Introduction: Vasculopathy, inflammatory fibrosis and functional autoantibodies (Abs) are major manifestations of systemic sclerosis (SSc). Abs directed against the angiotensin II type 1 receptor (AT₁R) and endothelin-1 type A receptor (ET_AR) are associated with characteristic disease features including vascular, inflammatory, and fibrotic complications indicating their role in SSc pathogenesis. Therefore, the impact of anti-AT₁R and anti-ET_AR Abs on initiation of inflammation and fibrosis was analyzed. Methods: Anti-AT₁R and anti-ET_AR Ab-positive immunoglobulin G (IgG) from SSc patients (SSc-IgG) was used for experiments. Healthy donor IgG served as a normal control, and AT₁R and ET_AR activation was inhibited by antagonists. Protein expression was measured with ELISA, mRNA expression with real time-PCR, endothelial repair with a scratch assay, and collagen expression with immunocytochemistry. Transendothelial neutrophil migration was measured with a culture insert system, and neutrophil ROS activation with immunofluorescence. Neutrophils in bronchoalveolar lavage fluids (BALFs) were analyzed microscopically after passive transfer of SSc-IgG or NC-IgG into naïve C57BL/6J mice. KC plasma levels were quantified by a suspension array system. Histologic analyses were performed by using light microscopy. Results: Anti-AT₁R and anti-ET_AR Ab-positive SSc-IgG induced activation of human microvascular endothelial cells (HMEC-1). Elevated protein and mRNA levels of the proinflammatory chemokine interleukin-8 (IL-8, CXCL8) and elevated mRNA levels of the vascular cell adhesion molecule-1 (VCAM-1) were induced in HMEC-1. Furthermore, activation of HMEC-1 with SSc-IgG increased neutrophil migration through an endothelial cell layer and activation of reactive oxygen species (ROS). SSc-IgG decreased HMEC-1 wound repair and induced type I collagen production in healthy donor skin fibroblasts. Effects of migration, wound repair, and collagen expression were dependent on the Ab-levels. Passive transfer of anti-AT₁R and anti-ET_AR Ab-positive SSc-IgG into naïve C57BL/6J mice increased neutrophil BALF counts. In parallel, increased levels of the murine functional IL-8 homologue, chemokine KC, were found in the plasma of SSc-IgG-treated mice as well as structural alterations of the lungs. Conclusions: We conclude that angiotensin and endothelin-receptor activation via anti-AT₁R and anti-ET_AR Abs mediate pathogenic effects, indicating their contribution to pathogenesis of SSc. Therefore, anti-AT₁R and anti-ET_AR Abs could provide novel targets for therapeutic intervention in the treatment of SSc.

Introduction: Spleen tyrosine kinase (Syk) mediates bronchoconstriction. However, its role in pulmonary vasoconstriction and pulmonary vascular hyperresponsiveness is unknown. Aims and objectives: We hypothesized that intracellular signal transduction via Syk is relevant for pulmonary arterial smooth muscle cell contraction with potential significance for the pathogenesis of pulmonary arterial hypertension (PAH). Methods: Vascular Syk expression was analyzed by immunofluorescence microscopy in human (PAH patients vs. donors) and mouse lungs. The effects of Syk inhibition (either with BAY 61-3606 or with BI 1002494) on pulmonary vasoconstriction were assessed in human precision-cut lung slices, in analgosedated ventilated pigs as well as in isolated perfused and ventilated mouse lungs. For mechanistic analyses, transgenic murine models (deficient in eNOS, PKCα, or mast cells) were assessed. A model of ovalbumin-induced pulmonary Th2 inflammation was used to study pulmonary vascular hyperresponsiveness. Results: Syk expression was detected in human and murine pulmonary arterial smooth muscle cells. Syk inhibition reduced pulmonary vasoconstriction in all analyzed species (human, pig, mouse). Syk-mediated vasoconstriction was independent of eNOS, PKCα, or mast cell expression. In vivo, in analgosedated ventilated pigs, Syk inhibitor BI 1002494 largely reduced hypoxic pulmonary vasoconstriction. Both pulmonary vascular hyperresponsiveness and pre-established vasoconstriction were diminished in isolated perfused mouse lungs following Syk inhibition. Conclusions: Syk is required for (hypoxic) pulmonary vasoconstriction, and may serve as a therapeutic target in PAH.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a chronic lung condition characterized by progressively deteriorating respiratory function. Exacerbations due to lung infections are thought to promote disease progression, and the presence of Streptococcus in the lung microbiome has been linked to progression of IPF. The aim of this study was to evaluate the impact of pulmonary fibrosis on susceptibility to pneumococcal pneumonia and subsequent bacteremia. Methods: The effect of subclinical low-dose infection with Streptococcus pneumoniae via the nasal route was studied using fos-related antigen-2 (Fra-2) transgenic (TG) mice, which develop spontaneous progressive pulmonary fibrosis. Two days after infection bacterial load was assessed in lung tissue, bronchoalveolar lavage (BAL), blood and spleen. Leukocyte subsets and cytokine levels were analyzed in BAL and blood. Additionally, lung compliance and arterial blood gases were assessed. Results: Low dose lung infection with Streptococcus pneumoniae in Fra-2 TG mice resulted in substantial pneumonia including weight loss, increased lung bacterial load and bacteremia, whereas WT mice remained mostly unaffected. Compared to WT mice BAL alveolar macrophages were reduced in Fra-2 TG mice. Proinflammatory cytokines and chemokines were elevated upon infection in BAL supernatant and plasma of Fra-2 TG mice. Following infection lung compliance was decreased in Fra-2 TG mice. Conclusion: Pulmonary fibrosis increases susceptibility to pneumococcal pneumonia and bacteremia possibly via impaired alveolar bacterial clearance.