Understanding macrophage activation in the adipose tissue: at the crossroads of immunology and metabolism

Macrophages and their monocyte precursors continuously patrol the bloodstream and tissues, ready to eliminate unwelcome visitors such as pathogens or foreign particles. Tissue-resident macrophages are crucial during development and for maintaining tissue homeostasis as well. The engulfment of dying or damaged tissue cells, a process called efferocytosis, is a central part of their role to maintain homeostasis, yet is accompanied by several other tissue-tailored functions. Accordingly, macrophages display great plasticity by adopting unique phenotypes to fulfil tissue-specific needs. This thesis is particularly devoted to macrophages residing in the adipose tissue. In lean conditions adipose tissue macrophages (ATMs) promote tissue and whole-body homeostasis by buffering lipids released by adipocytes and removing dead or damaged cells, and ensure tissue dynamics by promoting angiogenesis, adipogenesis, and extracellular matrix remodelling. Obese adipose tissue, however, is characterized by low-grade chronic inflammation reflective of homeostatic imbalance. Given their pivotal role for maintaining homeostasis in lean conditions, ATMs are considered key players in the development of adipose tissue inflammation during obesity. Indeed, during obesity ATMs sharply increase in number while simultaneously gaining a pro-inflammatory trait. This pro-inflammatory activation of ATMs is thought to importantly link obesity to the development of insulin resistance and, ultimately, Type 2 Diabetes. Notwithstanding the considerable progress made, the underlying causes of macrophage activation and phenotypical and functional characteristics of ATMs in obese adipose tissue have not yet been fully unravelled. In this thesis, we have investigated various aspects of activation of macrophage and their monocyte precursors. First, we have examined metabolic reprogramming in monocytes stimulated with various pathogenic stimuli (Chapter 2). This research adds to the growing evidence of intracellular metabolism as fundamental driver of immune cell functioning. In contrast to the majority of studies in the field that have focussed on one single stimulant, we have carefully evaluated intracellular metabolism upon activation with different pathogenic stimuli, including whole pathogen lysates and isolated Toll-like receptor (TLR) ligands. In line with the current paradigm, we found glycolysis to be a general characteristic of monocyte activation irrespective of the present stimulus. Interestingly, however, in contrast to the current paradigm, oxidative phosphorylation (OXPHOS), the alternative route for ATP production that occupies mitochondria, was found to be enhanced by most pathogenic stimuli as well. In fact, the most commonly used stimulant for activating monocytes and macrophages, being lipopolysaccharide (LPS), appeared unique in aggravating mitochondrial metabolism. Importantly, such stimulus-specific metabolic reprogramming appeared to have functional consequences, that we evaluated by comparing the two different TLR-ligands LPS (TLR4 ligand) and Pam3CysSK4 (P3C: TLR2 ligand). While glycolysis contributed to cytokine release by both LPS and P3C, OXPHOS only contributed to cytokine production in P3C-stimulated monocytes. Moreover, phagocytosis appeared to rely on OXPHOS but not glycolysis in monocytes stimulated with P3C. Probably consequential to their reduced mitochondrial activity, LPS-stimulated monocytes displayed low phagocytic capacity. Together these findings are suggestive of stimulus-tailored metabolic rearrangements fuelling functional output of monocytes. After reviewing various aspects of ATMs in Chapter 3 – including their origin, activation and function in obese versus lean conditions – we examined metabolic rearrangements in ATMs, and evaluated their contribution to the pro-inflammatory ATM trait apparent in obese adipose tissue (Chapter 4). Not surprisingly given the rather challenging environment provided by obese adipose tissue, ATMs were found to be strongly metabolically activated during obesity illustrated by enhanced activation of both glycolysis and OXPHOS. Interestingly, this metabolic activation appeared to be specific for ATMs, and was not manifested in macrophages isolated from the peritoneum of obese versus lean mice. In line with recent studies, we showed that both the metabolic and inflammatory trait of ATMs was pronouncedly different from that displayed by classically (LPS-)activated macrophages. Indeed, the ATM phenotype appeared dose-dependently induced by adipose tissue-derived factors. Using metabolic inhibitors, we identified various metabolic routes including fatty acid oxidation, glycolysis and glutaminolysis to contribute to cytokine release by ATMs isolated from lean mice. Glycolysis, however, contributed the most to cytokine production and was responsible for the increased release of inflammatory cytokines by ATMs from obese mice. Unexpectedly, however, HIF-1α, a key regulator of glycolysis and inflammatory activation, appeared not to be critically involved in the development of a pro-inflammatory ATM trait during obesity. Because lipids most likely play a central role in shaping the ATM phenotype, we evaluated the role of triglycerides (TGs) versus free fatty acids (FFAs) as driver of pro-inflammatory activation of ATMs in Chapter 5. First we confirmed lipid handling to be a fundamental characteristic of ATMs by showing that ATMs, but not other tissue macrophages or circulating monocytes from humans and mice, display enhanced expression of genes involved in lipid uptake and processing. This associated with increased expression of ER stress markers and inflammatory activation of macrophages, pointing to a relation between lipid loading and inflammatory activation of ATMs. Interestingly, both lipoprotein lipase (Lpl), that breaks down extracellular TGs into FAs that can be taken up, and its endogenous inhibitor angiopoietin-like 4 (Angptl4) were upregulated in macrophages in an adipose tissue environment, suggestive of the presence of a negative feedback mechanism to limit LPL activity and thus excessive uptake of FAs from TGs. Indeed, we observed ANGPTL4 to inhibit inflammatory activation of macrophages in an adipose tissue environment. Intriguingly, however, reduced inflammatory activation of Angptl4 knock-out macrophages in an adipose tissue environment appeared to be independent of lipid loading which most likely occurred through uptake of FFAs rather than TGs. In Chapter 6, we zoomed into a role for ATMs in efferoctysis of dead adipocytes, that may impose an important source of lipids for ATMs. Indeed, we found profound transcriptional regulation of the efferocytic machinery in ATMs isolated from obese versus lean adipose tissue accompanied by increased expression of genes involved in lipid handling and processing of lipid-derivatives. In vitro, dead adipocytes were readily taken up by macrophages and induced the expression of various genes involved in lipid handling, similar to what we found in ATMs in vivo. Interestingly, macrophages part of obese adipose tissue display pronounced down-regulation of Interferon (IFN)-signalling, whereas effective efferocytosis in vitro was characterized by enhanced IFN signalling. Accordingly, our data are suggestive of a link between impaired IFN signalling and dysfunctional, pro-inflammatory ATMs in obese adipose tissue. Lastly, in Chapter 7 we have evaluated a role for TLR10, the sole anti-inflammatory TLR family member, in adipose tissue of humans and mice. Because mice do not express functional TLR10, we fed mice expressing human TLR10 a high-fat diet for 16 weeks. Unexpectedly, TLR10 did not attenuate the development of adipose tissue inflammation during obesity. Interestingly, however, mice carrying human TLR10 had reduced adipose tissue weight and adipocyte size, suggestive of a role for TLR10 in adiposity. In humans, obese but not lean individuals carrying single nucleotide polymorphisms (SNPs) in TLR10 had or tended to have lower circulating leptin and macrophage numbers in the adipose tissue, reflective of a role for TLR10 in the adipose tissue at states of low-grade chronic inflammation specifically. In conclusion, we have revealed macrophage metabolic reprogramming to be stimulus-driven and location-specific and crucial for fuelling functional output in line with specific environmental demands. In the adipose tissue, lipid handling is central to macrophage functioning, yet ATMs appear to be overwhelmed by lipids during obesity. From a therapeutic point of view, we propose stimulation of FA oxidation to support ATM functioning according to increasing demands of the obese adipose tissue environment, while simultaneously driving them away from glycolysis that appeared to critically underlie their pro-inflammatory trait. Future studies, however, are warranted to clarify the therapeutic potential of raising mitochondrial FA oxidation in ATMs of obese individuals.</p

Rewiring cellular metabolism via the AKT/mTOR pathway contributes to host defence against Mycobacterium tuberculosis in human and murine cells

Contains fulltext : 171426.pdf (publisher's version ) (Open Access)Cells in homeostasis metabolize glucose mainly through the tricarboxylic acid cycle and oxidative phosphorylation, while activated cells switch their basal metabolism to aerobic glycolysis. In this study, we examined whether metabolic reprogramming toward aerobic glycolysis is important for the host response to Mycobacterium tuberculosis (Mtb). Through transcriptional and metabolite analysis we show that Mtb induces a switch in host cellular metabolism toward aerobic glycolysis in human peripheral blood mononuclear cells (PBMCs). The metabolic switch is TLR2 dependent but NOD2 independent, and is mediated in part through activation of the AKT-mTOR (mammalian target of rapamycin) pathway. We show that pharmacological inhibition of the AKT/mTOR pathway inhibits cellular responses to Mtb both in vitro in human PBMCs, and in vivo in a model of murine tuberculosis. Our findings reveal a novel regulatory layer of host responses to Mtb that will aid understanding of host susceptibility to Mtb, and which may be exploited for host-directed therapy

Adipose tissue macrophages : going off track during obesity

Inflammation originating from the adipose tissue is considered to be one of the main driving forces for the development of insulin resistance and type 2 diabetes in obese individuals. Although a plethora of different immune cells shapes adipose tissue inflammation, this review is specifically focused on the contribution of macrophages that reside in adipose tissue in lean and obese conditions. Both conventional and tissue-specific functions of adipose tissue macrophages (ATMs) in lean and obese adipose tissue are discussed and linked with metabolic and inflammatory changes that occur during the development of obesity. Furthermore, we will address various circulating and adipose tissue-derived triggers that may be involved in shaping the ATM phenotype and underlie ATM function in lean and obese conditions. Finally, we will highlight how these changes affect adipose tissue inflammation and may be targeted for therapeutic interventions to improve insulin sensitivity in obese individuals.(Table presented.)</p

Unique metabolic activation of adipose tissue macrophages in obesity promotes inflammatory responses

Aims/hypothesis: Recent studies have identified intracellular metabolism as a fundamental determinant of macrophage function. In obesity, proinflammatory macrophages accumulate in adipose tissue and trigger chronic low-grade inflammation, that promotes the development of systemic insulin resistance, yet changes in their intracellular energy metabolism are currently unknown. We therefore set out to study metabolic signatures of adipose tissue macrophages (ATMs) in lean and obese conditions. Methods: F4/80-positive ATMs were isolated from obese vs lean mice. High-fat feeding of wild-type mice and myeloid-specific Hif1α−/− mice was used to examine the role of hypoxia-inducible factor-1α (HIF-1α) in ATMs part of obese adipose tissue. In vitro, bone marrow-derived macrophages were co-cultured with adipose tissue explants to examine adipose tissue-induced changes in macrophage phenotypes. Transcriptome analysis, real-time flux measurements, ELISA and several other approaches were used to determine the metabolic signatures and inflammatory status of macrophages. In addition, various metabolic routes were inhibited to determine their relevance for cytokine production. Results: Transcriptome analysis and extracellular flux measurements of mouse ATMs revealed unique metabolic rewiring in obesity characterised by both increased glycolysis and oxidative phosphorylation. Similar metabolic activation of CD14+ cells in obese individuals was associated with diabetes outcome. These changes were not observed in peritoneal macrophages from obese vs lean mice and did not resemble metabolic rewiring in M1-primed macrophages. Instead, metabolic activation of macrophages was dose-dependently induced by a set of adipose tissue-derived factors that could not be reduced to leptin or lactate. Using metabolic inhibitors, we identified various metabolic routes, including fatty acid oxidation, glycolysis and glutaminolysis, that contributed to cytokine release by ATMs in lean adipose tissue. Glycolysis appeared to be the main contributor to the proinflammatory trait of macrophages in obese adipose tissue. HIF-1α, a key regulator of glycolysis, nonetheless appeared to play no critical role in proinflammatory activation of ATMs during early stages of obesity. Conclusions/interpretation: Our results reveal unique metabolic activation of ATMs in obesity that promotes inflammatory cytokine release. Further understanding of metabolic programming in ATMs will most likely lead to novel therapeutic targets to curtail inflammatory responses in obesity.<br/

Different pathogenic stimuli induce specific metabolic rewiring in human monocytes

Recent studies have demonstrated that upon encountering a pathogenic stimulus, robust metabolic rewiring of immune cells occurs. A switch away from oxidative phosphorylation to glycolysis, even in the presence of sufficient amounts of oxygen (akin the Warburg effect), is typically observed in activated innate and adaptive immune cells and is thought to accommodate adequate inflammatory responses. However, whether the Warburg effect is a general phenomenon applicable in human monocytes exposed to different pathogenic stimuli is unknown. Our results using human monocytes from healthy donors demonstrate that the Warburg effect only holds true for TLR4 activated cells. Although activation of other TLRs leads to an increase in glycolysis, no reduction or even an enhancement in oxidative phosphorylation is observed. Moreover, specific metabolic rewiring occurs in TLR4 vs. TLR2 stimulated cells characterized by altered gene expression profiles of pathways related to metabolism, changes in spare respiratory capacity of the cells and differential regulation of mitochondrial enzyme activity. Similarly, results from ex vivo and in vivo studies demonstrate metabolic rewiring of immune cells that is highly dependent on the type of pathogenic stimulus. Although the Warburg effect is observed in human monocytes after TLR4 activation, we propose that this typical metabolic response is not applicable to other inflammatory signalling routes including TLR2 in human monocytes. Instead, each pathogenic stimulus and subsequently activated inflammatory signalling cascade induces specific metabolic rewiring of the immune cell to accommodate an appropriate response

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

Microbial stimulation of different Toll-like receptor signalling pathways induces diverse metabolic programmes in human monocytes

Microbial stimuli such as lipopolysaccharide (LPS) induce robust metabolic rewiring in immune cells known as the Warburg effect. It is unknown whether this increase in glycolysis and decrease in oxidative phosphorylation (OXPHOS) is a general characteristic of monocytes that have encountered a pathogen. Using CD14+ monocytes from healthy donors, we demonstrated that most microbial stimuli increased glycolysis, but that only stimulation of Toll-like receptor (TLR) 4 with LPS led to a decrease in OXPHOS. Instead, activation of other TLRs, such as TLR2 activation by Pam3CysSK4 (P3C), increased oxygen consumption and mitochondrial enzyme activity. Transcriptome and metabolome analysis of monocytes stimulated with P3C versus LPS confirmed the divergent metabolic responses between both stimuli, and revealed significant differences in the tricarboxylic acid cycle, OXPHOS and lipid metabolism pathways following stimulation of monocytes with P3C versus LPS. At a functional level, pharmacological inhibition of complex I of the mitochondrial electron transport chain diminished cytokine production and phagocytosis in P3C- but not LPS-stimulated monocytes. Thus, unlike LPS, complex microbial stimuli and the TLR2 ligand P3C induce a specific pattern of metabolic rewiring that involves upregulation of both glycolysis and OXPHOS, which enables activation of host defence mechanisms such as cytokine production and phagocytosis

Top 10 genes that showed the highest amount of downregulation in FTO-4 mice per tissue.

<p>Top 10 genes that showed the highest amount of downregulation in FTO-4 mice per tissue.</p

Effect of FTO overexpression on m6A levels in RNA.

<p>FTO mRNA expression in wild-type (black bar) and FTO-4 (white bar) MEFs as the fold change compared to wild-type (A). m6A levels measured as a percentage of adenosine levels for mRNA and total RNA by LC/MS in wild-type (black bar) and FTO-4 (white bar) MEFs (B). Significance was tested using Student's t-tests to compare FTO-4 to WT data. **p<0.01.</p

Differences in gene expression between WT and FTO-4 mice based on microarray data.

<p>Expression of the indicated genes in the cerebellum (A), hypothalamus (B), WAT (C) and gastrocnemius (D) of wildtype (WT, black bars) and FTO-4 (white bars) mice. Data are expressed as the fold change compared to wildtype. Significance was tested using Student's t-test to compare FTO-4 to WT. ***p<0.001, **p<0.01, *p<0.05.</p