ACAT-1 Inhibition Limits iNOS in anIn VitroModel of Macrophage Activation

Abstract

Each year in the United States, there are 190,600 cases of acute lung injury (ALI), associated with a mortality rate of over 74,000 deaths. Data shows acyl ‐ coenzyme A acetyltransferase ‐ 1 (ACAT ‐ 1) inhibition improves pulmonary inflammation in an in vivo murine model of ALI. We hypothesize that ACAT ‐ 1 inhibition has anti ‐ inflammatory effects beyond its intended use to reduce cholesterol esterification. The purpose of this study is to establish an in vitro bone marrow ‐ derived macrophage (BMDM) model to investigate the effect of ACAT ‐ 1 inhibition in macrophage activation by inducing an inflammatory response through lipopolysaccharide (LPS), and selectively inhibiting ACAT ‐ 1 with K ‐ 604. This model will provide insight on target cell metabolism, reduce interference from whole ‐ body effects, and minimize animal use. Monocytes were harvested from the bone marrow of 6 ‐ 8 week old wild ‐ type mice C57BL/6J (Jackson Laboratory) and stimulated with M ‐ CSF on d0, 3, and 7 to induce macrophage differentiation. To examine if ACAT ‐ 1 inhibition limits macrophage activation, K ‐ 604 was co ‐ administered with M ‐ CSF. Then, the cells were treated with LPS on d7 and harvested after 24h. Nitrite, NOS2 expression, and iNOS protein were determined through nitrite colorimetric measurement, RT ‐ qPCR, and western blot, respectively. Nitrite was measured as a proxy for nitric oxide (NO) production and was observed to decrease in LPS ‐ stimulated cells chronically treated with K ‐ 604. NOS2 was also reduced in LPS and K ‐ 604 conjunctive treatment. Compared to LPS, iNOS was reduced with chronic K ‐ 604 as measured by iNOS protein. LPS ‐ induced macrophage activation was suppressed and NO production was hindered due to lack of the iNOS protein. This aligns with the in vivo model, where K ‐ 604