Mutations in surfactant SPA1 (Surfactant Protein A, encoded by SFTPA1) have been associated with pulmonary fibrosis (PF). Recently, necroptosis, an inflammatory type of cell death has emerged as potential contributing factor. We aimed to explore the potential role of necroptosis in an epithelial model of the A549 cells expressing SPA1.

The A549 alveolar epithelial cell line was transfected with a 500ng plasmid expressing receptor-interacting protein kinase 3 (RIPK3) and 500ng of pSPA1-WT or pSPA1-Y208H or with the empty vector (EV). Necroptosis was induced with 200ng/ml TNF?+2µg/ml of the caspace inhibitor Z-VAD+20µM doxorubicine, in the presence or absence of 1?g/ml necrostatin 10??, a necroptosis specific inhibitor. Protein expression of RIPK3, phosphorylated pRIPK3 and phosphorylated mixed lineage kinase domain like pseudokinase (pMLKL) were analysed by western blot. In parallel pMLKL was tested by immunohistochemistry in biopsies from patients with PF.

Our results showed that transfection with RIPK3 was important for necroptosis induction in A549. In cells transiently expressing the WT or mutated SPA protein, treatment with TNFa+zVAD+doxorubicin induced necroptosis as attested by the decreased cell number and the levels of pRIPK3 and pMLKL compared to EV. Necrostatin reduced the expression of pRIPK3 and pMLKL in both groups without affecting RIPK3. Interestingly pMLKL was present in alveolar type 2 epithelial cells and macrophages in biopsies of PF patients.

RIPK3 mediated necroptosis in lung epithelial cells could represent a potential mechanism in the pathogenesis of surfactant-related pulmonary fibrosis. Further studies are needed to confirm our results.