Modulation of innate immune cells with obesity and glucose intolerance

Obesity is a worldwide epidemic now recognized as a low-grade inflammatory disease, favouring the development of metabolic diseases and cancers. It is characterised by an expansion of the adipose tissue, accompanied by a release of fatty acids from dead adipocytes. Immunity is modified in this enlarged tissue, with macrophage enrichment (up to 50% increased) and a general shift towards pro-inflammatory immune cells. These changes get systemic with time, as we can see higher rates of circulating free fatty acids as well as circulating immune cells favouring the general low-grade inflammatory state. In this work, we tried to better understand the role of free fatty acids-induced changes in macrophages. We also attempted to bring a finer characterization of innate immune changes befitting obesity and glucose intolerance. Stearate, a saturated fatty acid, is able to reshape the transcriptome of human monocyte derived macrophages (MDM), in opposition to oleate, its unsaturated counterpart. Gene set enrichment analysis of the stearate-induced transcriptome highlights metabolic pathways such as glycolysis and fatty acid metabolism. Increase of glycolysis is confirmed by lactate assay, and seems to be mediated by mTORC1 pathway. Phospholipidomic profiling of stearate-treated MDM shows an increase of saturated phospholipids, which is characteristic of de novo lipogenesis. Seeing these changes in vitro, we asked ourselves if they were also observed with obesity and glucose intolerance. Phospholipidomic profiling of peripheral blood mononuclear cells underlines an increase of saturated and monounsaturated phospholipids with obesity, but more specifically with glucose intolerance. Flow cytometry experiments indicate higher amount of circulating inflammatory monocyte subtypes with obese glucose intolerant patients. Monocytes also uptake higher amounts of glucose with obesity, which might mirror a glycolytic change. Metabolic variation of immune cells is usually linked to activity alteration. No differences of monocyte phagocytosis are observed, whereas CCL2 and IL8 secretion vary for obese patients. In this work, we also studied variations for circulating NK cells. CD56bright NK cells levels increase with obesity and glucose tolerance where CD56dim NK cells decrease. CD56bright NK cells phenotype differs with obesity and glucose tolerance and these cells show variations of their activating receptors expression. NK cells from obese patients are less cytotoxic against cancer cells, which is not observed with glucose intolerant patients. These cells ability to secrete cytokines also decreases with obesity, showing a global restraining of NK cells with this disease. Altogether, our results indicate that saturated fatty acids induce a metabolic switch in human MDM, with increased glycolysis and fatty acid metabolism. Signs of metabolic changes are also observed in circulating immune cells, which show phenotype and activity variations with obesity and / or glucose intolerance

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

Investigation of the glycolytic switch induced by saturated fatty acid in human macrophages

Obesity is a worldwide epidemic now recognized as a low-grade inflammatory disease, favoring the development of metabolic diseases and cancers. This systemic inflammation results from an accumulation of pro-inflammatory macrophages in adipose tissue. Recent work shows that these adipose tissue macrophages (ATMs) are characterized by M1/M2 and metabolic markers. Free fatty acids released in excess in obese adipose tissue could be proposed as triggers in shaping the ATM phenotype. Indeed, the treatment with saturated fatty acids (SFAs) recapitulates many features of the polarization seen in obese ATM. The goals of this research are to further characterize ATMs in obese patients and understand underlying transcriptional programs by using the model of human monocyte-derived macrophages (MDMs) treated with a common SFA. We performed the sequencing of the whole transcriptome of MDMs treated with saturated (C18:0) or unsaturated fatty acid (C18:1) complexed to BSA or with BSA alone. C18:0 treatment induces 3345 differentially expressed genes (q<0,05) while C18:1 treatment produces a weak response. As expected, gene set enrichment analysis (GSEA) demonstrates that C18:0 activates the transcription of genes involved in several M1 pathways. Interestingly, GSEA shows the enrichment of hypoxia and glycolysis gene sets, suggesting SFA-mediated aerobic glycolysis. We confirmed the glycolytic switch in C18 :0-treated MDMs by showing up-regulation of glycolysis enzymes and metabolites transporters and by measuring lactate production. We are currently investigating upstream events and have preliminary data suggesting SFAs do not induce this glycolytic switch through the TLR4 binding but has to be activated into acylCoA inside cells. This research project should lead to a better understanding of the metabolic reprogramming that occurs in ATMs during obesity and fuels an obesity-associated « sterile » inflammation

IRE1 endoribonuclease plays a key role in activation of both HIF-1alpha and glucose metabolism in saturated fatty acid-treated human macrophages

peer reviewedIn obesity, adipose tissue infiltrating macrophages acquire a unique pro-inflammatory polarization, thereby playing a key role in the development of chronic inflammation and T2D. Increased saturated fatty acids (SFAs) levels have been proposed to drive this specific polarization. Accordingly, we investigated the immunometabolic reprogramming in SFA-treated human macrophages. The RNAseq highlighted a pro-inflammatory profile and signatures like UPR, glycolysis, hypoxia. Glycolysis activation, as estimated by glycolytic gene expression, glucose uptake, lactate production and GLUT1 levels, has been dependent on HIF-1alpha stabilization and has fueled the production of IL-1alpha. Concomitantly, SFAs have also induced a strong xbp-1 mRNA splicing via the endoribonuclease IRE1alpha, one ER stress sensor. Interestingly, the knockdown and pharmacological inhibition of IREalpha, unlike the knockdown of XBP1s, have prevented activation of both HIF-1alpha and glycolysis. These data suggest for the first time that IRE1alpha plays a key role through XBP1s-independent way in glucose metabolism activation in SFAs-treated macrophages

Obesity induces metabolic and phenotypic variations in blood monocytes and NK cells.

Introduction : Obesity is recognized as low-grade inflammatory disease, favoring the development of metabolic diseases and cancers. Methods : We investigated by flow cytometry obesity and glucose intolerance effects on monocytes and NK cells in lean and normo-glycemic or (pre)-diabetic obese patients. Results : Monocytes present increased glucose uptake and higher GLUT1 expression in all obese patients. This glucose uptake increase is specifically seen in classical monocytes. In normo-glycemic obese compared to lean patients, CD56dim NK cells are decreased while expression of their activating receptors such as CD16, NKp30, NKG2D and NKp46 is not affected. However CD56high NK cells are increased with obesity and their receptors are differentially modulated according to BMI and glycemia. Discussion : We are assessing monocytes and NK cells activity in different patients groups. We are also doing lipidomics on plasma and PBMC. These experiments will allow us to highlight new correlations between lipidomic, metabolic and immunologic parameters at both systemic and cellular levels

La modulation du phospholipidome dans les cellules immunitaires circulantes des patients obèses est associée à la résistance à l'insuline ou à l'état glycémique

This lipidomic study highlights for the first-time modulations of the phospholipidome of PBMCs in obese patients with prediabetes and type 2 diabetes. Such phospholipidome remodeling could disrupt the cell membranes and the lipid mediator’s levels, driving an immune cell dysfunction

Novel XBP1s-independent function of IRE1 RNase in HIF-1α-mediated glycolysis upregulation in human macrophages upon stimulation with LPS or saturated fatty acid

International audienceIn obesity, adipose tissue infiltrating macrophages acquire a unique pro-inflammatory polarization, thereby playing a key role in the development of chronic inflammation and Type 2 diabetes. Increased saturated fatty acids (SFAs) levels have been proposed to drive this specific polarization. Accordingly, we investigated the immunometabolic reprogramming in SFA-treated human macrophages. As expected, RNA sequencing highlighted a pro-inflammatory profile but also metabolic signatures including glycolysis and hypoxia as well as a strong unfolded protein response. Glycolysis upregulation was confirmed in SFA-treated macrophages by measuring glycolytic gene expression, glucose uptake, lactate production and extracellular acidification rate. Like in LPS-stimulated macrophages, glycolysis activation in SFA-treated macrophages was dependent on HIF-1α activation and fueled the production of pro-inflammatory cytokines. SFAs and LPS both induced IRE1α endoribonuclease activity, as demonstrated by XBP1 mRNA splicing, but with different kinetics matching HIF-1α activation and the glycolytic gene expression. Interestingly, the knockdown of IRE1α and/or the pharmacological inhibition of its RNase activity prevented HIF-1α activation and significantly decreased glycolysis upregulation. Surprisingly, XBP1s appeared to be dispensable, as demonstrated by the lack of inhibiting effect of XBP1s knockdown on glycolytic genes expression, glucose uptake, lactate production and HIF-1α activation. These experiments demonstrate for the first time a key role of IRE1α in HIF-1α-mediated glycolysis upregulation in macrophages stimulated with pro-inflammatory triggers like LPS or SFAs through XBP1s-independent mechanism. IRE1 could mediate this novel function by targeting other transcripts (mRNA or pre-miRNA) through a mechanism called regulated IRE1-dependent decay or RIDD. Deciphering the underlying mechanisms of this novel IRE1 function might lead to novel therapeutic targets to curtail sterile obesity- or infection-linked inflammation

New insights on the PBMCs phospholipidome in obesity demonstrate modulations associated with insulin resistance and glycemic status

Abstract: (1) Background: Obesity and type 2 diabetes have been suspected to impact both intrin-sic metabolism and function of circulating immune cells. (2) Methods: To further investigate this immunometabolic modulation, we profiled the phospholipidome of the peripheral blood mononu-clear cells (PBMCs) in lean, normoglycemic obese (OBNG) and obese with dysglycemia (OBDysG) individuals. (3) Results: The global PBMCs phospholipidome is significantly downmodulated in OBDysG unlike OBNG patients when compared to lean ones. Multiple linear regression analyses show a strong negative relationship between the global PBMCs phospholipidome and parameters assessing insulin resistance. Even though all classes of phospholipid are affected, the relative abundance of each class is maintained with the exception of Lyso-PC/PC and Lyso-PE/PE ratios that are downmodulated in PBMCs of OBDysG compared to OBNG individuals. Interestingly, the percentage of saturated PC is positively associated with glycated hemoglobin (HbA1c). Moreover, a few lipid species are significantly downmodulated in PBMCs of OBDysG compared to OBNG individuals, making possible to distinguish the two phenotypes. (4) Conclusions: This lipidomic study highlights for the first-time modulations of the PBMCs phospholipidome in obese patients with prediabetes and type 2 diabetes. Such phospholipidome remodeling could disrupt the cell membranes and the lipid mediator’s levels, driving an immune cell dysfunction