Phenotypic switching of adipose tissue macrophages with obesity is generated by spatiotemporal differences in macrophage subtypes

OBJECTIVE—To establish the mechanism of the phenotypic switch of adipose tissue macrophages (ATMs) from an alternatively activated (M2a) to a classically activated (M1) phenotype with obesity.RESEARCH DESIGN AND METHODS—ATMs from lean and obese (high-fat diet–fed) C57Bl/6 mice were analyzed by a combination of flow cytometry, immunofluorescence, and expression analysis for M2a and M1 genes. Pulse labeling of ATMs with PKH26 assessed the recruitment rate of ATMs to spatially distinct regions.RESULTS—Resident ATMs in lean mice express the M2a marker macrophage galactose N-acetyl-galactosamine specific lectin 1 (MGL1) and localize to interstitial spaces between adipocytes independent of CCR2 and CCL2. With diet-induced obesity, MGL1⁺ ATMs remain in interstitial spaces, whereas a population of MGL1⁻CCR2⁺ ATMs with high M1 and low M2a gene expression is recruited to clusters surrounding necrotic adipocytes. Pulse labeling showed that the rate of recruitment of new macrophages to MGL1⁻ ATM clusters is significantly faster than that of interstitial MGL1⁺ ATMs. This recruitment is attenuated in Ccr2^−/− mice. M2a- and M1-polarized macrophages produced different effects on adipogenesis and adipocyte insulin sensitivity in vitro.CONCLUSIONS—The shift in the M2a/M1 ATM balance is generated by spatial and temporal differences in the recruitment of distinct ATM subtypes. The obesity-induced switch in ATM activation state is coupled to the localized recruitment of an inflammatory ATM subtype to macrophage clusters from the circulation and not to the conversion of resident M2a macrophages to M1 ATMs in situ.Obesity activates inflammatory pathways in leukocytes that contribute to the pathogenesis of obesity-associated diseases, such as type 2 diabetes and atherosclerosis. In particular, adipose tissue macrophages (ATMs) have been identified as the primary source of inflammatory cytokine production in adipose tissue and a key component in the progression to insulin resistance with obesity. ATMs are increased in visceral fat depots with obesity and correlate with measures of insulin resistance (1). A range of mouse models with loss-of-function mutations in genes important in macrophage recruitment (Ccr2), inflammatory cytokine production (Tnfα), and proinflammatory activation (Ikkβ) have demonstrated protection from high-fat diet–induced insulin resistance (2–4).Despite these advances, the function of ATMs in lean and obese states is poorly understood. It has been suggested that ATMs influence a range of processes in adipose tissue, including adipogenesis, angiogenesis, and the response to hypoxia (5–7). One potential clue to understanding these diverse functions emerges from the evidence that macrophages can exist in different activation states with distinct properties (8). On stimulation with lipopolysaccharide (LPS) and interferon-γ, macrophages assume a proinflammatory classical activation profile also known as M1. However, under the influence of T_H2 cytokines interleukin (IL)-4 or IL-13, macrophages assume an alternative activation state (M2a) and produce immunosuppressive factors, such as IL-10, IL-1RA, and arginase (9). We have previously demonstrated that ATMs in lean mice are polarized toward an alternatively activated state (M2a), whereas ATMs in obese mice have an M1 profile (10). The importance of this M1/M2a balance in glucose metabolism has been illustrated in macrophage-specific Pparg^−/− mice that have deficient M2a polarization, increased adipose tissue inflammation, and worse insulin resistance (11,12).How does obesity switch ATMs from an M2a to an M1 activation state? One hypothesis is that resident M2a ATMs are uniformly converted to an M1 state. A second hypothesis is that M1 ATMs are generated by the obesity-induced trafficking of a subset of inflammatory CCR2⁺Ly6G^hiCX3CR1^low monocytes to fat similar to what is seen in atherosclerotic lesions (13). Currently, there is insufficient information to differentiate between these two models. In this study, we provide evidence for the latter model by characterizing subtypes of ATMs in lean and obese mice. We identified a population of resident ATMs expressing the M2a marker macrophage galactose N-acetyl-galactosamine specific lectin 1 (MGL1/CD301) that localizes to interstitial spaces between adipocytes. High-fat diet feeding does not alter the presence of MGL1⁺ ATMs but instead induces the spatially restricted accumulation of M1-polarized MGL1⁻ ATMs in clusters surrounding dead adipocytes. Kinetic studies show that the rate of recruitment of these subtypes into adipose tissue differs significantly and support a model in which the M2a-to-M1 switch in ATMs is coupled to differential monocyte/macrophage recruitment.     

Involvement of Cbl-b-mediated macrophage inactivation in insulin resistance

Aging and overnutrition cause obesity in rodents and humans. It is well-known that obesity causes various diseases by producing insulin resistance(IR). Macrophages infiltrate the adipose tissue(AT) of obese individuals and cause chronic low-level inflammation associated with IR. Macrophage infiltration is regulated by the chemokines that are released from hypertrophied adipocytes and the immune cells in AT. Saturated fatty acids are recognized by toll-like receptor 4(TLR4) and induce inflammatory responses in AT macrophages(ATMs). The inflammatory cytokines that are released from activated ATMs promote IR in peripheral organs, such as the liver, skeletal muscle and AT. Therefore, ATM activation is a therapeutic target for IR in obesity. The ubiquitin ligase Casitas b-lineage lymphoma-b(Cbl-b) appears to potently suppress macrophage migration and activation. Cbl-b is highly expressed in leukocytes and negatively regulates signals associated with migration and activation. Cbl-b deficiency enhances ATM accumulation and IR in aging- and diet-induced obese mice. Cbl-b inhibits migration-related signals and SFA-induced TLR4 signaling in ATMs. Thus, targeting Cbl-b may be a potential therapeutic strategy to reduce the IR induced by ATM activation. In this review, we summarize the regulatory functions of Cbl-b in ATMs.     

Regulatory Mechanisms for Adipose Tissue M1 and M2 Macrophages in Diet-Induced Obese Mice

OBJECTIVE

To characterize the phenotypic changes of adipose tissue macrophages (ATMs) under different conditions of insulin sensitivity.

RESEARCH DESIGN AND METHODS

The number and the expressions of marker genes for M1 and M2 macrophages from mouse epididymal fat tissue were analyzed using flow cytometry after the mice had been subjected to a high-fat diet (HFD) and pioglitazone treatment.

RESULTS

Most of the CD11c-positive M1 macrophages and the CD206-positive M2 macrophages in the epididymal fat tissue were clearly separated using flow cytometry. The M1 and M2 macrophages exhibited completely different gene expression patterns. Not only the numbers of M1 ATMs and the expression of M1 marker genes, such as tumor necrosis factor-α and monocyte chemoattractant protein-1, but also the M1-to-M2 ratio were increased by an HFD and decreased by subsequent pioglitazone treatment, suggesting the correlation with whole-body insulin sensitivity. We also found that the increased number of M2 ATMs after an HFD was associated with the upregulated expression of interleukin (IL)-10, an anti-inflammatory Th2 cytokine, in the adipocyte fraction as well as in adipose tissue. The systemic overexpression of IL-10 by an adenovirus vector increased the expression of M2 markers in adipose tissue.

CONCLUSIONS

M1 and M2 ATMs constitute different subsets of macrophages. Insulin resistance is associated with both the number of M1 macrophages and the M1-to-M2 ratio. The increased expression of IL-10 after an HFD might be involved in the increased recruitment of M2 macrophages.Obesity and insulin resistance are closely associated with a state of low-grade inflammation in adipose tissue, where resident macrophages play important roles (1–9). Adipose tissue macrophages (ATMs) consist of at least two different phenotypes (i.e., classically activated M1 macrophages and alternatively activated M2 macrophages). A recent report (10) proposed that M1 or M2 ATMs are distinguished by the presence or the absence of CD11c, an M1 macrophage marker. M1 ATMs produce proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1, thus contributing to the induction of insulin resistance (11–13). On the other hand, M2 ATMs, which are the major resident macrophages in lean adipose tissue, are reported to have a different gene expression profile, characterized by the relatively high expression of CD206, arginase-1, MglI, and IL-10, which are involved in the repair or remodeling of tissues (10–14).Recent studies have demonstrated the involvement of M1/M2 ATMs in the regulation of insulin sensitivity. The deletion of M1 marker genes such as TNF-α (15) and C-C motif chemokine receptor (CCR) 2 (8) or the ablation of CD11c-positive cells resulted in the normalization of insulin sensitivity (16). On the other hand, the mice with the macrophage-specific knockout of peroxisome proliferator–activated receptor (PPAR) γ or PPARβ/δ displayed insulin resistance with reduced number and impaired function of M2 macrophages (17–20). The latter studies also indicated that IL-4 or IL-13 from adipocytes or hepatocytes promotes the expression of PPARγ and PPARβ/δ in monocytes, resulting in the differentiation of M2 macrophages. Although it is generally accepted that M1 ATMs induce insulin resistance by secreting a variety of proinflammatory cytokines, how M2 ATMs contribute to the amelioration of insulin resistance is currently unknown.Recently, Lumeng et al. (10) used CD11c as an M1 marker in a flow cytometry analysis and reported that high-fat diet (HFD)-induced obesity causes a shift in ATMs from an M2 polarized state in lean animals to an M1 proinflammatory state. However, the precise mechanism of how the increased ratio of M1 macrophages is induced in diet-induced obese mice is not fully understood (e.g., are M1 macrophages newly recruited from circulating monocytes, or do M2 macrophages present in lean adipose tissue differentiate into M1 macrophages?)To evaluate the changes in the number as well as the gene expression of M1 and M2 markers more precisely, we analyzed mouse ATMs by flow cytometry using CD206 as an M2 marker in addition to using CD11c as an M1 marker. Here, we show that the CD11c-positive/CD206-negative M1 ATMs and the CD206-positive/CD11c-negative M2 ATMs constituted distinct populations. The number of M1 macrophages and the M1-to-M2 ratio are related to the development of insulin resistance. Interestingly, the overexpression of IL-10 by adenovirus vector increased the markers of M2 ATMs in adipose tissue, suggesting that enhanced IL-10 expression by an HFD and/or pioglitazone treatment may be involved in a phenotypic switch in ATMs.     

Adipose Tissue Macrophages Function As Antigen-Presenting Cells and Regulate Adipose Tissue CD4+ T Cells in Mice

The proinflammatory activation of leukocytes in adipose tissue contributes to metabolic disease. How crosstalk between immune cells initiates and sustains adipose tissue inflammation remains an unresolved question. We have examined the hypothesis that adipose tissue macrophages (ATMs) interact with and regulate the function of T cells. Dietary obesity was shown to activate the proliferation of effector memory CD4⁺ T cells in adipose tissue. Our studies further demonstrate that ATMs are functional antigen-presenting cells that promote the proliferation of interferon-γ–producing CD4⁺ T cells in adipose tissue. ATMs from lean and obese visceral fat process and present major histocompatibility complex (MHC) class II–restricted antigens. ATMs were sufficient to promote proliferation and interferon-γ production from antigen-specific CD4⁺ T cells in vitro and in vivo. Diet-induced obesity increased the expression of MHC II and T-cell costimulatory molecules on ATMs in visceral fat, which correlated with an induction of T-cell proliferation in that depot. Collectively, these data indicate that ATMs provide a functional link between the innate and adaptive immune systems within visceral fat in mice.Obesity-induced inflammation contributes to the development of type 2 diabetes, metabolic syndrome, and cardiovascular disease (1–3). Accumulation of activated leukocytes in metabolic tissues is a driving force for obesity-associated metabolic inflammation (metainflammation) and insulin resistance (3,4). In adipose tissue, a vast array of leukocytes have been identified and reported to contribute to obesity-induced metainflammation. How adipose tissue leukocytes interact to shape the inflammatory environment within fat is an important unresolved gap in our current understanding of metabolic disease.In humans and rodent models, F4/80⁺ adipose tissue macrophages (ATMs) are the predominant leukocyte found in metabolically healthy and insulin-resistant fat (5). Resident (type 2) ATMs are distributed between adipocytes in healthy adipose tissue throughout development, express anti-inflammatory markers typical of “alternatively activated” or M2 polarized macrophages, and promote tissue homeostasis (6,7). Disruption of macrophage M2 polarization increases the susceptibility to insulin resistance induced by a high-fat diet (HFD) (8–10). Obesity triggers the accumulation of F4/80⁺ ATMs that coexpress the dendritic cell (DC) marker CD11c as well as genes typically expressed by “classically activated” or proinflammatory M1 polarized macrophages (11–13). M1 ATMs form multicellular lipid-laden clusters, known as crown-like structures (CLS), around dead adipocytes in obese fat (6,14,15) and produce inflammatory cytokines (e.g., interleukin [IL]-1β, IL-6, and tumor necrosis factor-α [TNF-α]) that can impair insulin action in adipocytes (16,17). Current models suggest that obesity promotes metainflammation in part by altering the balance between type 2 and type 1 ATMs in visceral fat (13,18).In addition to ATMs, adipose tissue contains lymphocytes (e.g., natural killer T cells [NKTs], conventional CD4⁺ T cells [Tconvs], regulatory CD4⁺ T cells [Tregs], cytotoxic CD8⁺ T cells, and B cells) that are also regulated by metabolic status (19–24). Treg content in visceral fat is inversely correlated with measures of insulin resistance and inflammation (19,25,26), suggesting that Tregs are anti-inflammatory. In contrast, T helper 1 (Th1) CD4⁺ T cells and CD8⁺ adipose tissue T cells (ATTs) accumulate in fat during obesity, promoting IFN-γ and TNF-α production and insulin resistance (20,21,27). Thus, analogous to ATMs, the imbalance between anti-inflammatory Tregs and proinflammatory CD4⁺/CD8⁺ ATTs contributes to metainflammation.The mechanisms that regulate ATTs in adipose tissue are largely unknown. Spectratyping experiments suggest that CD4⁺ ATTs (but not CD8⁺ ATTs) undergo monoclonal expansion within fat and have an effector-memory (CD44^High CD62L^Low) phenotype (19,21,28). This implies that ATT activation and expansion may be an adaptive immune response to an obesity-induced antigen. T-cell activation depends on an intricate relationship between T cells and antigen-presenting cells (APCs) (29). Classically, APCs (specifically, macrophages and DCs) shape CD4⁺ T-cell activation by three signals: 1) presentation of peptide antigens on major histocompatibility complex (MHC) class II (MHC II) molecules (signal 1), 2) expression of T-cell costimulatory molecules (e.g., CD40, CD80, and CD86) (signal 2), and 3) production of cytokines (e.g., transforming growth factor-β, IL-10, or IL-12) (signal 3). These three signals shape the differentiation of naïve CD4⁺ T cells into effector T-cell subsets (e.g., Th1, Th2, Th17, Treg).The APCs that interact with ATTs in fat have not been well characterized but could include ATMs, adipose tissue DCs, adipose tissue B cells, mast cells, and neutrophils (24,30–34). Quantitative changes in ATTs can precede the accumulation of type 1 CD11c⁺ ATMs in visceral fat in obese mice, suggesting that APCs present in lean and obese fat could trigger an adaptive immune response. Because ATMs are the predominant leukocyte population in lean and obese fat and ATMs from obese mice and humans express MHC II molecules (35–37), we tested the hypothesis that ATMs (CD11b⁺ F4/80⁺) are capable of functioning as APCs to regulate CD4⁺ ATT activation and proliferation. We report that ATMs within visceral fat from mice phagocytose and process antigens for presentation, express costimulatory molecules, and induce antigen-specific CD4⁺ T-cell proliferation in vitro and in situ. Furthermore, we found proliferating ATTs localized with ATMs in fat-associated lymphoid clusters (FALCs) where antigen-specific T-cell activation and proliferation may be initiated. Our data indicate that ATMs meet the functional definition of APCs and suggest that MHC II-restricted antigens presented by ATMs in visceral fat regulate Tregs and Tconvs CD4⁺ ATTs in mice.     

Increased inflammatory properties of adipose tissue macrophages recruited during diet-induced obesity

Although recent studies show that adipose tissue macrophages (ATMs) participate in the inflammatory changes in obesity and contribute to insulin resistance, the properties of these cells are not well understood. We hypothesized that ATMs recruited to adipose tissue during a high-fat diet have unique inflammatory properties compared with resident tissue ATMs. Using a dye (PKH26) to pulse label ATMs in vivo, we purified macrophages recruited to white adipose tissue during a high-fat diet. Comparison of gene expression in recruited and resident ATMs using real-time RT-PCR and cDNA microarrays showed that recruited ATMs overexpress genes important in macrophage migration and phagocytosis, including interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and C-C chemokine receptor 2 (CCR2). Many of these genes were not induced in ATMs from high-fat diet-fed CCR2 knockout mice, supporting the importance of CCR2 in regulating recruitment of inflammatory ATMs during obesity. Additionally, expression of Apoe was decreased, whereas genes important in lipid metabolism, such as Pparg, Adfp, Srepf1, and Apob48r, were increased in the recruited macrophages. In agreement with this, ATMs from obese mice had increased lipid content compared with those from lean mice. These studies demonstrate that recruited ATMs in obese animals represent a subclass of macrophages with unique properties.     

Increased macrophage migration into adipose tissue in obese mice

Macrophage-mediated inflammation is a key component of insulin resistance; however, the initial events of monocyte migration to become tissue macrophages remain poorly understood. We report a new method to quantitate in vivo macrophage tracking (i.e., blood monocytes from donor mice) labeled ex vivo with fluorescent PKH26 dye and injected into recipient mice. Labeled monocytes appear as adipose, liver, and splenic macrophages, peaking in 1-2 days. When CCR2 KO monocytes are injected into wild-type (WT) recipients, or WT monocytes given to MCP-1 KO recipients, adipose tissue macrophage (ATM) accumulation is reduced by ~40%, whereas hepatic macrophage content is decreased by ~80%. Using WT donor cells, ATM accumulation is several-fold greater in obese recipient mice compared with lean mice, regardless of the source of donor monocytes. After their appearance in adipose tissue, ATMs progressively polarize from the M2- to the M1-like state in obesity. In summary, the CCR2/MCP-1 system is a contributory factor to monocyte migration into adipose tissue and is the dominant signal controlling the appearance of recruited macrophages in the liver. Monocytes from obese mice are not programmed to become inflammatory ATMs but rather the increased proinflammatory ATM accumulation in obesity is in response to tissue signals.     

Aberrant accumulation of undifferentiated myeloid cells in the adipose tissue of CCR2-deficient mice delays improvements in insulin sensitivity

OBJECTIVE

Mice with CCR2 deficiency are protected from insulin resistance but only after long periods of high-fat diet (HFD) feeding, despite the virtual absence of circulating inflammatory monocytes. We performed a time course study in mice with hematopoietic and global CCR2 deficiency to determine adipose tissue–specific mechanisms for the delayed impact of CCR2 deficiency on insulin resistance.

RESEARCH DESIGN AND METHODS

Mice with global or hematopoietic CCR2 deficiency (CCR2^−/− and BM-CCR2^−/−, respectively) and wild-type controls (CCR2^+/+ and BM-CCR2^+/+, respectively) were placed on an HFD for 6, 12, and 20 weeks. Adipose tissue myeloid populations, degree of inflammation, glucose tolerance, and insulin sensitivity were assessed.

RESULTS

Flow cytometry analysis showed that two different populations of F4/80⁺ myeloid cells (CD11b^loF4/80^lo and CD11b^hiF4/80^hi) accumulated in the adipose tissue of CCR2^−/− and BM-CCR2^−/− mice after 6 and 12 weeks of HFD feeding, whereas only the CD11b^hiF4/80^hi population was detected in the CCR2^+/+ and BM-CCR2^+/+ controls. After 20 weeks of HFD feeding, the CD11b^loF4/80^lo cells were no longer present in the adipose tissue of CCR2^−/− mice, and only then were improvements in adipose tissue inflammation detected. Gene expression and histological analysis of the CD11b^loF4/80^lo cells indicated that they are a unique undifferentiated monocytic inflammatory population. The CD11b^loF4/80^lo cells are transiently found in wild-type mice, but CCR2 deficiency leads to the aberrant accumulation of these cells in adipose tissue.

CONCLUSIONS

The discovery of this novel adipose tissue monocytic cell population provides advances toward understanding the pleiotropic role of CCR2 in monocyte/macrophage accumulation and regulation of adipose tissue inflammation.Obesity is an independent risk factor for type 2 diabetes, cardiovascular disease, fatty liver disease, atherosclerosis, and several cancers. Chronic adipose tissue inflammation is a cardinal feature of obesity that leads to other health complications such as insulin resistance (1–3). Recruitment of macrophages is an important factor in adipose tissue inflammation (4) and has been shown to be temporally associated with insulin resistance (5,6).CCR2 regulates monocyte chemotaxis through direct interactions with its ligands, monocyte chemoattractant protein (MCP)-1 and -3 (7). CCR2^−/− mice have an immune deficiency in Th1 responses characterized by low levels of interferon-γ (IFN-γ) production and delayed macrophage recruitment to sites of inflammation (8). A study by Tsou et al. (9) found that CCR2^−/− mice have a defect in the egress of inflammatory monocytes from the bone marrow, resulting in a dramatic reduction of these cells in the circulation (9,10). Thus, CCR2^−/− mice have a decreased pool of circulating inflammatory monocytes and reduced numbers of differentiated myeloid cells recruited to sites of inflammation.The role of CCR2 and MCP-1 in macrophage recruitment to adipose tissue and the liver and their contribution to insulin resistance have been evaluated by various groups (11–16). One study showed that obese CCR2^−/− mice have a modest decrease in macrophage marker expression and macrophage number in adipose tissue after 24 weeks on a 60% fat diet compared with wild-type controls (11). Furthermore, a bone marrow transplant (BMT) study showed that hematopoietic CCR2 deficiency leads to decreased F4/80 expression in the adipose tissue of obese ob/ob mice (13). These experiments indicate that substantial obesity (i.e., long periods of high-fat diet [HFD] feeding and/or a genetically morbidly obese model) is required before differences in macrophage recruitment and improvements in insulin sensitivity are observed in CCR2^−/− mice, despite their striking reduction in circulating inflammatory monocytes. We set out to investigate the mechanism responsible for the delayed protection in adipose tissue inflammation and macrophage recruitment in global and hematopoietic models of CCR2 deficiency (CCR2^−/− and BM-CCR2^−/−, respectively).In this study, we analyzed the changes in myeloid populations that take place in the adipose tissue of CCR2^−/− and BM-CCR2^−/− mice during HFD-induced obesity and contrasted them with changes in CCR2^+/+ and BM-CCR2^+/+ controls. Our data showed that global and hematopoietic CCR2 deficiency leads to the accumulation of CD11b^loF4/80^lo myeloid cells during early periods of HFD feeding. Further analysis of the CD11b^loF4/80^lo cells showed that they have a monocytic and inflammatory gene expression profile characterized by significantly elevated expression of Itgax, Il8rb, Ccl5, Nos2, and Csf1. In addition, no differences in inflammation or glucose tolerance were observed between BM-CCR2^+/+ and BM-CCR2^−/− mice until after substantial obesity was reached during extended periods of HFD feeding—a condition that corresponds temporally with the absence of CD11b^loF4/80^lo cells.     

Reduced Adipose Tissue Macrophage Content Is Associated With Improved Insulin Sensitivity in Thiazolidinedione-Treated Diabetic Humans

Obesity is associated with increased adipose tissue macrophage (ATM) infiltration, and rodent studies suggest that inflammatory factors produced by ATMs contribute to insulin resistance and type 2 diabetes. However, a relationship between ATM content and insulin resistance has not been clearly established in humans. Since thiazolidinediones attenuate adipose tissue inflammation and improve insulin sensitivity, we examined the temporal relationship of the effects of pioglitazone on these two parameters. The effect of 10 and 21 days of pioglitazone treatment on insulin sensitivity in 26 diabetic subjects was assessed by hyperinsulinemic-euglycemic clamp studies. Because chemoattractant factors, cytokines, and immune cells have been implicated in regulating the recruitment of ATMs, we studied their temporal relationship to changes in ATM content. Improved hepatic and peripheral insulin sensitivity was seen after 21 days of pioglitazone. We found early reductions in macrophage chemoattractant factors after only 10 days of pioglitazone, followed by a 69% reduction in ATM content at 21 days and reduced ATM activation at both time points. Although markers for dendritic cells and neutrophils were reduced at both time points, there were no significant changes in regulatory T cells. These results are consistent with an association between adipose macrophage content and systemic insulin resistance in humans.Obesity is an important causal factor in the global diabetes epidemic (1,2). Adipose tissue generates substantial amounts of proinflammatory molecules believed to contribute to insulin resistance (3). Obesity is associated with increased adipose tissue macrophage (ATM) infiltration in rodents and humans (4–6). Inflammatory cells, including macrophages, appear to be the main source of various fat-derived inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1b (7,8), and many rodent models suggest that increased ATM content is associated with insulin resistance (9–12). However, human studies have not universally shown a relationship between ATM content and insulin resistance, raising questions about the role of ATMs in the metabolic consequences of obesity in humans (13,14).Increased local production of macrophage chemoattractant protein-1 (MCP-1) appears to recruit circulating monocytes/macrophages through interaction with the MCP-1 receptor chemokine (C-C motif) receptor 2 (CCR2) (9–11), and MCP-1 expression is increased in human adipose tissue from obese subjects (6). Adipocytes in obesity also appear to express increased amounts of hyaluronan and its receptor CD44, thereby recruiting more monocytes into adipose tissue (15–18). In addition, a number of studies have suggested that regulatory T cells (Tregs) oppose recruitment of proinflammatory macrophages, thereby improving insulin sensitivity (19–21), although a recent study showed that Treg markers were paradoxically upregulated and correlated with inflammation in adipose tissue of obese human subjects (22). Furthermore, rodent studies suggest a role for additional adipose inflammatory cells, including neutrophils and dendritic cells, in macrophage recruitment and insulin resistance (23,24).Therefore, many unanswered questions remain regarding the relationships among ATMs, chemoattractant factors, and insulin action in humans. Because thiazolidinediones have been shown to reduce insulin resistance and inflammatory factors in subjects with type 2 diabetes (25,26) and to reduce ATM content in subjects with impaired glucose tolerance (27), we used pioglitazone to prospectively study the temporal sequence of its effects on hepatic and peripheral insulin sensitivity, subcutaneous ATM content, chemoattractant factors, and immune cell populations. We conducted hyperinsulinemic-euglycemic pancreatic clamp studies along with adipose tissue biopsies after 10 and 21 days of pioglitazone treatment in individuals with type 2 diabetes.     

Heme Oxygenase-1 Regulates Myeloid Cell Trafficking in AKI

Renal ischemia-reperfusion injury is mediated by a complex cascade of events, including the immune response, that occur secondary to injury to renal epithelial cells. We tested the hypothesis that heme oxygenase-1 (HO-1) expression, which is protective in ischemia-reperfusion injury, regulates trafficking of myeloid-derived immune cells in the kidney. Age-matched male wild-type (HO-1^+/+), HO-1–knockout (HO-1^−/−), and humanized HO-1–overexpressing (HBAC) mice underwent bilateral renal ischemia for 10 minutes. Ischemia-reperfusion injury resulted in significantly worse renal structure and function and increased mortality in HO-1^−/− mice. In addition, there were more macrophages (CD45⁺ CD11b^hiF4/80^lo) and neutrophils (CD45⁺ CD11b^hi MHCII⁻ Gr-1^hi) in HO-1^−/− kidneys than in sham and HO-1^+/+ control kidneys subjected to ischemia-reperfusion. However, ischemic injury resulted in a significant decrease in the intrarenal resident dendritic cell (DC; CD45⁺MHCII⁺CD11b^loF4/80^hi) population in HO-1^−/− kidneys compared with controls. Syngeneic transplant experiments utilizing green fluorescent protein–positive HO-1^+/+ or HO-1^−/− donor kidneys and green fluorescent protein–negative HO-1^+/+ recipients confirmed increased migration of the resident DC population from HO-1^−/− donor kidneys, compared to HO-1^+/+ donor kidneys, to the peripheral lymphoid organs. This effect on renal DC migration was corroborated in myeloid-specific HO-1^−/− mice subjected to bilateral ischemia. These mice also displayed impaired renal recovery and increased fibrosis at day 7 after injury. These results highlight an important role for HO-1 in orchestrating the trafficking of myeloid cells in AKI, which may represent a key pathway for therapeutic intervention.     

Attenuated pik3r1 expression prevents insulin resistance and adipose tissue macrophage accumulation in diet-induced obese mice

Obese white adipose tissue (AT) is characterized by large-scale infiltration of proinflammatory macrophages, in parallel with systemic insulin resistance; however, the cellular stimulus that initiates this signaling cascade and chemokine release is still unknown. The objective of this study was to determine the role of the phosphoinositide 3-kinase (PI3K) regulatory subunits on AT macrophage (ATM) infiltration in obesity. Here, we find that the Pik3r1 regulatory subunits (i.e., p85α/p55α/p50α) are highly induced in AT from high-fat diet-fed obese mice, concurrent with insulin resistance. Global heterozygous deletion of the Pik3r1 regulatory subunits (αHZ), but not knockout of Pik3r2 (p85β), preserves whole-body, AT, and skeletal muscle insulin sensitivity, despite severe obesity. Moreover, ATM accumulation, proinflammatory gene expression, and ex vivo chemokine secretion in obese αHZ mice are markedly reduced despite endoplasmic reticulum (ER) stress, hypoxia, adipocyte hypertrophy, and Jun NH(2)-terminal kinase activation. Furthermore, bone marrow transplant studies reveal that these improvements in obese αHZ mice are independent of reduced Pik3r1 expression in the hematopoietic compartment. Taken together, these studies demonstrate that Pik3r1 expression plays a critical role in mediating AT insulin sensitivity and, more so, suggest that reduced PI3K activity is a key step in the initiation and propagation of the inflammatory response in obese AT.     

Vascular-Resident CD169-Positive Monocytes and Macrophages Control Neutrophil Accumulation in the Kidney with Ischemia-Reperfusion Injury

Monocytes and kidney-resident macrophages are considered to be involved in the pathogenesis of renal ischemia-reperfusion injury (IRI). Several subsets of monocytes and macrophages are localized in the injured tissue, but the pathologic roles of these cells are not fully understood. Here, we show that CD169⁺ monocytes and macrophages have a critical role in preventing excessive inflammation in IRI by downregulating intercellular adhesion molecule-1 (ICAM-1) expression on vascular endothelial cells. Mice depleted of CD169⁺ cells showed enhanced endothelial ICAM-1 expression and developed irreversible renal damage associated with infiltration of a large number of neutrophils. The perivascular localization of CD169⁺ monocytes and macrophages indicated direct interaction with blood vessels, and coculture experiments showed that the direct interaction of CD169⁺ cell-depleted peripheral blood leukocytes augments the expression levels of ICAM-1 on endothelial cells. Notably, the transfer of Ly6C^lo monocytes into CD169⁺ cell-depleted mice rescued the mice from lethal renal injury and normalized renal ICAM-1 expression levels, indicating that the Ly6C^lo subset of CD169⁺ monocytes has a major role in the regulation of inflammation. Our findings highlight the previously unknown role of CD169⁺ monocytes and macrophages in the maintenance of vascular homeostasis and provide new approaches to the treatment of renal IRI.     

Weight Cycling Increases T-Cell Accumulation in Adipose Tissue and Impairs Systemic Glucose Tolerance

Obesity is one of the leading causes of morbidity in the U.S. Accumulation of proinflammatory immune cells in adipose tissue (AT) contributes to the development of obesity-associated disorders. Weight loss is the ideal method to counteract the negative consequences of obesity; however, losses are rarely maintained, leading to bouts of weight cycling. Fluctuations in weight have been associated with worsened metabolic and cardiovascular outcomes; yet, the mechanisms explaining this potential correlation are not known. For determination of whether weight cycling modulates AT immune cell populations, inflammation, and insulin resistance, mice were subjected to a diet-switch protocol designed to induce weight cycling. Weight-cycled mice displayed decreased systemic glucose tolerance and impaired AT insulin sensitivity when compared with mice that gained weight but did not cycle. AT macrophage number and polarization were not modulated by weight cycling. However, weight cycling did increase the number of CD4⁺ and CD8⁺ T cells in AT. Expression of multiple T helper 1–associated cytokines was also elevated subsequent to weight cycling. Additionally, CD8⁺ effector memory T cells were present in AT of both obese and weight-cycled mice. These studies indicate that an exaggerated adaptive immune response in AT may contribute to metabolic dysfunction during weight cycling.Obesity is associated with an increased risk for the development insulin resistance (IR), type 2 diabetes, and cardiovascular disease (1). Many of the metabolic consequences of obesity are the result of adipose tissue (AT) dysfunction. Recent findings have implicated immune cell accumulation in AT as a key contributor to obesity-associated inflammation. It is well established that innate immune cells, including macrophages, accumulate in AT during obesity and are a major source of AT-derived inflammatory cytokines/chemokines (2–4).In addition to innate immune cells, recent evidence points to the involvement of the adaptive immune system in the initiation of AT inflammation during obesity. Upon high-fat diet (HFD) feeding, the proportion of AT-resident anti-inflammatory lymphocytes, including CD4⁺ regulatory T cells (5) and T helper (Th)2 cells (6), is decreased. Furthermore, obesity promotes the influx of proinflammatory lymphocytes such as B-2 cells (7), natural killer T cells (8,9), interferon-γ (IFN-γ)–secreting CD4⁺ Th1 (6,10–12), and CD8⁺ cytotoxic T cells (10,11,13,14) into AT. The accumulation of T-cells in AT appears to be antigen driven (6,14) and is also characterized by the formation of memory cells (10,11). Interestingly, preventing the accumulation of proinflammatory T-cell subsets in AT during obesity improves systemic glucose tolerance (6,14), indicating that a shift toward a Th1 immune response contributes to the development of AT inflammation and IR during obesity.Weight loss is the ideal approach to counteract the negative consequences of obesity. Lifestyle or surgical interventions that promote weight loss decrease AT macrophage (ATM) number, reduce inflammation, and improve insulin sensitivity (15–17). However, even when weight loss is achieved, losses are rarely maintained (18). These bouts of weight regain lead to weight cycling. Although the literature regarding the impact of weight cycling on metabolic health remains controversial (19–22), multiple studies indicate that weight cycling increases the risk of developing type 2 diabetes and cardiovascular disease in humans (23–27). While the potentially deleterious effects of weight cycling are recognized, the mechanisms by which weight cycling increases metabolic dysfunction remain unknown. Additionally, it is not known whether weight cycling alters AT immune cell composition.In this study, mice were cycled between HFD and low-fat diet (LFD) to determine if weight cycling alters metabolic and immunological parameters when compared with mice that gain weight in the absence of cycling. We show that weight cycling impairs systemic glucose tolerance and decreases AT insulin sensitivity. Weight cycling did not alter the HFD-induced increase in ATM number or M1 polarization. However, both CD4⁺ and CD8⁺ T-cell numbers were increased in AT during weight cycling. In addition, CD8⁺ effector memory T-cell populations were increased during obesity and weight cycling. This intensified T cell–driven inflammatory response may contribute to the metabolic abnormalities associated with weight cycling.