Metabolic reprogramming of human monocytes/macrophages in obesity ; implications in obesity-linked inflammation and cancers.

Obesity is an ongoing worldwide epidemic now recognized as a low-grade inflammatory disease, favoring the development of metabolic diseases and cancers. This systemic inflammation results mainly from an excessive accumulation of M1-polarized macrophages in adipose tissue. Chronic, low-grade inflammation has long been associated with cancer initiation, promotion and progression. Therefore, adipose tissue macrophages (ATMs) may play a significant role in carcinogenesis, making them a good target for therapeutic strategies. Free fatty acids concentrations are strongly increased in adipose tissue and in the blood of obese patients. Since saturated fatty acids (SFAs), unlike unsaturated ones (UFAs), were shown to induce pro-inflammatory pathways, they are suspected of triggering this M1 macrophage polarization. Through a whole transcriptome sequencing (RNAseq), we demonstrated that stearate (C18 :0), a common saturated fatty acid, is able to induce M1-like phenotype in human monocytes-derived macrophages (MDMs). Interestingly, hypoxia and glycolysis pathways are significantly upregulated through Gene Set Enrichment Analysis (GSEA), suggesting that stearate could trigger a glycolytic switch. The objectives of this project are (i) to investigate the metabolic reprogramming in saturated fatty acid-stimulated MDMs in vitro and (ii) to assess the metabolic status of circulating monocytes in obese versus lean patients. The up-regulation of glycolysis genes was confirmed by qRT-PCR but we wanted to validate the glycolytic switch by other methods. First of all, we monitored the lactate released in the supernatant of FFAs-treated MDMs. We showed a significant increase of lactate production by MDMs treated by C18:0 compared to the BSA control. Currently, we are analyzing the 2-NBDG (…) uptake by FFAs-treated MDMs through flow cytometry. Preliminary results suggest an increase of 2-NBDG uptake by C18:0-stimulated MDMs compared to the BSA control. WB ….Finally, to validate the glycolytic switch and give further informations about the metabolism (OxPhos) in FFAs-treated MDMs, we plan to use the SeaHorse technology. Moreover, we would like to study molecular mechanisms underlying the glycolytic switch induced by SFAs in MDMs. Currently, we are testing various inhibitors on both lactate secretion and glycolysis enzymes up-regulation. Preliminary results suggest that C18:0 has to be activated by the acetyl CoA synthase (ACS) inside cells to be able to induce the glycolytic switch. We are investigating both ER stress and mTORC1 pathways highlighted in the GSEA. While ER stress is not involved, the mTORC1 pathway seems to play a role. The up-regulation of glycolysis genes was confirmed in C18:0-treated MDMs by qRT-PCR and Western blotting experiments but we wanted to validate the glycolytic switch by other methods. First of all, we showed a significant increase of lactate production by MDMs treated by C18:0 compared to the BSA control. Currently, we are analyzing the uptake of a fluorescent glucose analog (2-NBDG) by FFAs-treated MDMs through flow cytometry. Finally, to validate the glycolytic switch and give further informations about the metabolism (OxPhos) in FFAs-treated MDMs, we plan to use the SeaHorse technology. Moreover, we would like to study molecular mechanisms underlying the glycolytic switch in SFAs-treated MDMs. Currently, we are testing various inhibitors on both lactate secretion and glycolysis enzymes up-regulation. Preliminary results suggest that C18:0 has to be activated by the acetyl CoA synthase (ACS) inside cells to induce the glycolytic switch. We are also investigating the involvement of both ER stress and mTORC1 pathways highlighted in the GSEA. We think that this SFA-induced glycolytic switch, by providing metabolic intermediates and ATP, allows MDMs to rapidly produce pro-inflammatory cytokines. We plan to validate this hypothesis by studying the impact of the glycolysis inhibitor, 2-deoxy-D-glucose (2-DG) on the production of pro-inflammatory cytokines (IL-1β, TNFα, IL-6) by SFAs-treated MDMs. In parallel, we are performing different analyses on circulating monocytes from obese patients versus lean individuals. We demonstrated a significant increase in the 2-NBDG uptake by monocytes from obese compared to lean patients. The upregulation of glycolytic genes in circulating monocytes will be confirmed by qRT-PCR and flow cytometry experiments. We are currently carrying out activity tests on monocytes freshly isolated from blood of obese and lean patients (phagocytosis, cytokines secretion). This project should bring a better understanding of the metabolic reprogramming that occurs in ATMs and circulating monocytes and fuels sterile inflammation in obesity. It could lead to new therapeutic strategies that would not directly target the pro-inflammatory signaling cascades but rather the metabolic pathways of human monocytes/macrophage