Free Actin and Effects on Lung Macrophage Bacterial Defenses

Abstract

Actin release into the circulation and extracellular space, with subsequent removal by the actin scavenging protein plasma gelsolin (pGSN), has long been reported in injury and infection. Our laboratory’s recent finding that pGSN improved survival in a murine primary pneumococcal pneumonia model via macrophage nitric oxide synthase type III (NOS3) enhancement underscored pGSN’s therapeutic potential, but also suggested the possibility of a more significant role for exposed actin in lung macrophage host defenses. Inflamed and injured lungs are more prone to bacterial infection, yet the ability of extracellular actin released from damaged tissue and cells to depress host defense has not been studied. In patients following burn and inhalation injury, and in murine models of oxidizing injury, influenza, and postinfluenza secondary pneumococcal pneumonia, we detected free actin release into the lung alveolar fluid via Western Blotting. We further demonstrate that free actin impairs alveolar macrophage binding and uptake of bacteria by interfering with the function of the scavenger receptor MARCO. In CFU bacterial binding assays in vitro, a murine alveolar macrophage cell line exhibited reduced binding and uptake of Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli in the presence of free actin. Similarly, primary human alveolar macrophage host defenses were impaired in the presence of actin, as observed via reduced binding and uptake of fluorescent-S. aureus in assays analyzed using scanning cytometry. Bacterial binding and uptake were restored through addition of pGSN, a highly conserved member of the extracellular actin scavenging system (EASS). We then demonstrated a mechanism of free actin interaction with macrophages. A647-tagged fluorescent actin bound a murine lung macrophage derived cell line and primary human alveolar macrophages as observed via flow cytometry. Actin binding by AMs was reduced under pan-scavenger receptor inhibitors and in a MARCO / SRAI/II DKO cell line, pointing to scavenger receptor mediated actin binding. Ex vivo pan-scavenger receptor treated AMs obtained from MARCO -/- mice via bronchoalveolar lavage (BAL) exhibited even greater reductions in actin binding under flow cytometry, furthering implicating the MARCO receptor as an actin-binding AM partner. Free actin impairment of macrophage host defenses following release during injury and infection may be widespread and thus clinically relevant, further underscoring the importance of characterizing actin-macrophage interactions and the potential value of pGSN as a therapeutic immunomodulator.