Non-concentrated supernatants gave the same results by tendency, although less pronounced. intermediates. SP-CA116D expression further led to reduced cell viability and increased levels of the chaperones Hsp90, Hsp70, calreticulin and calnexin. Lipid analysis revealed decreased intracellular levels of phosphatidylcholine (PC) and increased lyso-PC levels. Treatment with methylprednisolone or hydroxychloroquine partially restored these lipid alterations. Furthermore, SP-CA116D cells secreted soluble factors into the medium that modulated surface expression of CCR2 or CXCR1 receptors on CD4+ lymphocytes and neutrophils, suggesting a direct paracrine effect of SP-CA116D on neighboring cells in the alveolar space. Conclusions We show that this A116D mutation leads to impaired processing of proSP-C in alveolar epithelial cells, alters cell viability and lipid composition, and also activates cells of the immune system. In addition, we show that some of the effects of the mutation on cellular homeostasis can be antagonized by application of pharmaceuticals commonly applied in ILD therapy. Our findings shed new light around the pathomechanisms underlying SP-C deficiency associated ILD and provide insight into the mechanisms by which drugs currently used in ILD therapy act. Background Pulmonary surfactant is usually a phospholipid/protein mixture secreted to the alveolar surface by alveolar type 2 (AT2) cells [1]. It reduces surface tension and prevents alveolar collapse at the end of expiration [2]. A normal composition and homeostasis of pulmonary surfactant is critical for its surface-tension-reducing properties NAD 299 hydrochloride (Robalzotan) and gas exchange in the alveoles of the lung. Surfactant protein C (SP-C) is usually a hydrophobic, lung-specific protein that coisolates with the phospholipid fraction of pulmonary surfactant [3]. SP-C is usually synthesized exclusively by AT2 cells as a 197 amino acid proprotein (proSP-C) and proteolytically processed into the 4.2 kDa mature protein by a sequence of proteolytic cleavages [4]. Mature SP-C is usually subsequently secreted together with lipids and other surfactant components to the alveolar surface [3,5]. AT2 cells contain specialized lysosome-derived organelles for the storage of surfactant prior to its secretion. Exocytosis is usually facilitated by fusion of these so-called lamellar bodies (LBs) with the plasma membrane [6]. The SNARE proteins syntaxin 2 and SNAP-23 are associated with the plasma membrane and to some degree with lamellar bodies and have been shown to be required for regulated surfactant secretion [7,8]. Interstitial lung diseases (ILD) are a heterogeneous group of respiratory disorders that can be classified into those with known and unknown etiologies [9]. ILD NAD 299 hydrochloride (Robalzotan) are characterized by deposition of cellular and non-cellular components into the lung parenchyma. They vary widely in regard to radiological presentation, histopathological features, and clinical course [10]. ILD are mostly chronic and associated with high morbidity and mortality. Typical features of ILD include dyspnoea, the presence of diffuse infiltrates on chest radiographs and abnormal pulmonary function assessments with evidence of a restrictive ventilatory defect and/or impaired gas exchange [11]. Mutations in the surfactant protein genes em SFTPB /em and em SFTPC /em as well as in the ABC-transporter coding gene em ABCA3 /em , all of them resulting in a disturbed lung surfactant homeostasis, have been identified as genetic causes in some forms of ILD [12-16]. While loss-of-function mutations in SP-B result in surfactant deficiency and fatal neonatal lung disease, NAD 299 hydrochloride (Robalzotan) consequences of mutations in SP-C tend to be less severe, ranging from fatal pulmonary surfactant deficiency to childhood ILD [17]. Most SP-C mutations cluster within the preprotein’s BRICHOS domain name and lead to misfolding of the preprotein, aberrant trafficking and processing [3]. To date, all affected individuals with BRICHOS domain Rabbit Polyclonal to OR56B1 name mutations have been heterozygous with no detectable mature SP-C in their lungs, suggesting a NAD 299 hydrochloride (Robalzotan) dominant-negative effect of the mutant allele [3,12]. Moreover, in cell lines NAD 299 hydrochloride (Robalzotan) expressing BRICHOS domain name mutations, proSP-C forms perinuclear aggregates, consistent with the cell’s inability to clear aggregates of misfolded protein and a toxic gain-of-function [12,18]. Accumulation of misfolded proSP-C may trigger several distinct pathological mechanisms, such as induction of endoplasmic reticulum (ER) stress, cytotoxicity, and caspase 3- and caspase 4-mediated apoptosis [14,19,20]. These factors might contribute to ILD by causing cell injury and apoptotic death of AT2 cells. Current treatment of ILD in children is usually unfortunately empirical. Since an inflammatory component is present in ILD, corticosteroids and immunosuppressive.