**2. Obesity-Induced Inflammation and Its Impact on Health**

#### *2.1. Obesity*

Obesity is defined as an increase in body mass fat, resulting in excessive calorie consumption associated with a high incidence in the development of cardiovascular disease, metabolic dysfunction, diabetes, liver damage, and even cancer. Obesity is characterized by the presence of low and systemic chronic inflammation, which leads to dysregulated adipocyte function, promoting hormonal changes that alter the regulation of food consumption. In mammals, adipose tissue is classified into white adipose tissue (WAT) and brown adipose tissue (BAT). WAT functions as a reservoir of triglycerides from which free fatty acids (FFAs) are released to fuel the energy demands. BAT is considered to be a lipid reserve for cold-induced adaptive thermogenesis and is characterized by an increased mitochondrial count, lipolysis, and expression of uncoupling protein-1 (UCP-1), a key protein involved in the regulation of energy expenditure and protection against oxidative stress [12]. WAT is divided into two main depots, the subcutaneous adipose tissue (SAT) found under the skin, and the visceral adipose tissue (VAT) located around the internal organs. Among these depots, two types of thermogenic adipocytes are known to exist—classical brown and beige, which have disparate developmental and anatomical characteristics. The classical brown adipocytes found in the BAT have an embryonic origin, whereas the inducible thermogenic beige adipocytes exist in WAT and are derived either through transdifferentiation of WAT or from beige adipocyte precursor cells expressing platelet-derived growth

factor receptor (PDGFR)-α [13]. This occasional development of beige adipocytes is referred to as the browning of WAT and was first identified in rodents, however, recent findings suggest the presence of beige adipocytes in humans as well. Browning is associated with resistance to HFD-induced obesity. In obese individuals, the conversion of triglycerides into FFAs through lipolytic enzymes such as adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), and the subsequent FFA β-oxidation are impaired, consequently affecting the browning of adipose tissues [14]. Adipose tissue is also recognized as an endocrine organ that regulates systemic energy homeostasis, releasing a repertoire of cytokines (referred to as adipokines), hormones, and lipokines [15,16]. Leptin, an adipokine secreted during food intake, plays a key role in maintaining metabolic balance by suppressing appetite. Leptin levels are highly upregulated in obesity, but obese individuals become "leptin resistant" by losing their ability to control food ingestion, despite the presence of high levels of leptin [17]. The adipokine adiponectin has anti-obesogenic functions and regulates glucose levels and lipid oxidation [18]. Interestingly, recent studies involving single cell sequencing, and metabolomic and proteomic analyses of human mesenchymal progenitors and WAT, identified adipocyte progenitors that developed into adipocyte subsets with distinct metabolic and endocrine functions [19,20]. These findings highlight the cellular heterogeneity of adipose tissues and the need to gain a better understanding of the adipocyte populations, its precursors and the regulatory mechanisms that define their role in obesity.

Adipogenesis is a multi-step process that involves the development of a multipotent mesenchymal stem cell into a precursor preadipocyte, which then further differentiates into a mature lipid-laden adipocyte [21]. In the mouse cell line 3T3-L1, a broadly used model of adipogenesis, mitotic clonal expansion (MEC) involving multiple cell divisions, precedes the terminal differentiation [22]. Adipogenesis is regulated by a cascade of transcription factors, including peroxisome proliferator-activated receptor gamma (PPARγ), CAAT/enhancer-binding proteins (C/EBP), and sterol regulatory element binding protein isoform (SREBP)-1c, which induce temporal changes of adipogenesis-regulatory genes [23–25]. A recent study using a single molecule 5 cap analysis of gene expression (CAGE) revealed dynamic patterns of gene expression profiles during adipocyte differentiation, in which the early stage involved an increase in genes related to structural remodeling and cell division, whereas genes in the later differentiation state were involved in the regulation of lipid metabolism and energy homeostasis characteristic to WAT [26]. Human transcriptome analyses reported several adipocyte-specific genes, such as leptin, adiponectin, fatty acid binding protein (FABP)-4 and ATGL, to be highly expressed in mature adipocytes [27].

In obesity, an imbalance in energy homeostasis causes adipose tissue remodeling, including adipocyte enlargement (hypertrophy) and an increase in numbers (hyperplasia) [28]. The adipose tissue constitutes adipocytes and stroma, which includes endothelial cells, pericytes, adipose stem cells, and immune cells (Figure 1). In lean conditions, macrophages, the predominant immune cell population accounting for 10% of all cells in the adipose tissue, are found in an alternatively activated M2 state, characterized by expressing CD206<sup>+</sup> CD163<sup>+</sup> CD301<sup>+</sup> surface receptors and are sparsely distributed [3]. Loss of M2 macrophages resulted in increased weight gain in myeloid-specific PPAR delta (δ) ablated mice fed with HFD, reflecting on the relevance of M2 ATMs in mitigating obesity [29]. In obese conditions, there is a significant increase in the number of ATMs, which are mainly found as classically activated M1 phenotype expressing CD11c<sup>+</sup> CD86<sup>+</sup> surface proteins responsible for promoting inflammatory conditions [30,31]. Notably, the main mechanism contributing to the increase of M1 phenotype is the recruitment of inflammatory monocytes (characterized by the presence of CCR2++ CX3CR1low Ly6Chigh CD11b<sup>+</sup> surface proteins) from the circulation, which on entering the adipose tissue differentiate into M1 ATMs [32]. The adipocyte secreted chemokine monocyte chemoattractant protein (MCP)-1 is known to be a key player in recruiting inflammatory monocytes to the adipose tissue [3,33]. Transgenic mice overexpressing MCP-1 showed an increased number of infiltrated macrophages in the adipose tissue, supporting the relevance of MCP-1 [34]. The recruitment of monocytes also requires CD11b integrin, as demonstrated using CD11b-deficient HFD-fed mice [35]. Several other adipocyte-induced chemokines such as colony stimulating factor (CSF)-1, C-X3-C motif

ligand (CX3CL)-1, leukotriene B4, and macrophage migration inhibitory factor (MIF) also promoted macrophage infiltration [3,36–38]. Additionally, it was suggested that the proliferation of resident M2 ATMs might also contribute to an increase in the ATM population at the early stages of obesity [39,40]. Recent findings revealed the presence of a distinct pool of proinflammatory metabolically activated macrophages (MMe), which were stimulated by palmitate and participated in the trafficking and lysosomal metabolism of lipids, yet, failed to express the typical M1 markers [41,42]. These initial findings suggest a higher ATM heterogeneity than expected and would require further investigation. Other immune cells also contribute to maintaining the metabolic homeostasis in adipose tissue. Tregs and eosinophils aid in the polarization of ATMs into an M2 phenotype, by releasing cytokines such as interleukin (IL)-4, IL-13, and IL-10 [43–46]. In addition, innate lymphoid cells (ILC)-2 seem to induce adipocyte browning and ameliorate obesity through IL-33 dependent upregulation of UCP-1 [47]. Recent metabolomic and lipidomic profiling studies reported differential metabolite and lipid signatures in human and animal models, revealing a significant increase in glycerol 1-phosphate, glycolic acid, uric acid, polysaturated fatty acids, and fatty acyl chains in obese groups, thereby engendering fatty livers [48–50]. These studies highlight the need for the identification of molecules that can be used as an early diagnostic and prognostic marker in obesity-induced inflammation.

**Figure 1.** Schematic representation of the cellular dynamics of adipose tissue associated with obesity. As obesity develops, hypertrophic adipocytes and changes in immune cell populations contribute to the development of a chronic inflammatory adipose microenvironment that leads to metabolic dysregulation.

#### *2.2. Inflammation and Its Link with Obesity*

Chronic inflammation is a prolonged and progressive response that is accompanied by an altered immune function, ultimately leading to tissue dysfunction. It plays a key role in the initiation and progression of the pathophysiological alterations that are characteristic of obesity. Although there is an indisputable link between inflammation and obesity, there are still unresolved questions pertaining to its trigger and causative factors. It was hypothesized that inflammation initially originates as a consequence of homeostatic stress due to energy imbalance in the adipocytes [51]. Obesity-induced increase in gut permeability can give rise to circulating intestinal stemmed Gram-negative bacterial lipopolysaccharide (LPS) levels, which provoke inflammatory responses by interacting with toll-like receptor (TLR)-4 in adipocytes and macrophages [52,53]. Additionally, dietary or adipose tissue-derived FFAs binding to TLR2 and TLR4 can trigger the inflammatory signaling pathways [54]. Hypertrophic adipocytes can stimulate local induction of the transcription factor hypoxia-inducing factor (HIF)-1α, as a result of excessive oxygen depletion and decreased perfusion, leading to the upregulation of proinflammatory genes, the FFAs plasma levels, and macrophage infiltration [55,56]. FFAs released from dying adipocytes also exacerbate inflammation by binding to macrophage TLR2/4 receptors triggering the activation of nuclear factor-kappaB (NF-κB) and NOD-like receptor (NLR)P3, through the damage-associated molecular proteins (DAMPs), with subsequent production of inflammatory cytokines [57,58]. Often obese adipose tissues are characterized by the elevated generation of mitochondrial reactive oxygen species (ROS), leading to mitochondrial deregulation, oxidative stress, and inflammation [59]. In response to these stimuli, metabolically dysregulated adipocytes secrete proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, which stimulate the neighboring adipocytes and the endothelial cells to secrete NF-κB regulated MCP-1 and other chemokines, thereby recruiting M1 ATMs [33]. High levels of MCP-1 were also implicated in mediating resident macrophage proliferation [60]. Transgenic and chemically induced mice models lacking macrophages conclusively supported the role of ATMs in promoting obesity-induced inflammation. Ablation of macrophages using clodronate liposomes (CL) treatment resulted in the reduction of HFD-induced adipose tissue inflammation [61]. Moreover, a significant decrease in inflammation and insulin resistance was observed in granulocyte macrophage colony stimulating factor (GM-CSF) knock-out mice fed with HFD, owing to the abatement of C-C motif chemokine receptor (CCR)-2-specific macrophage infiltration in adipose tissues, but had no effect on body weight [62]. Adipocyte-induced netrin-1 promoted the retention of inflammatory macrophages in obese adipose tissues by interacting with specific macrophage receptors [63,64]. The infiltrated M1 ATMs produce significantly large amounts of proinflammatory cytokines such as TNF-α, IL-6, IL-8, and IL-1β and attract more macrophages through MCP-1 secretion, creating a deleterious adipose microenvironment [31,34,65]. M1 macrophages can group around the dying adipocytes to form crown-like structures (CLS), which are indicative of an increase in hypoxia, hypertrophy, and stress [66]. This aberrant inflammatory environment creates further adipocyte dysregulation through increased secretion of leptin and lipolytic genes, and enhanced insulin resistance, eventually leading to adipocyte death.

Additional immune cells including mast cells, neutrophils, dendritic cells, T cells, B cells, and ILCs in the adipose tissue stroma participate in the onset of obesity (Figure 1). Nonetheless, how the interaction between these immune cells initiate and sustain adipose tissue inflammation remains elusive. In obesity, prior to ATM infiltration, there is a rise in the number of lymphocytes in the obese adipose tissues, which affects the sustenance of obesity-induced inflammation but is dispensable for the onset of obesity [4]. CD8<sup>+</sup> T cells, activated by hypertrophic adipocytes expressing major histocompatibility complex (MHC)-II, enhance ATM filtration and adipose tissue inflammation in HFD-fed mice, as confirmed by the CD8<sup>+</sup> T cells loss and gain of function in vivo studies [67–69]. CD8<sup>+</sup> T cells were found around CLS along with M1 ATMs in epididymal adipose tissues (EAT). Interestingly, CD4<sup>+</sup> T cells were identified to exhibit long-lasting obesity memory and induction of body mass regain in a weight gain–loss–regain C57BL/6J model, suggesting the potential role of an immune cell stimulated inflammatory condition in promoting obesity relapse [70]. B cells also stimulated secretion of proinflammatory cytokines, adipocyte hypertrophy and insulin resistance in obese mice. A decrease in systemic inflammation in obese B-cell-deficient mice was correlated with a significant reduction in Tregs, indicating the ability of B cells to regulate the T cell function in hypertrophic adipose tissue [71]. Additionally, B cell filtration in adipose tissues seems to precede other immune cells. B cells were infiltrated into the adipose tissues after 3 weeks of HFD, followed by T cell at week 6, and macrophage

infiltration at week 12. Importantly, there was a significant rise in ATMs and natural killer cells (NK) in lymphocyte-deficient Rag2-/- mice, suggesting that T cells and B cells are not essential for the initiation of obesity [72]. Additionally, it remains debatable whether the early lymphocyte accumulation is a protective response rather than a stimulus of inflammatory conditions. Loss and gain of function in HFD-fed and leptin-mutated genetically obese mice revealed that CD4<sup>+</sup> Foxp3<sup>+</sup> Tregs secreted anti-inflammatory cytokines like IL-10 and influenced the insulin sensitivity of adipose tissues [44]. Interestingly, single cell RNA-sequencing of SAT, identified crosstalk induced between the adipocytes and the IL-10 secreting immune cells, wherein beige-like metabolically active adipocytes exhibited an enhanced expression of IL-10Rα responsive thermogenic genes [73]. A potential role of mast cells in mediating systemic thermogenesis, macrophage recruitment, and insulin resistance in high cholesterol and HFD-fed mice was reported [74]. Eosinophils-induced IL-4 in WAT was found to promote M2 macrophage polarization [75,76]. Neutrophils promote inflammation in HFD-induced obese mice, through the secretion of proteases, such as elastase [77]. CD11c<sup>+</sup> CD64- expressing dendritic cells accumulate in the SAT of obese mice and in humans, which promotes the activation of CD4<sup>+</sup> T-cell polarization and proliferation through Th17-type responses, to trigger inflammation [78,79]. The role of ILCs in obesity remains controversial. ILC2 depletion was reported to decline eosinophils, M2 ATMs, and anti-inflammatory cytokines, such as IL-13 and IL-5 in the VAT, thereby inducing adiposity [80]. On the contrary, recent studies identified that the loss of ILC2 and ILC3 resulted in decreased weight-gain in HFD-fed mice, which was reversed through the adoptive transfer of small intestine ILC2 [81]. The discrepancies in these studies suggest that further investigations to evaluate the role of different immune cells are necessary. Collectively, these findings support the complex roles of innate and adaptive immune cells during the early stages of obesity-induced inflammation, contributing to adipose tissue remodeling.

Several molecular pathways induce inflammation in obesity (Figure 2). TLR-mediated polarization of macrophages into an M1 inflammatory phenotype involves different transcription factors, such as NF-κB, PU.1, C/EBP-α, activator protein-1 (AP-1), STAT1, and interferon regulatory factor (IRF)-5 [82]. FFAs, TNF-α, or LPS can activate TLRs to stimulate c-Jun N-terminal kinases (JNK) or NF-κB mediated inflammation, resulting in enhanced innate immunity, activation of NLRP3 inflammasomes, and production of proinflammatory cytokines [83–85]. IKKβ and TLR4 deficiency in macrophages protected from insulin resistance in mice when exposed to HFD, and also inhibited FFA induced upregulation of TNF-α and IL-6 [54,86]. Mice, with a deficiency in TGF-β–activated kinase 1 (TAK1), an upstream modulator of NF-κB, in adipocytes, displayed an increased M2 ATM count in WAT, along with an enhanced resistance to HFD or leptin-deficiency-induced obesity [87]. Thus, targeting the IKKβ/NF-κB pathway has become an appealing approach to ameliorate the devastating effects of inflammation induced by obesity [88]. The association of obesity with increased insulin resistance has been extensively studied. Several adipokines such as leptin and retinol-binding protein (RBP)-4 were increased in obese insulin-resistant mice [33,89]. JNK and NF-κB pathways in adipocytes and macrophages, activated in response to obesity-induced stimuli, directly inhibit insulin response [63,90]. FFAs trigger diacylglycerol (DAG) and fatty acyl-CoA in the adipocytes, leading to protein kinase C (PKC) activation, which further phosphorylates insulin receptor substrate (IRS)-1 to inhibit AKT and GLUT-4, causing impairment in the ability of the liver to take up glucose and consequentially increased the circulating glucose levels (Figure 2) [91,92]. Obesity-induced inflammation and related dysregulated metabolic homeostasis often impact the liver, leading to nonalcoholic fatty liver diseases and steatosis (fat accumulation in the liver). Obesity enhances the supply of FFAs to the liver from the adipocytes, causing upregulation of PPARγ-dependent fatty acid translocase protein CD36. This increase in lipid storage elevates the activation of inflammatory responses in resident Kupffer cells and the recruitment of inflammatory myeloid cells to the liver, in a CCR2/MCP1-dependent manner, thereby elevating the severity of hepatic damage [93]. Evidence on the close overlap between the functional roles of adipocytes and macrophages imply inflammation to be the linking hub in obesity [94]. Therefore,

defining, cell-specific regulators of obesity-induced inflammation can be promising in identifying therapeutic targets that can ameliorate the complications associated with obesity.

**Figure 2.** Adipocyte–macrophage crosstalk plays a key role in the induction and maintenance of obesity. Hypertrophic adipocytes release chemoattractants, promoting macrophage infiltration. Adipose-induced adipokines and free fatty acids (FFAs) stimulate adipose tissue macrophages (ATMs) into an M1 inflammatory stage to trigger JNK, NF-κB, and NLRP3-mediated pathways and inflammatory cytokines, which further induce adipocyte responses, including PPARγ and C/EBPs-regulated expressions of adipogenic, thermogenic, lipolytic, and lipogenic genes.
