Tissue fibrosis results when dysregulation of extracellular matrix (ECM) turnover favors deposition of collagen and other ECM proteins over degradation. Fibrosis may then lead to organ dysfunction and pathology as observed in systemic sclerosis (SSc). In the present study, we investigated the antifibrotic properties of proteasome blockade. A dose- and time-dependent reduction in type-I collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) production was observed in normal fibroblasts exposed to proteasome inhibitors (PI). In the same culture conditions, metalloproteinase-1 (MMP-1) protein and the collagenolytic activity on type I collagen was increased. The steady-state mRNA levels of COL1A1, TIMP-1, and MMP-1 paralleled protein levels. These effects were dominant over the profibrotic properties of TGF-beta and were observed with fibroblasts generated from normal and SSc skin. PI decreased type I collagen mRNA levels with kinetics similar to those observed with DRB, a specific RNA polymerase II inhibitor, thus indicating transcriptional inhibition. Of interest, PI induced c-Jun phosphorylation and c-Jun nuclear accumulation. The specific N-terminal Jun-kinase inhibitor SP-600125 selectively abrogated c-Jun phosphorylation and, in a dose-dependent fashion, the up-regulated synthesis of MMP-1 induced by PI. Finally, PI did not affect fibroblast viability. Thus, the coordinated down-regulation of collagen and TIMP-1 and up-regulation of MMP-1 renders proteasome blockade an attractive strategy for treating conditions as SSc, characterized by excessive fibrosis.

Objectives Altered signaling in B-cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signaling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterize the role of BANK1 and BLK in SLE, we performed a genetic interaction analysis hypothesizing that genetic interactions could reveal functional pathways relevant to disease pathogenesis. Methods We Used the method GPAT16 to analyze the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localization, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK. Results Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from Northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK. As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, we tested the possibility that BANK1 and BLK could also show a protein-protein interaction. We demonstrated co-immunoprecipitation and co-localization of BLK and BANK1. In a Daudi cell line and primary naïve B-cells the endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies. Conclusions Here, we show a genetic interaction between BANK1 and BLK, and demonstrate that these molecules interact physically. Our results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signaling pathway.

Objective. Previous studies have revealed the presence of IgG antifibroblast antibodies (AFAs) capable of binding to the surface of fibroblasts in systemic sclerosis (SSc) sera. Since chemokines may directly or indirectly affect the development of fibrosis, this study was undertaken to investigate the production of chemokines induced by AFAs in fibroblasts, and to characterize the signaling pathways and surface molecules involved.Methods. AFA-positive and AFA-negative IgG were tested on fibroblasts. Chemokine messenger RNA expression was screened by microarray and quantitative reverse transcription-polymerase chain reaction. Production of CCL2, CXCL8, and CXCL10 proteins was assessed by enzyme-linked immunosorbent assay. Pharmacologic inhibitors were used to study signal transduction, with results assessed by Western blotting and immunofluorescence analysis. Fibroblasts with defective expression of Toll-like receptors (TLRs) and anti-TLR monoclonal antibodies (mAb) were used to assess AFA specificity.Results. In human fibroblasts, AFA-positive IgG induced the preferential transcription of chemokines with profibrotic and proangiogenic potential, including, but not exclusively, CCL2, CXCL1, CXCL8, CKLF, and ECGF1, which were distinctly different from those induced by interferon-␥. Levels of CCL2 and CXCL8 proteins were increased in AFA-stimulated fibroblast culture supernatants. AFA binding to fibroblasts resulted in concomitant activation of ERK-1/2, c-Jun, and NF-B. CCL2 production was sensitive to inhibition of both proteasome and JNK, while CXCL8 production was sensitive only to inhibition of proteasome. AFAs failed to up-regulate CCL2 expression in TLR-4-deficient fibroblasts but not in TLR-6-or TLR-2-deficient fibroblasts. Moreover, anti-TLR-4 mAb, but not anti-TLR-2 mAb, partially inhibited the production of CCL2 induced by AFAs in human fibroblasts. Conclusion.Autoantibodies that bind to the surface of fibroblasts may contribute to the pathogenesis of SSc by up-regulating the fibroblast production of profibrotic and proangiogenic chemokines, in a proteasomeand TLR-4-dependent manner.

In systemic sclerosis (SSc), a disease characterized by fibrosis of the skin and internal organs, the occurrence of interstitial lung disease is responsible for high morbidity and mortality. We previously demonstrated that proteasome inhibitors (PI) show anti-fibrotic properties in vitro by reducing collagen production and favoring collagen degradation in a c-jun N-terminal kinase (JNK)-dependent manner in human fibroblasts. Therefore, we tested whether PI could control fibrosis development in bleomycin-induced lung injury, which is preceded by massive inflammation. We extended the study to test PI in TSK-1/+ mice, where skin fibrosis develops in the absence of overt inflammation. C57Bl/6 mice received bleomycin intratracheally and were treated or not with PI. Lung inflammation and fibrosis were assessed by histology and quantification of hydroxyproline content, type I collagen mRNA, and TGF-beta at Days 7, 15, and 21, respectively. Histology was used to detect skin fibrosis in TSK-1/+mice. The chymotryptic activity of 20S proteasome was assessed in mice blood. JNK and Smad2 phosphorylation were evaluated by Western blot on lung protein extracts. PI reduced collagen mRNA levels in murine lung fibroblasts, without affecting their viability in vitro. In addition, PI inhibited the chymotryptic activity of proteasome and enhanced JNK and TGF-beta signaling in vivo. PI failed to prevent bleomycin-induced lung inflammation and fibrosis and to attenuate skin fibrosis in TSK-1/+mice. In conclusion, our results provide direct evidence that, despite promising in vitro results, proteasome blockade may not be a strategy easily applicable to control fibrosis development in diseases such as lung fibrosis and scleroderma.

The coronavirus disease (COVID-19) is a respiratory tract infection caused by the new virus SARS-CoV-2. The acute phase of the infection may in certain individuals be followed by another longer phase of disease (long COVID) of unknown etiology probably associated in certain cases with autoimmune activation. It has been shown that COVID-19 can trigger autoantibody production and in genetically predisposed patients may cause the onset or exacerbation of autoimmune diseases. We are reporting a case of mild COVID-19 infection complicated by autoantibody production and cutaneous and gastrointestinal symptoms and subsequently diagnosed with systemic sclerosis (SSc). A 47-year-old man with no history of any autoimmune diseases and in good health became sick together with his family on the 12th of November with mild symptoms: tiredness, fever, cough, and sore throat. Oropharyngeal swab for SARS-CoV-2 tested positive. He was isolated at home and did not require hospitalization. Three weeks later he presented with clinical manifestation compatible with suspicion of SSc. He briefly presented with skin rush, periorbital edema and conjunctivitis, vomiting, dysphagia, burning sensation in the skin, above all in the fingertips and around the mouth, puffy fingers, Raynaud’s phenomenon, pain at the fingertip of the middle finger where a depressed area was noticed without a clear ulceration. ANA showed a strongly positive nucleolar pattern. Anti-PM/Scl 75 and PM/Scl 100 resulted positive. High-resolution computed tomography (HCRT) showed early stage of interstitial lung disease (ILD). The patient was diagnosed with SSc based on the persistence of autoantibodies and the clinical and radiological pictures according to the ACR/EULAR classification (scores: puffy finger, 2; ILD, 2; Raynaud’s phenomenon, 3; SSc related antibodies, 3; total 10). There are several cases described in the medical literature of possible new onset of SLE after COVID-19 infection. This is the first case that describes a possible new onset of SSc. Conclusion: SARS-CoV-2 may trigger systemic sclerosis.