|Title||Understanding macrophage activation in the adipose tissue: at the crossroads of immunology and metabolism|
|Source||Wageningen University. Promotor(en): Sander Kersten; M.G. Netea, co-promotor(en): Rinke Stienstra. - Wageningen : Wageningen University - ISBN 9789463438896 - 240|
Chair Nutrition Metabolism and Genomics
|Publication type||Dissertation, internally prepared|
|Availibility||Full text available from 2019-03-23|
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.