Abstract TMP74: HIF-1a And MTORC2 Co-regulate Macrophage Inflammation And Metabolism In Intracerebral Hemorrhage

Abstract

Introduction: Recruited macrophages are known to spur secondary inflammation-induced damage in intracerebral hemorrhage (ICH), and targeted metabolic modulation of these macrophages could limit inflammation. Recent macrophage transcriptomic work has associated increased expression of HIF-1a, master regulator of the hypoxia response, and mTORC2, the more recently-discovered mTOR kinase complex, with good patient outcomes. The goal of this work was to identify the inflammatory and metabolic effects of these factors, providing insight into their putatively beneficial role in acute brain injury. Methods: Human monocyte-derived macrophages were stimulated either with ICH-associated danger signals (DAMP) (S100A9 (2 ug/mL, R&D) + IL-1β (10 ng/mL, Biolegend)), or 1 ng/mL LPS (Invivogen). Echinomycin (Sigma Aldrich), NR1 (Aobious), JR-AB2-011 (Aobious), and Torin-1 (LC Labs) were used to inhibit HIF-1, mTORC1, mTORC2, and mTOR signaling respectively. Cytokine levels were quantified using cytometric bead array (BD) and prostaglandin-E2 (PGE 2 ) levels were quantified using ELISA (Cayman). Metabolic flux was measured using the Seahorse platform (Agilent). Results/Conclusions: HIF-1a was necessary for the production of inflammatory and reparative cytokines, consistent with previous literature. Inhibition of mTORC1 or mTORC2 had little effect on cytokine levels, while broad inhibition of mTOR resulted in a step-wise decrease in chemokine secretion. mTORC2 was required for the production of PGE 2 (p<0.01; Figure 1A), a lipid mediator highly expressed in ICH patients with good outcomes. This effect was dependent on HIF-1a signaling (p<0.01; Figure 1A) and resulted in an increase in glycolytic flux (p<0.001; Figure 1B), suggesting a neuroprotective mTORC2-HIF-PGE 2 axis. Future research will explore the role of HIF-1a and mTORC2 in murine ICH models using pharmacological inhibition and LysM-Cre HIF-1a-floxed mixed bone marrow chimeras.