ between patients with SLE, pSS and healthy controls. However, patients with a high type I IFN signature expressed elevated levels of Fas on ILC2s and ILC3s, which coincided with decreased frequencies of these cells in blood. Conclusion: The presence of a type I IFN signature is related to Fas expression and frequencies of circulating ILC2s and ILC3s in patients with SLE and pSS, potentially altering the homeostatic balance of ILCs

Increased expression of Fas on group 2 and 3 innate lymphoid cells is associated with an interferon signature in systemic lupus erythematosus and Sjögren's syndrome

The role of innate lymphoid cells (ILCs) in the pathophysiology of rheumatic diseases is emerging. Evidence from animal studies implicate type I IFN, produced by plasmacytoid dendritic cells, to be involved in regulating the survival of group 2 and group 3 ILCs (ILC2s and ILC3s) via the upregulation of Fas (CD95) expression. For the first time, we explored the frequency and phenotype of circulating ILCs in SLE and primary Sjögren's syndrome (pSS) in relationship to the IFN signature. Methods: Frequencies and phenotypes of ILC subsets and plasmacytoid dendritic cells were assessed by flow cytometry in peripheral blood of patients with SLE (n = 20), pSS (n = 20) and healthy controls (n = 17). Patients were stratified by the presence or absence of an IFN signature as assessed by RT-qPCR on circulating mononuclear cells. Results: ILC1 frequencies were increased in peripheral blood of patients with SLE as compared with healthy controls and correlate with disease activity in pSS patients. Overall, the frequencies of ILC2s or ILC3s did not differ between patients with SLE, pSS and healthy controls. However, patients with a high type I IFN signature expressed elevated levels of Fas on ILC2s and ILC3s, which coincided with decreased frequencies of these cells in blood. Conclusion: The presence of a type I IFN signature is related to Fas expression and frequencies of circulating ILC2s and ILC3s in patients with SLE and pSS, potentially altering the homeostatic balance of ILCs

Systemic and local granzyme B levels are associated with disease activity, kidney damage and interferon signature in systemic lupus erythematosus

Objectives: Granzymes (Grs) are serine proteases that eliminate virally infected or tumour cells by inducing apoptosis. GrB has been shown to be associated to the pathophysiology of SLE, whereas the role of the other Grs in SLE remain unknown. Methods: Gr levels were determined in the serum of SLE patients and controls and linked to SLE activity parameters, including the IFN signature. In addition, GrB expression was investigated in LN biopsies and correlated to kidney function parameters and disease severity. Results: Serum GrK and GrM levels were not elevated in SLE and did not correlate with disease activity. In contrast, GrB was increased in SLE serum, which correlated to both the SLEDAI and IFN signature. GrB expression was detected in LN tissue biopsies. The number of GrB-positive cells in tissue correlated to several kidney function parameters (e.g. serum creatinine, proteinuria) and to the LN chronicity index. Conclusion: GrB, but not GrK and GrM, is increased in the serum and kidney of patients with SLE and correlates with measures of poor prognosis in LN. These data suggest that GrB may contribute to the pathogenesis of SLE/LN, which indicates the possibility that GrB might be used as a biomarker and/or a therapeutic target

Systemic and local granzyme B levels are associated with disease activity, kidney damage and interferon signature in systemic lupus erythematosus

Objectives: Granzymes (Grs) are serine proteases that eliminate virally infected or tumour cells by inducing apoptosis. GrB has been shown to be associated to the pathophysiology of SLE, whereas the role of the other Grs in SLE remain unknown. Methods: Gr levels were determined in the serum of SLE patients and controls and linked to SLE activity parameters, including the IFN signature. In addition, GrB expression was investigated in LN biopsies and correlated to kidney function parameters and disease severity. Results: Serum GrK and GrM levels were not elevated in SLE and did not correlate with disease activity. In contrast, GrB was increased in SLE serum, which correlated to both the SLEDAI and IFN signature. GrB expression was detected in LN tissue biopsies. The number of GrB-positive cells in tissue correlated to several kidney function parameters (e.g. serum creatinine, proteinuria) and to the LN chronicity index. Conclusion: GrB, but not GrK and GrM, is increased in the serum and kidney of patients with SLE and correlates with measures of poor prognosis in LN. These data suggest that GrB may contribute to the pathogenesis of SLE/LN, which indicates the possibility that GrB might be used as a biomarker and/or a therapeutic target