Abstract 1756 Molecular Mechanism of Macrophage Plasticity Mediated by Dexamethasone and Aldosterone

Synthetic glucocorticoids (GCs) and mineralocorticoids (MCs) have been utilized within the medical field for over 70 years for their broad versatility in modulating the immune response. Previous research has observed their respective nuclear ligand-dependent protein receptors GR and MR to participate in the regulation of the immune response, yet current research lacks knowledge on how these pathways may interplay with each other. During the COVID-19 pandemic, an estimated 40% of patients infected were reported to develop a form of acute respiratory distress syndrome (ARDS) while other patients developed a progressive pulmonary fibrotic disease state. Given GCs known anti-inflammatory modulation, such synthetic steroid hormones became a mainstream treatment for respiratory hyperinflammation though the inflammatory response is still not entirely known. The mechanisms underlying the immune systems' exact role and involvement in pulmonary fibrotic diseases remain poorly understood due to the vast heterogeneity observed in disease pathologies and currently have no cure. During fibrotic progression, reports of high numbers of monocyte-derived macrophages were observed to infiltrate the alveoli in an inflammation-dependent nature. Moreover, monocyte-derived macrophages were also shown to have high heterogeneity in their gene expression patterns making them an important immune cell to study. Thus, an exploration into the molecular mechanism of the immune response could provide more insights and potential therapeutic avenues for lung fibrotic pathogenesis. To elucidate how synthetic glucocorticoid (Dexamethasone; DEX) and mineralocorticoid (Aldosterone) affect macrophage polarization and mediate fibrotic pathogenesis, the study aims to utilize a THP-1 monocyte-macrophage-derived, conditioned-media, culture-system with human lung alveolar-epithelial cells (A549) to investigate the nature of macrophage-mediated profibrotic induction. Several techniques are being used to identify THP-1-macrophage pro(M1)/anti(M2)-inflammatory phenotypes including RT-qPCR to measure the expression of signature genes related to THP-1 macrophage phenotypes (M1: NOS2/iNOS, IL-1β, PTPN6, and TNF )(M2: MRC1, CD163, and CD2000R1, LGALS3) 2) Western Blot Analysis of promising candidate genes from gene profiling and A549 alveolar lung epithelial cell fibrotic protein expression via ⍺-Smooth Muscle Actin (SMA), Collagen 1, E-cadherin, and Vimentin in A549 cells. 4) Imaging of fibrotic stress fibers via phalloidin and SMA staining. RT-qPCR analysis revealed that DEX induced a classic anti-inflammatory response with up-regulation of signature genes CD163, and GILZ, and subsequent downregulation of IL-1β. Aldosterone-induced signature pro-inflammatory gene expression is directly inverse of DEX-mediated gene regulation. The combinatory treatment group revealed synergistic gene regulation of GILZ and PTPN6, while also revealing unique signaling mechanisms for GR and MR within CD163 and IL-1β. Western blot experiments will be performed to assess fibrotic changes within lung epithelial cells exposed to monocyte-macrophage-derived conditioned media. The resulting gene expression patterns reveal insights into the possibility of GR and MR cross-talk-mediated gene regulation, specifically within the immune response. Such synergistic and dominating regulation aids in expanding current research on macrophage plasticity.