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Title: Myeloid Slc2a1-deficient murine model revealed macrophage activation and metabolic phenotype are fueled by GLUT1

Author
item FREEMERMAN, ALEX - UNIVERSITY OF NORTH CAROLINA
item ZHAO, LIYANG - UNIVERSITY OF NORTH CAROLINA
item PINGILI, AJEETH - UNIVERSITY OF TENNESSEE
item TENG, BIN - UNIVERSITY OF TENNESSEE
item COZZO, ALYSSA - UNIVERSITY OF NORTH CAROLINA
item FULLER, ASHLEY - UNIVERSITY OF NORTH CAROLINA
item JOHNSON, AMY - UNIVERSITY OF NORTH CAROLINA
item MILNER, JUSTIN - UNIVERSITY OF NORTH CAROLINA
item LIM, MAILI - UNIVERSITY OF NORTH CAROLINA
item GALANKO, JOSEPH - UNIVERSITY OF NORTH CAROLINA
item BECK, MELINDA - UNIVERSITY OF NORTH CAROLINA
item BEAR, JAMES - UNIVERSITY OF NORTH CAROLINA
item ROTTY, JEREMY - UNIVERSITY OF NORTH CAROLINA
item BEZAVADA, LAVANYA - UNIVERSITY OF TENNESSEE
item SMALLWOOD, HEATHER - UNIVERSITY OF TENNESSEE
item PUCHOWICZ, MICHELLE - UNIVERSITY OF TENNESSEE
item LIU, JUAN - DUKE UNIVERSITY SCHOOL OF MEDICINE
item LOCASALE, JASON - UNIVERSITY OF NORTH CAROLINA
item LEE, DOUGLAS - OMIC INSIGHT, INC.
item Bennett, Brian
item ABEL, DALE - UNIVERSITY OF IOWA
item RATHMELL, JEFF - VANDERBILT UNIVERSITY
item MAKOWSKI, LIZA - UNIVERSITY OF NORTH CAROLINA

Submitted to: Journal of Immunology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2018
Publication Date: 2/15/2019
Citation: Freemerman, A.J., Zhao, L., Pingili, A.K., Teng, B., Cozzo, A.J., Fuller, A.M., Johnson, A.R., Milner, J.J., Lim, M.F., Galanko, J.A., Beck, M.A., Bear, J.E., Rotty, J.D., Bezavada, L., Smallwood, H.S., Puchowicz, M.A., Liu, J., Locasale, J.W., Lee, D., Bennett, B.J., Abel, D.E., Rathmell, J.C., Makowski, L. 2019. Myeloid Slc2a1-deficient murine model revealed macrophage activation and metabolic phenotype are fueled by GLUT1. Journal of Immunology. 202(4):1265-1286. https://doi.org/10.4049/jimmunol.1800002.
DOI: https://doi.org/10.4049/jimmunol.1800002

Interpretive Summary: Macrophages are cells of the innate immune system that play critical roles in a myriad of processes including development, tissue homeostasis, host defense, and tumor growth. A better understanding of Macrophage metabolism bears potential to inform pathological basis of disease and how diet influences inflammation. Several laboratories have demonstrated that metabolic substrate preference (e.g., glucose vs. fatty acids vs. amino acids) plays pivotal roles in Macrophage phenotype and function, including inflammatory responses. The glucose transporter GLUT1 (encoded by the gene Slc2a1) is a critical modulator of Macrophage glucose metabolism. Since GLUT1 is the primary receptor for glucose uptake in macrophages, we hypothesized that restricting glucose uptake through GLUT1 gene ablation could impact biosynthetic and energy metabolic pathways. Using gene targeting techniques in mice, we deleted the expression of GLUT1 in macrophages and tested how reduced glucose utilization affects macrophage function in diet induced obesity and atherosclerosis. Together, these studies illuminate for the first time a role for macrophage GLUT1-mediated glucose metabolism in directing inflammatory potential in diet driven diseases (obesity and atherosclerosis).

Technical Abstract: Macrophages (MFs) are metabolically flexible and highly heterogeneous, exhibiting a range of phenotypes from pro-inflammatory to immunoregulatory depending on their tissue microenvironment. Upon classical pro-inflammatory immune activation, MF metabolic reprogramming entails a redirection of substrates from oxidative ATP generation toward biosynthetic and bioenergetic demand pathways, to produce metabolites necessary for classical MF immune function. A key response to classical activation through pro- inflammatory receptors is increased flux through glycolysis and the pentose phosphate pathway (PPP) along with a downregulation of mitochondrial oxidative metabolism. However, increased glycolytic flux is also essential for alternative MF activation. Appreciating the degree of flexibility in MF metabolic phenotype and its influence on immune function is critical for understanding how immunometabolism can impact disease. Therefore, we created a novel murine model in which the glucose transporter GLUT1 (Slc2a1) was deleted in a myeloid-specific manner, to test dependence of the classical and alternative MF phenotypes on glucose metabolism. Our studies employed mice displaying lysozyme M (LysM) promoter-driven Cre-mediated excision of Slc2a1 (Slc2a1M-/-) and littermate floxed controls (Slc2a1fl/fl). Bone marrow-derived MFs (BMDM) and thioglycollate-elicited peritoneal MFs from Slc2a1M-/- mice failed to uptake glucose ex vivo. Bioenergetic, biochemical, and metabolomic analyses demonstrated a profound substrate switch in Slc2a1M-/-BMDMs, as evidenced by reduced glycolysis and PPP activity, elevated fatty acid and glutamine metabolism, and an overall mixed metabolic phenotype. Surprisingly, metabolites associated with classical activation and glycolysis, such as citrate, succinate, and fumarate, remained elevated in Slc2a1M-/- compared to Slc2a1fl/fl BMDMs, suggesting that these metabolites were derived from anaplerotic substrates rather than glucose-derived pyruvate. On the other hand, metabolites associated with alternative MF activation were increased in the absence of GLUT1, including itaconate, polyamines, and palmitoleate. These metabolic shifts were associated with a mixed inflammatory phenotype demonstrated by reduced expression of classically activated pro-inflammatory markers iNOS (Nos2), TNFa (Tnfa), IL-1ß (Il1b), and MF chemotactic protein (MCP-1 (Ccl2)), combined with reduced arginase 1 (Arg1), a marker of alternative MF activation. Moreover, Slc2a1M-/- MFs displayed reduced oxidative stress and increased capacity to buffer from oxidative insult. Thus, lack of GLUT1 led to a reduction in glycolysis and PPP without complete reprogramming to an alternate phenotype. Instead a mixed metabolic and inflammatory phenotype that resembled neither classically or alternatively activated MFs resulted, suggesting that GLUT1 is critical to many aspects of immunometabolism. To determine the effects of myeloid-specific Slc2a1 deletion in vivo, we next employed two models of MF-associated disease: diet- induced obesity and atherosclerosis. Obese mice lacking myeloid GLUT1 displayed elevated adipose tissue MCP-1 and MF infiltration. Surprisingly, obese Slc2a1M-/- mice were protected from concomitant defects typically associated with higher adipose MF infiltration, exhibiting no significant GLUT1-mediated differences in body weight, local cytokine concentrations, glycemic control, or glucose and insulin tolerance. Next, to investigate atherogenesis in the absence of myeloid GLUT1, Ldlr-/- mice were transplanted with bone marrow from Slc2a1M-/- and Slc2a1fl/fl mice and fed an atherogenic diet. Atherosclerotic lesions in Slc2a1M-/- Ldlr-/- mice were larger, necrotic, and unstable compared to Slc2a1fl/fl recipient mice (Slc2a1fl/flLdlr-/-), despite a lack of major systemic alterations. Importantly, defective phagocytic capacity was detected i