View Abstract

A4486 - Apoptotic Cell Engulfment Triggers Anti-Inflammatory Metabolic Reprogramming of Macrophages
Author Block: A. McCubbrey, P. M. Henson, W. J. Janssen; National Jewish Health, Denver, CO, United States.
Rationale: Apoptotic cell engulfment, also called efferocytosis, is a critical biologic process performed by phagocytes, particularly macrophages. Efferocytosis reprograms inflammatory macrophages towards a resolving phenotype, suppressing the production of pro-inflammatory cytokines and upregulating the production of anti-inflammatory mediators. Defects in efferocytosis have been shown to worsen outcomes in animal models of acute and chronic lung inflammation, and have been observed in patients with COPD and IPF, concurrent with increased numbers of inflammatory macrophages. However, the mechanisms by which efferocytosis orchestrates macrophage reprogramming are poorly understood. Recent work has connected macrophage reprogramming to a shift in macrophage metabolism. We examined how efferocytosis altered macrophage metabolism and whether this was required for anti-inflammatory reprogramming.
Methods: Murine peritoneal and bone marrow-derived macrophages were used for all studies. Jurkat cells killed with UV radiation were used as apoptotic targets. Apoptotic targets were co-cultured with macrophages in vitro. Uningested apoptotic targets were removed after 1 hour of co-culture, and macrophages were collected 0-16 hours later. 150 intracellular metabolites were measured using liquid chromatography-mass spectrometry. Flow cytometry and microscopy were used to assess levels of efferocytosis and the rate of target cell degradation. RT-PCR was used to measure changes in gene expression. Experiments used both wild type macrophages and macrophages deficient in arginase 1 (Arg1); for some experiments inflammatory macrophages were generated by stimulation with LPS.
Results: Efferocytosis triggered a prolonged, delayed metabolic shift that occurred in parallel with the degradation of engulfed apoptotic cells. Maximal change from baseline was observed 8 hours after efferocytosis. Among many changes, efferocytosis induced a significant increase in intracellular polyamines, a family of anti-inflammatory arginine metabolites. The induction of polyamines in response to efferocytosis was abolished when macrophages were deficient in Arg1. Separately, polyamines and efferocytosis have both been shown to suppress expression of IL-1beta. However, we found that LPS-polarized macrophages deficient in Arg1 were unable to suppress IL-1beta expression in response to efferocytosis, although the level of efferocytosis remained the same. Conclusions: Efferocytosis significantly alters macrophage metabolism. Efferocytosis induces the production of polyamines through a mechanism that involves Arg1. This switch in metabolism proceeds inflammatory macrophage reprogramming; blocking polyamine production through Arg1-deficiency prevented the downregulation of IL-1beta expression in response to efferocytosis. We identify macrophage metabolism as a critical mechanism by which efferocytosis orchestrates macrophage reprogramming. This is important for our understanding of persistent inflammatory macrophages in chronic lung disease and suggests a novel avenue for therapeutic intervention.