Adipose tissue & Immunity: The basics 1

Obesity has been, and its still seen, as primarily a metabolic disease. Obesity results from increased energy intake and decreased energy expenditure, that is, a positive energy balance for a prolonged time. By this logic, to cure obesity and associated diseases, one must restrict calories and/or do more exercise. While this approach works (calorie restriction being the key player), it does not solve the cause of obesity in the first place. This has been shown by numerous evidence that finds that even after weight loss, there is still some metabolic dysfunction in previously obese people. Some obese people can't fully recover. This underscores a common problem in modern health science: clinicians and health practitioners only focus on the proximate cause and not in the ultimate cause (for an interesting read on the subject, see this article). Moreover, obesity is just the tip of the iceberg. We have evolved mechanisms to prevent the development of a rather unadvantageous phenotype. Obesity occurs when these mechanisms start to fail, such as when pathological insulin resistance and leptin resistance develop. 

The obvious cause of obesity is the storage of excess energy as fat tissue. In this manner, excess energy causes an increased fat mass and problems start to arise due to the accumulation of excess body fat. While this statement is true, there is recent evidence that suggests that energy excess has also peripheral effects in cells that were previously unrelated to obesity; in particular, immune cells. 

Immunometabolism

Immunometabolism refers to "the interplay between immunological and metabolic processes" (1). Traditionally, the immune system and metabolic processes have been viewed as different, non-related systems. Now, research findings suggest that this is not the case, on the contrary, both are very related and understanding their interplay is essential for preventing and treating metabolic disorders.

The hypothesis that the immune system is involved in the pathogenesis of obesity started from the findings that targeting proteins which are part of inflammatory cellular pathways ameliorated or prevented the development of obesity and insulin resistance. For instance, Uysal, et al. (2) showed that a null mutation of the TNF-alpha and its two receptor genes improved insulin sensitivity in diet-induced obesity and in ob/ob mice. This confirmed previous in vitro evidence linking TNF-alpha with insulin resistance in adipocytes (3). Interest began to increase with the discovery and characterization of adipocytokines, as well as the finding that the adipose tissue secretes inflammatory cytokines. Adipocytokines are cytokines produced mainly (but not exclusively) in the adipose tissue, and include adiponectin, leptin, resistin and visfatin; being the first two the main adipocytokines produced. Other cytokines secreted by adipocytes are TNF-alpha, IL-6, IL-1 and CCL2; as well as other proteins, including PAI-1 and some complement factors. The table below shows some immune and metabolic effects of the main cytokines discovered, as well as their potential role on inflammation (modified from Tilg & Moschen, 2006). 

Adipocytokine	Effect on inflammation	Levels in obesity	Immunity	Metabolism
Adiponectin	Anti-inflammatory	Decreased	↓NFκB ↓TNF ↓Phagocytic activity (macrophages) ↑IL-10 ↑IL-1RA ↓IFN-γ	↓Hyperglycemia ↓FFA ↑Insulin sensitivity ↑β-oxidation ↓SREBP1c ↑AMPK
Leptin	Pro-inflammatory	Increased	↑TNF ↑IL-6 ↑IL-12 ↑CCL2 ↑Th1 (IL-2, IFN-γ) ↓Th2 (IL-4) ↑ROS ↑Chemotaxis ↑NK-cell function ↑Lymphopoiesis ↑Thymocyte survival ↑T-cell proliferation	↑Energy expenditure ↑Satiety ↑Insulin sensitivity
Resistin	Pro-inflammatory	Increased	↑NFκB ↑TNF ↑IL-6 ↑IL-1 ↑IL-12 ↑CCL2 ↑VCAM1 ↑ICAM1	↑Hepatic insulin resistance
Visfatin	Pro-inflammatory	Increased	↑IL-6 ↑IL-8 ↑IL-1β ↑TNF ↑ICAM-1 ↑IL-10, IL-1Ra (high concentrations)	↓Insulin resistance

The adipose tissue as an immune organ

Besides the role of pro-inflammatory cytokines produced by the adipose tissue, research has shown that obesity alters the function of several immune cells. In an elegant study, Caspar-Bauguil, et al. (4) found that immune cells are present in adipose tissue from mice, but their characteristics are different from other tissues, sharing some common ancestral features with hepatic immune cells. Specifically, the adipose tissue (levels of specific cell populations varying in different anatomical sites) shows both innate and adaptive features, such as the presence of Natural Killer cells (NK), NKT cells and delta-gamma T cells for the former and the presence of lymph nodes, B-cells and alpha-beta T-cells for the latter. This led the authors to propose that the adipose tissue (specially the epididymal adipose tissue) is an ancestral immune organ, due to the fact that delta-gamma T cells are thought to represent an evolutionary and functional bridge between the innate and adaptive immune systems. In addition, inguinal fat contained more adaptive immune cells. Not surprisingly, inducing obesity produced some changes in immune cells: NK cells in epididymal fat were decreased, whereas delta-gamma T cells were increased in inguinal fat and lymph nodes. 

The adipose tissue has site-specific properties and adipocytes interact in a paracrine fashion with adjacent lymphoid cells. Adipocytes near a lymph node are called "perinodal", and show differences from adipocytes far from lymph nodes (5, 6):

• They are smaller and size increases in a gradient manner from the node.
• Lipids extracted from perinodal adipose tissue contain proportionately more PUFAs and less SFA than those further from nodes or nodeless depots, proportion which is not significantly affected by diet (perinodal adipose tissue still has more PUFAs than nodeless adipose tissue). 
• Perinodal adipocytes influence the lipid composition of dendritic cells (and other lymphoid cells) found in lymph nodes, which suggests that perinodal adipocytes provide energy to immune cells for their activity.
• Following chronic immune stimulation, ratios of omega-6/omega-3 PUFA converge in perinodal adipocytes, probably for providing more substrates for ecosanoid and docosanoid synthesis.  
• Perinodal adipocytes are very sensitive to cytokines and noradrenaline, compared to adipocytes from other sites. 

Diet can influence the activity of perinodal adipocytes and associated immune cells. For example, Mattacks, et al. (7) compared the effect of feeding beef suet (mostly saturated and monounsaturated fat), sunflower oil (mostly omega-6 PUFA) and fish oil (mostly omega-3 PUFA) on the response of mesenteric, omental, popliteal and perirenal adipocytes to experimentally-induced local inflammation in guinea pigs. They found that basal lipolysis from sunflower oil-fed pigs was higher and lipolysis from perinodal adipocytes after incubation with noradrenaline was increased, compared with the other groups. The same authors found that the addition of sunflower seed oil (20%) to chow increased the number of dendritic cells in all adipose tissue samples, after stimulation with LPS (8).

Infiltration of immune cells to adipose tissue is now an accepted phenomenon during obesity. It seems that CD4+ T lymphocytes are recruited to adipose tissue first, coinciding with the appearance of glucose intolerance and reduced insulin sensitivity, while macrophages accumulate at late stages of obesity-induced insulin resistance (9, 10). Infiltration of B-cells occur rapidly in mice, before any significant change in body fat mass (10).

Innate immunity and adipose tissue

As has been mentioned, some adipose tissue show the presence of innate immune cells. One striking fact is that adipocytes and macrophages show similar characteristics. Weisberg, et al. (11) found that the expression of 1,304 transcripts in perigonadal adipose tissue from different mice correlated significantly with body mass. Of the 100 most significantly correlated genes, 30% encoded macrophage specific proteins. In mice, the adipose tissue is a major source of IL-6 during systemic inflammation produced by LPS (12). The tight relationship between adipocytes and monocytes/macrophages is exemplified by C3a. After activation of the alternative complement pathway, C3a induces mast cell degranulation and an immune response. This protein is also produced by adipocytes and the N-terminal cleavage of its alpha chain through the interaction of complement factors B and adipsin, followed by C-terminal arginine cleavage by serum carboxipeptidase N produces acylation stimulating protein (ASP) or C3adesArg, which is an important regulator of triglyceride synthesis. Moreover, C3a (ASP precursor) can also have metabolic effects: its receptor, C3aR, is expressed on both monocytes-macrophages and adipocytes. C3aR null mice are transiently resistant to diet-induced obesity, and are protected from diet-induced insulin resistance and hepatic steatosis, showing improved insulin sensitivity compared to wild-type mice (13). This was accompanied by a decrease in macrophage infiltration to adipose tissue, plasma cytokine levels and a polarization of macrophages towards a M1 phenotype (see below).

Toll-like receptors (TLR) are pattern recognition molecules with an essential function recognizing pathogens via pathogen-associated molecular patterns (PAMPs). Several types of TLRs are expressed by pre- and mature murine adipocytes, but mature adipocytes seem to be more responsive to a broader spectrum of TRL ligands (14). LPS triggers the secretion of IL-6 and different chemokines (CCL2, CCL5 and CCL11) and this inflammatory response appears to be based mainly on preadipocytes. The ultimate result of the activation of TLRs in adipocytes is the secretion of inflammatory cytokines via activation of NFkB signaling. Accordingly, LPS increases the expression of TLR2, TRAF-6 and NFkB in human adipose tissue, and increased levels of these markers, as well as LPS, is observed in type 2 diabetic patients (15). The finding that mice with defects on different TLRs are protected from obesity and insulin resistance supports the role of TRLs in the development of metabolic dysregulation (16, 17, 18, 19). Additionally, it suggests that different inflammatory stimuli act in the adipose tissue via different TLRs.

Macrophages infiltrating the adipose tissue can have two potential sources: those differentiated from bone-marrow-derived monocytes which reach the adipose tissue from the systemic circulation or by trans-differentiation from local adipose tissue preadipocytes and mesenchymal stem cells (14). Diapedesis of monocytes is stimulated by chemoattractants secreted by adipocytes (CCL2, CCL5, MIF and MIP1a) and locally produced macrophage colony stimulating factor (M-CSF) supports differentiation and maturation of monocytes into macrophages. On the other hand, both adipocyte and macrophage differentiation and function is controlled by PPAR-gamma. In addition, adipocytes also express macrophage-specific genes (20). This suggests that both cell types arise from a common precursor cell, and trans-differentiation of adipocytes into macrophages is supported by the findings of Charriere, et al. (21): preadipocytes are able to convert into macrophage-like cells, judging by specific antigens and the phagocytic index. Similar findings have been observed in other studies (22, 23), which report that preadipocytes can phagocyte and kill micro-organisms. 

Macrophages can show different activities depending on their phenotype. Classically activated macrophages (M1) respond to products derived from or associated with bacterial infections, like LPS and IFN-gamma. These macrophages are characterized by a highly inflammatory phenotype, displaying high phagocytic and bactericidal potential. Alternatively activated macrophages (M2) are induced in response to products from or associated with parasitic infections, such as Schistosoma egg antigen and Th2-type cytokines like IL-4 and IL-13 (24). M2 stimulate tissue repair and remodeling. Contrary to what was thought, adipose tissue from lean animals does have macrophages. However, obesity, besides promoting infiltration and migration of macrophages, induces a shift in macrophage balance towards M1 phenotype (25). In fact, obesity shifts the adipose M2:M1 ratio  from 4:1 in normal mice to 1.2:1 (26). Th2-type cytokines derived from the adipose tissue (IL-13 and IL-4) regulate macrophage polarization, favoring alternative activation (27). M2 development is also promoted by IL-10 (28). 

The role of other innate cells is not well characterized. It has been seen that peripheral natural killer (NK) cell levels in unhealthy obese patients is reduced compared with healthy obese, and NK cells from these patients show increased levels of inhibitory markers (CD158b and NKB1), but expression of CD69, a marker of NK activation (29). This suggests that although activated, NK cells from unhealthy obese patients cannot function properly. In visceral adipose tissue from obese subjects, there is an increase in NK cells compared to subcutaneous fat (30). Another type of innate cells, natural killer T (NKT) cells , which are T-like innate cells capable of producing both Th-1 and Th-2 type cytokines have been implicated in the inflammatory environment seen in obesity. Mice lacking NKT cells show reduced macrophage infiltration in response to a high-fat diet, and activation of NKT cells by alpha-galactosylceramide exacerbates glucose intolerance, macrophage infiltration and cytokine gene expression in diet-induced obese mice (31). However, the effects of NKT cells on obesity and insulin resistance seem to be dependent on CD8+ T cells (32). 

Summary

Interrelationship between the adipose tissue and the immune system. The adipose tissue (AT) secretes  adipocytokines (adiponectin, resistin, leptin, visfatin, among others) and conventional cytokines (TNF-a, IFN-gamma. IL-6, etc.), which influence metabolism and immunity. Cells from the innate and adaptive immune system are present in AT, and are fueled by perinodal adipocytes. Adipocytokines influence the function of immune cells, and cytokines secreted both by adipocytes and immune cells regulate the inflammatory milieu of the AT. Nutrition, by regulating fat mass and lipid composition, has direct effects on the function of immune cells.

Schäffler A, & Schölmerich J (2010). Innate immunity and adipose tissue biology. Trends in immunology, 31 (6), 228-35 PMID: 20434953 

Pond CM (2005). Adipose tissue and the immune system. Prostaglandins, leukotrienes, and essential fatty acids, 73 (1), 17-30 PMID: 15946832