An exploratory human study investigating the influence of type 2 diabetes on macrophage phenotype after myocardial infarction.

Background: Myocardial infarction (MI) is the primary cause of death in subjects with type 2 diabetes (T2D) and their in-hospital mortality after MI is still elevated compared with those without T2D. Therefore, it is of crucial importance to identify possible mechanisms of worse clinical outcomes and mortality in T2D subjects. Monocyte/macrophage-mediated immune response plays an important role in heart remodelling to limit functional deterioration after MI. Indeed, first pro-inflammatory macrophages digest damaged tissue, then anti-inflammatory macrophages become prevalent and promote tissue repair. Here, we hypothesize that the worse clinical outcomes in patients with T2D could be the consequence of a defective or a delayed polarization of macrophages toward an anti-inflammatory phenotype. Methods and results: In an exploratory human study, circulating monocytes from male patients with or without T2D at different time-points after MI were in vitro differentiated toward pro- or anti-inflammatory macrophages. The results of this pilot study suggest that the phenotype of circulating monocytes, as well as the pro- and anti-inflammatory macrophage polarization, or the kinetics of the pro- and anti-inflammatory polarization, is not influenced by T2D. Conclusion: Further studies will be necessary to understand the real contribution of macrophages after MI in humans.

Macrophage death in atherosclerosis: potential role in calcification.

Cell death is an important aspect of atherosclerotic plaque development. Insufficient efferocytosis of death cells by phagocytic macrophages leads to the buildup of a necrotic core that impacts stability of the plaque. Furthermore, in the presence of calcium and phosphate, apoptotic bodies resulting from death cells can act as nucleation sites for the formation of calcium phosphate crystals, mostly in the form of hydroxyapatite, which leads to calcification of the atherosclerotic plaque, further impacting plaque stability. Excessive uptake of cholesterol-loaded oxidized LDL particles by macrophages present in atherosclerotic plaques leads to foam cell formation, which not only reduces their efferocytosis capacity, but also can induce apoptosis in these cells. The resulting apoptotic bodies can contribute to calcification of the atherosclerotic plaque. Moreover, other forms of macrophage cell death, such as pyroptosis, necroptosis, parthanatos, and ferroptosis can also contribute by similar mechanisms to plaque calcification. This review focuses on macrophage death in atherosclerosis, and its potential role in calcification. Reducing macrophage cell death and/or increasing their efferocytosis capacity could be a novel therapeutic strategy to reduce the formation of a necrotic core and calcification and thereby improving atherosclerotic plaque stability.

Atherosclerosis Calcification: Focus on Lipoproteins.

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipids in the vessel wall, leading to the formation of an atheroma and eventually to the development of vascular calcification (VC). Lipoproteins play a central role in the development of atherosclerosis and VC. Both low- and very low-density lipoproteins (LDL and VLDL) and lipoprotein (a) (Lp(a)) stimulate, while high-density lipoproteins (HDL) reduce VC. Apolipoproteins, the protein component of lipoproteins, influence the development of VC in multiple ways. Apolipoprotein AI (apoAI), the main protein component of HDL, has anti-calcific properties, while apoB and apoCIII, the main protein components of LDL and VLDL, respectively, promote VC. The role of lipoproteins in VC is also related to their metabolism and modifications. Oxidized LDL (OxLDL) are more pro-calcific than native LDL. Oxidation also converts HDL from anti- to pro-calcific. Additionally, enzymes such as autotaxin (ATX) and proprotein convertase subtilisin/kexin type 9 (PCSK9), involved in lipoprotein metabolism, have a stimulatory role in VC. In summary, a better understanding of the mechanisms by which lipoproteins and apolipoproteins contribute to VC will be crucial in the development of effective preventive and therapeutic strategies for VC and its associated cardiovascular disease.

Trem2 promotes foamy macrophage lipid uptake and survival in atherosclerosis.

Atherosclerosis is driven by the expansion of cholesterol-loaded 'foamy' macrophages in the arterial intima. Factors regulating foamy macrophage differentiation and survival in plaque remain poorly understood. Here we show, using trajectory analysis of integrated single-cell RNA sequencing data and a genome-wide CRISPR screen, that triggering receptor expressed on myeloid cells 2 (Trem2) is associated with foamy macrophage specification. Loss of Trem2 led to a reduced ability of foamy macrophages to take up oxidized low-density lipoprotein (oxLDL). Myeloid-specific deletion of Trem2 showed an attenuation of plaque progression, even when targeted in established atherosclerotic lesions, and was independent of changes in circulating cytokines, monocyte recruitment or cholesterol levels. Mechanistically, we link Trem2-deficient macrophages with a failure to upregulate cholesterol efflux molecules, resulting in impaired proliferation and survival. Overall, we identify Trem2 as a regulator of foamy macrophage differentiation and atherosclerotic plaque growth and as a putative therapeutic target for atherosclerosis.

Diabetes-Induced Changes in Macrophage Biology Might Lead to Reduced Risk for Abdominal Aortic Aneurysm Development.

Type 2 diabetes patients are less likely to develop an abdominal aortic aneurysm (AAA). Since macrophages play a crucial role in AAA development, we hypothesized that this decrease in AAA risk in diabetic patients might be due to diabetes-induced changes in macrophage biology. To test this hypothesis, we treated primary macrophages obtained from healthy human volunteers with serum from non-diabetic vs. diabetic AAA patients and observed differences in extracellular acidification and the expression of genes involved in glycolysis and lipid oxidation. These results suggest an increase in metabolism in macrophages treated with serum from diabetic AAA patients. Since serum samples used did not differ in glucose content, these changes are not likely to be caused by differences in glycemia. Macrophage functions have been shown to be linked to their metabolism. In line with this, our data suggest that this increase in macrophage metabolism is accompanied by a shift towards an anti-inflammatory state. Together, these results support a model where diabetes-induced changes in metabolism in macrophages might lead to a reduced risk for AAA development.

Gene Doping with Peroxisome-Proliferator-Activated Receptor Beta/Delta Agonists Alters Immunity but Exercise Training Mitigates the Detection of Effects in Blood Samples.

Synthetic ligands of peroxisome-proliferator-activated receptor beta/delta (PPARß/δ) are being used as performance-enhancing drugs by athletes. Since we previously showed that PPARß/δ activation affects T cell biology, we wanted to investigate whether a specific blood T cell signature could be employed as a method to detect the use of PPARß/δ agonists. We analyzed in primary human T cells the in vitro effect of PPARß/δ activation on fatty acid oxidation (FAO) and on their differentiation into regulatory T cells (Tregs). Furthermore, we conducted studies in mice assigned to groups according to an 8-week exercise training program and/or a 6-week treatment with 3 mg/kg/day of GW0742, a PPARß/δ agonist, in order to (1) determine the immune impact of the treatment on secondary lymphoid organs and to (2) validate a blood signature. Our results show that PPARß/δ activation increases FAO potential in human and mouse T cells and mouse secondary lymphoid organs. This was accompanied by increased Treg polarization of human primary T cells. Moreover, Treg prevalence in mouse lymph nodes was increased when PPARß/δ activation was combined with exercise training. Lastly, PPARß/δ activation increased FAO potential in mouse blood T cells. Unfortunately, this signature was masked by training in mice. In conclusion, beyond the fact that it is unlikely that this signature could be used as a doping-control strategy, our results suggest that the use of PPARß/δ agonists could have potential detrimental immune effects that may not be detectable in blood samples.

Roles of Nuclear Receptors in Vascular Calcification.

Vascular calcification is defined as an inappropriate accumulation of calcium depots occurring in soft tissues, including the vascular wall. Growing evidence suggests that vascular calcification is an actively regulated process, sharing similar mechanisms with bone formation, implicating both inhibitory and inducible factors, mediated by osteoclast-like and osteoblast-like cells, respectively. This process, which occurs in nearly all the arterial beds and in both the medial and intimal layers, mainly involves vascular smooth muscle cells. In the vascular wall, calcification can have different clinical consequences, depending on the pattern, localization and nature of calcium deposition. Nuclear receptors are transcription factors widely expressed, activated by specific ligands that control the expression of target genes involved in a multitude of pathophysiological processes, including metabolism, cancer, inflammation and cell differentiation. Some of them act as drug targets. In this review we describe and discuss the role of different nuclear receptors in the control of vascular calcification.

Regulation of Monocytes/Macrophages by the Renin-Angiotensin System in Diabetic Nephropathy: State of the Art and Results of a Pilot Study.

Diabetic nephropathy (DN) is characterized by albuminuria, loss of renal function, renal fibrosis and infiltration of macrophages originating from peripheral monocytes inside kidneys. DN is also associated with intrarenal overactivation of the renin-angiotensin system (RAS), an enzymatic cascade which is expressed and controlled at the cell and/or tissue levels. All members of the RAS are present in the kidneys and most of them are also expressed in monocytes/macrophages. This review focuses on the control of monocyte recruitment and the modulation of macrophage polarization by the RAS in the context of DN. The local RAS favors the adhesion of monocytes on renal endothelial cells and increases the production of monocyte chemotactic protein-1 and of osteopontin in tubular cells, driving monocytes into the kidneys. There, proinflammatory cytokines and the RAS promote the differentiation of macrophages into the M1 proinflammatory phenotype, largely contributing to renal lesions of DN. Finally, resolution of the inflammatory process is associated with a phenotype switch of macrophages into the M2 anti-inflammatory subset, which protects against DN. The pharmacologic interruption of the RAS reduces albuminuria, improves the trajectory of the renal function, decreases macrophage infiltration in the kidneys and promotes the switch of the macrophage phenotype from M1 to M2.

Invalidation of the Transcriptional Modulator of Lipid Metabolism PPARß/δ in T Cells Prevents Age-Related Alteration of Body Composition and Loss of Endurance Capacity.

Anti-inflammatory regulatory T cells (Tregs) are the most metabolically flexible CD4+ T cells by using both glycolysis and fatty acid oxidation (FAO) which allow them to migrate in tissues. With aging, Tregs accumulate in secondary lymphoid organs and are involved in impairment of skeletal muscle (SKM) regeneration and mass maintenance. In this study, we showed that a deletion of a FAO modulator, peroxisome proliferator-activated receptor beta/delta (PPARß/δ), specifically in T cells (KO-T PPARß/δ), increased the number of CD4+ T cells at day 2 following a cardiotoxin-induced SKM regeneration. Older KO-T PPARß/δ mice maintained a Tregs prevalence in lymph nodes similar to young mice. Surprisingly, KO-T PPARß/δ mice were protected from the effects of age on lean and fat mass and endurance capacity. Our results lead us to propose an original potential role of T cell metabolism in the effects of aging on the maintenance of body composition and endurance capacity.

Alpha-lipoic acid supplementation increases the efficacy of exercise- and diet-induced obesity treatment and induces immunometabolic changes in female mice and women.

The decrease in the regulatory T cells (Tregs) population is highly involved in adipose tissue inflammation and insulin resistance in obesity. Tregs depend on fatty acids via ß-oxidation for immunosuppressive function adapting their antioxidant systems to allow survival to oxidative stress. In this study, we have hypothesized that a dietary supplementation with alpha-lipoic acid (ALA), a powerful antioxidant, would improve immunometabolism when added to the classical strategy of obesity treatment. First, we showed by in vitro experiments that ALA favors the polarization of mice CD4 + T cells toward Tregs. Next, we have carried out a translational study where female obese mice and women were supplemented with ALA or vehicle/placebo (mice: 2.5 gALA /kgfood ; 6 weeks; women: 600 mgALA /day, 8 weeks) while following a protocol including regular exercise and a change in diet. Fatty acid oxidation potential and activity of nuclear erythroid-related factor 2 (NRF2) of mouse secondary lymphoid tissues were improved by ALA supplementation. ALA reduced visceral adipose tissue (VAT) mass and preserved Tregs in VAT in mice. In women, ALA supplementation induced significant metabolic changes of circulating CD4 + T cells including increased oxidative capacity and fatty acid oxidation, ameliorated their redox status, and improved the reduction of visceral fat mass. While appropriate biological markers are still required to be used in clinics to judge the effectiveness of long-term obesity treatment, further studies in female mice and women are needed to determine whether these immunometabolic changes would reduce VAT mass-associated risk for secondary health issues arising from obesity.

Nuclear receptors in abdominal aortic aneurysms.

Abdominal aortic aneurysms (AAA) pose a considerable health burden and at present are only managed surgically since there is no proven pharmacotherapy that will retard their expansion or reduce the incidence of fatal rupture. This pathology shares several pathophysiological mechanisms with atherosclerosis, such as macrophage infiltration, inflammation, and degradation of extracellular matrix. Therefore, therapeutic targets proven effective in the treatment of atherosclerosis could also be considered for treatment of AAA. Different members of the nuclear receptor (NR) superfamily have been extensively studied as potential targets in the treatment of cardiovascular disease (CVD) and therefore might also be suited for AAA treatment. In this context, this review summarizes the role of different NRs in CVD, mostly atherosclerosis, and discusses in detail the current knowledge of their implications in AAA. From this overview it becomes apparent that NRs that were attributed a beneficial or adverse role in CVD have similar roles in AAA. Together, this overview provides compelling evidence to consider several NRs as attractive targets for future treatment of AAA.

Complementary Immunometabolic Effects of Exercise and PPARß/δ Agonist in the Context of Diet-Induced Weight Loss in Obese Female Mice.

Regular aerobic exercise, independently of weight loss, improves metabolic and anti-inflammatory states, and can be regarded as beneficial in counteracting obesity-induced low-grade inflammation. However, it is still unknown how exercise alters immunometabolism in a context of dietary changes. Agonists of the Peroxisome Proliferator Activated-Receptor beta/delta (PPARß/δ) have been studied this last decade as "exercise-mimetics", which are potential therapies for metabolic diseases. In this study, we address the question of whether PPARß/δ agonist treatment would improve the immunometabolic changes induced by exercise in diet-induced obese female mice, having switched from a high fat diet to a normal diet. 24 mice were assigned to groups according to an 8-week exercise training program and/or an 8-week treatment with 3 mg/kg/day of GW0742, a PPARß/δ agonist. Our results show metabolic changes of peripheral lymphoid tissues with PPARß/δ agonist (increase in fatty acid oxidation gene expression) or exercise (increase in AMPK activity) and a potentiating effect of the combination of both on the percentage of anti-inflammatory Foxp3+ T cells. Those effects are associated with a decreased visceral adipose tissue mass and skeletal muscle inflammation (TNF-α, Il-6, Il-1ß mRNA level), an increase in skeletal muscle oxidative capacities (citrate synthase activity, endurance capacity), and insulin sensitivity. We conclude that a therapeutic approach targeting the PPARß/δ pathway would improve obesity treatment.

GAPDH Overexpression in the T Cell Lineage Promotes Angioimmunoblastic T Cell Lymphoma through an NF-κB-Dependent Mechanism.

GAPDH is emerging as a key player in T cell development and function. To investigate the role of GAPDH in T cells, we generated a transgenic mouse model overexpressing GAPDH in the T cell lineage. Aged mice developed a peripheral Tfh-like lymphoma that recapitulated key molecular, pathological, and immunophenotypic features of human angioimmunoblastic T cell lymphoma (AITL). GAPDH induced non-canonical NF-κB pathway activation in mouse T cells, which was strongly activated in human AITL. We developed a NIK inhibitor to reveal that targeting the NF-κB pathway prolonged AITL-bearing mouse survival alone and in combination with anti-PD-1. These findings suggest the therapeutic potential of targeting NF-κB signaling in AITL and provide a model for future AITL therapeutic investigations.

Decrease in αß/γδ T-cell ratio is accompanied by a reduction in high-fat diet-induced weight gain, insulin resistance, and inflammation.

The implication of αß and γδ T cells in obesity-associated inflammation and insulin resistance (IR) remains uncertain. Mice lacking γδ T cells show either no difference or a decrease in high-fat diet (HFD)-induced IR, whereas partial depletion in γδ T cells does not protect from HFD-induced IR. αß T-cell deficiency leads to a decrease in white adipose tissue (WAT) inflammation and IR without weight change, but partial depletion of these cells has not been studied. We previously described a mouse model overexpressing peroxisome proliferator-activated receptor ß (PPAR-ß) specifically in T cells [transgenic (Tg) T-PPAR-ß] that exhibits a partial depletion in αß T cells and no change in γδ T-cell number. This results in a decreased αß/γδ T-cell ratio in lymphoid organs. We now show that Tg T-PPAR-ß mice are partially protected against HFD-induced weight gain and exhibit decreased IR and liver steatosis independently of animal weight. These mice display an alteration of WAT-depots distribution with an increased epididymal-WAT mass and a decreased subcutaneous WAT mass. Immune cell number is decreased in both WAT-depots, except for γδ T cells, which are increased in epididymal-WAT. Overall, we show that decreasing αß/γδ T-cell ratio in WAT-depots alters their inflammatory state and mass repartition, which might be involved in improvement of insulin sensitivity.-Le Menn, G., Sibille, B., Murdaca, J., Rousseau, A.-S., Squillace, R., Vergoni, B., Cormont, M., Niot, I., Grimaldi, P. A., Mothe-Satney, I., Neels, J. G. Decrease in αß/γδ T-cell ratio is accompanied by a reduction in high-fat diet-induced weight gain, insulin resistance, and inflammation.

Regulation of Immune Cell Function by PPARs and the Connection with Metabolic and Neurodegenerative Diseases.

Increasing evidence points towards the existence of a bidirectional interconnection between metabolic disease and neurodegenerative disorders, in which inflammation is linking both together. Activation of members of the peroxisome proliferator-activated receptor (PPAR) family has been shown to have beneficial effects in these interlinked pathologies, and these improvements are often attributed to anti-inflammatory effects of PPAR activation. In this review, we summarize the role of PPARs in immune cell function, with a focus on macrophages and T cells, and how this was shown to contribute to obesity-associated inflammation and insulin resistance, atherosclerosis, and neurodegenerative disorders. We address gender differences as a potential explanation in observed contradictory results, and we highlight PPAR-induced metabolic changes as a potential mechanism of regulation of immune cell function through these nuclear receptors. Together, immune cell-specific activation of PPARs present a promising therapeutic approach to treat both metabolic and neurodegenerative diseases.

Peroxisome Proliferator Activated Receptor Beta (PPARß) activity increases the immune response and shortens the early phases of skeletal muscle regeneration.

Peroxisome Proliferator-Activated Receptor Beta (PPARß) is a transcription factor playing an important role in both muscle myogenesis and remodeling, and in inflammation. However, its role in the coordination of the transient muscle inflammation and reparation process following muscle injury has not yet been fully determined. We postulated that activation of the PPARß pathway alters the early phase of the muscle regeneration process, i.e. when immune cells infiltrate in injured muscle. Tibialis anteriors of C57BL6/J mice treated or not with the PPARß agonist GW0742 were injected with cardiotoxin (or with physiological serum for the contralateral muscle). Muscle regeneration was monitored on days 4, 7, and 14 post-injury. We found that treatment of mice with GW0742 increased, at day 4 post-damage, the recruitment of immune cells (M1 and M2 macrophages) and upregulated the expression of the anti-inflammatory cytokine IL-10 and TGF-ß mRNA. Those effects were accompanied by a significant increase at day 4 of myogenic regulatory factors (Pax7, MyoD, Myf5, Myogenin) mRNA in GW0742-treated mice. However, we showed an earlier return (7 days vs 14 days) of Myf5 and Myogenin to basal levels in GW0742- compared to DMSO-treated mice. Differential effects of GW0742 observed during the regeneration were associated with variations of PPARß pathway activity. Collectively, our findings indicate that PPARß pathway activity shortens the early phases of skeletal muscle regeneration by increasing the immune response.

A role for Peroxisome Proliferator-Activated Receptor Beta in T cell development.

Metabolism plays an important role in T cell biology and changes in metabolism drive T cell differentiation and fate. Most research on the role of metabolism in T lymphocytes focuses on mature T cells while only few studies have investigated the role of metabolism in T cell development. In this study, we report that activation or overexpression of the transcription factor Peroxisome Proliferator-Activated Receptor ß (PPARß) increases fatty acid oxidation in T cells. Furthermore, using both in vivo and in vitro models, we demonstrate that PPARß activation/overexpression inhibits thymic T cell development by decreasing proliferation of CD4-CD8- double-negative stage 4 (DN4) thymocytes. These results support a model where PPARß activation/overexpression favours fatty acid- instead of glucose-oxidation in developing T cells, thereby hampering the proliferative burst normally occurring at the DN4 stage of T cell development. As a consequence, the αß T cells that are derived from DN4 thymocytes are dramatically decreased in peripheral lymphoid tissues, while the γδ T cell population remains untouched. This is the first report of a direct role for a member of the PPAR family of nuclear receptors in the development of T cells.

α-Lipoic acid up-regulates expression of peroxisome proliferator-activated receptor ß in skeletal muscle: involvement of the JNK signaling pathway.

We hypothesized that α-lipoic acid (α-LA) might interact with the transcriptional control of peroxisome proliferator-activated receptor (PPAR)ß in skeletal muscle. Molecular mechanisms were investigated using differentiated C2C12 myotubes treated with α-LA and/or PPARß agonist GW0742. In vivo studies with 3-mo-old C57Bl6 mice were realized: voluntary wheel running (VWR) training (7 wk), and a 6 wk diet containing (or not) α-LA (0.25% wt/wt). This last condition was combined with (or not) 1 bout of treadmill exercise (18 m/min for 1 h). Using a reporter assay, we demonstrate that α-LA is not an agonist of PPARß but regulates PPARß target gene expression through an active PPARß pathway. GW0742-induced pyruvate dehydrogenase kinase 4 mRNA is potentiated by α-LA. In C2C12, α-LA lowers the activation of the JNK signaling pathway and increases PPARß mRNA and protein levels (2-fold) to the same extent as with the JNK inhibitor SP600125. Similarly to VWR training effect, PPARß expression increases (2-fold) in vastus lateralis of animals fed an α-LA-enriched diet. However, α-LA treatment does not further stimulate the adaptive up-regulation of PPARß observed in response to 1 bout of exercise. We have identified a novel mechanism of regulation of PPARß expression/action in skeletal muscle with potential physiologic application through the action of α-LA, involving the JNK pathway.

Physiological functions of peroxisome proliferator-activated receptor ß.

The peroxisome proliferator-activated receptors, PPARα, PPARß, and PPARγ, are a family of transcription factors activated by a diversity of molecules including fatty acids and fatty acid metabolites. PPARs regulate the transcription of a large variety of genes implicated in metabolism, inflammation, proliferation, and differentiation in different cell types. These transcriptional regulations involve both direct transactivation and interaction with other transcriptional regulatory pathways. The functions of PPARα and PPARγ have been extensively documented mainly because these isoforms are activated by molecules clinically used as hypolipidemic and antidiabetic compounds. The physiological functions of PPARß remained for a while less investigated, but the finding that specific synthetic agonists exert beneficial actions in obese subjects uplifted the studies aimed to elucidate the roles of this PPAR isoform. Intensive work based on pharmacological and genetic approaches and on the use of both in vitro and in vivo models has considerably improved our knowledge on the physiological roles of PPARß in various cell types. This review will summarize the accumulated evidence for the implication of PPARß in the regulation of development, metabolism, and inflammation in several tissues, including skeletal muscle, heart, skin, and intestine. Some of these findings indicate that pharmacological activation of PPARß could be envisioned as a therapeutic option for the correction of metabolic disorders and a variety of inflammatory conditions. However, other experimental data suggesting that activation of PPARß could result in serious adverse effects, such as carcinogenesis and psoriasis, raise concerns about the clinical use of potent PPARß agonists.

A role for 5-lipoxygenase products in obesity-associated inflammation and insulin resistance.

There is a growing amount of evidence that obesity-induced low-grade inflammation is an important causative link between obesity and many of its associated pathologies such as type 2 diabetes and atherosclerosis. In the quest to identify the triggers of obesity-associated inflammation of adipose tissue, our laboratory recently demonstrated that adipocytes can secrete leukotrienes, and that these pro-inflammatory lipid mediators contribute to obesity-associated inflammation and insulin resistance in mice. Together with other recent studies, our recent findings identify an important role for the enzyme 5-lipoxygenase and its products in the induction and resolution of adipose tissue inflammation. Therefore, pharmaceutical approaches that target this enzyme or its products should be considered as novel treatments aimed at preventing or resolving obesity-induced inflammation and its associated pathologies.