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Received: 03-Apr-2015; Revised: 07-Jul-2015; Accepted: 22-Jul-2015
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The “Big Bang” in obese fat:
events initiating obesity-induced adipose tissue inflammation
Felix M. Wensveen1,21,41,4, Tamara Turk Wensveen31*
1Department of Histology & Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
2Department of Experimental Immunology, Amsterdam Medical Centre, Amsterdam, The
Netherlands
3Department of Internal Medicine, University Hospital Rijeka, Rijeka, Croatia
4These authors contributed equally
*Correspondence to: bojan.polic@medri.uniri.hr, tel. +385 51 651 171, fax. +385 51 651 176
Abstract
Obesity is associated with the accumulation of pro-inflammatory cells in visceral adipose
tissue (VAT), which is an important underlying cause of insulin resistance and progression to
diabetes mellitus type 2 (DM2). Whereas the role of pro-inflammatory cytokines in disease
development is established, the initiating events leading to immune cell activation remain elusive.
Lean adipose tissue is predominantly populated with regulatory cells, such as eosinophils and type 2
innate lymphocytes. These cells maintain tissue homeostasis through the excretion of type 2
cytokines, such as IL-4, IL-5 and IL-13, which keep adipose tissue macrophages (ATMs) in an anti-
inflammatory, M2-like state. Diet-induced obesity is associated with the loss of tissue homeostasis
and development of type 1 inflammatory responses in VAT, characterized by IFN-
shift of ATMs towards an M1 phenotype. Recent studies show that obesity-induced adipocyte
hypertrophy results in upregulated surface expression of stress markers. Adipose stress is detected
by local sentinels, such as NK cells and CD8+ T cells, which produce IFN-, driving M1 ATM
polarization. A rapid accumulation of pro-inflammatory cells in VAT follows, leading to inflammation.
In this review, we provide an overview of events leading to adipose tissue inflammation, with a
special focus on adipose homeostasis and the obesity-induced loss of homeostasis which marks the
initiation of VAT-inflammation.
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Key Words: Obesity, inflammation, adipose tissue, IFN-, TNF, adiponectin, NK cells, macrophages,
insulin resistance, diabetes mellitus type 2
Introduction
Over the past few decades, we have seen a dramatic worldwide increase in the incidence of
obesity and its associated pathologies, such as insulin resistance, which contributes to the
development of metabolic syndrome and diabetes mellitus type 2 (DM2)[1]. Metabolic syndrome is
a cluster of conditions such as elevated blood glucose, elevated blood pressure, excess body fat and
abnormal lipid (cholesterol) levels, which together increase the risk of diabetes mellitus and
cardiovascular disease. DM2 is characterized by high blood glucose levels, which is a direct result of
reduced systemic sensitivity to the anabolic hormone insulin. An important underlying cause of
obesity-induced insulin resistance is chronic low-grade systemic inflammation (reviewed in [2]). The
long-term presence of pro-inflammatory cytokines in the blood blunts the signal transduction
capacity of the insulin receptor in insulin-sensitive tissues [3]. Obesity-induced systemic
inflammation is thought to originate predominantly in adipose tissue. The human body contains
various types of fat depots, generally divided in white and brown fat. The role of brown adipose
tissue is to produce body heat and in humans is mostly found in newborns, even though adults do
have small amounts of this type of fat [4]. The role of white adipose tissue (WAT) is to store
nutrients in the form of a single large fat droplet. In addition, WAT is an important sensor of the
metabolic state of an organism and is therefore one of the main endocrine organs in the body [5].
WAT can be found at multiple sites in the body and can be further subdivided based on location and
on differences in precursor cells that give rise to these organs [6]. For example, retroperitoneal WAT
is derived from a precursor that requires expression of the transcription factors Myf5 and Pax3,
whereas these genes are not essential for the development of mesenteric WAT. In contrast, male
perigonadal WAT does not require Myf5, but is partially dependent on Pax3 for its development in
mice [6].
Of these various fat tissues, the intra-abdominal fat depots, collectively referred to as
visceral adipose tissue (VAT), have been shown to be the predominant source of chronic systemic
inflammation and most important for the development of DM2 [2]. When the perigonadal fat pads
of mice were surgically removed two weeks before initiation of HFD feeding, a significant reduction
of glucose intolerance and insulin resistance was observed in mouse models of obesity-induced DM2
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[7]. VAT contains a relatively large population of immune cells, which changes dramatically in its
composition during the development of obesity [2]. Recently, various studies have provided further
insight in the earliest changes that occur within the immune cell composition of VAT in response to
diet-induced obesity (DIO). In this review, we provide a brief overview of the events that ultimately
result in adipose tissue inflammation and systemic insulin resistance. We focus on the initial stages
of immune cell activation, which represent the equivalent of the astronomical Bfor adipose
tissue inflammation. Whereas many of these events will (partially) overlap, we present them as
consecutive events to facilitate understanding.
Adipose tissue in homeostasis
The immune system plays an important role in the control of adipose tissue structure and
homeostasis. Two main functions of the immune system in fat can be distinguished: (i) inhibition of
tissue inflammation and (ii) tissue (re)modeling. Much attention has been given to regulation of
immune cell activation in VAT, since loss of immune cell inhibitory mechanisms drives the tissue
inflammation that contributes to insulin resistance. However, various immunological deficiencies
(described below) result either in reduced or increased adipose tissue mass, with obvious
implications for the endocrine function of fat. Therefore, a better understanding of tissue
remodeling by the immune system may have important implications for the current pandemic of
obesity.
Inhibition of immune cell activation in adipose tissue
Under lean conditions, adipose tissue is populated by a number of immune cells (Figure 1).
These cells either inhibit immune cell activation or promote a Th2-type response, characterized by
the production of cytokines such as IL-4, IL-5 and IL-13 [8]. In lean tissue, macrophages have been
shown to be a dominant immune cell population, and the majority of these cells have an M2-like (or
alternatively activated) phenotype (Figure 1) [9]. Lean adipose tissue macrophages (ATMs) express
arginase-1, which inhibits iNOS activity, and produce anti-inflammatory molecules, such as IL-10 and
IL-1Ra [9]. Importantly, under non-obese conditions, macrophages have been shown to play a key
role in inhibiting immune cell activation in murine fat. In mouse models where ATMs fail to respond
to M2-polarizing stimuli, an increase of pro-inflammatory cytokines, such as TNF and IL-, has been
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observed. [10, 11]. The M2 phenotype of ATMs is maintained by a number of immune cells, as well
as by adipocytes. IL-4 has been shown to be an important cytokine that drives M2 polarization [10].
In adipose tissue, eosinophils are the dominant source of IL-4. Deficiency of these cells in dblGATA
mice, or hyper-eosinophilia in IL-5 transgenic animals, has been shown to result in reduced or
increased numbers of M2-like macrophages respectively [11]. VAT-resident eosinophils depend on
IL-5 for their survival, which is mainly produced by type 2 innate lymphoid cells (ILC2s) in this organ
[12]. Elimination of any component of the ILC2/eosinophil/M2-ATM axis has been shown to result in
an increase in pro-inflammatory cytokines in VAT and increased sensitivity to obesity-induced
development of insulin resistance, making this particular axis one of the dominant regulatory
mechanisms of adipose tissue homeostasis [11-13].
Invariant-chain natural killer T (iNKT) cells represent a second immune cell population which
sustains M2 ATMs in VAT (Figure 1). These cells are present at relatively high frequency in lean fat
and recognize lipid antigens in the context of CD1d [14]. Lack of iNKT cells as a result of
CD1d deficiency in mice leads to reduced adipose tissue levels of IL-4 and IL-13, as well as to
increased pro-inflammatory ATM numbers (reviewed in [15]). The role of iNKT cells may change in
response to obesity, as it has been reported that these cells can also promote insulin resistance
following DIO [15]. The exact mechanism via which iNKT cells inhibit adipose tissue inflammation
under homeostasis, yet promote it in models of DIO, has yet to be resolved.
Regulatory T (Treg) cells represent a second T-cell subset that is directly involved in the
inhibition of adipose tissue inflammation. CD4+ T cells are the most predominant T cells in adipose
tissue and compared to other tissues, a very large fraction of these is Foxp3positive, which was
shown to depend on the cytokine IL-33 [16] [17]. Regulatory T cells
in VAT repress immune cell activation through production of the anti-inflammatory cytokine IL-10
[18]. Experimental ablation of Treg cells by injection of diphtheria toxin in mice expressing the
diphtheria toxin receptor under the Foxp3 promoter was shown to result in an increase of pro-
inflammatory cytokines such as TNF, IL-6 and RANTES in fat [18]. Importantly, elimination of Treg
cells acutely reduced insulin sensitivity in these animals [18], whereas transfer of Treg cells into T-
cell-deficient animals improved insulin sensitivity upon DIO [19].
In addition to immune cells, adipocytes and adipose stroma contribute to tissue
homeostasis. Adipose tissue excretes a number of factors, generally referred to as adipokines,
which play an important role in the regulation of systemic metabolism [20]. Many of these factors
share homologies with cytokines and have profound impact on immune cell behavior. One of the
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most well characterized adipokines is adiponectin (Figure 1). This molecule shares functional
homology with insulin, and has been shown to impair gluconeogenesis in the liver and promote
glucose uptake [21]. In addition, adiponectin has a strong anti-inflammatory effect [22]. In vitro
stimulation assays showed that the adiponectin receptor is expressed at relatively high levels on M2
macrophages, whereas M1 polarization results in its downregulation, explaining the stronger effect
of adiponectin on the former ATM subset [22]. Adiponectin inhibits NF-B activation and promotes
IL-10 and IL-1Ra production by macrophages. Moreover, adiponectin suppresses TLR4 signaling [22],
which has been shown to be important for diet-induced insulin resistance [23].
Immunological control of adipose tissue structure
Various immune cell subsets have been implicated in the control of adipose tissue
remodeling. KitW-sh mice, which lack mature mast cells, have been shown to have strikingly less
adipose tissue mass than wild type animals [24]. In addition, KitW-sh mice show reduced adipose
tissue expansion upon feeding with a high-fat diet, when compared with that in wild type controls
[24]. Mast cells were shown to promote micro-vessel growth by excreting IL-6 and IFN-
appear to be essential for healthy adipose tissue formation and expansion [24]. In contrast, mice
deficient for IL-17 demonstrated increased adipose tissue mass gain in response to HFD [25]. A
normal, low-fat diet did not result in altered adipose tissue mass in these animals, suggesting that
adipogenesis induced by the abundant presence of nutrients differs from basic ontogenesis of
adipose organs. Immune cell-derived stimuli are also capable of inhibiting adipogenesis. IL-5
transgenic animals, which suffer from eosinophilia, have been shown to have reduced adipose tissue
mass, whereas mice, which lack eosinophils, have larger fat pads than wild type animals,
both under lean and obese conditions [11]. Deletion of iNKT cells, as a result of genetic deficiency for
an increase of adipose tissue mass only after high-fat feeding [26, 27]. The
mechanisms via which these cells control white adipose tissue proliferation is currently unclear.
A cytokine of particular interest for adipose tissue homeostasis, because it affects both
immune cell behavior and adipose tissue remodeling, is IL-33 [28]. IL-33 is a member of the IL-1
superfamily and binds to the receptor ST2, which is highly expressed on mast cells, Th2 CD4+ T cells
and ILC2s [29]. Under homeostatic conditions IL-33 is mainly expressed by epithelial cells and tissue
stroma [30]. Upon infection, IL-33 expression is highly induced in many tissues and it was therefore
originally classified as a pro-inflammatory mediator which drives anti-helminth immune responses
[29]. Under non-inflammatory conditions, IL-33 sustains type 2 immune cells, including ILC2s and M2
macrophages, in order to maintain tissue homeostasis (reviewed in [29]). In VAT, IL-33 is abundantly
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expressed in adipose tissue stroma, predominantly by endothelial cells and fibroblast-like reticular
cells [29, 30]. Adipose tissue Treg cells express high levels of ST2 and deficiency of this receptor
results in a specific loss of Treg cells in fat, but not in other organs [16]. IL-33 also plays a role in the
maintenance of adipose tissue eosinophils, as blocking of ST2 was shown to result in a significant
reduction in the number of these cells, independently of ILC2s [31].
Of particular interest is the effect of IL-33 on ILC2s. Exogenous administration of IL-33
increases the number of ILC2s in VAT, with a concomitant increase of IL-5 levels and eosinophil cell
numbers [12]. Strikingly, in response to IL-33, ILC2s regulate adipocyte phenotype and function. For
example, IL-33 deficiency results in increased body mass and increased formation of glucose
intolerance in response to high-fat feeding, due to a lack of Type 2 ILCs [16, 32]
(Brown in white) cell numbers, which are UCP1-expressing adipocytes in white fat with a brown
adipocyte phenotype, were shown to be decreased in these animals [32, 33]. Two recent studies
have shown that IL-33 activates ILC2s to produce IL-13 and the endogenous opioid Met-enkephalin,
which drives adipocyte precursors to differentiate into Brite cells
[32, 33]. Future studies are required to show whether other cells that produce IL-13 in fat, such as
iNKT cells, are also capable of skewing pre-adipocyte differentiation. Nevertheless, since the role of
IL-33 in beiging is most prominent in subcutaneous fat, it comprises most likely a different biological
mechanism than its anti-diabetic and anti-adipogenic effects on VAT. In addition, the biological
significance of beiging requires further study, as it is currently unclear whether it protects against
development of insulin resistance following DIO.
Reaching critical mass: obesity-driven immune cell accumulation in VAT
The early phases of DIO in visceral adipose fat are characterized by an increase in the
amount of fat per adipocyte and by an accumulation of immune cells which initially are of limited
inflammatory capacity. The most commonly used animal model for the induction of obesity and
obesity-induced insulin resistance is by feeding animals, usually mice, with a high-fat diet (HFD).
Within the first weeks after start of HFD feeding, mice accumulate neutrophils, macrophages and NK
cells in VAT. Data showing the biological relevance of these cells in VAT will be discussed below.
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Neutrophils
Already in the first days after initiation of HFD feeding in mice, neutrophil numbers rapidly increase
in adipose tissue and produce the proteolytic enzyme elastase [34]. Even after long term (>3
months) HFD feeding, however, neutrophils remain a minor fraction of adipose tissue leukocytes.
Nevertheless, elastase deficiency or chemical inhibition of elastase was shown to result in enhanced
insulin sensitivity compared to wild type mice upon 12 weeks of HFD feeding. In vivo administration
of exogenous elastase resulted in acute reduction of insulin sensitivity in hepatocytes [34]. These
experiments demonstrate the importance of elastase in metabolic disease, even though it is
currently unclear how this enzyme contributes to adipose tissue inflammation. The immediate
trigger for neutrophil infiltration into the VAT following DIO is also unknown. It has been shown in
humans that acute lipid overload induces an inflammatory boost, as demonstrated by an increase of
circulating MCP1 and C-reactive protein [35]. Alternatively, HFD is thought to induce acute changes
in the adipose tissue micro-environment, such as alterations in oxygen consumption, following a
brief surge in adipocyte precursor proliferation [36]. This acute stress may be involved in recruiting
neutrophils to this site in the first days following initiation of high-fat feeding.
Macrophages and NK cells
Compared with that of neutrophils, the increase in the numbers of adipose tissue
macrophages and NK cells appears to be delayed, occurring in weeks, rather than days after the start
of HFD feeding. Whereas neutrophils in VAT are thought to be primarily of peripheral origin [34],
ATMs and NK cells appear to increase partially or completely through proliferation of tissue resident
populations [7, 37, 38]. When mice fed a HFD were intravenously injected with labeled NK cells,
tracing of the labeled cells demonstrated that only a small number of NK cells reach the adipose
tissues from the periphery. In contrast, BrdU labeling, as a marker of proliferation, was significantly
increased in adipose-tissue resident NK cells, but not in splenic NK cells following HFD [7]. This
suggests that the increase in NK cells in VAT is due to proliferation of the tissue-resident population
rather than influx from the periphery. The origin of increased macrophage populations in the VAT in
response to HFD appears to be more complex. One study demonstrates that local proliferation of
these cells is the dominant source of cellular increase [38]. Other studies, in which the capacity of
macrophages to respond to chemo-attractants was blocked, showed reduced macrophage cell
numbers in VAT due to decreased peripheral influx [37, 39]. Possibly, both depots contribute to the
ATM pool in the case of chronic inflammation in VAT.
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Regulatory cells: ILC2s, Treg cells and eosinophils
In addition to the accumulation of pro-inflammatory cells in VAT, DIO is also associated with
a decrease in anti-inflammatory immune cells. In humans and mice, ILC2s were shown to be
reduced in adipose tissue of obese individuals, both relative to the total number of leukocytes and
per gram of adipose tissue [32]. This decrease appears to involve the inhibition of ILC2s to respond
to IL-33 in the presence of the type 1 cytokine IFN-[40]. Similarly, Treg cells decrease in number per
gram of fat following DIO, and especially relative to the number of effector CD8+ and CD4+ T cells.
The relative inhibitory capacity of this cell population therefore decreases in response to DIO, which
was shown to be an important contributor to VAT inflammation [16, 18, 19]. Finally, eosinophils also
decrease in number following DIO, possibly as a result of the decreased numbers of ILC2s, which
produce the IL-5 required for the maintenance of this population [11, 12]. Thus, the regulatory
capacity of inhibitory immune cell subsets in VAT decreases due to a relative decrease in the
contribution of these cells per gram of fat and as a percentage of the overall immune cell pool.
Adipose tissue
The crucial trigger for the increase of immune cells in adipose tissue in response to DIO is still
unclear, but is likely to be derived from adipocytes. Via lineage tracing of adipocyte precursors, it
was shown that the increase in adipose tissue mass early after HFD feeding is predominantly the
result from adipocyte hypertrophy [36, 41]. Despite an early surge in pre-adipocyte proliferation,
only prolonged exposure to HFD (> 4 weeks) increases mature adipocyte cell numbers (hyperplasia)
[36, 41]. With the increase of adipose tissue mass, several changes occur in adipokine production.
Most notably, adiponectin production drops, which results in decreased glucose uptake and reduced
anti-inflammatory regulation of the local tissue environment [22]. Instead, adipocytes start
producing more leptin. Leptin affects the hypothalamus, where it triggers satiety signals, thereby
directly inhibiting the effects of ghrelin, a hormone produced in the gut that stimulates hunger [22].
Leptin levels in the blood closely correlate with the amount of adipose tissue mass, both in humans
and mice [42]. In addition to its central effects, leptin has a profound effect on the immune system.
The leptin receptor, also known as LEP-R or CD295, is expressed on most immune cells including
neutrophils, macrophages and NK cells. LEP-R shares structural homology with the IL-6 receptor and
also signals through Stat3 [43]. Mice that are deficient for leptin (ob/ob) or for LEP-R (db/db) are
obese due to increased food intake as a result of never feeling sated. In addition, they have a strong
This article is protected by copyright. All rights reserved. 9
reduction in functional immune cells, such as (regulatory) T cells [44], NK cells and dendritic cells
(reviewed in [45]). Importantly, T cells and B cells have been shown to increase expression of LEP-R
in response to activation [46]. Moreover, survival of activated lymphocytes was enhanced when
leptin was added to the cell culture [46]. Increased leptin production by adipose tissue may be an
important initial trigger for immune cell increase in VAT in response to obesity. Indeed, in the first
weeks after start of HFD feeding, macrophage and NK-cell numbers and functionality are specifically
increased in VAT, whereas they are reduced in ob/ob mice [7, 47]. Moreover, leptin has been shown
to promote NK-cell survival in the bone marrow [48] and intravenous administration of leptin
resulted in a specific increase of granulocytes, monocytes and NK cells in the circulation of rats [49].
In addition to leptin, adipose tissue produces other adipokines that affect immune cell
numbers in VAT, most notably monocyte chemo-attractant protein-1 (MCP-1) and IL-6 (Figure 2).
MCP-1 is a potent chemokine that recruits monocytes and its levels are increased in the serum of
DM2 patients [50]. Mice deficient for MCP-1 have a significant reduction in adipose tissue
inflammation and a delayed onset of insulin resistance in response to DIO [37]. The role of IL-6 in
obesity appears to be more complex and shares parallels with IL-33; IL-6 is generally assumed to be a
pro-inflammatory mediator and its levels have been shown to be significantly increased in obese
individuals [51]. The IL-6 receptor consists of the gp130 and IL-6R subunits. Although IL-6 can bind
directly to this receptor complex, IL-6 can also be bound to a soluble form of gp130 and be trans-
presented to cells that express only the IL-6R subunit [52]. Trans-presentation of IL-6 bound to
gp130 has been shown to be crucial for the accumulation of macrophages in adipose tissue [53].
Paradoxically, IL-6-deficient mice develop late-onset glucose tolerance [54]. IL-6 has been shown to
stimulate IL-4R expression on macrophages, thereby promoting M2 polarization of these cells [10],
and macrophages express high levels of the IL-6 receptor. Therefore, similarly to IL-33, under
homeostatic conditions IL-6 appears to function as an anti-inflammatory mediator by preventing M1
macrophage formation. In response to DIO, IL-6 levels increase [55], allowing other, pro-
inflammatory cells to be activated by this cytokine. Therefore, the effects of IL-6 on the development
of DM2 appear to be time- and concentration dependent.
In addition to the factors mentioned above, there is an increasingly large number of
adipokines that has been shown to mediate immune cell function in adipose tissue, such as resistin,
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visfatin and apelin [56]. The exact role of these molecules in the promotion or inhibition of obesity-
induced VAT-inflammation and the development of insulin resistance is mostly unclear. However,
the sheer number of immune-modulatory molecules derived from adipose tissue clearly illustrates
the extensive communication of fat with its tissue-resident immune system.
The “Big Bang”: Initiation of inflammation in VAT
Whereas the accumulation of immune cells predisposes an organ to tissue inflammation, in
itself this local accumulation is insufficient for the activation of the immune system. A key event in
the induction of obesity-induced VAT inflammation and development of insulin resistance appears to
be the polarization of macrophages from an M2 to a pro-inflammatory M1-like state [9, 13]. In
evidence of this, depletion of M1 macrophages in both humans and mice has been shown to cause
significant improvement of systemic insulin resistance [13, 57]. Pro-inflammatory licensing of
macrophages is therefore crucial for the development of VAT inflammation, as they appear to be the
primary source of pro-inflammatory cytokines that are found in the circulation of subjects with
diabetes [55]. In addition, macrophages potently recruit T cells via the production of chemokines
such as CXCL16 [58], a molecule of which the expression is greatly increased in adipose tissue in
response to obesity [59]. Thus, macrophages contribute both directly and indirectly to the excretion
of pro-inflammatory cytokines.
Systemic sources of immune activation
Various stimuli for M1 macrophage priming have been proposed, including free fatty acids
(FFAs) and microbial components [23, 60]. FFAs are normal components of our food, but they are
also the vehicle by which triacylglycerol stored in adipose tissue is transported through the
bloodstream [61]. FFAs are used as a source of fuel by many tissues, including skeletal muscle and
the brain [61]. Uncontrolled (i.e. non-treated) diabetes patients have increased amounts of FFAs in
circulation [62]. Importantly, FFAs were shown to be able to stimulate TLR4 on macrophages, but
only when bound to the protein carrier Fetuin-A [23, 63]. Palmitate, a FFA found in many food
sources, including palm oil, cheese and meat, has been shown to drive activation of the
inflammasome, the protein complex responsible for the formation of pro-inflammatory cytokines IL-
-18 [64]. Recently, the role of FFAs as initiators of inflammation is being questioned.
This article is protected by copyright. All rights reserved. 11
In large cohort studies of obese non-diabetic individuals, the levels of FFAs in the serum did not
correlate with BMI [65, 66]. Importantly, meta-analysis shows that serum FFA levels are not
predictive for the formation of DM2 [61]. FFA uptake from the circulation is controlled by insulin and
the increase of FFA levels in the blood of uncontrolled DM2 patients is therefore thought to be a
result of insulin resistance rather than its cause [61]. Indeed, well-controlled DM2 patients do not
have higher serum levels of FFA than healthy subjects [61, 67]. Notably, in vitro studies demonstrate
that FFAs can only induce pro-inflammatory cytokine excretion in macrophages when they are first
primed with LPS or IFN- [64]. Thus, before a free fatty acid can function as a pro-inflammatory
mediator, polarization of ATMs is first required. This observation in turn triggered the hypothesis
that obesity-induced gut dysbiosis drives adipose tissue inflammation, through the leakage of
microbial components (or pathogen-associated molecular patterns (PAMPs)) into the blood stream
(Figure 2). Whereas large-scale, metagenome-wide association studies on microbial gut DNA
revealed only mild dysbiosis in the intestines of DM2 patients [68], several micro-organisms could
indeed be associated with markers of metabolic syndrome [69]. Animal studies indicate that a HFD
increases endotoxin levels in the circulation [60, 70]. However, this could only be achieved when
food with a very high fat content was used, whereas more commonly used high-fat diets failed to do
so [60]. Nevertheless, studies in humans indicate that HFDs acutely increase systemic endotoxin
levels [71]. More importantly, a large prospective cohort study showed that systemic endotoxin
levels are a risk factor for the development of DM2 [72]. Thus, there is clear evidence that systemic
factors, especially diet-induced endotoxemia, play a role in the development of insulin resistance.
Whether and how these factors affect chronic systemic inflammation has yet to be elucidated.
Local sources of VAT inflammation
Systemic endotoxemia does not explain, however, why VAT macrophages specifically would
be primed towards an M1 phenotype. Rather, a local trigger is required to initiate macrophage
polarization in particularly this tissue. IFN- is a potent inducer of M1 polarization [73]. Mice
deficient for this cytokine have reduced M1 macrophage accumulation in the VAT and improved
insulin sensitivity in response to HFD feeding [74-76]. Therefore, obesity-induced IFN- production
has been suggested to be an important initiating event in VAT inflammation.
NK cells are sentinels and browse tissues looking for signs of cellular stress,
activating the immune system in response to viral infection or oncogenic transformation. NK cells
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are armed with a broad repertoire of activating and inhibitory receptors that recognize self-ligands
on target cells. In the absence of inhibi and/or in the presence of
cells are triggered to induce a cytolytic response, or produce
cytokines such as IFN- [77]. In several models, NK cells have been shown to drive M1 macrophage
polarization through the production of IFN- [73, 78].
Hypertrophy is a well-known stress factor for adipocytes, leading to micro-hypoxia, ER stress
and extracellular matrix confinement [79, 80]. Indeed, we have observed that in response to HFD
feeding, murine adipocytes of the visceral fat induce expression of stress ligands which bind to the
activating NK-cell receptor NKp46 (NCR1 in mice) (Figure 2) [7]. Early after initiation of HFD feeding,
the number of IFN--producing cells increase, and in particular the number of NK cells [7]. Antibody-
mediated depletion of NK cells or deficiency of NCR1 in mice was shown to result in a significant
reduction of M1-polarized ATMs upon HFD feeding, whereas the total number of ATMs was not
greatly affected [7, 81]. Furthermore, the development of diet-induced insulin resistance could be
delayed by preventing NK-cell activation using soluble NCR1 [7]. These findings are in line with
several recent studies in humans and mice. VAT from obese humans shows a significant increase in
the number of NK cells compared with that in subcutaneous fat, and these cells produce higher
levels of IFN-[76]. In addition, human adipocytes can stimulate NK cells to produce IFN- in vitro
[82].
In addition to NK cells, T cells are an important source of IFN-[7, 19]. Both CD4+ and
CD8+ T-cell numbers strongly increase in response to DIO and ultimately become the most prevalent
lymphocytes in VAT, albeit with delayed kinetics compared to NK cells [7, 83]. In the first weeks after
DIO, CD4+ T cells retain a TH2 phenotype and contribute to the inhibition of VAT-inflammation [19].
CD8+ T cells on the other hand, have been shown to recruit macrophages through the production of
MCP-1 and their deletion in mice results in a significant decrease in systemic inflammation and
insulin resistance upon DIO [84]. It is currently unclear whether CD4+ and CD8+ T cells are activated
in VAT through engagement of their T-cell receptor (TCR), or whether they are recruited in a
chemokine-dependent fashion, independent of TCR engagement. Obese, but not lean adipose tissue
was shown to be able to induce proliferation of CD8+ T cells in vitro [84]. However, whether this is
also the way via which CD8+ T-cell numbers increase in obese VAT in vivo remains to be investigated.
Finally, NKT cells can be a source of IFN- in VAT. Whereas iNKT cells initially have an anti-
inflammatory role in VAT through the production of IL-4 and IL-13, in response to HFD the number of
TNF and IFN--producing NKT cells strongly increases [7, 83]. Whether and how these cells
This article is protected by copyright. All rights reserved. 13
contribute to initiation of VAT inflammation is still a matter of debate, especially since people using
the same mouse models deficient for iNKT cells found opposing effects on the development of
insulin resistance following DIO [14, 83].
In summary, the increase in macrophage cell numbers in VAT in response to HFD feeding is
promoted by various factors, both derived from local and systemic sources. However, in order to
achieve their full pro-inflammatory potential, licensing of ATMs by NK cells through immune cell
derived cytokines such as IFN-Figure 2). The observation that IFN- deficient mice still
develop insulin resistance in response to HFD feeding, though delayed compared to wild type
animals, also indicates that this cytokine is not the only factor that drives VAT inflammation [75].
An additional layer of complexity has recently been added with the surprising observation
that recruitment of pro-inflammatory macrophages in VAT is not absolutely required for the
development of obesity-induced insulin resistance. Mice in which IL-6 trans signaling was impaired
showed reduced recruitment of macrophages into the VAT, but still developed glucose intolerance
[53]. Possibly, redundancy between pro-inflammatory mediators ensures recruitment of pro-
inflammatory cells to the VAT even in the absence of macrophages.
Inflation of inflammation in the VAT
The later stages of adipose tissue inflammation are well studied and have been elaborately
described in excellent reviews [2, 15, 85]. Therefore, we will describe this phase only briefly here.
Priming of macrophages in adipose tissue makes them susceptible to a range of pro-inflammatory
stimuli that are associated with obesity, described below. In response to obesity, adipocytes induce
expression of enzymes that convert arachidonic acid into pro-inflammatory mediators, including
leukotrienes [39, 86]. Deficiency in key components of leukotriene biology has been shown to
prevent cytokine secretion by macrophages and obesity-induced insulin resistance [39, 86]. In
addition, obesity has been shown to induce necrotic cell death of adipocytes as a result of micro-
hypoxia [79]. Necrosis results in the release of danger-associated molecular patterns (DAMPs), such
as double-stranded DNA and heat shock proteins, which drives macrophages to produce pro-
inflammatory cytokines [87]. Advanced obesity is therefore characterized by adipocytes in VAT that
are surrounded by a ring of pro-inflammatory macrophages, known as crown-like structures [88]. In
addition, priming makes murine macrophages susceptible to activation by FFAs [63]. These pro-
inflammatory stimuli drive the activation of the JNK and NF-B signaling cascades and of the
This article is protected by copyright. All rights reserved. 14
inflammasome in macrophages [87, 89, 90]. The inflammasome is a protein complex, which converts
pro-IL-1 and pro-IL-18 into IL--18, both potent activators of the immune system[91]. Either
genetic deficiency or chemical inhibition for components of the JNK, NF-B or inflammasome
pathways has been shown to strongly reduce obesity-induced insulin resistance in humans and mice
[87, 89, 90].
The activation of macrophages in VAT and the generation of an inflammatory environment
in this tissue subsequently drives the recruitment of a plethora of pro-inflammatory cells, mostly
with a TH1 signature. TH1 effector CD4 T cells, CD8 T cells and B cells most strongly increase during
the later stages of adipose tissue inflammation [2, 15, 19, 85]. Not only are these cells important
sources of IFN- that further drive ATM polarization, they also contribute to chronic systemic
inflammation by producing pro-inflammatory cytokines such as TNF (Figure 2). Ultimately, it is the
chronic leakage of these pro-inflammatory cytokines into the blood stream that forms a strong risk
factor for the development of systemic insulin resistance by dampening the signaling capacity of the
insulin receptor in local tissues [3].
Concluding remarks
Although the immunological complexity of VAT inflammation is becoming increasingly clear,
many questions remain unanswered. Subcutaneous adipose tissue is comparable in many aspects to
its visceral counterpart, including the capacity to produce leptin and adiponectin [92], yet it
accumulates much less pro-inflammatory macrophages in response to DIO [93]. Adipose tissue
stroma is an important source of adipokines and has been shown to activate CD8+ T cells in HFD-fed
animals [84, 92]. Therefore, differences between the connective tissues that support visceral and
subcutaneous adipocytes are likely to be important. In addition, the chronic systemic presence of
pro-inflammatory mediators cannot in itself explain the development of insulin resistance. Whereas
some inflammatory diseases, such as psoriasis, ulcerative colitis and systemic vasculitis are
ry
arthritis and systemic autoimmune disorders (e.g SLE, scleroderma) do not [94]. Clearly,
communication between metabolic and immunological regulators is required for the development
of DM2.
Finally, a better understanding of the underlying mechanisms behind adipose tissue
inflammation holds great promise as a target for future therapeutic intervention. Current clinical
This article is protected by copyright. All rights reserved. 15
practice mainly focuses on the reduction of blood glucose levels in DM2 patients[95], which is a
consequence of insulin resistance, whereas obesity-induced VAT inflammation is crucial for the
development this disease. Several phase II and III clinical trials have been initiated to inhibit key
immunological processes of VAT inflammation in DM2 patients, such as NF- , IL-
function or arachidonic acid metabolism, with promising results [85]. Thus, targeting of
inflammatory processes in VAT of obese patients appears to be a promising future strategy for
prophylaxis against diabetes development.
Acknowledgments:
This work is supported by the European Foundation for the Study of Diabetes (New Horizons
Program), the Unity through Knowledge Fund (15/13 to B.P.), the University of Rijeka (13.06.1.1.03
to B.P.), the European Social Fund - ES (HR.3.2.01-0263 to B.P.), the Netherlands Organization for
Scientific Research (91614029 to F.M.W.) and the European Commission (PCIG14-GA-2013-630827
to F.M.W.).
Conflict of interest
The authors declare no financial or commercial conflict of interest.
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Figure 1. Control of adipose tissue homeostasis under lean conditions. Healthy adipose tissue is
populated by a Type 2-polarized immune system, which maintains ATMs in an M2-like state.
Dominant immune cell subsets under these conditions are eosinophils, iNKT cells and Treg cells,
which produce IL-4, IL-13 and IL-10 respectively. Adipocytes also contribute to the Type 2 immune
response through the production of adiponectin. ILC2s regulate eosinophil numbers through the
production of IL-5. Type 2 immune cells are supported by a stromal structure which promotes
immune cell viability through the production of several cytokines, most importantly IL-33. In
addition to sustaining a Type 2 immune cell environment, adipose tissue cells engage in extensive
cross-talk to (re)model adipose tissue structure and phenotype. One example is the formation of
beiging through the production of IL-4, IL-13
and Met-Enkephalin by the ILC2/eosinophil axis. NKT = Invariant-Chain Natural Killer T cell, TReg =
regulatory T cell, Eo = Eosinophil, M2 = M2 Macrophage, ILC2 = Innate Lymphocyte 2, AP = adipocyte
Stroma = adipose tissue stroma. MetEnk = Met-
enkephalin.
M2
NKT
Treg WA
AP
Brite
IL-10
IL-33
IL-33
IL-33
IL-25
TSLP
IL-4
IL-4
IL-5
IL-13
Adiponectin
MetEnk
IL-13
Stroma
?
ILC2
Eo
This article is protected by copyright. All rights reserved. 23
Figure 2. Model for development of obesity-induced adipose tissue inflammation. In response to
HFD, adipocytes initially become hypertrophic and later hyperplastic (indicated by increase in size
and number of adipocytes respectively). This is associated with a shift in adipokine production from
adiponectin to leptin/MCP-1 and an increase in the number of ATMs, neutrophils and NK cells in
visceral fat. As obesity persists, adipocyte stress drives CD8+ T-cell and NK-cell activation through
NKp46, resulting in local production of IFN-coming from the periphery, this
locally-produced IFN- -inflammatory M1 state. This makes these cells
sensitive to a range of pro-inflammatory stimuli, such as leukotrienes, FFA-Fetuin-A complexes and
DAMPs from necrotic adipocytes. As a result, ATMs produce pro-inflammatory cytokines, such as
TNF and IL- and recruit more pro-inflammatory cells, including CD4+ T cells, CD8+ T cells and B
cells, into the VAT to amplify the immune response. The chronic systemic presence of pro-
inflammatory cytokines derived from this response ultimately contributes to the development of
insulin resistance. M1 = M1 Macrophage, M2 = M2 Macrophage,
cell, IFN-
DAMP = danger associated molecular pattern, TNF = tumor necrosis factor.
CIRCULATION
M2 M2
M2
M2
NK
NK
NK
M1
M1
M1
M1
NK
CD4
CD8
B
Insulin
Resistance
DAMP
TNF
IL-1β
Leukotrienes
Adiponectin
Leptin, MCP-1
Adiponectin
Leptin, MCP-1
IFNγ
CD8
PAMP
Nφ
FFA-
FetuinA
?
