Intracellular complement protein C3 promotes cell survival via Factor B (FB).

Abstract

Abstract Intracellular complement proteins such as C3 and FB play protective role during stress-induced cell death. C3−/−and Cfb−/−mice had worse Pseudomonas aeruginosa (P.a.)-induced acute lung injury at 24 h post infection compared to wildtype (WT) mice. However, the mechanism by which C3 exerts these functions is unclear. We performed CRISPR-induced deletion of C3and CFBin BEAS-2B cells, a human tracheobronchial airway epithelial cell line and assessed key findings in primary human airway cells. We also tested our observations in bone-marrow derived macrophages (BMDM) from Cfb−/−versus WT mice. Measures of acute lung injury were quantified in C3−/−, Cfb−/−and WT mice infected with PA57, a clinical P.a.strain. We observed that C3 and FB-deficient airway cells have increased death overtime compared to their parent clones in the setting of both oxidative stress and P.a.infection in vitro. An increased autophagic stress response, along with increased apoptosis, was observed in both C3 and FB deficiency. On further evaluation, both C3 and FB deficiency resulted in significantly lower AKT-mTOR signaling in P.a.infection. FB deficiency resulted in lower phospho-P70 S6 kinase levels, and higher LC3-II levels in P.a.infection. A similar phenotype was observed in infected BMDMs from Cfb−/−compared to WT mice. FB-deficient BEAS-2B cells and Cfb−/−mouse BMDMs had higher intracellular bacterial burden compared to FB-sufficient controls, suggesting that a lack of intracellular FB altered intracellular processing of P.a. The protective effect of C3 was present even when its secretion was inhibited but was abolished in FB deficiency. We conclude that intracellular complement protein C3 plays an essential role in cell survival via FB.