HILPDA Uncouples Lipid Droplet Accumulation in Adipose Tissue Macrophages from Inflammation and Metabolic Dysregulation

Obesity leads to a state of chronic, low-grade inflammation that features the accumulation of lipid-laden macrophages in adipose tissue. Here, we determined the role of macrophage lipid-droplet accumulation in the development of obesity-induced adipose-tissue inflammation, using mice with myeloid-specific deficiency of the lipid-inducible HILPDA protein. HILPDA deficiency markedly reduced intracellular lipid levels and accumulation of fluorescently labeled fatty acids. Decreased lipid storage in HILPDA-deficient macrophages can be rescued by inhibition of adipose triglyceride lipase (ATGL) and is associated with increased oxidative metabolism. In diet-induced obese mice, HILPDA deficiency does not alter inflammatory and metabolic parameters, despite markedly reducing lipid accumulation in macrophages. Overall, we find that HILPDA is a lipid-inducible, physiological inhibitor of ATGL-mediated lipolysis in macrophages and uncouples lipid storage in adipose tissue macrophages from inflammation and metabolic dysregulation. Our data question the contribution of lipid droplet accumulation in adipose tissue macrophages in obesity-induced inflammation and metabolic dysregulation.</p

Fuelling the fire : Immunometabolism of monocytes and macrophages in obesity & diabetes

The prevalence of obesity has tripled since 1975 and is projected to exponentially increase in the coming years. Obesity increases the risk of diabetes mellitus and various other complications, which may be partly driven by aberrations in innate immune cell functioning. Intracellular metabolism can be an important driver of functional properties in innate immune cells. By driving immune cell dysfunction, it may also contribute to disease progression during obesity and diabetes mellitus. For the development of diabetes mellitus and the progression of its long-term complications, dysfunctional innate immune cells form a common denominator. In this thesis, we aimed to explore the role of immunometabolism in monocytes and macrophages in the development of diabetes mellitus and associated complications. Furthermore, we investigated whether immunometabolism could be targeted in these cells to avert disease development or progression.Increased aerobic glycolysis is a metabolic hallmark of immune cell activation. To understand the close interplay between glycolytic metabolism and functional output in humans, we define the contribution of aerobic glycolysis to the production of cytokines in chapter 2. Our results demonstrate that although different PAMPs lead to robust upregulation of cytokine and lactate production, the strength of the association between these two parameters can differ. Additionally, the production of specific cytokines is differentially associated with the upregulation of aerobic glycolysis, with stronger associations for IL-10 and IL-1RA. Inter-individual variations in lactate production revealed groups with different glycolytic flexibility, linked to the magnitude of cytokine responses. In patients with T1DM, associations between lactate and cytokine production were generally similar, although slightly attenuated, compared with healthy subjects. Overall, intra-individual differences in immune cell responses could be driven by differences in aerobic glycolysis, although specific TLRs and cytokines differentially rely on the use of aerobic glycolysis.Many diabetes-related complications, including increased susceptibility to infections and increased cardiovascular risk, suggest an inadequate functioning of innate immune cells. Several studies have indeed demonstrated innate immune dysfunction in diabetes mellitus. However, a link to metabolic rewiring of immune cells in the diabetic microenvironment driving these functional alterations was lacking. In chapter 3, we examined diabetes-dependent alterations in monocyte function and metabolism. We revealed that a high glycemic burden, reflected by high HbA1c levels, was coupled to reduced cytokine secretion in stimulated monocytes from patients with T1DM. Interestingly, decreased cytokine secretion was associated with increased relative glycolytic rates. High HbA1c levels were subsequently linked to a pro-inflammatory transcriptional signature in circulating monocytes. This observation suggests the existence of immune tolerance in monocytes from patients with a high glycemic burden, where chronic inflammatory activation of circulating monocytes may lead to immune dysfunction upon acute activation. Together, these findings could partly explain the increased risk of infections and cardiovascular disease in patients with diabetes mellitus.Besides driving diabetes-related complications, aberrations in innate immune responses can also contribute to the development of diabetes mellitus itself. Obesity-driven inflammation of adipose tissue can promote peripheral and systemic resistance to insulin, advancing the development of T2DM. In chapter 4, we set out to elucidate the role of UCP2 in adipose tissue macrophages in the context of adipose tissue inflammation. We confirmed the importance of UCP2 in regulating both the inflammatory response and metabolism in LPS-activated macrophages. Deletion of UCP2 resulted in a generally attenuated pro-inflammatory response to LPS, whereas glycolytic and oxidative metabolism were upregulated. However, the metabolic differences were normalized in a lipid-rich environment, potentially representative for the adipose tissue. These findings suggest that UCP2 is not a crucial component in controlling macrophage metabolism in a lipid-rich environment. Hence, in the context of obesity, deletion of UCP2 did not affect the development of adipose tissue inflammation or insulin resistance.Metabolic activation of ATMs in obese adipose tissue leads to a unique rewiring of lipid metabolism, allowing macrophages to cope with a lipid-enriched environment. Eventually, macrophages residing in obese adipose tissue develop into foam cells characterized by excessive lipid droplet formation. To determine whether lipid accumulation itself contributes to ATM dysfunction and the development of adipose tissue inflammation or insulin resistance, we studied the function of lipid droplet-related protein HILPDA in macrophages in chapter 5. We found HILPDA expression to be strongly upregulated in obese adipose tissue, where it colocalized with crown-like structures. Fatty acids and triglycerides induced HILPDA expression, and specific myeloid deletion of HILPDA led to the abolishment of lipid droplet accumulation in macrophages after lipid loading. From a mechanistic perspective, our data revealed that HILPDA is a direct inhibitor of ATGL, and lack of lipid accumulation after HILPDA deletion was caused by enhanced ATGL-mediated lipolysis. The decreased accumulation of lipid droplets after HILPDA deletion was confirmed in adipose tissue macrophages isolated from obese adipose tissue. However, decreased lipid droplet accumulation did not lead to altered secretion of cytokines from ATMs, nor did it affect the development of adipose tissue inflammation and insulin resistance. Based on these data, we show that excessive lipid droplet accumulation in ATMs is not the sole driver of adipose tissue inflammation.Besides the lipid-rich environment of the obese adipose tissue, the accumulation of triglycerides in lipid droplets also characterizes the metabolic response of macrophages toward classical inflammatory stimuli. In chapter 6, we studied triglyceride accumulation in pro-inflammatory macrophages and the involvement of HILPDA and ATGL in this process. The expression of HILPDA in response to inflammatory TLR ligands corresponded with the visual accumulation of lipid droplets, especially after treatment with LPS. Decreased expression of ATGL was found to be an important contributor to lipid droplet accumulation after the inflammatory activation of macrophages. Our data revealed that HILPDA is directly involved in enhancing the proteasomal degradation of ATGL. Specific deletion of HILPDA in macrophages emphasized their inflammatory phenotype in response to LPS, characterized by increased production of PGE2 and IL-6 both ex vivo and in vitro. Together, our findings suggest that ATGL-mediated lipolysis is partly responsible for the production of PGE2, which can, in turn, enhance the production of IL-6 and regulate the inflammatory response in macrophages.&nbsp

An integrated toolbox to profile macrophage immunometabolism

International audienceMacrophages are plastic immune cells that can adopt several activation states. Fundamental to these functional activation states is the regulation of cellular metabolic processes. Especially in mice, the metabolic alterations underlying pro-inflammatory or homeostatic phenotypes have been assessed thoroughly using various techniques. However, researchers new to the field may encounter ambiguity in choosing which combination of techniques is best suited to profile immunometabolism. To guide readers, we provide a toolbox to assess cellular metabolism in a semi-highthroughput 96-well-plate-based format. We applied the approach to activated mouse and human macrophages by metabolically pre-screening cells, followed by measuring extracellular fluxes, mitochondrial mass and membrane potential, glucose and lipid uptake, along with the application of SCENITH. Hereby, we not only validated established activation-induced metabolic rewiring in mouse macrophages, but also uncovered new insights in human macrophage immunometabolism. By thoroughly discussing each techniques, we guide readers with a practical workflow to interrogate immunometabolism

Glycolytic activity in human immune cells: inter-individual variation and functional implications during health and diabetes

An increase in glucose uptake driving aerobic glycolysis is a robust hallmark of immune cell activation. The glycolytic response supports functional alterations of the innate immune cells including the production and release of cytokines. Large inter-individual differences in the magnitude of this cytokine response are known to exist. In addition, the presence of disease is known to impact on immune cell function. Whether variation in metabolic responses of immune cells exist between individuals during health or disease is currently unknown. Here, we explore inter-individual differences in the glycolytic rate of immune cells using lactate production as readout upon activation using a variety of different stimuli. Glycolytic responses are subsequently associated to functional immune cell responses in healthy humans. In addition, we determined the glycolytic rate of immune cells and its association with immune function using patients diagnosed with diabetes mellitus. Based on the relative increase in lactate production after activation, distinct clusters of low, intermediate, and high responders could be identified, illustrating the existence of variation in glycolytic responses in healthy subjects. Interestingly, the production of cytokines mirrored these high-, intermediate-, and low-lactate patterns after pathogenic stimulation. In patients with diabetes mellitus, a reduced correlation was found between lactate and cytokine production, specifically for IL-6. Furthermore, based on the relative increase in lactate production, variability in the glycolytic response was reduced compared to healthy subjects. In conclusion, our results show a specific association between the glycolytic rate and function in human immune cells after stimulation with different pathogens. In addition to demonstrating the existence of glycolytic variability and specificity depending on the type of stimulus, the association between glycolysis and function in innate immune cells is altered during the presence of diabetes

Characterization of ANGPTL4 function in macrophages and adipocytes using Angptl4-knockout and Angptl4-hypomorphic mice

ANGPTL4 regulates plasma lipids, making it an attractive target for correcting dyslipidemia. However, ANGPTL4 inactivation in mice fed a high fat diet causes chylous ascites, an acute-phase response, and mesenteric lymphadenopathy. Here, we studied the role of ANGPTL4 in lipid uptake in macrophages and in the above-mentioned pathologies using Angptl4-hypomorphic and Angptl4-/- mice. Angptl4 expression in peritoneal and bone marrow-derived macrophages was highly induced by lipids. Recombinant ANGPTL4 decreased lipid uptake in macrophages, whereas deficiency of ANGPTL4 increased lipid uptake, upregulated lipid-induced genes, and increased respiration. ANGPTL4 deficiency did not alter LPL protein levels in macrophages. Angptl4-hypomorphic mice with partial expression of a truncated N-terminal ANGPTL4 exhibited reduced fasting plasma triglyceride, cholesterol, and non-esterified fatty acid levels, strongly resembling Angptl4-/- mice. However, during high fat feeding, Angptl4-hypomorphic mice showed markedly delayed and attenuated elevation in plasma serum amyloid A and much milder chylous ascites than Angptl4-/- mice, despite similar abundance of lipid-laden giant cells in mesenteric lymph nodes. In conclusion, ANGPTL4 deficiency increases lipid uptake and respiration in macrophages without affecting LPL protein levels. Compared with the absence of ANGPTL4, low levels of N-terminal ANGPTL4 mitigate the development of chylous ascites and an acute-phase response in mice

HILPDA Uncouples Lipid Droplet Accumulation in Adipose Tissue Macrophages from Inflammation and Metabolic Dysregulation

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