Innate lymphoid type 2 cells maintain eosinophils and alternatively activated macrophages in visceral fat via the production of IL-5 and IL-13.
Eosinophils in visceral adipose tissue (VAT) have been implicated in metabolic homeostasis and the maintenance of alternatively activated macrophages (AAMs). The absence of eosinophils can lead to adiposity and systemic insulin resistance in experimental animals, but what maintains eosinophils in adipose tissue is unknown. We show that interleukin-5 (IL-5) deficiency profoundly impairs VAT eosinophil accumulation and results in increased adiposity and insulin resistance when animals are placed on a high-fat diet. Innate lymphoid type 2 cells (ILC2s) are resident in VAT and are the major source of IL-5 and IL-13, which promote the accumulation of eosinophils and AAM. Deletion of ILC2s causes significant reductions in VAT eosinophils and AAMs, and also impairs the expansion of VAT eosinophils after infection with Nippostrongylus brasiliensis, an intestinal parasite associated with increased adipose ILC2 cytokine production and enhanced insulin sensitivity. Further, IL-33, a cytokine previously shown to promote cytokine production by ILC2s, leads to rapid ILC2-dependent increases in VAT eosinophils and AAMs. Thus, ILC2s are resident in VAT and promote eosinophils and AAM implicated in metabolic homeostasis, and this axis is enhanced during Th2-associated immune stimulation.
The formation of the atherosclerotic lesion is a complex process influenced by an
array of inflammatory and lipid metabolism pathways. We previously demonstrated
that NR4A nuclear receptors are highly induced in macrophages in response to
inflammatory stimuli and modulate the expression of genes linked to inflammation
in vitro. Here we used mouse genetic models to assess the impact of NR4A
expression on atherosclerosis development and macrophage polarization.
Transplantation of wild-type, Nur77−/−, or
Nor1−/− null hematopoetic precursors into LDL
receptor (LDLR)−/− recipient mice led to comparable
development of atherosclerotic lesions after high-cholesterol diet. We also
observed comparable induction of genes linked to M1 and M2 responses in
wild-type and Nur77-null macrophages in response to lipopolysaccharides and
interleukin (IL)-4, respectively. In contrast, activation of the nuclear
receptor liver X receptor (LXR) strongly suppressed M1 responses, and ablation
of signal transductor and activator of transcription 6 (STAT6) strongly
suppressed M2 responses. Recent studies have suggested that alterations in
levels of Ly6Clo monocytes may be a contributor to inflammation and
atherosclerosis. In our study, loss of Nur77, but not Nor1, was associated with
decreased abundance of Ly6Clo monocytes, but this change was not
correlated with atherosclerotic lesion development. Collectively, our results
suggest that alterations in the Ly6Clo monocyte population and bone
marrow NR4A expression do not play dominant roles in macrophage polarization or
the development of atherosclerosis in mice.
Nur77; Ly6C; nuclear receptor
The cholinergic antiinflammatory pathway (CAP), which terminates in the spleen, attenuates postoperative cognitive decline (PCD) in rodents. Surgical patients with metabolic syndrome exhibit exaggerated and persistent PCD that is reproduced in postoperative rats selectively bred for easy fatigability and that contain all features of metabolic syndrome (low-capacity runners [LCRs]). We compared the CAP and lipoxin A4 (LXA4), another inflammation-resolving pathway in LCR, with its counterpart high-capacity runner (HCR) rats. Isoflurane-anesthetized LCR and HCR rats either underwent aseptic trauma involving tibial fracture (surgery) or not (sham). At postoperative d 3 (POD3), compared with HCR, LCR rats exhibited significantly exaggerated PCD (trace fear conditioning freezing time 43% versus 57%). Separate cohorts were killed at POD3 to collect plasma for LXA4 and to isolate splenic mononuclear cells (MNCs) to analyze CAP signaling, regulatory T cells (Tregs) and M2 macrophages (M2 Mφ). Under lipopolysaccharide (LPS) stimulation, tumor necrosis factor (TNF)-α produced by splenic MNCs was 117% higher in LCR sham and 52% higher in LCR surgery compared with HCR sham and surgery rats; LPS-stimulated TNF-α production could not be inhibited by an α7 nicotinic acetylcholine receptor agonist, whereas inhibition by the β2 adrenergic agonist, salmeterol, was significantly less (−35%) than that obtained in HCR rats. Compared to HCR, sham and surgery LCR rats had reduced β2 adrenergic receptor–expressing T lymphocytes (59%, 44%), Tregs (47%, 54%) and M2 Mφ (45%, 39%); surgical LCR rats’ hippocampal M2 Mφ was 66% reduced, and plasma LXA4 was decreased by 120%. Rats with the metabolic syndrome have ineffective inflammation-resolving mechanisms that represent plausible reasons for the exaggerated and persistent PCD.
Efficient execution of apoptotic cell death followed by efficient clearance mediated by professional macrophages is a key mechanism in maintaining tissue homeostasis. Removal of apoptotic cells usually involves three central elements: (1) attraction of phagocytes via soluble `find me' signals, (2) recognition and phagocytosis via cell surface presenting `eat me' signals, and (3) suppression or initiation of inflammatory responses depending on additional innate immune stimuli. Suppression of inflammation involves both direct inhibition of pro-inflammatory cytokine production and release of anti-inflammatory factors, which all contribute to the resolution of inflammation. In the present study, using wild type and adenosine A2A receptor (A2AR) null mice, we investigated whether A2ARs, known to mediate anti-inflammatory signals in macrophages, participate in the apoptotic cell-mediated immunosuppression. We found that macrophages engulfing apoptotic cells release adenosine in sufficient amount to trigger A2ARs, and simultaneously increase the expression of A2ARs, possibly via activation of activation of liver X receptor and peroxisome proliferators activated receptor δ. In macrophages engulfing apoptotic cells, stimulation of A2ARs suppresses the NO-dependent formation of neutrophil migration factors, such as macrophage inflammatory protein-2, using the adenylate cyclase / protein kinase A pathway. As a result, loss of A2ARs results in elevated chemoattractant secretion. This was evident as pronounced neutrophil migration upon exposure of macrophages to apoptotic cells in an in vivo peritonitis model. Altogether our data indicate that adenosine is one of the soluble mediators released by macrophages that mediate engulfment-dependent apoptotic cell suppression of inflammation.
Adenosine; Macrophages; Phagocytosis; Proinflammatory cytokines
Vertebrate tissues comprise precise admixtures of parenchymal and hematopoietic cells, whose interactions are vital to proper tissue function. By regulating this interaction, vertebrates are able to mitigate environmental stress and coordinate dramatic physiologic adaptations. For instance, under conditions of chronic nutrient excess, leukocyte recruitment and activation increase in an effort to decrease excess nutrient storage and alleviate adipocyte stress. While basal equilibria may be reestablished upon normalization of nutrient intake, a new set point characterized by insulin resistance and chronic inflammation is established if the stress persists. Consequently, although this response is adaptive in settings of acute overfeeding and infection, it has catastrophic health consequences in the modern context of obesity. Understanding how leukocyte set points (numbers and activation status) are established, maintained, and regulated in tissues is, thus, critical to our understanding of, and intervention in, chronic metabolic diseases, such as obesity and diabetes.
Metabolism and immunity are two fundamental systems of metazoans. The presence of immune cells, such as macrophages, in metabolic tissues, suggests dynamic, on-going crosstalk between these two regulatory systems. Here, we discuss how changes in recruitment and activation of macrophages contribute to metabolic homeostasis. In particular, we focus our discussion on the pathogenic and protective functions of classically (M1) and alternatively (M2) activated macrophages, respectively, in experimental models of obesity and metabolic disease.
Chronic inflammation is now recognized as a key step in the pathogenesis of obesity-induced insulin resistance and type 2 diabetes mellitus. This low-grade inflammation is mediated by the inflammatory (classical) activation of recruited and resident macrophages that populate metabolic tissues, including adipose tissue and liver. These findings have led to the concept that infiltration and activation of adipose tissue macrophages is causally linked to obesity-induced insulin resistance. Studies have shown, however, that alternatively activated macrophages taking residence in adipose tissue and liver perform beneficial functions in obesity-induced metabolic disease. By attenuating tissue inflammation and increasing oxidative metabolism in liver and skeletal muscle, alternatively activated macrophages have lessened insulin resistance in obese mice. The discovery that distinct subsets of macrophages are involved in the promotion or attenuation of insulin resistance suggests that pathways controlling macrophage activation can potentially be targeted to treat these co-morbidities of obesity. Thus, this Review focuses on the stimuli and mechanisms that control classical and alternative activation of tissue macrophages, and how these macrophage activation programs modulate insulin action in peripheral tissues. The functional importance of macrophage activation is further discussed in the context of host defense to highlight the crosstalk between innate immunity and metabolism.
alternatively activated macrophages; inflammation; insulin resistance; obesity; peroxisome proliferator-activated receptors
Obesity and its attendant metabolic disorders represent the great public health challenge of our time. Recent evidence suggests that onset of inflammation in metabolic tissues pathogenically links obesity to insulin resistance and type 2 diabetes. In this review, we briefly summarize the extant literature with special attention to the central role of the tissue-associated macrophage in the initiation of metabolic inflammation. We argue that rather than simple inflammatory disease, obesity and metabolic syndrome represent derangements in macrophage activation with concomitant loss of metabolic coordination. As such, the sequelae of obesity are as much products of the loss of positive macrophage influences as the presence of deleterious inflammation. The therapeutic implications of this conclusion are profound because they suggest that pharmacologic targeting of macrophage activation, rather than purely inflammation, might be efficacious in treating this global epidemic.
obesity; insulin resistance; inflammation; diabetes; PPAR
All homeotherms utilize thermogenesis to maintain core body temperature, ensuring that cellular functions and physiologic processes can ensue in cold environments1-3. In the prevailing model, when the hypothalamus senses cold temperatures, it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue (BAT) and white adipose tissue (WAT)4,5. Acting via the β3-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes6, whereas it stimulates the expression of thermogenic genes, such as PPARγ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1), and acyl-CoA synthetase long-chain family member 1 (Acsl1), in brown adipocytes7-9. However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report an unexpected requirement for the interleukin 4 (IL4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Cold exposure rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in BAT and lipolysis in WAT. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL4 increased thermogenic gene expression, fatty acid mobilization, and energy expenditure, all in a macrophage-dependent manner. We have thus discovered a surprising role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold.
The escalating epidemic of obesity has driven the prevalence of both type 1 and 2 diabetes mellitus to historically high levels. Chronic low-grade inflammation, which is present in both type 1 and type 2 diabetics, contributes to the pathogenesis of insulin resistance. The accumulation of activated innate immune cells in metabolic tissues results in release of inflammatory mediators, in particular, IL-1β and TNFα, which promote systemic insulin resistance and β-cell damage. In this article, we discuss the central role of innate immunity and, in particular, the macrophage in insulin sensitivity and resistance, β-cell damage, and autoimmune insulitis. We conclude with a discussion of the therapeutic implications of this integrated understanding of diabetic pathology.
Expanding waistlines have blurred the distinction between type 1 and type 2 diabetes. Chronic low-grade inflammation, caused by the activation of macrophages in obese individuals, may underlie all forms of diabetes.
Eosinophils are associated with helminth immunity and allergy, often in conjunction with alternatively activated macrophages (AAMs). Adipose tissue AAMs are necessary to maintain glucose homeostasis and are induced by the cytokine interleukin-4 (IL-4). Here, we show that eosinophils are the major IL-4-expressing cells in white adipose tissues of mice, and, in their absence, AAMs are greatly attenuated. Eosinophils migrate into adipose by an integrin-dependent process and reconstitute AAMs through an IL-4/IL-13-dependent process. Mice on high-fat diet develop increased body fat, impaired glucose tolerance and insulin resistance in the absence of eosinophils, and helminth-induced adipose eosinophilia enhances glucose tolerance. Our results suggest that eosinophils play an unexpected role in metabolic homeostasis through maintenance of adipose AAMs.
Macrophages, a key component of the innate defense against pathogens, participate in the initiation and resolution of inflammation, and in the maintenance of tissues. These diverse and at times antithetical functions of macrophages are executed via distinct activation states, ranging from classical to alternative to deactivation. Because the dysregulation of macrophage activation is pathogenically linked to various metabolic, inflammatory and immune disorders, regulatory proteins controlling macrophage activation have emerged as important new therapeutic targets. Here, the mechanisms by which Peroxisome Proliferator Activated Receptors (PPARs) transcriptionally regulate macrophage activation in health and disease states, including obesity, insulin resistance and cardiovascular disease, will be reviewed.
Peroxisome proliferator–activated receptors (PPARs; PPAR-α, PPAR-δ, and PPAR-γ) comprise a family of nuclear receptors that sense fatty acid levels and translate this information into altered gene transcription. Previously, it was reported that treatment of mice with a synthetic ligand activator of PPAR-δ, GW0742, ameliorates experimental autoimmune encephalomyelitis (EAE), indicating a possible role for this nuclear receptor in the control of central nervous system (CNS) autoimmune inflammation. We show that mice deficient in PPAR-δ (PPAR-δ−/−) develop a severe inflammatory response during EAE characterized by a striking accumulation of IFN-γ+IL-17A− and IFN-γ+IL-17A+ CD4+ cells in the spinal cord. The preferential expansion of these T helper subsets in the CNS of PPAR-δ−/− mice occurred as a result of a constellation of immune system aberrations that included higher CD4+ cell proliferation, cytokine production, and T-bet expression and enhanced expression of IL-12 family cytokines by myeloid cells. We also show that the effect of PPAR-δ in inhibiting the production of IFN-γ and IL-12 family cytokines is ligand dependent and is observed in both mouse and human immune cells. Collectively, these findings suggest that PPAR-δ serves as an important molecular brake for the control of autoimmune inflammation.
Obesity is associated with infiltration of white adipose tissue (WAT) by macrophages, which contributes to the development of insulin resistance. In this issue of the JCI, Kosteli and colleagues demonstrate that weight loss is unexpectedly also associated with rapid, albeit transient, recruitment of macrophages to WAT and that this appears to be related to lipolysis.
A major component of obesity-related insulin resistance is the establishment of a chronic inflammatory state with invasion of white adipose tissue by mononuclear cells. This results in the release of pro-inflammatory cytokines, which in turn leads to insulin resistance in target tissues such as skeletal muscle and liver. To determine the role of insulin action in macrophages and monocytes in obesity-associated insulin resistance, we conditionally inactivated the insulin receptor (IR) gene in myeloid lineage cells in mice (IRΔmyel-mice). While these animals exhibit unaltered glucose metabolism on a normal diet, they are protected from the development of obesity-associated insulin resistance upon high fat feeding. Euglycemic, hyperinsulinemic clamp studies demonstrate that this results from decreased basal hepatic glucose production and from increased insulin-stimulated glucose disposal in skeletal muscle. Furthermore, IRΔmyel-mice exhibit decreased concentrations of circulating tumor necrosis factor (TNF) α and thus reduced c-Jun N-terminal kinase (JNK) activity in skeletal muscle upon high fat feeding, reflecting a dramatic reduction of the chronic and systemic low-grade inflammatory state associated with obesity. This is paralleled by a reduced accumulation of macrophages in white adipose tissue due to a pronounced impairment of matrix metalloproteinase (MMP) 9 expression and activity in these cells. These data indicate that insulin action in myeloid cells plays an unexpected, critical role in the regulation of macrophage invasion into white adipose tissue and in the development of obesity-associated insulin resistance.
Obesity represents a major health burden with steadily increasing incidence. While it is associated with numerous co-morbidities, type 2 diabetes mellitus represents one of the major life-threatening, obesity-related conditions. Over the last years, it has become clear that during the course of obesity development not only does fat mass increase, but also fat composition changes qualitatively, leading to an influx of inflammatory cells, such as macrophages, into adipose tissue. Macrophages in turn secrete inflammatory mediators, which inhibit insulin action in skeletal muscle, liver, and even the central nervous system to ultimately cause insulin-resistant diabetes mellitus. However, the effect of insulin action and resistance in these inflammatory cell types themselves has not been addressed. To this end, we have generated and analyzed mice with inactivation of the insulin receptor specifically in myeloid cell-derived, inflammatory cells. Surprisingly, these animals are protected from the development of obesity-associated deterioration of glucose metabolism, thereby defining insulin action in inflammatory cells as a novel and promising target for therapeutic intervention against obesity-associated diabetes mellitus.
Macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses against self antigens. Although factors participating in recognition and engulfment of apoptotic cells have been identified, the transcriptional basis for the sensing and silently disposing of apoptotic cells is unknown. Here we show that peroxisome proliferator activated receptor-δ (PPAR-δ) is induced when macrophages engulf apoptotic cells and functions as a transcriptional sensor of dying cells. Genetic deletion of PPAR-δ decreases expression of opsonins, such as C1qb, resulting in impairment of apoptotic cell clearance and reduction in anti-inflammatory cytokine production. This increases autoantibody production and predisposes global and macrophage-specific PPARd−/− mice to autoimmune kidney disease, a phenotype resembling the human disease systemic lupus erythematosus. Thus, PPAR-δ plays a pivotal role in orchestrating the timely disposal of apoptotic cells by macrophages, ensuring that tolerance to self is maintained.
Macrophage infiltration and activation in metabolic tissues underlie obesity-induced insulin resistance and type 2 diabetes. While inflammatory activation of resident hepatic macrophages potentiates insulin resistance, the functions of alternatively activated Kupffer cells in metabolic disease remain unknown. Here we show that, in response to the Th2 cytokine interleukin-4 (IL-4), peroxisome proliferator activated receptor δ (PPARδ) directs expression of the alternative phenotype in Kupffer cells and adipose tissue macrophages of lean mice. However, adoptive transfer of PPARδ null bone marrow into wild type mice only diminishes alternative activation of hepatic macrophages, causing hepatic dysfunction and systemic insulin resistance. Suppression of hepatic oxidative metabolism is recapitulated by treatment of primary hepatocytes with conditioned media from PPARδ null macrophages, indicating direct involvement of Kupffer cells in liver lipid metabolism. Taken together, these data suggest an unexpected beneficial role for alternatively activated Kupffer cells in metabolic syndrome and type 2 diabetes.
Progesterone-induced Xenopus laevis oocyte maturation is mediated via a plasma membrane-bound receptor and does not require gene transcription. Evidence from several species suggests that the relevant progesterone receptor is a G-protein coupled receptor (GPCR) and that a second receptor—GPR3 and/or GPR12 in mammals—tonically opposes the progesterone receptor. We have cloned a novel Xenopus laevis GPCR, GPRx, which may play a similar role to GPR3/GPR12 in amphibians and fishes. GPRx is related to but distinct from GPR3, GPR6, and GPR12; GPRx orthologs are present in Xenopus tropicalis and Danio rerio, but apparently not in birds or mammals. Xenopus laevis GPRx is mainly expressed in brain, ovary, and testis. The GPRx mRNA increases during oogenesis, persists during oocyte maturation and early embryogenesis, and then falls after the midblastula transition. Microinjection of GPRx mRNA increases the concentration of cAMP in oocytes and causes the oocytes to fail to respond to progesterone, and this block is reversed by co-injecting GPRx with morpholino oligonucleotides. Morpholino injections did not cause spontaneous maturation of oocytes, but did accelerate progesterone-induced maturation. Thus, GPRx contributes to the maintenance of G2-arrest in immature Xenopus laevis oocytes.
Obesity and insulin resistance, cardinal features of metabolic syndrome, are closely associated with a state of low-grade inflammation1,2. In adipose tissue chronic overnutrition leads to macrophage infiltration, resulting in local inflammation that potentiates insulin resistance3,4. For instance, transgenic expression of Mcp1 in adipose tissue increases macrophage infiltration, inflammation, and insulin resistance5,6. Conversely, disruption of Mcp1 or its receptor, Ccr2, impairs migration of macrophages into adipose tissue, thereby lowering adipose tissue inflammation and improving insulin sensitivity5,7. These findings together suggest a correlation between adipose tissue macrophage content (ATM) and insulin resistance. However, resident macrophages in tissues display tremendous heterogeneity in their activities and functions, primarily reflecting their local metabolic and immune microenvironment8. While Mcp-1 directs recruitment of pro-inflammatory classically activated macrophages to sites of tissue damage5,8, resident macrophages, such as those present in adipose tissue of lean mice, display the alternatively activated phenotype9. Despite their higher reparative capacity10, the precise role of alternatively activated macrophages in obesity-induced insulin resistance remains unknown. Using mice with macrophage-specific deletion of peroxisome proliferator activated receptor-γ (PPARγ), we show here that PPARγ is required for maturation of alternatively activated macrophages. Disruption of PPARγ in myeloid cells impairs alternative macrophage activation, thereby predisposing these animals to development of diet-induced obesity, insulin resistance, and glucose intolerance. Furthermore, gene expression profiling revealed that downregulation of oxidative phosphorylation gene expression in skeletal muscle and liver leads to decreased insulin sensitivity in these tissues. Together, our findings demonstrate that resident alternatively activated macrophages have a beneficial role in regulating nutrient homeostasis and suggest that macrophage polarization towards the alternative state might be a useful strategy for treating type 2 diabetes.
Macrophages participate in physiologic and pathologic processes through elaboration of distinct activation programs. Studies with macrophage cell systems have revealed much concerning the importance of this pleiotropic cell; however, these studies are inherently limited by three factors: heterogeneity of the target cell population, poor capacity to elaborate various activation programs, and lack of a genetically tractable model system for loss- and gain-of-function studies. Although definitive, hematopoietic lineages can be isolated from embryonic stem (ES) cells, these isolation procedures are inefficient and time-consuming and require elaborate cell-sorting protocols. We, therefore, examined whether myeloid precursors, capable of differentiating into macrophages, could be conditionally expanded in vitro. Here, we report methods for selective isolation and immortalization of ES cell-derived myeloid precursors by estrogen-regulated HoxA9 protein. Using this new macrophage differentiation system, an unlimited number of custom-designed macrophages with defined functional characteristics can be generated from any targeted ES cell. In combination with knockout or small interfering RNA knockdown technologies, this macrophage differentiation system provides a powerful tool for high throughput analysis of regulatory mechanisms controlling macrophage activation in health and disease.
Complex interplay between T helper (Th) cells and macrophages contributes to the formation and progression of atherosclerotic plaques. While Th1 cytokines promote inflammatory activation of lesion macrophages, Th2 cytokines attenuate macrophage-mediated inflammation and enhance their repair functions. In spite of its biologic importance, the biochemical and molecular basis of how Th2 cytokines promote maturation of anti-inflammatory macrophages is not understood. We show here that in response to interleukin-4 (IL-4), signal transducer and activator of transcription 6 (STAT6) and PPARγ-coactivator-1β (PGC-1β ) induce macrophage programs for fatty acid oxidation and mitochondrial biogenesis. Transgenic expression of PGC-1β primes macrophages for alternative activation and strongly inhibits proinflammatory cytokine production, whereas inhibition of oxidative metabolism or RNAi-mediated knockdown of PGC-1β attenuates this immune response. These data elucidate a molecular pathway that directly links mitochondrial oxidative metabolism to the anti-inflammatory program of macrophage activation, suggesting a potential role for metabolic therapies in treating atherogenic inflammation.