Innate Immune Modulation Induced by EBV Lytic Infection Promotes Endothelial Cell Inflammation and Vascular Injury in Scleroderma.

Microvascular injury is considered an initial event in the pathogenesis of scleroderma and endothelial cells are suspected of being the target of the autoimmune process seen in the disease. EBV has long been proposed as a trigger for autoimmune diseases, including scleroderma. Nevertheless, its contribution to the pathogenic process remains poorly understood. In this study, we report that EBV lytic antigens are detected in scleroderma dermal vessels, suggesting that endothelial cells might represent a target for EBV infection in scleroderma skin. We show that EBV DNA load is remarkably increased in peripheral blood, plasma and circulating monocytes from scleroderma patients compared to healthy EBV carriers, and that monocytes represent the prominent subsets of EBV-infected cells in scleroderma. Given that monocytes have the capacity to adhere to the endothelium, we then investigated whether monocyte-associated EBV could infect primary human endothelial cells. We demonstrated that endothelial cells are infectable by EBV, using human monocytes bound to recombinant EBV as a shuttle, even though cell-free virus failed to infect them. We show that EBV induces activation of TLR9 innate immune response and markers of vascular injury in infected endothelial cells and that up-regulation is associated with the expression of EBV lytic genes in infected cells. EBV innate immune modulation suggests a novel mechanism mediating inflammation, by which EBV triggers endothelial cell and vascular injury in scleroderma. In addition, our data point to up-regulation of EBV DNA loads as potential biomarker in developing vasculopathy in scleroderma. These findings provide the framework for the development of novel therapeutic interventions to shift the scleroderma treatment paradigm towards antiviral therapies.

Epstein-Barr virus lytic infection promotes activation of Toll-like receptor 8 innate immune response in systemic sclerosis monocytes.

BACKGROUND: Monocytes/macrophages are activated in several autoimmune diseases, including systemic sclerosis (scleroderma; SSc), with increased expression of interferon (IFN)-regulatory genes and inflammatory cytokines, suggesting dysregulation of the innate immune response in autoimmunity. In this study, we investigated whether the lytic form of Epstein-Barr virus (EBV) infection (infectious EBV) is present in scleroderma monocytes and contributes to their activation in SSc. METHODS: Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) depleted of the CD19+ cell fraction, using CD14/CD16 negative-depletion. Circulating monocytes from SSc and healthy donors (HDs) were infected with EBV. Gene expression of innate immune mediators were evaluated in EBV-infected monocytes from SSc and HDs. Involvement of Toll-like receptor (TLR)8 in viral-mediated TLR8 response was investigated by comparing the TLR8 expression induced by infectious EBV to the expression stimulated by CL075/TLR8/agonist-ligand in the presence of TLR8 inhibitor in THP-1 cells. RESULTS: Infectious EBV strongly induced TLR8 expression in infected SSc and HD monocytes in vitro. Markers of activated monocytes, such as IFN-regulated genes and chemokines, were upregulated in SSc- and HD-EBV-infected monocytes. Inhibiting TLR8 expression reduced virally induced TLR8 in THP-1 infected cells, demonstrating that innate immune activation by infectious EBV is partially dependent on TLR8. Viral mRNA and proteins were detected in freshly isolated SSc monocytes. Microarray analysis substantiated the evidence of an increased IFN signature and altered level of TLR8 expression in SSc monocytes carrying infectious EBV compared to HD monocytes. CONCLUSION: This study provides the first evidence of infectious EBV in monocytes from patients with SSc and links EBV to the activation of TLR8 and IFN innate immune response in freshly isolated SSc monocytes. This study provides the first evidence of EBV replication activating the TLR8 molecular pathway in primary monocytes. Immunogenicity of infectious EBV suggests a novel mechanism mediating monocyte inflammation in SSc, by which EBV triggers the innate immune response in infected cells.

B cell depletion with rituximab in patients with diffuse cutaneous systemic sclerosis.

OBJECTIVE: To determine the safety of rituximab, to provide preliminary data regarding the potential efficacy of rituximab, and to investigate the effects of rituximab on autoimmunity and fibrosis in patients with diffuse cutaneous systemic sclerosis (dcSSc). METHODS: Fifteen patients with dcSSc, all of whom experienced their first non-Raynaud's disease-associated disease manifestation within 18 months of trial entry, were recruited to receive 2 intravenous doses of rituximab (1,000 mg), administered 2 weeks apart. Safety, clinical, and exploratory outcomes were evaluated at baseline and at 6 months. The primary outcome was the change in the modified Rodnan skin thickness score (MRSS) at 6 months compared with baseline. RESULTS: Adverse events included frequent infusion reactions and rare infections (urinary tract infection and dental abscess occurred in 1 patient each). The mean change in the MRSS between baseline and 6 months was not significant. Results of pulmonary function tests and other measures of major organ involvement were stable. The modest B cell infiltrates that were present in most skin biopsy specimens at baseline were completely depleted at 6 months in most patients. Autoantibody titers showed only modest and variable changes after treatment. CONCLUSION: In this pilot study, treatment with rituximab appeared to be safe and well tolerated among patients with dcSSc. Rituximab treatment resulted in both depletion of circulating B cells and depletion of dermal B cells but had little effect on the levels of SSc-associated autoantibodies. Rituximab treatment did not appear to result in a significant beneficial effect on skin disease. The potential efficacy of rituximab in other organs such as the lung could not be clearly evaluated in this small open-label trial.