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.
Data availability
Microarray data of ATMs isolated from obese or lean mice have been submitted to the Gene Expression Omnibus (accession no. GSE84000).
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, yet the pathogenesis of NAFLD is only partially understood. Here, we investigated the role of the gut bacteria in ...NAFLD by stimulating the gut bacteria via feeding mice the fermentable dietary fiber, guar gum (GG), and suppressing the gut bacteria via chronic oral administration of antibiotics. GG feeding profoundly altered the gut microbiota composition, in parallel with reduced diet-induced obesity and improved glucose tolerance. Strikingly, despite reducing adipose tissue mass and inflammation, GG enhanced hepatic inflammation and fibrosis, concurrent with markedly elevated plasma and hepatic bile acid levels. Consistent with a role of elevated bile acids in the liver phenotype, treatment of mice with taurocholic acid stimulated hepatic inflammation and fibrosis. In contrast to GG, chronic oral administration of antibiotics effectively suppressed the gut bacteria, decreased portal secondary bile acid levels, and attenuated hepatic inflammation and fibrosis. Neither GG nor antibiotics influenced plasma lipopolysaccharide levels. In conclusion, our data indicate a causal link between changes in gut microbiota and hepatic inflammation and fibrosis in a mouse model of NAFLD, possibly via alterations in bile acids.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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.
Highlights
• Macrophages play a significant role in regulating adipose tissue functioning during health and disease
• In addition to conventional functions such as clearing cellular debris and participating in tissue immune surveillance, lipid buffering is an important function of ATMs
• Obesity-induced inflammation, characterised by an elevated number of proinflammatory macrophages in adipose tissue, has been suggested to contribute to systemic insulin resistance
• Their origin, as well as a combination of peripheral changes and adipose tissue-derived stressors, probably contribute to ATM dysfunction and inflammatory traits during obesity
• Identification of transcriptional differences between ATMs from lean vs obese adipose tissue at several key points during the development of obesity and insulin resistance may reveal upstream triggers, regulatory factors and intracellular pathways that shape ATM function
• Targeting metabolic capacity rather than the inflammatory phenotype of ATMs may hold potential to restore ATM function and adipose tissue homeostasis in obese individuals
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Aims/hypothesis
Obesity induces macrophages to drive inflammation in adipose tissue, a crucial step towards the development of type 2 diabetes. The tricarboxylic acid (TCA) cycle intermediate ...succinate is released from cells under metabolic stress and has recently emerged as a metabolic signal induced by proinflammatory stimuli. We therefore investigated whether succinate receptor 1 (SUCNR1) could play a role in the development of adipose tissue inflammation and type 2 diabetes.
Methods
Succinate levels were determined in human plasma samples from individuals with type 2 diabetes and non-diabetic participants. Succinate release from adipose tissue explants was studied.
Sucnr1
−
/−
and wild-type (WT) littermate mice were fed a high-fat diet (HFD) or low-fat diet (LFD) for 16 weeks. Serum metabolic variables, adipose tissue inflammation, macrophage migration and glucose tolerance were determined.
Results
We show that hypoxia and hyperglycaemia independently drive the release of succinate from mouse adipose tissue (17-fold and up to 18-fold, respectively) and that plasma levels of succinate were higher in participants with type 2 diabetes compared with non-diabetic individuals (+53%;
p
< 0.01).
Sucnr1
−
/−
mice had significantly reduced numbers of macrophages (0.56 ± 0.07 vs 0.92 ± 0.15 F4/80 cells/adipocytes,
p
< 0.05) and crown-like structures (0.06 ± 0.02 vs 0.14 ± 0.02, CLS/adipocytes
p
< 0.01) in adipose tissue and significantly improved glucose tolerance (
p
< 0.001) compared with WT mice fed an HFD, despite similarly increased body weights. Consistently, macrophages from
Sucnr1
−
/−
mice showed reduced chemotaxis towards medium collected from apoptotic and hypoxic adipocytes (−59%;
p
< 0.05).
Conclusions/interpretation
Our results reveal that activation of SUCNR1 in macrophages is important for both infiltration and inflammation of adipose tissue in obesity, and suggest that SUCNR1 is a promising therapeutic target in obesity-induced type 2 diabetes.
Data availability
The dataset generated and analysed during the current study is available in GEO with the accession number GSE64104,
www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64104
.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Angiopoietin-like protein (ANGPTL)4 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 NEFAs, 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.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Monocytes are innate immune cells that play a pivotal role in antifungal immunity, but little is known regarding the cellular metabolic events that regulate their function during infection. Using ...complementary transcriptomic and immunological studies in human primary monocytes, we show that activation of monocytes by Candida albicans yeast and hyphae was accompanied by metabolic rewiring induced through C-type lectin-signaling pathways. We describe that the innate immune responses against Candida yeast are energy-demanding processes that lead to the mobilization of intracellular metabolite pools and require induction of glucose metabolism, oxidative phosphorylation and glutaminolysis, while responses to hyphae primarily rely on glycolysis. Experimental models of systemic candidiasis models validated a central role for glucose metabolism in anti-Candida immunity, as the impairment of glycolysis led to increased susceptibility in mice. Collectively, these data highlight the importance of understanding the complex network of metabolic responses triggered during infections, and unveil new potential targets for therapeutic approaches against fungal diseases.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Single nucleotide polymorphisms in the first intron of the fat-mass-and-obesity-related gene FTO are associated with increased body weight and adiposity. Increased expression of FTO is likely ...underlying this obesity phenotype, as mice with two additional copies of Fto (FTO-4 mice) exhibit increased adiposity and are hyperphagic. FTO is a demethylase of single stranded DNA and RNA, and one of its targets is the m6A modification in RNA, which might play a role in the regulation of gene expression. In this study, we aimed to examine the changes in gene expression that occur in FTO-4 mice in order to gain more insight into the underlying mechanisms by which FTO influences body weight and adiposity. Our results indicate an upregulation of anabolic pathways and a downregulation of catabolic pathways in FTO-4 mice. Interestingly, although genes involved in methylation were differentially regulated in skeletal muscle of FTO-4 mice, no effect of FTO overexpression on m6A methylation of total mRNA was detected.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Recognition of Mycobacterium tuberculosis via TLR‐2 drives activation of the mammalian target of rapamycin (mTOR)/AKT signaling cascade. This contributes to the upregulation of glycolysis and the metabolic changes needed to fuel energy demands and drive transcriptional changes that account for functional outputs like cytokine production.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
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 proinflammatory 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 stimulusdriven 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.
•Toll-like receptors are promoting development of obesity-induced inflammation.•TLR10 is the only TLR with anti-inflammatory properties.•hTLR10 transgenic mice show an up-regulation of hTLR10 ...expression in gWAT upon HFD.•Obese individuals have more pro-inflammatory macrophages in the adipose tissue.•Obese individuals bearing TLR10 SNPs have less macrophages in the adipose tissue.•TLR10 SNPs can have anti-inflammatory properties in obesity-induced inflammation.
Toll like receptors (TLRs) are expressed in adipose tissue and promote adipose tissue inflammation during obesity. Recently, anti-inflammatory properties have been attributed to TLR10 in myeloid cells, the only member of the TLR family with inhibitory activity. In order to assess whether TLR10-induced inhibition of inflammation may be protective during the development of obesity and metabolic abnormalities we used transgenic human TLR10 mice (hTLR10tg) and wild type (WT) controls on a C57B6J background. HFD-feeding enhanced TLR10 expression in the adipose tissue, and HFD-fed hTLR10tg mice displayed reduced adipocyte size, adipose tissue weight, and a trend toward lower plasma insulin levels compared to WT mice.
In humans, obese individuals with polymorphisms in the TLR10 gene displayed reduced macrophage infiltration in the adipose tissue accompanied by a trend to lower leptin levels and higher adiponectin levels in plasma. In healthy individuals with the same polymorphisms in the TLR10 gene we did not observe any difference in plasma concentrations of leptin and adiponectin.
We conclude that TLR10 impacts adipose tissue morphology in obesity. Larger studies in humans are warranted to assess its potential value as therapeutic target in metabolic syndrome and type 2 diabetes.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
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