Assessment of different selective inhibitors of autophagy on inflammation in macrophages

Abstract

Title: Assessment of different selective inhibitors of autophagy on inflammation in macrophages Autophagy is a self-eating process responsible for the sequestering and lysosomal degradation of damaged organelles and proteins in response to stress. Accumulating evidence indicates that defects in autophagy lead to increased inflammatory responses. Statins are hydroxy-3-methylglutaryl-coenzyme A or HMG Co-A reductase inhibitors that have many beneficial effects on cells including anti-inflammatory and anti-oxidant roles. Intensive studies have shown that autophagy is involved in different inflammatory conditions such as infectious diseases, neurodegenerative disorders, and cancer. Our goal is to investigate the mechanisms involved in the anti-inflammatory effects of Rho kinase (ROCK) specific inhibitor Y27632. We aimed to develop in culture a cellular system of macrophages where autophagy is diminished using potential autophagy inhibitors. Methods. Murine macrophages were prepared from bone marrow of C57BL-6j mice activated with LPS. Enzyme linked immunosorbent assay (ELISA) was used to determine the levels of proinflammatory cytokines, IL-6 and TNF-α. Prostaglandin E2 (PGE2) was measured using enzyme linked immunoassay (EIA). Western blotting was performed to detect the expression of LC3, a known autophagy marker. Quantitative polymerase chain reaction (qPCR) was conducted to detect the expression of anti-inflammatory genes. Results. Bone marrow derived macrophages (BMDM) were prepared from C57BL-6j mice and tested for their response. Lipopolysaccharide (LPS) increased IL-6 and TNF-α by 132 and 200-fold, respectively, compared to the control (p0.05). Under these conditions, inhibitors of ULK (unc-51-like) 1-2, an important kinase in the initiation steps of macroautophagy decreased the levels of proinflammatory cytokines in response to LPS. These data suggest that inhibition of ULK in BMDM has an additional effect to the inhibition of autophagy. In contrast, inhibitors of VPS34, a PI3K-III isofor