WAT inflammation in obesity
The ability of adipocytes to increase in size (adipocyte hypertrophy) during lipogenesis was believed to be the only mechanism by which adult WAT expands upon insulin stimulation. However, it is now widely accepted that an increase in adipocytes number - or adipose tissue hyperplasia - also contributes to WAT mass gain through the recruitment and differentiation of adipose progenitors, in a process known as adipogenesis. Therefore, the ability of WAT to expand during life in response to metabolic needs depends not only on adipocytes, but also on the adipogenic potential of adipose progenitors. Other factors such as vasculature and extracellular matrix remodeling also contribute to the plasticity of adipose tissue and influence adipocyte hypertrophy and adipogenesis from stem cells.
During the development of obesity, WAT expands to an extent that leads to chronic tissue inflammation, which is associated with an increased risk of type-2 diabetes and cardiovascular disease. The first functional connection between obesity and inflammation was the observation that obese WAT secretes large amounts of the proinflammatory cytokine tumor necrosis factor (TNF)-α, and that this cytokine had a direct role in obesity-induced insulin resistance[89,90]. As well as increased levels of proinflammatory cytokines, obese WAT also exhibits low level of anti-inflammatory mediators[89,91]. The discovery that obesity is characterized by macrophage accumulation in adipose tissue added a new dimension to our understanding of how obesity propagates inflammation, as macrophage recruitment is an important factor in promoting insulin resistance[62,63]. A clue to the origin of these recruited macrophages came from the observation that, in CD45.2 mice transplanted with bone marrow cells from CD45.1 mice, 85% of the adipose tissue macrophage (F4/80+) cell population had the CD45.1 marker. Therefore, during obesity development, the expanding WAT secretes chemoattractants (such as the mouse chemoattractant protein-1, MCP-1, and the macrophage inflammatory protein-1α, MIP-1α) that recruit monocytes from the bone marrow to adipose tissue[62,63].
In obesity, the infiltrating macrophages adopt a proinflammatory (“M1”) phenotype, becoming a source of proinflammatory cytokines such as IL-1β and TNF-α, which trigger local and systemic insulin resistance. These infiltrating macrophages differ from adipose tissue resident (“M2”) macrophages, which exhibit anti-inflammatory characteristics[92,93]. In mice, high-fat diets turn the secretion pattern of M2 macrophages into M1, by the reduction of IL-10 and arginase levels, and the increase in TNF-α and iNOS levels. Diet-induced obesity increases the expression of the M1 marker CD11c in WAT, while decreasing CD206 expression, which is typical of M2 macrophages.
The poorly-defined mechanisms that initiate inflammation and connect the inflammatory scenario of obese WAT to other diseases are the subject of intense investigation, in a research area known as “metabolic inflammation”. Metabolically altered adipose tissue cells may interact with immune cells to initiate the inflammatory process. Interactions between immune and metabolic cells occurs in other metabolic tissues and organs (liver, muscle and pancreas) in obese individuals, suggesting that metabolic inflammation could be a systemic feature of obesity.
Immune-metabolic interactions occur in obesity between adipocytes or SVF cells and macrophages. Indeed, adipocyte hypertrophy is a potential trigger for macrophage accumulation in WAT. In association with the large increase in protein synthesis, hypertrophied adipocytes display mitochondrial and endoplasmic reticulum stress, which could lead to the activation of inflammatory signaling pathways[99-101]. In line with this hypothesis, hypertrophied adipocytes in obese individuals change their intrinsic secretion profile towards a proinflammatory phenotype (characterized by high TNF-α and low adiponectin levels)[19,102,103]. TNF-α could stimulate pre-adipocytes and endothelial cells to secrete MCP-1, attracting monocytes from the bone marrow[62,63]. In addition, pro-inflammatory cytokines and fatty acids secreted by hypertrophic adipocytes can lead recruited macrophages towards an M1 proinflammatory phenotype. Moreover, groups of hypoxic and hypertrophic adipocytes undergo necrosis, and are cleared by macrophage phagocytosis. Indeed, macrophages form crown-like structures around necrotic adipocytes in obese WAT, in a typical chronic inflammatory response[95,105].
While the M1 profile is pro-inflammatory, the potentiation of M2 pathways in macrophages appears to reduce metabolic inflammation (or “metainflammation”), improving insulin sensitivity. The M2 phenotype of resident adipose-tissue macrophages is maintained by the paracrine action of lymphocytes and eosinophils; however, in obesity, the recruitment of these cells to WAT is suppressed[106,107]. Tolerogenic CD4+ T-regulatory cells (Tregs) are also downregulated in WAT during obesity, which could lead to metainflammation[108,109]. Aside from Tregs, other leukocytes, including NK, NKT and mast cells, have a yet poorly-defined role in metainflammation[110-112]. Further studies on the temporal and spatial immune-metabolic interactions between leukocytes and WAT cells should shed light on the mechanisms underlying inflammation in obesity, to identify potential targets for clinical intervention.
Complex molecular signaling pathways may link metabolic challenges (e.g., excessive fat storage) with inflammation in obesity, including pathways involving the NLRP3 inflammasome, a cytoplasmic protein complex that promotes the conversion of pro-cytokines into active cytokines, which are then secreted. NLRP3 inflammasome activity can be modulated by several metabolites, including fatty acids, and the activation of this complex can interfere with insulin signaling[115,116]. Inflammasome activity can be triggered by endogenous or exogenous stress signals (e.g., cytokines, free fatty acids, glucose, reactive oxygen species, ATP), which function as “pathogen-associated molecular patterns” that interact with pattern recognition receptors, especially toll-like receptors (TLRs), in WAT cells. The interaction of stress signals with TLR4, for example, activates the nuclear factor-κB pathway, which increases NLRP3 expression[116-118].
Adipose progenitors could be key regulators of macrophage recruitment and activation in WAT. Indeed, human ASC express active TLRs, including TLR4, whose activation results in the secretion of the pro-inflammatory cytokines IL-6 and IL-8. Moreover, adipose progenitors express molecules that favor immune differentiation, such as osteopontin, which was identified as one of the factors involved in macrophage accumulation during diet-induced obesity. In line with this notion, we showed that human ASC secrete MCP-1 in vitro, and that mouse ASC populations enriched in pre-adipocytes (CD34+ ASC) could be responsible for most of the MCP-1 secretion in mice. In addition, we observed that ASC can support in vitro hematopoiesis, with a tendency to generate macrophages from hematopoietic progenitors. Moreover, while adipocytes are the main source of hormones that regulate energy metabolism (such as adiponectin and leptin), inflammatory cytokines are mostly secreted by cells from the SVF. Therefore, adipose progenitors can be key players in the regulation of the metabolic inflammation established during obesity, acting as a key source of secreted immune-mediators in adipose tissue, both in normal and in pathological conditions.
Although macrophage infiltration in obese adipose tissue potentiates inflammation and favors the development of comorbidities, the pro-inflammatory cytokines secreted by infiltrating macrophages with an M1-phenotype could also decrease WAT mass by stimulating adipocyte lipolysis and inhibiting adipogenesis. In fact, classically activated M1 macrophages impair insulin signaling and adipogenesis in adipocytes, by both direct and paracrine signals. The immune and metabolic interactions that occur within WAT may have evolved as a mechanism to regain homeostasis, in order to prevent the obesity-associated mobility impairment that makes animals more vulnerable to predators.
The mechanisms regulating adipogenesis and inflammatory responses from stromal cells have been the subject of several studies, using various in vivo and in vitro model systems[126-129]. These studies have shown that the TNF-receptor superfamily molecule CD40 is expressed during adipogenic differentiation and interacts with surrounding immune cells, modulating adipocyte inflammatory responses and insulin resistance[127,128]. Additionally, a study by Tous et al identified sphingosine kinase-1 as a potential therapeutic target to attenuate chronic inflammation in obesity and related metabolic diseases, as this molecule regulates the pro-inflammatory response in adipose progenitors.
Impact of inflammation induction on ASC functionality
ASC functionality is directly affected by obesity-induced inflammation[121,130]. Some studies have reported an inverse correlation between the body-mass index (BMI, a commonly used obesity indicator) and ASC differentiation capacity[130-132]. In agreement with these data, our studies and those of others demonstrated that ASC from obese subjects have decreased ability to differentiate into adipocytes in vitro, when compared with those from lean subjects, as assessed by intracellular lipid accumulation and/or the expression of adipogenic genes[121,130-133]. Isakson et al suggested that the inflammatory state in adipose tissue may be responsible for the impaired adipocyte differentiation observed in obesity. Indeed, inflammatory cytokines are anti-adipogenic, and it is possible that ASC from obese patients carry a “memory” of differentiation inhibition from the inflammatory environment in vivo, and which manifests itself as impaired adipogenesis in vitro. Pro-inflammatory macrophages secrete factors that impair human adipogenesis from ASC in vitro[136,137], and there is a negative correlation between the adipogenic capacity of obese ASC and the up-regulation of inflammatory genes[130,138]. In contrast, some studies reported that ASC from obese donors showed higher expression of adipogenic genes, suggesting that obese ASC are more potent in adipogenesis[138,139]. A recent study showed that ASC from obese pigs (given a high-fat diet) exhibited increased adipogenic potential relative to those from lean pigs, at the onset of obesity. The discrepancies between studies on the impact of inflammation on the adipogenic potential of ASC could be due to differences in the methods used to evaluate adipogenesis, or to the use of donors with different adiposity grades, or at different stages of obesity development.
The pro-angiogenic potential of ASC is also altered in obesity. ASC from morbidly obese individuals have higher mRNA and protein expression of the anti-angiogenic factor TSP-1 than ASC from lean individuals. In addition, “lean” ASC (i.e., those differentiated from adipose tissue of lean individuals) had increased capacity to form tube-like networks while “obese” ASC (derived from obese individuals) were not responsive to angiogenic stimuli, showing a reduced capacity to form capillary-like structures. Moreover, extracellular vesicles from obese ASC exhibited lower levels of angiogenic-related factors and, consequently, reduced angiogenic potential compared with those derived from lean ASC.
The ASC differentiation capacity is also disrupted in patients with type-2 diabetes mellitus. Global gene expression profiling revealed that ASC from type-2 diabetes donors have low levels of adipogenic genes compared with those from non-diabetic donors, indicating a decreased potential for adipogenic differentiation in diabetes. Additionally, ASC from diabetic rats were less effective at forming microvessels in vivo than those from non-diabetic animals.
Obesity also alters the immunomodulatory properties of ASC, and their ability to secrete chemical mediators. ASC isolated from patients with different adiposity grades exhibit different secretion patterns[146,147]. In particular, we demonstrated that ASC from morbidly obese patients secrete more proinflammatory cytokines, such as IL-6 and IL-8, which is in agreement with data from other groups showing that obese ASC display up-regulation of inflammatory genes (including IL-6, IL-8, IL-10 and MCP-1) compared with lean ASC[138,148]. In addition to the increased expression of inflammatory markers, obese ASC had increased migration and phagocytosis capacity compared with lean ASC. Besides, ASC from obese individuals show reduced capacity to activate the M2 macrophage phenotype and to suppress lymphocyte proliferation. Therefore, the immunomodulatory properties of ASC are altered in obesity, which may be related to the role of adipose progenitors as key regulators of the immune response during obesity development. As well as in obesity, alterations in immunomodulatory properties are observed in patients with type-2 diabetes mellitus, and global gene expression profiling revealed that genes involved in inflammation are upregulated in ASC from type-2 diabetes patients. Recently, Liu and colleagues showed that ASC derived from mice with type-2 diabetes are less effective at restricting CD4+T lymphocyte proliferation and pro-inflammatory “polarization” (during pro-inflammatory immune phenotype acquisition) than ASC from lean mice.
Collectively, these data show that obesity and other immune metabolic pathologies disrupt ASC/adipose progenitor functionality, favoring a pro-inflammatory response. This response, in turn, impairs ASC adipogenic capacity, which may reduce the ability of adipose progenitors to generate new adipocytes in WAT depots, ultimately leading to ectopic fat storage. Overall, evidence from a large number of studies indicate that ASC/adipose progenitors are key regulators of the immune response in obesity and other metabolic disorders, highlighting the potential of ASC use in cell-based regenerative therapies.