Immune cell cholinergic signaling in adipose thermoregulation and immunometabolism

Abstract

Research focusing on adipose immunometabolism has been expanded from inflammation in white fat during obesity development to immune cell function regulating thermogenic fat, energy expenditure, and systemic metabolism. This opinion discusses our current understanding of how resident immune cells within the thermogenic fat niche may regulate whole-body energy homeostasis. Furthermore, various types of immune cells can synthesize acetylcholine and regulate important physiological functions. Thus, we highlight a unique subset of cholinergic macrophages within subcutaneous adipose tissue, termed cholinergic adipose macrophages; these macrophages interact with beige adipocytes through CHRNA2 signaling to induce adaptive thermogenesis. We posit that these newly identified thermoregulatory macrophages may broaden our view of immune system functions for maintaining metabolic homeostasis and potentially treating obesity and metabolic disorders.

Thermogenic fat: a potential target for cardiometabolic diseases

Recent clinical observations have found that human thermogenic fat is associated with improved metabolic conditions and reduced risks of cardiovascular disorders, even in obese populations [1–3]. Moreover, studies using genetically engineered human thermogenic adipocytes have provided further mechanistic insights into how these metabolically active cells might directly contribute to the prevention and intervention of obesity and metabolic diseases [4, 5]. Indeed, accumulating evidence highlights the potential of aiming at thermogenic fat as a promising putative therapeutic target for cardiovascular and metabolic dysfunctions [6, 7].

In contrast to white fat that serves as a site for energy storge, thermogenic fat is used by mammals to mediate energy dissipation. Our understanding of different types and subtypes of thermogenic adipocytes has been constantly evolving and was recently reviewed elsewhere [8, 9]. One simplified view is the classification of thermogenic fat based on distinct developmental lineages, separating classical brown fat from inducible beige fat (see Glossary) [9–11]. In human and rodents, brown fat is characterized by multilocular lipid droplets and high expression of uncoupling protein 1 (UCP1), whereas beige fat shares the feature of white fat which is presented with the unilocular lipid droplet and low expression of UCP1 [9–11]. Upon thermogenic stimulation, beige fat undergoes dramatic remodeling and exhibits similar characteristics to brown fat [10]. Activation and deactivation of thermogenic fat through approaches such as cold exposure/high calorie diet have shown to greatly affect systemic metabolism [9–11]. Moreover, both brown and beige adipocytes can be influenced through paracrine mechanisms by other cell types within the tissue niche, including various types of resident immune cells in fat [7, 12]. In this opinion article, we will discuss the effects of adipose immune cells on thermoregulation, the roles of cholinergic immune cells under physiological and pathological conditions, with particular focus on the impact of cholinergic adipose macrophages (termed as ChAMs) on beige fat. We posit that a better understanding of resident immune cells in fat may enhance our understanding of beige fat regulation, informing the development of immune-based therapeutic strategies against cardiometabolic diseases.

An integrated and coordinated regulatory network of immune cells and thermogenic adipocytes

The role of immunity in thermoregulation of adipose tissue has been increasingly appreciated in recent years [12–14]. The comprehensive atlas of adipose immune cells and their cellular dynamics upon environmental and metabolic changes have been investigated and evaluated in several recent studies using single-cell RNA sequencing (scRNA-seq) approaches [15–21].

Myeloid cells and thermogenesis

Much has been reported regarding the thermoregulatory role of resident myeloid cells in human and murine fat, including macrophages, monocytes, eosinophils, and mast cells. Arguably, the most prominently studied adipose immune cell population is the macrophage, both in the context of metabolic maintenance in general and energy homeostasis in particular (Figure 1, Key figure). Even though now considered overly simplified and misleading, traditionally, macrophages have been grouped into pro-inflammatory M1 (classical activation) and anti-inflammatory M2 (alternative activation) macrophages [22]. For clarity purposes, we still use here the “M1/M2-like macrophage” terminology to refer to pro- vs anti-inflammatory phenotypes, as how the cells were referred to in the original studies.

Figure 1. Different subpopulations of macrophages mediate distinct functions and can either repress or activate thermogenesis in fat. (Key Figure).

Macrophages dampen thermogenic activity of murine brown fat through release of PDGFcc [39] and inhibition of sympathetic innervation mediated by PLXNA4 [31]. Sympathetic neuron-associated macrophages import and degrade NE in thermogenic fat in both mouse and human [32]. M1-like macrophages negatively regulate thermogenesis of beige adipocytes through direct interaction mediated by integrin α4 and VCAM1, observed in both human and mouse subcutaneous fat [38]. CXCL14 secreted from rodent brown adipocytes [57] and ADIPOQ secreted from rodent beige adipocytes [26] recruit and activate M2-like macrophages, promoting thermogenesis. Rodent studies have shown that M2-like macrophages are important in the removal of damaged mitochondria ejected from brown adipocytes [35, 36], regulation of the innervation in cancer cachexia [29] and TH activity of sympathetic nerve in beige fat through SLIT3 [28]. NE released from SNS binds to β2 AR on the surface of ChAMs in murine subcutaneous fat, releasing acetylcholine, and activating beige adipocytes through CHRNA2 signaling [40, 41, 81]. In mouse models, IL27 regulates thermogenesis of beige adipocytes through its receptor [37]. Abbreviations: ACh, acetylcholine; ADIPOQ, adiponectin; β2 AR, β2 adrenergic receptor; ChAM, cholinergic adipose macrophage; CHRNA2, cholinergic receptor nicotinic alpha 2 subunit; CXCL14, C-X-C motif chemokine ligand 14; IL27, interleukin 27; NE, norepinephrine; PDGFcc, platelet-derived growth factor cc; PLXNA4, plexin A4; ROBO1, roundabout 1; SLIT3, slit guidance ligand 3; SNS, sympathetic nervous system; TH, tyrosine hydroxylase; VCAM1, vascular cell adhesion molecule 1.

M2-like macrophages were implicated in the activation of thermogenic fat in both mice and humans in multiple studies [16, 23–29]. Although studies now challenge whether macrophages themselves produce catecholamine [30], a recent study revealed that in Slit3^fl/fl;Lyz2-Cre mice, myeloid-specific deletion of slit guidance ligand 3 (Slit3), a cytokine secreted by M2-like macrophages, significantly impaired sympathetic innervation and thermogenic capacity of beige adipocytes, suggesting an alternative mechanism for murine adipose M2-like macrophages stimulates sympathetic innervation and therefore promotes ‘beiging’ of fat cells [28]. Also, the pro-sympathetic innervation effects in adipose tissue of alternatively activated macrophages were observed in the context of cancer-associated cachexia (CAC), in multiple mouse models with syngeneic allografts of lung or colon carcinoma cells [29], suggesting that targeting M2-like macrophages may help to reverse the negative energy balance in CAC and its detrimental effects. In contrast, a macrophage subpopulation harboring chemokine receptor CX3CR1 in murine brown fat tissue was reported to repress sympathetic innervation through plexin A4, a receptor for Sema6A expressed on sympathetic nerves, indicating that different subpopulations of macrophages might exert opposite effects on thermoregulation [31]. Moreover, the modulation of adipose tissue sympathetic tone in human and mice via macrophages can also be mediated through removal of norepinephrine by a subpopulation of macrophages called sympathetic neuron-associated macrophages (SAMs) [32, 33]. This catabolic process within macrophages might be, at least partially, regulated by the action of the enhancer of trithorax and polycomb gene, Asxl2, as it is shown that Inhibition of the expression of Asxl2 in macrophages might promote thermogenesis by extending the half-life of norepinephrine in fat tissue [34].

Macrophages can also influence energy expenditure in murine adipocytes by either uptaking mitochondria from adjacent adipocytes in a heparan-sulfate dependent mechanism or removing the dysfunctional mitochondria ejected by brown adipocytes [35, 36]. Interleukin 27 and Interleukin 27 receptorα (IL27-IL27Rα) signaling has been reported to promote thermogenesis [37]. It was proposed that IL27 secreted by CX3CR1⁺ myeloid cells might be responsible as diminished thermogenesis was only observed in Il27p28^fl/fl;Cx3cr1-Cre mice but not Il27p28^fl/fl;Lyz2-Cre or Il27p28^fl/fl;Itgax-Cre mice; however, the exact producers of IL27 in this context remains to be elucidated [37]. Recruited monocytes in murine brown adipose tissue play an important role during tissue expansion [19]. Infiltration of classically activated macrophages into subcutaneous fat depots induced by inflammation can dampen beige adipogenesis in both mice and humans through direct contact of macrophages and adipocytes, which is mediated by integrin α4 and vascular cell adhesion molecule 1 (VCAM1) [38]. Of note, platelet-derived growth factor cc (PDGFcc) production from adipose macrophages is elevated in high fat diet fed obese mice, resulting in decreased thermogenic activity in brown fat, which further exacerbates body weight gain and its associated metabolic disorders [39]. Last by not least, as detailed in later sections, the activation of beige adipocytes is also mediated by so called cholinergic adipose macrophages [40, 41].

From another angle, eosinophils have been reported to activate murine beige adipocytes through the function of a hormone meteorin-like and a transcription factor Krüppel-like factor 3 [24, 42]. Two independent studies have demonstrated that inhibition of serotonin secretion from mast cells increases thermogenesis in beige adipocytes and protects mice from diet-induced obesity [43, 44]. Overall, myeloid cells resident in fat tissue niche, of different types and subtypes, and in various activation states, impact the thermogenic capacity in a complex manner. Future studies focusing on specific activation and deactivation of selected subsets are the key for harnessing the therapeutic potential of these cells.

Lymphocytes and thermogenesis

A clear role in adipose thermoregulation of lymphocytes, including B, T, regulatory T (Tregs) and γδ T cells, as well as invariant natural killer T cells (iNKT cells) and type 2 innate lymphoid cells (ILC2s), has also been reported [12, 14]. IL10 secreted from B and T cells has been shown to repress thermogenesis of murine beige fat in subcutaneous adipose tissue [15, 45]. The inhibitory effects on thermogenesis of T cells have also been demonstrated with the observation that compared to their wild-type (WT) counterparts, Rag1^−/− mice and Cd8^−/− mice have enhanced energy dissipation; furthermore, transfer of CD8⁺ T cells into Rag1^−/− mice impedes beige adipogenesis [46]. In addition, T cell specific deletion of disulfide-bond A oxidoreductase-like protein (DsbA-L), a mitochondrial chaperone initially found in the matrix of mitochondrion, resulted in elevated energy expenditure through reduction of interferon-gamma (IFN-γ) production in Th1 and CD8⁺ T cells [47]. These data suggested that at least a subpopulation of T cells, mainly CD8⁺, negatively impact adipose thermogenesis.

Regarding Tregs and thermogenesis, acute cold exposure facilitated human Foxp3⁺ Tregs induction from naïve CD4⁺ T cells in vivo, potentially suggesting that Tregs might be positive contributors to the adaptive thermogenesis, although this remains to be rigorously tested [48]. Consistently, the number of CD4⁺CD25⁺Foxp3⁺ Tregs is increased in brown fat and subcutaneous fat of WT mice after cold exposure and pharmacological activation of β3 adrenergic receptor by CL-316,243 and deletion of Tregs in Stat6 KO mice has been reported to impair adaptive thermogenesis [49]. Indeed, two distinct subpopulations (or two states) of adipose Tregs respectively labeled as CD73^highST2^low and CD73^lowST2^high, have been identified by integrated omics [20]. CD73^highST2^low Tregs, regulated by PPARγ, can activate beige adipocytes and can protect mice from diet-induced metabolic disorders [20].

The two key effector cytokines produced by γδ T cells, IL17A and IL17F, have both been shown to boost energy expenditure [50, 51]. A subpopulation of γδ T cells, labeled as CD3ε^highCD27⁻PLZF⁺, secreting IL17A, has been shown to increase thermogenic capacity of murine beige and brown adipocytes when compared with CD3ε^lowCD27⁺PLZF⁻ counterparts [50]. The potential thermogenic effects of IL17A have also been observed in cultured human adipocytes [50]. IL17F secreted from γδ T cells can control sympathetic innervation in murine fat tissue through its receptor IL17RC in adipocytes, as evidenced by significantly impaired cold tolerance of Il17f^−/− mice and compromised adipose sympathetic innervation in adipocyte-specific IL17RC KO mice when compared with control counterparts [51]. However, whether IL17A-producing and IL17F-producing γδ T cells are distinct remains to be investigated. Another potential thermogenesis positive influencer is iNKT cells. In addition to its known role in supporting M2-like macrophages and Tregs expansion [52], iNKT cells can be a source of fibroblast growth factor 21 in human and murine subcutaneous fat, which can directly increases the thermogenic activity of beige adipocytes [53]. Furthermore, early studies revealed that ILC2s can lead to the activation of beige adipocytes, as evidenced from both gain-of-function studies with activation of ILC2s with its inducer IL33 and loss-of-function mouse models with disruption of ILC2s [25, 54]. Further research demonstrated that adipocyte progenitors and beige adipocytes in subcutaneous fat may serve as a potential source of IL33 [55, 56], suggesting a positive feedforward regulatory mechanism may be involved.

When considering the totality of the adipose tissue microenvironment, the crosstalk between immune cells and thermogenic adipocytes is bidirectional (Figure 1) [12–14, 26, 55, 57]. Adiponectin, secreted by murine subcutaneous beige adipocytes after cold exposure, has been suggested to promote the proliferation of alternatively activated macrophages, leading to further activation of beige adipocytes [26]. Similarly, thermogenic activity of murine adipocytes can be enhanced by M2-like macrophages recruited by brown fat-secreted chemokine C-X-C motif chemokine ligand 14 (CXCL14), as much lower number of M2-like macrophages observed in Cxcl14^−/− mice compared with WT mice [57]. Signaling through a newly-identified adipokine, chemerin, and its receptor chemerin chemokine-like receptor 1 (CMKLR1) negatively regulates beige fat function through decreasing the production of IL33 from beige adipocytes and reducing ILC2 accumulation in murine fat tissue [55]. Furthermore, inactivation of CMKLR1 increased IL33 production in cultured human adipocyte [55], suggesting that chemerin-CMKLR1 signaling might be conserved in both mice and humans. The complex nature of the interconnection between adipocytes and adipose immune cells can be further demonstrated by the multifaceted role of IL6 [58, 59]. IL6 can be secreted from adipocytes, muscle and myeloid cells and the source of IL6 may dictate its impact on metabolism including energy homeostasis [59]. It is reasonable to anticipate that the ever-evolving role of immune cells in thermoregulation will continuously be delineated with advances in technologies. Overall, understanding the molecular nexus within adipose tissue niche will be useful in strategies aiming at improving systemic energy homeostasis.

Cholinergic machinery in immune cells

The mammalian immune system can also regulate metabolism and whole-body health through acetylcholine (ACh), a molecule best known for its role as a neurotransmitter. The immunoregulatory role of cholinergic signaling was well demonstrated by studies investigating cholinergic receptor nicotinic alpha 7 subunit (CHRNA7) [60, 61]. It was proposed that vagus nerve mediated reflex stimulates CHRNA7 in human and murine macrophages and leads to the reduction of inflammation and this protective effect is blunted in Chrna7^−/− mice. This signaling pathway later was termed as the cholinergic anti-inflammatory pathway and has been thoroughly investigated [60, 62–64].

Using various approaches, including multiple reporter mouse lines such as ChAT-eGFP mice that express enhanced green fluorescence protein (eGFP) under the control of choline acetyltransferase (Chat, the rate limiting enzyme for ACh biosynthesis) promoter, many studies have now convincingly shown that non-neuronal cells, including various types of immune cells, have the capacity of synthesizing ACh [40, 41, 65–80]. Functionally, murine T cells expressing ChAT have been shown to inhibit cytokine release [67]. It was later further demonstrated that ChAT⁺ T cells regulate immunological defense against enteric bacterial infection in mice [75], and are essential to sustain murine antiviral response through an IL21 dependent mechanism and inactivation of Chat expression specifically in T cells in ChAT^fl/fl;Cd4-Cre mice disrupts viral control [76]. ACh-producing T cells have also been shown to regulate systemic blood pressure [72] and coronary vasodilation in mice [77]. In human mononuclear leukocytes, the expression of ChAT was increased after treatment of phytohemagglutinin, a T cell activator [65], suggesting that ChAT⁺ T cells may also be present and functional in humans.

ChAT-expressing B cells have been reported to diminish the recruitment of neutrophils in mice, regulating local innate immunity [70]. ACh released from ChAT⁺ B cells in bone marrow curbs myelopoiesis and in mice with Chat specific deleted in B cells, it was revealed that increased accumulation of inflammatory myeloid cells may worsen symptoms of myocardial infarction [80]. Expression of ChAT has been observed in murine macrophages in several studies [70, 78], although its physiological function has not been elucidated except for ChAMs in regulating beige fat as discussed in the next section. It is worth noting that ChAT-expressing macrophages have also been found from human peripheral blood and lung samples [68]. Future investigation will help to reveal distinct roles of ChAT⁺ macrophages residing in various metabolic organs and whether these functions are conserved between rodents and human.

In the mouse model of multiple sclerosis, ChAT⁺ natural killer (NK) cells have been shown to reduce the accumulation of CCR2⁺Ly6C^high monocytes in the central nervous system, alleviating neural injury [73]. Two independent recent studies revealed that the expression of ChAT was induced in murine ILC2s by the parasite infection of helminth, and ChAT-deletion in ILC2s in ChAT^fl/fl;Il7r-Cre mice compromised these immune cells’ ability to expel helminth [78, 79]. The rapid growth of this area in the last couple of years, particularly the large number of functional studies, strongly indicates an essential role of these cholinergic immune cells in physiology (Figure 2).

Figure 2. Acetylcholine-producing immune cells in physiological and pathological conditions.

ChAT-expressing immune cells have been found in multiple tissues and organs in both human and mouse. Rodent studies have revealed that acetylcholine secreted from these subpopulations of immune cells plays an important role in regulating blood pressure and coronary vasodilation [72, 77], the response to enteric bacterial [75] and parasite infection [78, 79], inhibition of myelopoiesis in bone marrow [80], regulation of cytokine production and viral control [67, 76], and activation of beige adipocytes [40, 41]. In addition, accumulation of monocytes in the central nervous system is attenuated by acetylcholine-producing NK cells in human biopsies and mouse models of multiple sclerosis [73]. Abbreviations: ChAM, cholinergic adipose macrophage; ChAT, choline acetyltransferase; CHRNA2, cholinergic receptor nicotinic alpha 2 subunit; CHRNA7, cholinergic receptor nicotinic alpha 7 subunit; ILC2, type 2 innate lymphoid cell; Mϕ, macrophage; NK, natural killer; TNFα, tumor necrosis factor α.

ChAM: a unique subpopulation of macrophages in beige thermoregulation

An adipose non-neuronal cholinergic circuitry was recently uncovered in exploring unique signaling pathways that regulate beige fat function [40, 41, 81, 82]. A global transcriptional profiling approach was taken to search for factors with an enriched expression pattern in activated beige adipocytes, from which, a subunit of nicotinic acetylcholine receptors (nAChRs), cholinergic receptor nicotinic alpha 2 subunit (CHRNA2) was identified. Since subunits of nAChRs form ligand-gated ion channels, further investigation using calcium imaging assay revealed that CHRNA2 functions in a beige fat-selective fashion, as evidenced that only UCP1⁺ beige adipocytes from subcutaneous fat depot, but not brown or white adipocytes, respond to nAChR agonists. More importantly, CHRNA2 signaling is conserved in both murine and human adipocytes [40, 81].

Because murine subcutaneous fat lacks parasympathetic innervation [83], it was hypothesized that the endogenous ligand for CHRNA2 in subcutaneous fat tissue might be from non-neuronal sources. Indeed, immunostaining and fluorescence-activated cell sorting (FACS) approaches indicated that ChAT-expressing cells within subcutaneous fat were predominantly immune cells [40]. The production and secretion of acetylcholine from the subcutaneous adipose stromal vascular fraction (SVF) were directly measured by mass spectrometry and were abolished in those from ChAT^fl/fl;Vav-iCre mice where Chat was deleted within the hematopoietic lineage [40, 41]. Three-dimensional whole adipose tissue imaging and FACS analyses revealed that ChAT⁺ cells were interspersed throughout the subcutaneous inguinal fat pad and the lymph nodes, with ChAT⁺ macrophages being more prominent in adipose tissue rather than lymph nodes [41], suggesting a close interaction between these ChAT⁺ immune cells and neighboring adipocytes.

The inducibility and regulation of ChAT were manifested as the percentage of ChAT⁺ cells was increased in subcutaneous fat following cold exposure, which is attributed to the doubling number of ChAT⁺ macrophages with little changes in the abundance of ChAT⁺ T cells or B cells [41]. In addition, increased acetylcholine secretion from SVF isolated from WT mice was seen after cold exposure when compared with that from control mice housed at room temperature, but this increase was absent in SVF from ChAT^fl/fl;LysM-Cre mice in which Chat was deleted in the myeloid lineage [41]. Together, these data suggested that ChAT⁺ macrophages residing within the subcutaneous fat niche in mice, were responsible for the increase of acetylcholine secretion responding to cold and the thermoregulation of CHRNA2 signaling in beige adipocytes.

Subsequently, transcriptomic profiling comparing ChAMs with ChAT⁻ adipose macrophages, demonstrated that ChAM is a distinctive subpopulation of macrophages, which expresses crucial genes in ACh synthesis and secretion in mice [41]. Further examination using a reporter mouse line (Cx3cr1^CreER;tdTomato^LSL;ChAT-eGFP) that has been shown to differentially label tissue resident myeloid cells and macrophages derived from circulating monocytes [84], revealed that most ChAMs consist of tissue-resident myeloid cells [41]. Mechanistically, Adrb2, encoding β2 adrenergic receptor (β2 AR), was expressed at a significant level in macrophages isolated from inguinal subcutaneous fat; moreover, pharmacological activation of β2 AR in macrophages via treatments of specific agonists formoterol and terbutaline induced ChAT expression in vitro, and increased the abundance of adipose ChAT⁺ immune cells and ChAMs in vivo [41]. Of note, upon cold exposure, norepinephrine is released through sympathetic nerves and activates, adrenergic receptors, β3 adrenergic receptor in particular, in human and murine thermogenic brown and beige fat [10]. Therefore, it is conceivable that a parallel pathway via β2 AR in ChAMs and CHRNA2 in beige fat has evolved, and can be simultaneously activated by catecholamine upon cold exposure.

Ablation of Chrna2 globally in Chrna2^−/− mice or selectively in the adipocytes in Chrna2^fl/fl;Adipoq-Cre mice impairs thermogenic capacity of beige fat after cold exposure with no impact on brown fat [40, 81]. Consistently, deletion of ChAT either within the hematopoietic lineage in ChAT^fl/fl;Vav-iCre mice or in macrophages in ChAT^fl/fl;LysM-Cre mice compromises the thermogenic capacity of subcutaneous fat but not brown fat [40, 41]. These results from loss-of-function mouse models targeting either the receptor (CHRNA2 in fat) or the ligand (ChAT in macrophages), collectively demonstrated an essential role of ChAM-CHRNA2 signaling in beige thermoregulation. Furthermore, mouse model lacking Chrna2 exhibits dysfunctional glycemic control and insulin response when mice are challenged with high-fat diet feeding [40, 81]. Thus, pending further and robust investigation, we argue that small molecules that can influence the activity of this newly-defined metabolic circuitry within subcutaneous fat might present a promising therapeutic approach for treating obesity and metabolic disorders.

Concluding remarks

Acetylcholine as the first identified neurotransmitter had been the center of the heated debate between chemical and electrical synaptic transmission in the nervous system [85]. A century after Dale and Loewi’s Nobel prize awarded discovery, renewed excitement towards cholinergic regulation can be witnessed at the front line against many human diseases [86]. Inhibitors of acetylcholinesterase/butyrylcholinesterase (AChE/BChE), enzymes that control acetylcholine degradation, have been used clinically to treat certain neurodegenerative diseases for decades [87]. In terms of metabolic disorders and obesity, we posit that the interface between cholinergic macrophages and thermogenic beige adipocytes within subcutaneous adipose tissue is quite important. Compared with much smaller mammals, such as rodents, both common and distinct thermoregulatory mechanisms have been observed in humans [10]. Further investigation is warranted to reveal whether this ChAM-CHRNA2 signaling is present and regulated similarly in humans as that observed in rodents (see Outstanding questions). In addition, future studies will likely provide precise mechanistic insights and further establish the functional relevance of this crosstalk in regulating energy homeostasis during health and disease conditions.

Outstanding questions:

• It has been shown that adrenergic signaling in adipocytes becomes blunted in obesity and under other disease conditions in both mice and humans. Does catecholamine resistance also affect response through β2 AR in ChAMs?

• Studies using various genetically engineered mouse models have revealed that cholinergic immune cells play an important role in regulating antiviral and anti-inflammatory response. Do cholinergic immune cells within adipose tissue, not only ChAMs, but also cholinergic adipose lymphocytes, influence inflammation within fat tissue? If so, how does this further impact systemic metabolic health?

• The molecular characteristics and physiological function of macrophages vary significantly based on the tissue environment they reside in. Comparing ChAMs with cholinergic macrophages within other metabolic organs, what are the common and distinct, regulatory and downstream signaling pathways? How do the environmental or endocrine signals regulate these cells to coordinate whole body physiological function?

• Are cholinergic adipose macrophages with thermoregulatory function in adipose tissue present in humans, in particular within the individuals with high thermogenic fat content and activities?

Significance:

Recently discovered cholinergic adipose macrophages and acetylcholine secreted from this subpopulation of macrophages activate thermogenic beige adipocytes through CHRNA2 signaling. This paracrine immunometabolic interface may represent appealing new therapeutic targets combating obesity and cardiometabolic diseases.

Highlights:

• Subpopulations of immune cells are involved in non-neuronal cholinergic signaling in both mice and humans, contributing to the maintenance of whole-body health.

• A distinct population of adipose macrophages expresses choline acetyltransferase (ChAT) and synthesizes acetylcholine, referred to as cholinergic adipose macrophages (ChAMs).

• Investigation using mouse models uncovered that β2 adrenergic receptors (β2 ARs) in ChAMs are activated upon cold, increasing acetylcholine secretion, which, in turn, stimulates beige fat through cholinergic receptor nicotinic alpha 2 subunit (CHRNA2).

• The immunometabolic pathway, ChAM-CHRNA2 axis in the subcutaneous fat, modulates energy homeostasis and systemic metabolism in response to environmental changes, including cold exposure or high calorie diet, presenting potential new therapeutic opportunities against human metabolic diseases.

Glossary:

Acetylcholinesterase/butyrylcholinesterase (AChE/BChE)

the enzymes that catalyze the rapid hydrolysis of acetylcholine, a neurotransmitter secreted by neurons and non-neuronal cells, including immune cells, into choline and acetic acid. The brief duration of acetylcholine action can be extended by inhibitors of AChE/BChE, which have been shown to increase acetylcholine level and been used clinically to treat neurodegenerative diseases

β2 adrenergic receptor (β2 AR)

a receptor that belongs to the superfamily of the seven-transmembrane receptors. Activated by norepinephrine, it has been shown to regulate cholinergic immune cell function

Beige fat

a distinct type of adipocytes in human and mouse, which has a different developmental origin and molecular expression pattern from either brown or white fat. Beige fat shares similar morphological architecture as white fat under the basal conditions, but upon stimulation, such as cold exposure, thermogenic capacity of beige fat can be drastically increased

Cachexia

a complex syndrome characterized by extreme loss of body weight and muscle mass with or without fat mass loss, and often observed in the late stage of serious chronic diseases, such as cancer. Activation of thermogenic fat has been implicated in the negative energy balance state in cachexia

Cholinergic adipose macrophage (ChAM)

a subpopulation of macrophages in the subcutaneous adipose tissue that express choline acetyltransferase (ChAT), the rate-limiting enzyme for the synthesis of acetylcholine. Acetylcholine secreted from ChAMs is increased upon environmental stimulation, such as cold exposure, and activates thermogenic beige fat

Cold exposure/high calorie diet

environmental influences that affect energy homeostasis in mammals. Widely used experimental approaches to change the balance between energy expenditure and energy storage through acting on many metabolic organs, including thermogenic fat

Nicotinic acetylcholine receptors (nAChRs)

the cation-selective and ligand-gated ion channels that allow the flow of Na⁺, K⁺, and Ca²⁺ across the cell membrane after binding of the ligands, such as acetylcholine and nicotine. These pentameric receptors consisting of one or more of the subunits of nAChRs, are expressed in both the nervous system and non-neuronal tissues. Notably, cholinergic receptor nicotinic alpha 2 subunit (CHRNA2), a subunit of nAChRs, regulates beige fat function and influences systemic metabolism

Paracrine

a type of cellular communication in which a cell produces secretory molecules to diffuse over relatively short distance to a nearby cell and influences its function. Acetylcholine, with an estimated half-life of milliseconds, mediates non-neuronal cholinergic signaling in peripheral tissues mostly through paracrine mechanisms

Single-cell RNA sequencing (scRNA-seq)

one of the popular next-generation sequencing (NGS) technologies that examines the transcriptome at the single cell resolution. scRNA-seq can be used to explore the heterogeneity of complex tissues and identify rare cell types. Metabolic states of each cell subpopulations and how they respond to various physiological or pathological stimuli are also often evaluated using scRNA-seq approaches