SARA RUSSO MACROPHAGE METABOLIC REPROGRAMMING IN CHRONIC DISEASES
Macrophage metabolic reprogramming in chronic diseases Sara Russo
The research described in this thesis was carried out in the Department of Analytical Biochemistry and Molecular Pharmacology (Groningen Research Institute of Pharmacy, University of Groningen, The Netherlands) according to the requirements of the Graduate School of Science and Engineering, Faculty of Science and Engineering, University of Groningen, The Netherlands. The studies presented in this thesis were conducted in the context of the PROMINENT project of the Groningen University Institute for Drug Exploration (GUIDE). This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 754425. Printing of this thesis was financially supported by the University Library and the Graduate School of Science and Engineering, Faculty of Science and Engineering, University of Groningen, The Netherlands. Cover design: Ridderprint | www.ridderprint.nl Lay-out: Ridderprint | www.ridderprint.nl Print: Ridderprint | www.ridderprint.nl © Copyright 2024: Sara Russo, The Netherlands All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, by photocopying, recording, or otherwise, without the prior written permission of the author.
Macrophage metabolic reprogramming in chronic diseases PhD thesis to obtain the degree of PhD at the University of Groningen on the authority of the Rector Magnificus Prof. J.M.A. Scherpen and in accordance with the decision by the College of Deans. This thesis will be defended in public on Tuesday 26 March 2024 at 14:30 hours by Sara Russo born on 7 August 1992 in Pisa, Italy
Supervisors Prof. R.P.H. Bischoff Prof. B.N. Melgert Assessment Committee Prof. M. Schmidt Prof. G. Hopfgartner Prof. F.J. Dekker
TABLE OF CONTENTS Chapter 1 General Introduction 7 Chapter 2 Meta-inflammation and metabolic reprogramming of macrophages in diabetes and obesity: the importance of metabolites 23 Chapter 3 Effects of lysine deacetylase inhibitor treatment on LPS responses of alveolar-like macrophages 59 Chapter 4 Collagen Type I Alters the Proteomic Signature of Macrophages in a Collagen Morphology-Dependent Manner 103 Chapter 5 General Discussion 139 Appendix English summary 158 Nederlandse Samenvatting 160 Italian summary (Riassunto in Italiano) 162 Acknowledgements 164 Curriculum Vitae 169
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Chapter 1 Chapter 1 General Introduction General Introduction
8 Chapter 1 CHRONIC DISEASES Chronic obstructive pulmonary disease (COPD), cardiovascular disease, and diabetes are the most common chronic diseases worldwide. COPD is a progressive and debilitating respiratory condition characterized by persistent lung airflow limitation (1). It encompasses a group of chronic lung disorders, primarily chronic bronchitis, and emphysema (2). COPD gradually worsens over time, leading to significant respiratory symptoms, reduced lung function, and impaired quality of life. It is a global health concern, affecting millions of people worldwide and imposing a substantial burden on individuals, healthcare systems, and society as a whole. Understanding the causes, symptoms, diagnosis, and management of COPD is crucial in order to improve patient outcomes and alleviate the impact of this chronic respiratory condition. Diabetes mellitus is a chronic condition characterized by high blood sugar levels resulting from insulin deficiency or resistance. It can be classified into Type 1 diabetes, caused by a lack of insulin production, and Type 2 diabetes (T2DM), caused by insulin resistance. Globally, there are approximately 415 million diagnosed cases of diabetes (of which 87–91% have T2DM in high-income countries), a number expected to rise to 642 million by 2040 (3). Diabetes is a significant cause of morbidity and mortality, with complications including diabetic nephropathy, peripheral neuropathy, and cardiovascular diseases. Despite advancements in treatment and decreasing complication rates (4), the prevalence of newly diagnosed cases continues to rise (5). INFLAMMATION COPD is characterized by chronic inflammation that affects various tissues involved in respiratory function, including the lungs and airways. This persistent inflammation is marked by increased levels of pro-inflammatory cytokines and other immune mediators. In COPD, chronic inflammation is primarily triggered by long-term exposure to irritants, such as cigarette smoke or environmental pollutants. The inflammatory response in the lungs leads to structural changes, airway remodeling, and narrowing of the air passages, resulting in airflow limitation and respiratory symptoms. Pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β, and others, contribute to the perpetuation of the inflammatory process and tissue damage in COPD. This chronic inflammation not only worsens respiratory symptoms but also contributes to the development of comorbidities, such as cardiovascular disease. Understanding the role of chronic inflammation in COPD is crucial for the development of targeted therapies aimed
9 1 General Introduction at reducing inflammation, slowing disease progression, and improving the quality of life for individuals living with this chronic respiratory condition. T2DM is characterized by chronic low-grade inflammation in various tissues involved in energy regulation, including fat, liver, and pancreatic islets (6). This inflammation is marked by increased levels of pro-inflammatory cytokines and non-esterified free fatty acids (FFA), which contribute to insulin resistance and beta-cell dysfunction. In T2DM, pro-inflammatory cytokines such as TNF-α, IL-6, IL-1β, and IL-1α are produced in higher quantities, contributing to obesity-related inflammation and impairing insulin signaling (7). These cytokines also contribute to the development of microvascular complications associated with diabetes, including retinopathy, polyneuropathy, and nephropathy (8). The inflammatory effects of obesity were defined for the first time in 1993 when it was shown that adipose tissue from obese individuals expressed elevated levels of TNF-α, a proinflammatory cytokine, primarily secreted by macrophages (9). MACROPHAGES Macrophages are cells of the innate immune system derived from hematopoietic stem cells (HSCs) of the bone marrow or from erythro-myeloid progenitors (EMPs) of the fetal yolk sac (10). HSCs differentiate in myeloid (MPs) and lymphoid (LPs) committed precursors. MPs will then differentiate into monocytes, macrophages, and dendritic cell precursors (MDP). Two monocyte subsets (resident, lymphocyte antigen 6c negative, Ly6C-/low, and inflammatory Ly6C+/high) are released in the circulation, and, in case of infection, Ly6C+ can migrate into local inflammatory sites and differentiate into inflammatory macrophages (11,12). Macrophage polarization Macrophages, a type of immune cell, can be polarized into different groups based on their response to the microenvironment. The three main groups are M1 (classically activated) macrophages, M2 (alternatively activated) macrophages, and M2-like (regulatory) macrophages (13). The classification is determined by specific molecular markers, chemokine receptors, and cytokine production (14). M1 macrophages are activated by inflammatory stimuli and produce pro-inflammatory cytokines, promoting a Th1 immune response (15,16). They express markers like MHC II, CD68, CD80, and CD86 (17). M2 macrophages are associated with tissue remodeling and are activated by interleukins 4 and 13 (18–20). They express CD206 and transglutaminase 2, and have limited antigen-presenting capabilities. M2-like macrophages are induced by anti-inflammatory stimuli and secrete high levels of
10 Chapter 1 IL-10, exhibiting potent anti-inflammatory effects. These macrophages are also characterized by TGFβ production (21). Adipose tissue macrophages Obesity is associated with the development of type 2 diabetes (T2DM), and the immune system in adipose tissue plays a role in this process. Adipose tissue consists of white adipose tissue (WAT) and brown adipose tissue (BAT) (22). In obesity, WAT shows increased levels of pro-inflammatory cytokines, primarily secreted by macrophages (9). Adipose tissue macrophages (ATMs) significantly impact adipose tissue homeostasis. In lean individuals, ATMs resemble M2 macrophages, promoting insulin sensitivity through the production of anti-inflammatory cytokines (23). However, in obesity, excessive growth of WAT leads to lipid accumulation, cellular stress, and hypoxia (24,25), triggering the release of free fatty acids, proinflammatory cytokines, and reactive oxygen species (ROS) (26,27), resulting in impaired insulin sensitivity (28). Monocytes are recruited to adipose tissue in obesity, differentiating into M1-like macrophages that secrete inflammatory factors (29,30). Interestingly, macrophages in obesity display unique characteristics and do not align with the classical activation phenotype (31). They accumulate lipids, express fatty acid transporters such as CD36, and exhibit markers associated with metabolic activation (31,32). These changes in macrophage activation contribute to obesity-related meta-inflammation, characterized by elevated pro-inflammatory cytokines and insulin resistance. The dysregulation of the adipose tissue immune system and the accumulation of inflammatory macrophages play crucial roles in the development and progression of metabolic disorders. Lung macrophages The phenotype and transcriptional signature of alveolar macrophages (AMs), which are macrophages specific to the lung tissue, are shaped by the surrounding lung microenvironment (33). They reside within the alveolar niche, which is composed of type I and type II alveolar epithelial cells, capillary endothelial cells, and alveolar interstitial fibroblasts. This niche provides a cytokine-rich environment that supports the survival and function of AMs (34). In terms of abundance, the lung contains a significant number of AMs. For instance, the upper lobe of the human lung alone houses approximately 1.5 × 109 AMs. The majority of these macrophages are located in the diffusing area of the lung, where they are in close proximity to the gas-exchange region, while a smaller population is found in the conducting small airways (35). Traditionally, AMs were thought to be
11 1 General Introduction a relatively homogeneous population of cells. However, recent advances in singlecell sequencing have revealed the heterogeneity of AMs in healthy individuals. Studies have identified at least four superclusters of AMs with distinct subclusters, characterized by different gene expression profiles (36). One of the key factors contributing to the heterogeneity of AMs is the highly regulated production of specific combinations of chemokines, metallothioneins, interferon (IFN)-inducible genes, cholesterol-biosynthesis-related genes, and insulin-like growth factor 1 (IGF1) by different subsets of AMs. These subsets exhibit unique functional properties and may play specialized roles in immune responses and tissue homeostasis. Moreover, the observed heterogeneity of AMs is not limited to healthy individuals but extends to various lung diseases. Studies investigating diseases such as cystic fibrosis, COPD, and COVID-19 have consistently demonstrated the presence of these superclusters and subclusters across different individuals and disease conditions. This suggests that the heterogeneity of AMs is a universal feature and may have implications for understanding the pathogenesis and progression of lung diseases (37). In conclusion, lung macrophages, specifically alveolar macrophages, exhibit unique characteristics and heterogeneity in the lung microenvironment. They are highly abundant in the lung and display distinct gene expression profiles and functional properties. Understanding the heterogeneity of AMs has the potential to shed light on their diverse roles in lung health and disease. METABOLIC REPROGRAMMING OF MACROPHAGES Macrophages, as sentinel cells, need to respond rapidly to alterations in their microenvironment. They modify their metabolic pathways to ensure proper activation and function. One of the initial differences observed in macrophage metabolism during polarization is the alteration of amino acid metabolism. Classically activated macrophages convert arginine to nitric oxide (NO) and citrulline, while alternatively activated macrophages convert arginine to proline and polyamines (38). Furthermore, macrophages can reprogram their energy generation methods. Nonpolarized or alternatively activated macrophages use fatty acid beta-oxidation and mitochondrial oxidative phosphorylation (OXPHOS) to produce ATP (39). In contrast, pro-inflammatory stimuli induce a metabolic shift in macrophages towards aerobic glycolysis, similar to the Warburg effect observed in tumor cells (40). This metabolic reprogramming results in lactate secretion and the accumulation of citrate and succinate (41).
12 Chapter 1 The metabolic reprogramming of macrophages has several consequences. First, there is a breakpoint in the tricarboxylic acid (TCA) cycle after citrate due to lower expression of isocitrate dehydrogenase, leading to citrate accumulation. Citrate can be transported to the cytosol (42) and converted into acetyl-CoA (43), which can be used for fatty acid synthesis or lysine acetylation of proteins. Second, there is a breakpoint after succinate caused by the inhibition of succinate dehydrogenase by itaconate, resulting in succinate accumulation. Succinate accumulation leads to the stabilization of the transcription factor HIF-1α (44), promoting the switch to glycolysis and inducing the expression of glycolytic enzymes. This metabolic shift also activates the pentose phosphate pathway, generating NADPH for ROS production (45). Additionally, HIF-1α promotes the expression of lactate dehydrogenase (46), which converts pyruvate to lactate, and pyruvate dehydrogenase kinase 1, inhibiting mitochondrial function further (47). In summary, macrophages undergo metabolic reprogramming to adapt to different activation states. The shift towards glycolysis and altered metabolism of amino acids and fatty acids provide the necessary energy and biosynthetic precursors for macrophage activation and function during inflammatory responses (48). ANALYSIS OF METABOLITES Flow cytometry is commonly used to characterize macrophage phenotypes but does not provide information on cellular metabolism (49). Recent efforts have utilized flow cytometry with antibodies against metabolic enzymes to investigate single-cell metabolism, although quantitative insight into metabolite production and enzyme activity is still lacking (50). Other techniques such as extracellular flux analysis, colorimetric/fluorometric enzyme activity assays, and mass spectrometry (MS)- based metabolomics and flux analysis are used to measure the metabolic status of cells. Extracellular flux analyzers provide a functional readout of glycolytic or mitochondrial activity but do not directly measure individual metabolites (51). MS, coupled with gas or liquid chromatography, allows for detecting and quantifying a wide range of metabolites (52–54). Stable isotope labeling combined with MS can be used to study metabolic flux and pathway analysis (55). Targeted and untargeted MS methods can provide quantitative information on known metabolites as well as reveal new metabolites (56). Combining multiple analytical approaches, including metabolomics, lipidomics, fluxomics, transcriptomics, and proteomics, can help gain a comprehensive understanding of the mechanisms involved in macrophage metabolic reprogramming. This integrated approach has the potential to discover mechanistic links between inflammation and metabolic disturbances in chronic diseases.
13 1 General Introduction IMMUNOMETABOLISM AND LYSINE ACETYLATION The concept of immunometabolism involves the interaction between immune and metabolic processes (57). Recent research suggests that chronic inflammation is connected to changes in energy metabolism through lysine acetylation, a posttranslational modification of proteins. Lysine acetylation alters the behavior of acetylated proteins, affecting their interactions with other molecules, catalytic activity, subcellular localization, and stability (58). Lysine acetyltransferases (KATs) and lysine deacetylases (KDACs) regulate the precise stoichiometry of site-specific lysine acetylation (59). KDACs are classified into four groups, while KATs are divided into three. Lysine acetylation can also occur non-enzymatically, especially in alkaline environments like the mitochondrial matrix (58). Fluctuations in acetyl-CoA concentration, which vary in different cellular compartments, can influence the catalytic activity and selectivity of KATs (60). Reversible protein acetylation plays a role in gene expression, affecting histones, transcription factors, and enzymes involved in cellular energy metabolism (61). Changes in glycolysis, the tricarboxylic acid cycle (TCA), and fatty acid oxidation impact cellular acetyl-CoA levels, establishing a connection between energy metabolism, protein acetylation, and gene expression. Histone acetylation Chromatin is a complex of DNA and proteins called histones. The nucleosome is the fundamental subunit of chromatin, which is formed of an octamer of histones, an H3/H4 tetramer, and two H2A/H2B dimers, around which 146 bp of DNA is wrapped (62). The conformation of the chromatin changes to allow gene transcription due to changes in the histone acetylation stoichiometry and dynamics (63) Histone acetylation is a key component in the regulation of gene expression: during activation of gene transcription, chromatin conformation changes from tightly compacted to relaxed, allowing DNA binding proteins to interact with the DNA. The interaction of positively-charged epsilon amino groups in histones belonging to lysine residues with the negatively charged phosphate groups of DNA will decrease due to the removal of the positive charges on the histones upon acetylation. The fact that acetylation is a key component in the regulation of gene expression and that elevated levels of histone deacetylation are evident in several chronic human diseases has motivated the study of KDACs in relation to the often observed aberrant gene expression. The KDAC family consists of different classes of enzymes involved in the regulation of protein acetylation. Class I KDACs, including KDAC1, 2, 3, and 8, are primarily
14 Chapter 1 located in the nucleus due to the absence of a nuclear export signal sequence. KDAC3, however, has a nuclear export signal and can be found in both the cytoplasmic and nuclear compartments. Class I KDACs are widely distributed in various tissues (64). Class II KDACs, divided into IIA (KDAC4, 5, 7, 9) and IIB (KDAC6, 10), can shuttle between the nucleus and cytoplasm in response to cellular signals (65). Sirtuins, the third group of KDACs, rely on NAD hydrolysis for their deacetylase activity and are located in different subcellular compartments. For example, SIRT1, SIRT6, and SIRT7 are found in the nucleus, SIRT2 is primarily cytosolic, and SIRT3–5 are located in mitochondria. Recent research has highlighted the role of sirtuins in connecting deacetylation with cellular metabolism, as deacetylation is responsive to metabolic cues (66). Additionally, KDAC11, the sole member of its class, shares similarities with both Class I and II KDACs. KDAC11 is involved in regulating the protein stability of DNA replication factor CDT1 (67) and negatively regulates the expression of interleukin 10, leading to an inflammatory response when overexpressed (68). KDAC inhibitors Lysine deacetylases (KDACs) are regulated by protein-protein interactions, posttranslational modifications, and subcellular localization. Dysregulation of KDACs is associated with various human diseases, leading to the development of KDAC inhibitors (KDACi) as therapeutic agents. Clinical trials have resulted in the approval of vorinostat and romidepsin for the treatment of cutaneous T-cell lymphoma (69). Classical KDACi act on Class I, II, and Class IV HDACs by binding to the zinc ion in the catalytic pocket. They inhibit deacetylation by preventing the binding of the natural substrate. On the other hand, romidepsin inhibits KDAC enzymatic activity by interacting with the zinc ion through its reduced disulfide bond (70). However, these inhibitors are not highly specific and can cause side effects. Newer KDACi, such as MS-275 (entinostat), based on a benzamide group as a Zn2+ binder, offer improved specificity. Class III KDACs, which are NAD+-dependent, can be inhibited by compounds like nicotinamide and derivatives of NAD (71). SCOPE OF THIS THESIS The work described in this thesis aimed to improve our understanding of the different phenotypes and functional properties of macrophages in chronic inflammation, specifically in the different tissue niches. Particular attention was paid to the role of metabolism in macrophage characterization.
15 1 General Introduction In Chapter 2, macrophages’ role in the development of obesity and diabetes mellitus type II was reviewed. Insight into the different subsets associated with these diseases and how their metabolism changes depending on their microenvironment was provided. This chapter also describes different available possibilities for measuring cellular metabolites. In Chapter 3, we investigated whether the shown anti-inflammatory effects of KDACis in the COPD context apply also in our cell model of primary murine alveolar-like macrophages after lipopolysaccharide (LPS)-induced activation. We hypothesized that these anti-inflammatory effects may be associated with metabolic changes in macrophages. To validate this hypothesis, an unbiased and a targeted proteomic approach to investigate metabolic enzymes as well as LC- and GC-MS to quantify metabolites in combination with the measurement of functional parameters was used. While minimal metabolic changes were observed, KDAC inhibition reduced the production of inflammatory mediators. Interestingly, it specifically enhanced the expression of proteins involved in ubiquitination. The findings highlight the potential of KDAC inhibitors as anti-inflammatory drugs for diseases like COPD. In Chapter 4, primary murine alveolar-like macrophages to examine the impact of collagen morphology on macrophage marker expression, behaviour, and shape were used. Proteomic analysis revealed increased expression of glycolysis-related proteins, although this did not result in higher glycolytic activity, potentially due to reduced enzyme activity. Overall, our findings indicate that macrophages can detect collagen morphologies and adjust the expression and activity of metabolismrelated proteins. This suggests a significant interplay between macrophages and their microenvironment, which could be crucial in the progression of tissue repair to fibrosis in the lungs. Finally, in Chapter 5, the conclusions of this thesis are summarized and future implications are examined. The research presented in this thesis establishes a foundation for gaining a deeper understanding of macrophages and their intricate relationship with metabolism, as well as the implications of these metabolic changes in chronic diseases. These findings open up possibilities for therapeutic interventions targeting chronic inflammation.
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Chapter 2 Chapter 2 Meta-inflammation and metabolic reprogramming of macrophages in diabetes and obesity: the importance of metabolites Meta-inflammation and metabolic reprogramming of macrophages in diabetes and obesity: the importance of metabolites Sara Russo 1, Marcel Kwiatkowski 2, Natalia Govorukhina 1, Rainer Bischoff 1 †, Barbro N. Melgert 3,4 †* Frontiers in Immunology 2021; 12: 746151. Published online 2021 Nov 5. doi: 10.3389/fimmu.2021.746151
24 Chapter 2 ABSTRACT Diabetes mellitus type II and obesity are two important causes of death in modern society. They are characterized by low-grade chronic inflammation and metabolic dysfunction (meta-inflammation), which is observed in all tissues involved in energy homeostasis. A substantial body of evidence has established an important role for macrophages in these tissues during the development of diabetes mellitus type II and obesity. Macrophages can activate into specialized subsets by cues from their microenvironment to handle a variety of tasks. Many different subsets have been described and in diabetes/obesity literature two main classifications are widely used that are also defined by differential metabolic reprogramming taking place to fuel their main functions. Classically activated, pro-inflammatory macrophages (often referred to as M1) favor glycolysis, produce lactate instead of metabolizing pyruvate to acetyl-CoA, and have a tricarboxylic acid cycle that is interrupted at two points. Alternatively activated macrophages (often referred to as M2) mainly use beta-oxidation of fatty acids and oxidative phosphorylation to create energyrich molecules such as ATP and are involved in tissue repair and downregulation of inflammation. Since diabetes type II and obesity are characterized by metabolic alterations at the organism level, these alterations may also induce changes in macrophage metabolism resulting in unique macrophage activation patterns in diabetes and obesity. This review describes the interactions between metabolic reprogramming of macrophages and conditions of metabolic dysfunction like diabetes and obesity. We also focus on different possibilities of measuring a range of metabolites intra-and extracellularly in a precise and comprehensive manner to better identify the subsets of polarized macrophages that are unique to diabetes and obesity. Advantages and disadvantages of the currently most widely used metabolite analysis approaches are highlighted. We further describe how their combined use may serve to provide a comprehensive overview of the metabolic changes that take place intracellularly during macrophage activation in conditions like diabetes and obesity. Keywords: M1, M2, Metabolic syndrome, DMTII, alternatively activated, classically activated, Metabolite analysis, MS.
25 2 Macrophage Metabolic Reprogramming in Diabetes INTRODUCTION Diabetes mellitus type II (DMTII) is one of the main causes of death in modern society according to the World Health Organization (1). It correlates with long-term complications that include nephropathy, peripheral neuropathy, and cardiovascular disease. The International Diabetes Federation has estimated that globally the diagnosis of DMTII has been made in 415 million people and anticipates growth to up to 642 million by the year 2040 (2). Several factors can contribute to a higher risk of developing DMTII, but it has been proven that overweight or obesity are the most important ones (3). DMTII is often linked to obesity and both are associated with metabolic syndrome, which encompasses conditions such as high blood pressure, excess body fat around the waist, high blood sugar, high serum cholesterol or triglyceride levels, and low highdensity lipoprotein (HDL) cholesterol. Metabolic syndrome is characterized by low-grade chronic inflammation (meta-inflammation) (4) in all tissues involved in energy homeostasis, including adipose tissue, pancreatic islets, and liver (5). Studies have shown that the metabolic consequences of adipose tissue dysfunction increase mortality in patients with DMTII, emphasizing the importance of metainflammation in the context of DMTII (6) Macrophages are part of the innate immune system and are present in all tissues of our body, including adipose tissues (7). They play a crucial role in the first line of defense against microorganisms and other external or internal threats to homeostasis by initiating essential inflammatory responses (8). These inflammatory responses are facilitated by changes in macrophage cellular metabolism, with a focus on glycolysis that is induced in cells producing inflammatory mediators. The inflammatory response is counter-balanced by stimulation of tissue repair and antiinflammatory mechanisms once the threat has been overcome. At the same time, the cellular metabolism changes from glycolysis to oxidative phosphorylation to aid in tissue repair. Continuous exposure to pro-inflammatory stimuli, however, can shift the balance of inflammation and repair in favor of chronic inflammation and tissue damage. Excessive activation of macrophage inflammatory responses is seen in many diseases characterized by the continuous presence of pro-inflammatory stimuli, including DMTII, and explains in part the meta-inflammation found in this condition. Many studies have described how macrophages become activated by inflammatory stimuli (9,10) and there is increasing consensus that a particular macrophage activation state is associated with DMTII. Characterization of the different macrophage activation states is complicated, but in recent years has been aided by the development and use of novel techniques like multiparametric flow cytometry,
26 Chapter 2 single-cell RNA sequencing, and real-time extracellular flux analysis. Especially the latter has the potential to improve our understanding of how macrophages can switch between different types of responses. In DMTII and obesity, the changes in macrophage cellular metabolism coincide with profound changes in metabolism on a tissue and organism level, that probably interact and give rise to a specific DMTII-associated macrophage activation state (11). This review aims to summarize what is currently known about macrophage activation in DMTII-related meta- inflammation, how changes in intracellular metabolism are influenced by the changed presence in extracellular nutrients and metabolites, and how fluctuations in key metabolic intermediates could also play a role in cellular processes like gene expression. This overview emphasizes that profiling metabolites can help to characterize macrophages and their responses and to understand how changes in their intracellular metabolites affect DMTII progression. Therefore, we finish with a comparison between different approaches to metabolite analysis to provide an overview of the currently available methods and their pros and cons, highlighting metabolomics studies that have made use of these methods and have been central to characterizing macrophages. INSULIN RESISTANCE AND INFLAMMATION One of the key characteristics of DMTII is the altered insulin response. In healthy individuals, with a body mass index (BMI) in the normal range, pancreatic β cells produce insulin in response to circulating glucose levels. This will bind and activate insulin receptors on the cell membrane of different cell types, including macrophages, to lower blood glucose levels by enhancing its uptake by these cells. The binding of insulin to its receptor drives a cascade of events ultimately leading to uptake of glucose and further downstream effects (Figure 1). First glucose transporters, GLUT4 in most cell types and GLUT1 in macrophages, will either translocate from vesicles in the cytoplasm to the cell surface or their expression is upregulated, both increasing glucose influx into cells up to 10 times (12). Mammalian target of rapamycin (mTOR) will then be activated and protein synthesis will be induced. Furthermore, glycogen synthase kinase-3β (GSK3B) is inhibited allowing the activation of glycogen synthesis. When GSK3B is activated, it phosphorylates and inactivates glycogen synthase, decreasing glycogen synthesis, therefore GSK3B inhibition by Akt results in higher glycogen production. A change in gene transcription will also be initiated: expression of genes that favor either the synthesis of glycogen from glucose in the liver and muscles or of triglycerides from free fatty acids (FFA) in adipocytes will be induced and expression of genes that favor glycolysis will be transiently inhibited (13).
27 2 Macrophage Metabolic Reprogramming in Diabetes Figure 1: Regulation of glucose entrance through insulin signaling. Insulin receptors are tyrosine kinases consisting of two extracellular α-subunits and two transcellular β-subunits. In healthy individuals, insulin will bind the α subunit of the insulin receptor, causing a conformational change that leads to phosphorylation of tyrosine residues in its β subunit. The proteins insulin receptor substrates 1 or 2 (Irs-1/-2) will then bind to the tyrosinephosphorylated region of insulin receptors and be themselves phosphorylated. Phosphoinositide-3-kinase (PI3K) will bind to the phosphorylated IRS-1 or -2 and be activated, producing 3-phosphorylated polyphosphoinositides (PiP3) from phosphatidylinositol 4,5-bisphosphate (PiP2). PiP3 will recruit the serine/threonine kinase Akt (also known as protein kinase B) from the cytosol to the plasma membrane, where it will be phosphorylated and activated, leading to glycogen synthase kinase-3β (GSK3B) inhibition and therefore to higher glycogen synthesis. AKT is also responsible for the translocation of the glucose transporter (GLUT) to the plasma membrane, allowing glucose entry. DMTII is caused by the development of insulin resistance, meaning the inability of cells to respond to insulin due to a transmission blockage of the insulin receptor, mainly in muscle and liver cells. Pancreatic β-cells will at first try to compensate for the higher levels of glucose by increasing insulin production. This will eventually lead to lower glucose availability in combination with lower tissue insulin sensitivity resulting in loss of β-cell function. This will result in lower insulin secretion, which will consequently lead to a higher concentration of glucose in blood (14). Insulin resistance can be caused by many different factors, with obesity being the most important one (14). Elevated levels of circulating free fatty acids are one of the reasons for the development of insulin resistance in obese patients. These
28 Chapter 2 high levels of fatty acids are caused by increased basal lipolysis in adipose tissues and this elevated concentration has been proposed to serve as a stimulus for the entry and accumulation of macrophages in adipose tissue by increasing the local production and release of pro-inflammatory cytokines and chemokines (15). High concentrations of saturated free fatty acids will also induce pro-inflammatory effects through activation of Toll-like receptors (16). A consequence of this activation is the induction of the Jun N-terminal kinase and inhibitor of κB kinase (JNK/IKKκB) pathways, which is then followed by an inflammatory cascade. Both JNK and IKK are believed to promote insulin resistance because they phosphorylate serine/ threonine residues on insulin receptor substrate (IRS) proteins. By phosphorylating these residues, their phosphorylation by insulin receptors is blocked, which prevents the activation of insulin receptors by insulin. The result is inhibition of insulindriven signal transduction and downstream effects thus inhibiting glucose entry into the cell and its accumulation in the blood. MACROPHAGES AND INFLAMMATION IN OBESITY AND DMTII The inflammation related to obesity was first described in 1993 when Hotamisligil et al. showed that adipose tissue from obese rats expressed more tumor necrosis factor-α (TNF-α) (17) than adipose tissue from lean animals. Weisberg and colleagues further showed that TNF-α was not secreted by adipocytes but by macrophages and that the number of macrophages increased in adipose tissue during weight gain (10). Macrophages develop either from self-renewing fetal progenitors that can populate tissues before birth and maintain their numbers after birth or from circulating monocytes recruited to tissues after birth (18). Studies have shown a higher number of macrophages in white adipose tissue of obese subjects compared to people of normal BMI, going from 10% of total cells to more than 50% (19). The origin of these macrophages, either through local proliferation or monocyte recruitment, remains to be established in detail. A recent study in mice found that local proliferation of adipose tissue-resident macrophages at least contributes to macrophage accumulation during obesity too (20). Studies have shown that during obesity, triglyceride accumulation causes stress on adipocytes due to an increase in cell size and subsequent hypoxia (21). Capillary network development cannot keep up with fat mass expansion, resulting in adipocytes that are too far away from the vasculature to be efficiently supplied with oxygen (22). This leads to higher expression of hypoxia-inducible factor, adipocyte activation, and production and subsequent release of free fatty acids and pro-inflammatory mediators such as interleukin-1β (IL-1β), IL-6, macrophage migration inhibitory factor (MIF), monocyte chemoattractant protein 1 (MCP-1, also
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