Macrophage metabolic adaptation to heme detoxification involves CO-dependent activation of the pentose phosphate pathway.

Heme is an essential cofactor for numerous cellular functions, but release of free heme during hemolysis results in oxidative tissue damage, vascular dysfunction, and inflammation. Macrophages play a key protective role in heme clearance; however, the mechanisms that regulate metabolic adaptations that are required for effective heme degradation remain unclear. Here we demonstrate that heme loading drives a unique bioenergetic switch in macrophages, which involves a metabolic shift from oxidative phosphorylation toward glucose consumption. Metabolomic and transcriptional analysis of heme-loaded macrophages revealed that glucose is funneled into the pentose phosphate pathway (PPP), which is indispensable for efficient heme detoxification and is required to maintain redox homeostasis. We demonstrate that the metabolic shift to the PPP is controlled by heme oxygenase-dependent generation of carbon monoxide (CO). Finally, we show that PPP upregulation occurs in vivo in organ systems central to heme clearance and that PPP activity correlates with heme levels in mouse sickle cell disease (SCD). Together, our findings demonstrate that metabolic adaptation to heme detoxification in macrophages requires a shift to the PPP that is induced by heme-derived CO, suggesting pharmacologic targeting of macrophage metabolism as a novel therapeutic strategy to improve heme clearance in patients with hemolytic disorders.

Macrophages sensing oxidized DAMPs reprogram their metabolism to support redox homeostasis and inflammation through a TLR2-Syk-ceramide dependent mechanism.

OBJECTIVE: Macrophages control tissue homeostasis and inflammation by sensing and responding to environmental cues. However, the metabolic adaptation of macrophages to oxidative tissue damage and its translation into inflammatory mechanisms remains enigmatic. METHODS: Here we identify the critical regulatory pathways that are induced by endogenous oxidation-derived DAMPs (oxidized phospholipids, OxPL) in vitro, leading to formation of a unique redox-regulatory metabolic phenotype (Mox), which is strikingly different from conventional classical or alternative macrophage activation. RESULTS: Unexpectedly, metabolomic analyses demonstrated that Mox heavily rely on glucose metabolism and the pentose phosphate pathway (PPP) to support GSH production and Nrf2-dependent antioxidant gene expression. While the metabolic adaptation of macrophages to OxPL involved transient suppression of aerobic glycolysis, it also led to upregulation of inflammatory gene expression. In contrast to classically activated (M1) macrophages, Hif1α mediated expression of OxPL-induced Glut1 and VEGF but was dispensable for Il1ß expression. Mechanistically, we show that OxPL suppress mitochondrial respiration via TLR2-dependent ceramide production, redirecting TCA metabolites to GSH synthesis. Finally, we identify spleen tyrosine kinase (Syk) as a critical downstream signaling mediator that translates OxPL-induced effects into ceramide production and inflammatory gene regulation. CONCLUSIONS: Together, these data demonstrate the metabolic and bioenergetic requirements that enable macrophages to translate tissue oxidation status into either antioxidant or inflammatory responses via sensing OxPL. Targeting dysregulated redox homeostasis in macrophages could therefore lead to novel therapies to treat chronic inflammation.

Metabolism Plays a Key Role during Macrophage Activation.

Monocyte and macrophage diversity is evidenced by the modulation of cell surface markers and differential production of soluble mediators. These immune cells play key roles in controlling tissue homeostasis, infections, and excessive inflammation. Macrophages remove dead cells in a process named efferocytosis, contributing to the healthy tissue maintenance. Recently, it became clear that the main macrophage functions are under metabolic control. Modulation of glucose, fatty acid, and amino acid metabolism is associated with various macrophage activations in response to external stimuli. Deciphering these metabolic pathways provided critical information about macrophage functions.

Macrophage metabolism in atherosclerosis.

A key aspect of atherosclerosis is the maladaptive inflammatory response to lipoprotein accumulation in the artery. The failure to decrease lipid accumulation, to clear apoptotic cells, and to resolve inflammation ultimately leads to macrophage accumulation within the vascular wall [Thorp EB (2010) Apoptosis15, 1124-1136; Moore K et al. (2013) Nat Rev Immunol 13, 709-721; Moore KJ and Tabas I (2011) Cell 145, 341-355; Ley K et al. (2011) Arterioscler Thromb Vasc Biol 31, 1506-1516]. Several subsets of macrophages are found inside atherosclerotic plaques [Chinetti-Gbaguidi G et al. (2015) Nat Rev Cardiol 12, 10-17; Leitinger N and Schulman IG (2013) Arterioscler Thromb Vasc Biol 33, 1120-1126; Mantovani A et al. (2009) Arterioscler Thromb Vasc Biol 29, 1419-1423]: Proinflammatory M1-like macrophages potentially participate in atherosclerosis initiation and progression; M2-like macrophages are thought to be protective due to their anti-inflammatory and profibrotic properties, presumably stabilizing the plaque [Chistiakov DA et al. (2015) Int J Cardiol 184, 436-445; Gordon S (2003) Nat Rev Immunol 3, 23-35]; Mox macrophages develop in response to oxidized phospholipids and present a glutathione- and potentially redox-regulating phenotype [Kadl A et al. (2010) Circ Res 107, 737-746]; Mhem macrophages are found in areas of plaque hemorrhage [Boyle JJ et al. (2009) Am J Pathol 174, 1097-1108; Boyle JJ et al. (2012) Circ Res 110, 20-33] where they are involved in heme clearance. Recent evidence suggests that the relative abundance of these macrophage subsets is a better indicator of plaque progression and stability than the total number of lesion macrophages [Chinetti-Gbaguidi G et al. (2015) Nat Rev Cardiol 12, 10-17]. Over the last few years, findings in the area of immunometabolism established a link between the metabolic state of the different macrophage phenotypes and their functions [O'Neill LAJ and Pearce EJ (2016) J Exp Med 213, 15-23]. However, the effect of metabolic changes in macrophages on atherosclerotic plaque progression and stability is not well understood and an area of intensive study. In this review, we will summarize and critically discuss recent developments in the field of macrophage metabolism in the context of atherosclerosis to guide future investigation in this area.

Liver X receptor activation stimulates iron export in human alternative macrophages.

RATIONALE: In atherosclerotic plaques, iron preferentially accumulates in macrophages where it can exert pro-oxidant activities. OBJECTIVE: The objective of this study was, first, to better characterize the iron distribution and metabolism in macrophage subpopulations in human atherosclerotic plaques and, second, to determine whether iron homeostasis is under the control of nuclear receptors, such as the liver X receptors (LXRs). METHODS AND RESULTS: Here we report that iron depots accumulate in human atherosclerotic plaque areas enriched in CD68 and mannose receptor (MR)-positive (CD68(+)MR(+)) alternative M2 macrophages. In vitro IL-4 polarization of human monocytes into M2 macrophages also resulted in a gene expression profile and phenotype favoring iron accumulation. However, M2 macrophages on iron exposure acquire a phenotype favoring iron release, through a strong increase in ferroportin expression, illustrated by a more avid oxidation of extracellular low-density lipoprotein by iron-loaded M2 macrophages. In line, in human atherosclerotic plaques, CD68(+)MR(+) macrophages accumulate oxidized lipids, which activate LXRα and LXRß, resulting in the induction of ABCA1, ABCG1, and apolipoprotein E expression. Moreover, in iron-loaded M2 macrophages, LXR activation induces nuclear factor erythroid 2-like 2 expression, thereby increasing ferroportin expression, which, together with a decrease of hepcidin mRNA levels, promotes iron export. CONCLUSIONS: These data identify a role for M2 macrophages in iron handling, a process regulated by LXR activation.

Human adipose tissue macrophages display activation of cancer-related pathways.

Obesity is associated with a significantly increased risk for cancer suggesting that adipose tissue dysfunctions might play a crucial role therein. Macrophages play important roles in adipose tissue as well as in cancers. Here, we studied whether human adipose tissue macrophages (ATM) modulate cancer cell function. Therefore, ATM were isolated and compared with monocyte-derived macrophages (MDM) from the same obese patients. ATM, but not MDM, were found to secrete factors inducing inflammation and lipid accumulation in human T47D and HT-29 cancer cells. Gene expression profile comparison of ATM and MDM revealed overexpression of functional clusters, such as cytokine-cytokine receptor interaction (especially CXC-chemokine) signaling as well as cancer-related pathways, in ATM. Comparison with gene expression profiles of human tumor-associated macrophages showed that ATM, but not MDM resemble tumor-associated macrophages. Indirect co-culture experiments demonstrated that factors secreted by preadipocytes, but not mature adipocytes, confer an ATM-like phenotype to MDM. Finally, the concentrations of ATM-secreted factors related to cancer are elevated in serum of obese subjects. In conclusion, ATM may thus modulate the cancer cell phenotype.

Impaired alternative macrophage differentiation of peripheral blood mononuclear cells from obese subjects.

Visceral obesity is a chronic, low-grade inflammatory disease that predisposes people to the metabolic syndrome, type 2 diabetes and its cardiovascular complications. Adipose tissue is not a passive storehouse for fat, but an endocrine organ synthesizing and releasing a variety of bioactive molecules, some of which are produced by infiltrated immune-inflammatory cells including macrophages. Two different subpopulations of macrophages have been identified in adipose tissue: pro-inflammatory 'classical' M1 and anti-inflammatory 'alternative' M2 macrophages, and their ratio is suggested to influence the metabolic complications of obesity. These macrophages derive primarily from peripheral blood mononuclear cells (PBMCs). We hypothesised that obesity and the metabolic syndrome modulate PBMC functions. Therefore, alteration of the monocyte response, and more specifically their ability to differentiate toward alternative anti-inflammatory macrophages, was assessed in PBMCs isolated from lean and obese subjects with or without alterations in glucose homeostasis. Our results indicate that PBMCs from obese subjects have an altered expression of M2 markers and that their monocytes are less susceptible to differentiate toward an alternative phenotype. Thus PBMCs in obesity are programmed, which may contribute to the inflammatory dysregulation and increased susceptibility to inflammatory diseases in these patients.

Human atherosclerotic plaque alternative macrophages display low cholesterol handling but high phagocytosis because of distinct activities of the PPARγ and LXRα pathways.

RATIONALE: A crucial step in atherogenesis is the infiltration of the subendothelial space of large arteries by monocytes where they differentiate into macrophages and transform into lipid-loaded foam cells. Macrophages are heterogeneous cells that adapt their response to environmental cytokines. Th1 cytokines promote monocyte differentiation into M1 macrophages, whereas Th2 cytokines trigger an "alternative" M2 phenotype. OBJECTIVE: We previously reported the presence of CD68(+) mannose receptor (MR)(+) M2 macrophages in human atherosclerotic plaques. However, the function of these plaque CD68(+)MR(+) macrophages is still unknown. METHODS AND RESULTS: Histological analysis revealed that CD68(+)MR(+) macrophages locate far from the lipid core of the plaque and contain smaller lipid droplets compared to CD68(+)MR(-) macrophages. Interleukin (IL)-4-polarized CD68(+)MR(+) macrophages display a reduced capacity to handle and efflux cellular cholesterol because of low expression levels of the nuclear receptor liver x receptor (LXR)α and its target genes, ABCA1 and apolipoprotein E, attributable to the high 15-lipoxygenase activity in CD68(+)MR(+) macrophages. By contrast, CD68(+)MR(+) macrophages highly express opsonins and receptors involved in phagocytosis, resulting in high phagocytic activity. In M2 macrophages, peroxisome proliferator-activated receptor (PPAR)γ activation enhances the phagocytic but not the cholesterol trafficking pathways. CONCLUSIONS: These data identify a distinct macrophage subpopulation with a low susceptibility to become foam cells but high phagocytic activity resulting from different regulatory activities of the PPARγ-LXRα pathways.