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Purine catabolism as a modulator of macrophage activation Starchuk, Lucas

Abstract

Macrophages are multifunctional innate immune cells that can promote or dampen inflammation. Various stimuli including cytokines and pathogen- or damage-associated molecular patterns activate macrophages, reprogramming them into a diverse array of possible subtypes. The signals guiding the differing functional states of macrophages are of particular interest in the search to provide improved treatment of diseases in which macrophage-mediated inflammation is implicated. During macrophage activation, metabolic reprogramming is crucial to support various pro- and anti-inflammatory activation states. Xanthine oxidoreductase (XOR) is a metabolic enzyme which catalyzes the final two steps in purine catabolism, producing uric acid from hypoxanthine and xanthine. XOR activity has previously been shown to support pro-inflammatory IL-1β production in macrophages, however the broader role of XOR activity on macrophage activation remains to be fully elucidated. We therefore aimed to investigate the role of XOR activity on macrophage activation and function. Bone marrow-derived macrophages were generated from adult C57BL/6 mice and using LPS(+IFNγ) or IL-4 in the presence of the XOR inhibitor febuxostat. Global metabolic profiles were assessed using the Seahorse assay, which uses proxies to measure mitochondrial and glycolytic metabolism. Expression of key markers of macrophage activation were assessed by flow cytometry. Levels of signaling mediators for Toll-like receptor 4 (TLR4, which recognizes LPS), components of LPS-induced pro-inflammatory complex NLRP3 inflammasome, and its product IL-1β, were measured by immunoblotting. We found that inhibition of XOR resulted in a preservation of mitochondrial respiratory function in LPS+IFNγ-stimulated macrophages, which are known to progressively lose this function. XOR inhibition resulted in blunted upregulation of canonical markers of activation in both LPS+IFNγ- and IL-4-stimulated macrophages. LPS-stimulated macrophages contained lower levels of IL-1β when XOR was inhibited, consistent with observed changes in TLR4 signaling mediators, including a decrease in the adaptor protein MyD88, and differential modulation of mitogen-activated protein kinase activity. XOR activity appears to support both LPS(+IFNγ)- and IL-4-induced macrophage activation. Further elucidation of the mechanism linking purine catabolism and macrophage activation could uncover novel targets for the treatment of diseases in which macrophages are implicated.

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Attribution-NonCommercial-NoDerivatives 4.0 International