Immunometabolism

Obesity has reached epidemic proportions globally, with an estimated 1.9 billion adults being overweight or obese. Obesity-related diseases, which include not only type 2 diabetes (T2D) and heart disease but also cancer, threaten to shorten the human lifespan by 5-20 years. Obesity causes a chronic low-grade inflammation which plays a role in the development of insulin resistance. This inflammation originates from the recruitment of macrophages constituting up to 50% of all adipose tissue cells and secreting pro-inflammatory cytokines.

Obesity is a heterogeneous disease, some patients are obese but "metabolically healthy" while others develop metabolic abnormalities related to insulin resistance. The protective mechanisms by which these "healthy" obese individuals escape the harmful consequences of obesity are not yet fully understood. The topography and the different pro-inflammatory activities of adipose tissue seem to play a role. A more in-depth comparison of the two obesity phenotypes would allow to better understand the mechanisms involved in the development of metabolic abnormalities related to obesity in order to find new therapeutic options.

Obesity is not only associated with chronic inflammation but has been also linked to an imbalance in the immune system. Indeed, the dysfunction in systemic metabolism seen in obesity, by disturbing the plasma levels of lipids, glucose, insulin,…, can impair both intrinsic metabolism and function of circulating immune cells. Such immune dysfunction could play a role in the development of the obesity-related comorbidities.

We are currently focusing on cellular and molecular mechanisms underlying obesity-linked inflammation and immune dysfunction.

NLRP3 inflammasome in obesity-linked inflammation and metabolic disorders

Upon activation, NLRP3 drives the formation of the multi-protein platform called inflammasome, allowing caspase-1 activation and processing of the pro-IL-1β and pro-IL-18 into their biologically active forms. Compelling evidences showed that NLRP3 inflammasome plays a key role in obesity-related inflammation and T2D pathogenesis. We demonstrated that activation of the NLRP3 inflammasome in visceral adipose tissue macrophages (ATMs) is correlated with the development of metabolic disorders in obese people [1-3].

Unique among the inflammasomes, NLRP3 has been reported to sense a large variety of homeostasis-altering molecular processes (HAMPs). To identify the trigger(s) of NLRP3 inflammasome activation in obesity, we turned to free fatty acids (FFA) whose concentrations are increased in adipose tissue and plasma of obese people. We showed that the two main saturated fatty acids (SFA, palmitate C16:0 and stearate C18:0) are able to induce activation of NLRP3 inflammasome in human monocytes/macrophages [4, 5]. Interestingly, unsaturated fatty acids (UFAs, C18:1, C18:2) prevent SFA-mediated NLRP3 inflammasome activation [4, 5].

We then focused on the molecular mechanisms involved in the FFA-mediated NLRP3 modulation. We have shown that SFAs need to be incorporated into phosphatidylcholine, the main phospholipids of mammalian membranes, resulting in saturation and loss of membrane fluidity, followed by a disruption of the Na, K-ATPase pump and a K⁺ efflux essential for the activation of the NLRP3 inflammasome. Interestingly, the simultaneous addition of UFA redirects the SFAs flux towards neutral lipids (triacylglycerol) in lipid droplets, preserving membrane and Na, K-ATPase homeostasis and thus preventing the drop of intracellular K⁺ concentrations and NLRP3 inflammasome activation [6, 7 + 8 in revision].

[1] Esser N, L'homme L, De Roover A, Kohnen L, Scheen AJ, Moutschen M, Piette J, Legrand-Poels S*, Paquot N*. Obesity phenotype is related to NLRP3 inflammasome activity and immunological profile of visceral adipose tissue. Diabetologia (2013); 56(11):2487-97. * Sylvie Legrand-Poels and Nicolas Paquot contributed equally to this work.

[2] Esser N, L'homme L, De Roover A, Kohnen L, Scheen AJ, Moutschen M, Piette J, Legrand-Poels S*, Paquot N*. Increased Inflammasome and Caspase-1 Activation in Visceral adipose tissue from Metabolically Unhealthy Obese compared to Metabolically Healthy Obese subjects. Diabetes (2013), 62/suppl. 1, A555.

* Sylvie Legrand-Poels and Nicolas Paquot contributed equally to this work.

[3] Esser N, Legrand-Poels S, Piette J, Scheen AJ, Paquot N. Inflammation as a link between obesity, metabolic syndrome and type 2 diabetes. Diabetes Res Clin Pract. (2014); 105(2):141-50.

[4] L'homme L, Esser N, Riva L, Scheen A, Paquot N, Piette J, Legrand-Poels S. Unsaturated fatty acids prevent activation of NLRP3 inflammasome in human monocytes/macrophages. J Lipid Res. (2013); 54(11):2998-3008.

[5] Legrand-Poels S, Esser N, L'homme L, Scheen A, Paquot N, Piette J. Free fatty acids as modulators of the NLRP3 inflammasome in obesity/type 2 diabetes. Biochem Pharmacol (2014); 92:131–141.

[6] Gianfrancesco, M., Dehairs, J., Bloch, K., L'homme, L., Piette, J., Swinnen, J., Paquot, N., Esser, N., & Legrand, S. Involvement of membrane remodelling induced by fatty acids in the regulation of the NLRP3 inflammasome activity in human macrophages. Diabetologia (2016), 59 (Supplement 1): S291.

[7] Gianfrancesco MA, Paquot N, Piette J, Legrand-Poels S. Lipid bilayer stress in obesity-linked inflammatory and metabolic disorders. Biochem Pharmacol. (2018); 153:168-183.

[8] Gianfrancesco, M., Dehairs, J., L'homme, L., Herinckx, G., Esser, N., Jansen, O., Habraken, Y., Lassence, C., Swinnen, J., Rider, MH, Piette, J., Paquot, N. and Legrand-Poels S. Saturated fatty acids induce NLRP3 activation in human macrophages through K+ efflux resulting from phospholipid saturation and Na, K-ATPase disruption. BBA- Molecular and Cell Biology of Lipids (2019), in revision.

Polarization of adipose tissue macrophages in obesity

Adipose tissue macrophages (ATMs) of obese individuals adopt a pro-inflammatory polarization that seems distinct from LPS-induced M1 polarization and results from a lipid-rich environment. To further characterize ATMs polarization in human obesity and understand underlying mechanisms, we performed a sequencing of the whole transcriptome of FFA-treated human macrophages. Unlike unsaturated, saturated fatty acids (SFA) induced a strong transcriptional reprogramming.

First, we compared the SFAs effects on human Mϕ phenotype with respect to M1 versus M2 polarization. Despite a 50% similarity between SFA and M1 polarization regarding cytokines and receptors, SFA induced a unique profile. Analysis of SFA-exclusive genes in human WAT revealed an increased expression of these factors in obese compared to lean patients, suggesting that SFAs polarize macrophages towards a unique phenotype similar to adipose tissue macrophage in obesity. Among the SFA-specific factors, GDF-15 was the most upregulated cytokine. GDF-15 was recently described for its role in obesity. GDF15 expression correlated with Mϕ infiltration markers in human WAT. Obesity increased GDF15 expression in Mϕ and to a lesser extent in adipocytes, demonstrating the contribution of Mϕ for GDF-15 production during obesity. In vitro, SFA-induced GDF15 expression was dependent on ER stress, involved the PERK pathway, and induced the direct binding of CHOP transcription factor to the GDF15 promoter [L’homme et al., in preparation, collaboration with Bart Staels and David Dombrowicz, “Récepteurs nucléaires, maladies cardiovasculaires et diabète”, U1011, Univ. Lille, INSERM, CHU Lille, Institut Pasteur de Lille, EGID, France].

Gene set enrichment analysis highlighted the NF-kB-dependent pro-inflammatory signalling but also a prominent ER stress signature and a metabolic reprogramming (glycolytic switch, lipid metabolism,…). We are currently confirming the metabolic reprogramming and investigating underlying molecular mechanisms. We plan to study the role of these metabolic reprogramming in the pro-inflammatory activation of SFAs-treated macrophages.

All the results obtained with the model of SFA-polarized human macrophages will be confirmed in visceral adipose tissue macrophages from obese people.

This new knowledge might shift interventional approaches away from targeting the inflammatory traits of ATMs, towards targeting their metabolic programming.

Immunometabolic modulations of PBMCs in obesity

In this project, we compare the phenotype, the activities and the intrinsic metabolism of different subclasses of peripheral blood monocytic cells (PBMC) between healthy lean individuals and « healthy » or « unhealthy » obese patients. We mainly focus on innate immune cells like NK cells and monocytes that remain poorly studied in the context of obesity.

Profiling of monocytes demonstrated both a pro-inflammatory activation and an increased glucose uptake in obese patients compared to lean individuals. Lipidomic analyses were performed on the plasma and PBMCs of each patient (collaboration with J. Swinnen, KUL). Interestingly, the abundance of several species of glycerophospholipids is significantly modulated in PBMCs from glucose-intolerant obese patients but not from healthy obese compared to controls, suggesting that the lipid metabolism is differentially modulated according to the obesity phenotype. These results will be completed by ex vivo activity tests, qRT-PCR experiments and lipidomic analyses on isolated monocytes.

Interestingly, we also observed significant modulations in the expression of certain markers on monocytes from glucose–intolerant obese patients but not from healthy obese compared to lean individuals. We plan to further investigate the origins and consequences of such modulations to better understand the mechanisms involved in the development of metabolic abnormalities related to obesity in order to find new therapeutic options.

We also observed an impact of obesity on NK cells phenotype and cytotoxic activities. Since a decline in immunosurveillance has been proposed to contribute to the higher susceptibility of obese patients to developing certain cancers, we intend to further investigate NK cell dysfunction in obesity, notably by focusing on NK cells immunoregulatory activities.

 

 

Collaborations

• Johan Swinnen, Department of Oncology, Laboratory of Lipid Metabolism and Cancer, KULeuven, Belgium.

• Laurent L’homme, Bart Staels and David Dombrowicz, “Récepteurs nucléaires, maladies cardiovasculaires et diabète”, U1011, Univ. Lille, INSERM, CHU Lille, Institut Pasteur de Lille, EGID, France.

• Pascal de Tullio, Center for Interdisciplinary Research on Medicines (CIRM), Department of Medicinal Chemistry, University of Liege, Belgium.