Dario A. Gutierrez

Adipose tissue recruitment of leukocytes.

Purpose of review In December of 2003, two seminal articles describing the presence of macrophages in obese adipose tissue were published. These adipose tissue macrophages (ATMs) are inflammatory and promote local and systemic insulin resistance. Due to the continuing rise in obesity around the world, understanding how these ATMs contribute to metabolic disorders is of much interest. Recent findings Chemokines have been extensively studied for their role in ATM recruitment. Deficiency or antagonism of chemokine receptors that interact with multiple chemokine ligands reduces ATM accumulation. ATMs are now defined as either classically (M1) or alternatively (M2) activated. Peroxisome proliferator-activated receptor activation and adiponectin promote an M2-polarized state resulting in improved insulin sensitivity. Finally, recent studies have provided evidence that T lymphocytes, natural killer T cells, mast cells, and B cells also enter adipose tissue and may interact with macrophages and adipocytes. Summary Literature published during the past year has shown that macrophage recruitment to adipose tissue is only one of the important mediators of obesity-related insulin resistance. The phenotype of ATMs and recruitment of other immune cells to the adipose tissue play key roles in the overall contribution of adipose tissue to systemic metabolic outcomes of obesity.

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.

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 (CD11bloF4/80lo and CD11bhiF4/80hi) accumulated in the adipose tissue of CCR2−/− and BM-CCR2−/− mice after 6 and 12 weeks of HFD feeding, whereas only the CD11bhiF4/80hi population was detected in the CCR2+/+ and BM-CCR2+/+ controls. After 20 weeks of HFD feeding, the CD11bloF4/80lo 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 CD11bloF4/80lo cells indicated that they are a unique undifferentiated monocytic inflammatory population. The CD11bloF4/80lo 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.

Of Mouse Models of Mast Cell Deficiency and Metabolic Syndrome

Since the original discovery that immune-mediated inflammation contributes to development and/or progression of obesity and diabetes (reviewed in Gregor and Hotamisligil, 2011), the identification of the individual immune cell types involved in these diseases remains of broad interest. Mast cells are found at low levels in lean adipose tissue, but their numbers strongly increase in obese adipose tissue. Mast cells are potential sources of proinflammatory factors (including TNF-α), immune-polarizing cytokines (including IL-4), the acute phase protein IL-6, and many tissue-modulating factors, including mast cell proteases, heparin, and histamine, making these cells potential proinflammatory mediators in adipose tissue and in metabolic diseases.

CCR2 deficiency leads to increased eosinophils, alternative macrophage activation, and type 2 cytokine expression in adipose tissue

Adipose tissue (AT) inflammation during obesity is mediated by immune cells and closely correlates with systemic insulin resistance. In lean AT, eosinophils are present in low but significant numbers and capable of promoting alternative macrophage activation in an IL‐4/IL‐13‐dependent manner. In WT mice, obesity causes the proportion of AT eosinophils to decline, concomitant with inflammation and classical activation of AT macrophages. In this study, we show that CCR2 deficiency leads to increased eosinophil accumulation in AT. Furthermore, in contrast to WT mice, the increase in eosinophils in CCR2−/− AT is sustained and even amplified during obesity. Interestingly, a significant portion of eosinophils is found in CLSs in AT of obese CCR2−/− mice, which is the first time eosinophils have been shown to localize to these inflammatory hot spots. CCR2−/− bone marrow precursors displayed increased expression of various key eosinophil genes during in vitro differentiation to eosinophils, suggesting a potentially altered eosinophil phenotype in the absence of CCR2. In addition, the proportion of eosinophils in AT positively correlated with local expression of Il5, a potent eosinophil stimulator. The increase in eosinophils in CCR2−/− mice was detected in all white fat pads analyzed and in the peritoneal cavity but not in bone marrow, blood, spleen, or liver. In AT of CCR2−/− mice, an increased eosinophil number positively correlated with M2‐like macrophages, expression of the Treg marker Foxp3, and type 2 cytokines, Il4, Il5, and Il13. This is the first study to link CCR2 function with regulation of AT eosinophil accumulation.

Type 1 Diabetes in NOD Mice Unaffected by Mast Cell Deficiency

Mast cells have been invoked as important players in immune responses associated with autoimmune diseases. Based on in vitro studies, or in vivo through the use of Kit mutant mice, mast cells have been suggested to play immunological roles in direct antigen presentation to both CD4+ and CD8+ T cells, in the regulation of T-cell and dendritic cell migration to lymph nodes, and in Th1 versus Th2 polarization, all of which could significantly impact the immune response against self-antigens in autoimmune disease, including type 1 diabetes (T1D). Until now, the role of mast cells in the onset and incidence of T1D has only been indirectly tested through the use of low-specificity mast cell inhibitors and activators, and published studies reported contrasting results. Our three laboratories have generated independently two strains of mast cell–deficient nonobese diabetic (NOD) mice, NOD.Cpa3Cre/+ (Heidelberg) and NOD.KitW-sh/W-sh (Leuven and Boston), to address the effects of mast cell deficiency on the development of T1D in the NOD strain. Our collective data demonstrate that both incidence and progression of T1D in NOD mice are independent of mast cells. Moreover, analysis of pancreatic lymph node cells indicated that lack of mast cells has no discernible effect on the autoimmune response, which involves both innate and adaptive immune components. Our results demonstrate that mast cells are not involved in T1D in the NOD strain, making their role in this process nonessential and excluding them as potential therapeutic targets.

Impact of Macrophage Inflammatory Protein-1α Deficiency on Atherosclerotic Lesion Formation, Hepatic Steatosis, and Adipose Tissue Expansion

Macrophage inflammatory protein-1α (CCL3) plays a well-known role in infectious and viral diseases; however, its contribution to atherosclerotic lesion formation and lipid metabolism has not been determined. Low density lipoprotein receptor deficient (LDLR−/−) mice were transplanted with bone marrow from CCL3−/− or C57BL/6 wild type donors. After 6 and 12 weeks on western diet (WD), recipients of CCL3−/− marrow demonstrated lower plasma cholesterol and triglyceride concentrations compared to recipients of C57BL/6 marrow. Atherosclerotic lesion area was significantly lower in female CCL3−/− recipients after 6 weeks and in male CCL3−/− recipients after 12 weeks of WD feeding (P<0.05). Surprisingly, male CCL3−/− recipients had a 50% decrease in adipose tissue mass after WD-feeding, and plasma insulin, and leptin levels were also significantly lower. These results were specific to CCL3, as LDLR−/− recipients of monocyte chemoattractant protein−/− (CCL2) marrow were not protected from the metabolic consequences of high fat feeding. Despite these improvements in LDLR−/− recipients of CCL3−/− marrow in the bone marrow transplantation (BMT) model, double knockout mice, globally deficient in both proteins, did not have decreased body weight, plasma lipids, or atherosclerosis compared with LDLR−/− controls. Finally, there were no differences in myeloid progenitors or leukocyte populations, indicating that changes in body weight and plasma lipids in CCL3−/− recipients was not due to differences in hematopoiesis. Taken together, these data implicate a role for CCL3 in lipid metabolism in hyperlipidemic mice following hematopoietic reconstitution.

Haematopoietic leptin receptor deficiency does not affect macrophage accumulation in adipose tissue or systemic insulin sensitivity

The adipokine leptin is primarily produced by white adipose tissue (AT) and is a potent monocyte/macrophage chemoattractant in vitro. The long form of the leptin receptor (LepR) is required for monocyte/macrophage chemotaxis towards leptin. In this study, we examined the effects of haematopoietic LepR as well as LepR with C-C chemokine receptor 2 (CCR2) deficiency (double knockout (DKO)) on macrophage recruitment to AT after two different periods of high fat diet (HFD) feeding. Briefly, 8-week-old C57BL/6 mice were transplanted with bone marrow (BM) from Lepr(+/+), Lepr(-/-) or DKO donors (groups named BM-Lepr(+/+), BM-Lepr(-/-) and BM-DKO respectively), and were placed on an HFD for 6 or 12 weeks. At the end of the study, macrophage infiltration and the inflammatory state of AT were evaluated by real-time RT-PCR, histology and flow cytometry. In addition, glucose and insulin tolerance were assessed at both time points. Our results showed no differences in macrophage accumulation or AT inflammatory state between the BM-Lepr(+/+) and BM-Lepr(-/-) mice after 6 or 12 weeks of HFD feeding; any effects observed in the BM-DKO were attributed to the haematopoietic deficiency of CCR2. In addition, no changes in glucose or insulin tolerance were observed between groups after either period of HFD feeding. Our findings suggest that although leptin is a potent chemoattractant in vitro, haematopoietic LepR deficiency does not affect macrophage accumulation in AT in early to moderate stages of diet-induced obesity.

What Have We Really Learned About Macrophage Recruitment to Adipose Tissue

As the rates of obesity continue to rise around the world, many in the scientific community are seeking to understand mechanisms by which obesity increases the risk of different diseases. In particular, the contribution of the immune system to metabolic processes has come to the forefront in the exciting new field of immunometabolism (1). Although this field encompasses interactions of innate and adaptive immune cells in multiple organs, at its core lays a unique interaction between macrophages and adipocytes. Although the interface of resident macrophages and adipocytes in lean mice is necessary for adipose tissue development (2), expansion (3), and homeostasis, in obesity, the recruitment of inflammatory macrophages is thought to impair adipose tissue function (4, 5). Thus, much research has focused on molecules responsible for macrophage recruitment to adipose tissue. In this issue of Endocrinology, Dib et al (6) report their findings that the adipokine leptin may be a key mediator of this recruitment. They show that mice transplanted with bone marrow from leptin receptor-deficient (db/db) mice, when placed on a high-fat diet, have reduced weight gain and adiposity, decreased macrophage infiltration, and subsequently diminished adipose tissue inflammation. Adipocyte-released adipokines, chemokines, and fatty acids are known to modulate macrophage phenotype and function. For example, the adipokine adiponectin has been shown to promote a more M2-like phenotype of adipose tissue macrophages (ATMs), and thus, it helps to maintain adipose tissue homeostasis (7). More studies, however, have focused on the pathological mechanisms by which adipocytes increase macrophage recruitment to adipose tissue in obesity. An emphasis on various chemokines and their receptors has prevailed. It has been shown that chemokines, such as CC chemokine ligand 2 (CCL2), CCL5, and CCL3, as well as their receptors, CC chemokine receptor 2 (CCR2), and CCR5, are important for recruitment of macrophages to adipose tissue (8–10). In addition, mechanisms by which adipocyte-secreted fatty acids and adipokines promote a proinflammatory M1-like phenotype have been extensively studied. For example, it has been shown that activation of inflammatory signaling pathways, such as Toll-like receptor 4, contribute to the inflammatory status of ATMs (11). However, despite the convincing nature of these studies from many different groups, there are equally convincing data showing that these same chemokines, receptors, and inflammatory mediators do not influence macrophage infiltration or inflammation in adipose tissue (12–16). With respect to leptin and the current report by Dib et al (6), we had previously used an identical bone marrow transplantation technique and shown that recipients of db/db bone marrow were not protected from weight gain or macrophage infiltration into adipose tissue (17). Moreover, an additional study by Fantuzzi and coworkers (18) had shown no effects of hematopoietic leptin receptor deficiency on body weight or adipose tissue inflammation in lean mice. Taken together, these cumulative data raise the question of why similar experimental designs from different laboratories yield contrasting results. This is a critical question, because deciphering the answer may yield important biological information regarding macrophage recruitment and function in adipose tissue. We suggest that there are key elements that should be considered when designing, analyzing, and reviewing imjmunometabolic studies. First, the background strain is very important, because even slight genetic drift could impact the results obtained. This is highlighted by elegant studies of Dr Atties group mapping out the genes involved in susceptibility and protection from obesity and metabolic defects in mice (19). Thus, the use of littermate controls is ideal, even if not always possible. Second, an accumulating body of literature suggests that gut microbiota may influence systemic metabolic responses far more than is currently appreciated (20). To account for this, cohousing of experimental and control mice should be performed. Third, matching the experimental and control groups for body weight at the beginning of the study is critical. Even small differences at baseline can become exaggerated over time for reasons related to inherent susceptibility to weight gain, rather than because the manipulation has had an important physiological effect. Fourth, even seemingly unimportant details may be critical. Some examples include: 1) sometimes certain cages of mice do not gain weight as well as other cages; 2) a single short event of cage flooding could stunt the growth of mice and influence their metabolic phenotype for their entire lives; 3) mice housed individually may not thrive as well as those that are group housed; and 4) mice from first litters of a dam may have different metabolic characteristics than those born to multiparous dams (21). Certainly, all investigators do their best to control for these variables. However, as a field, it is important to acknowledge that these factors can impact our results in ways that make the underlying biology more difficult to decipher. The question still remains, “Does leptin influence macrophage recruitment and function in adipose tissue?” It is well established that leptin-deficient, ob/ob, and leptin receptor-deficient, db/db, mice have altered immune system function (22). A first line of evidence of the involvement of leptin in macrophage recruitment to adipose tissue is that ob/ob and db/db mice have lower numbers of ATMs than might be expected based upon their body weight (4). With regards to the influence of leptin on macrophages, it has been shown that high concentrations of leptin increase endothelial cell adhesion molecule expression and promote macrophage adherence (23). Furthermore, we had previously reported that at lower concentrations, leptin acts as a monocyte and macrophage chemoattractant (24). Finally, plasma leptin levels are positively correlated with the number of macrophages in adipose tissue (17). Thus, testing the hypothesis that leptin could mediate macrophage recruitment to adipose tissue was logical for Dib et al (6), Gove et al (18), and our laboratory (17) to perform. The dissimilar results obtained could have been due to the age of the mice at transplantation (5 wk by Dib et al vs 8 wk by our group). However, Gove et al (18) used the same transplantation age as Dib et al (6), and they found no differences in body weight and adiposity. Second, the 45% percent fat in the diet used by Dib et al vs the 60% diet used in our study, as well as the length of diet feeding (12 vs 16 wk), could account for the difference. Finally, it is possible that the slight bias toward a reduced baseline body weight in the recipients of db/db bone marrow in Dibs study, although not statistically significant, could have had biological significance for the ultimate differences in weight and, thus, adipose tissue inflammation (6). Taken together, the conflicting data available document the complexity of leptin effects on macrophage recruitment and function in the adipose tissue, which might be influenced (even in a subtle manner) by a number of genetic and environmental factors and is probably more complex than initially anticipated. In the past decade, the field of immunometabolism has made great strides in discovering the complex interactions of the immune system with metabolic processes. In the future, to continue the advancement of this field, publication of both “positive” and “negative” results from well-designed experiments, is critical if we hope to fully disseminate new discoveries. Perhaps, with the whole picture available, we will all be able to advance the current knowledge regarding adipose tissue inflammation more effectively.