Accepted Manuscript

HMGB1, an innate alarmin, plays a critical role in chronic inflammation of adipose

tissue in obesity

Jing Zhang, Lei Zhang, Shu Zhang, Qilin Yu, Xiong Fei, Kun Huang, Cong-Yi Wang,
Ping Yang

PII: S0303-7207(17)30339-8
DOI: 10.1016/j.mce.2017.06.012
Reference: MCE 9984

To appear in: Molecular and Cellular Endocrinology

Received Date: 28 March 2017

Revised Date: 17 May 2017
Accepted Date: 12 June 2017

Please cite this article as: Zhang, J., Zhang, L., Zhang, S., Yu, Q., Fei, X., Huang, K., Wang, C.-Y.,
Yang, P., HMGB1, an innate alarmin, plays a critical role in chronic inflammation of adipose tissue in
obesity, Molecular and Cellular Endocrinology (2017), doi: 10.1016/j.mce.2017.06.012.

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 ACCEPTED MANUSCRIPT
HMGB1, an innate alarmin, plays a critical role in chronic inflammation of adipose tissue in
obesity

Jing Zhang1, Lei Zhang1, Shu Zhang1, Qilin Yu1, Xiong Fei1, Kun Huang2, Cong-Yi Wang1,*, and
Ping Yang1,*

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The Center for Biomedical Research, Key Laboratory of Organ Transplantation, Ministry of
Education and Ministry of Health, Tongji Hospital, Tongji Medical College, Huazhong University
of Science & Technology, 1095 Jiefang Ave., Wuhan, 430030, China;

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2
Tongji School of Pharmacy, Huazhong University of Science & Technology, Wuhan 430030,

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China.

*All correspondence should be addressed to Drs. Cong-Yi Wang (wangcy@tjh.tjmu.edu.cn) and

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Ping Yang (yangping@tjh.tjmu.edu.cn), the Center for Biomedical Research, Tongji Hospital,
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Tongji Medical College, Huazhong University of Science & Technology, 1095 Jiefang Ave.,
Wuhan, 430030, China. Tel: 0086-27-8366-3485.
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Abstract

Obesity has emerged as an imminent global public health concern over the past several decades. It
has now become evident that obesity is characterized by the persistent and low-grade
inflammation in the adipose tissue, and serves as an independent risk factor for many metabolic
disorders such as diabetes and cardiovascular disease. Particularly, adipocytes originated from
obese mice and humans likely predominate necrosis upon stressful insults, leading to passive

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release of cellular contents including the high mobility group box 1 (HMGB1) into the
extracellular milieu. Extracellular HMGB1 acts as an innate alarmin to stimulate the activation of

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resident immune cells in the adipose tissue. Upon activation, those resident immune cells actively
secrete additional HMGB1, which in turn activates/recruits additional immune cells, and induces
adipocyte death. This review summarizes those novel discoveries in terms of HMGB1 in the

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initiation and maintenance of chronic inflammatory state in adipose tissue in obesity, and
discusses its potential application in clinical settings.

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Key words: HMGB1, obesity, alarmin, DC, macrophage, adipocyte, RAGE, TLRs
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Abbreviations: HMGB1, high mobility group box 1; DC, dendritic cell; RAGE, receptor for
advanced glycation endproducts; TLRs, Toll-like receptors; VAT, visceral adipose tissue; WAT,
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white adipose tissue; PAMPs, pathogen-associated molecular patterns; DAMPs,

damage-associated molecular patterns; NLS, nuclear localization sequence; SMCs, smooth muscle
cells; JAK, Janus Kinase; STAT1, Signal Transducer and Activator of Transcription; ER,
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endoplasmic reticulum; FFA, free fatty acid; ROS, reactive oxygen species; ASCs,
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adipose-derived stem cells; JNK, c-Jun N-terminal kinase; NLRP3, NLR family pyrin domain
containing 3; HFD, high-fat diet; AGEs, advanced glycation endproducts; NFκB, nuclear factor
κB; T2D, type 2 diabetes; BMI, Body Mass Index
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1. Introduction

The ongoing obesity epidemic and its associated complications of chronic diseases, exert
formidable challenges and burden to human health (Cheng et al. 2016). Obesity leads not only to
an increase in adipose tissue mass, but also to the infiltration of proinflammatory cells and
secretion of inflammatory cytokines. Therefore, obesity is characterized by a low-grade
inflammation in local and systemic sites as demonstrated by the robust secretion of

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proinflammatory cytokines (e.g., IL-6), and active recruitment of leukocytes (Feng et al. 2016).

HMGB1 is one of the most evolutionarily conserved proteins in the eukaryotic kingdom (Li et al.

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2011). It was originally identified as a chromosomal protein facilitating the binding of
transcription factors to their cognate DNA sequences (Bianchi et al. 1989). Around two decades

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ago, HMGB1, however, was re-discovered as an innate “danger signal” (alarmin) adopted by the
innate immune system during evolution for mediating adaptive immune responses (Wang et al.
1999; Andersson and Tracey 2004; Izuishi et al. 2006; Barkauskaite et al. 2007; Popovic et al.

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2005). More recently, it was interestingly noted that subjects with obesity are characterized by the
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increase of plasma HMGB1 levels (Giacobbe et al. 2015; Arrigo et al. 2013; Wang et al. 2015),
suggesting that HMGB1 could also be a critical player for initiating and maintaining chronic
inflammatory state in adipose tissue in obesity and insulin resistance. This review intends to
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summarize the role of HMGB1 in the initiation and maintenance of chronic inflammation state in
adipose tissue in obesity, and discuss its potential application in clinical settings.
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2. Obesity and adipose chronic inflammation

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In general, visceral adipose tissue (VAT) is considered as a site of cells and molecules to control
metabolism and immune interplay relevant to energy homeostasis (Mathis 2013). In 1993,
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Hotamisligil and his colleagues first noted that TNF-α mRNA and protein were elevated in
adipose tissue from four different rodent models of obesity and diabetes, and blockade of TNF-α
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in obese fa/fa rats resulted in a significant increase of peripheral glucose uptake in response to
insulin (Hotamisligil et al. 1993), suggesting that hyperglycemia could be exacerbated by cytokine
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overproduction. Ten years later, two groups independently reported a marked upregulation of
many inflammatory genes along with accumulation of F4/80+ macrophages in the adipose tissue
of obese mice and humans (Weisberg et al. 2003; Xu et al. 2003). These studies catalyzed the
emerging notion that chronic and low-grade inflammation is a common feature in obese
individuals, which serves as an underlying etiological factor for obesity-related chronic diseases
such as type 2 diabetes, fat-liver disease, and cardiovascular disease (Eichelmann et al. 2016).

Over past few years, our understanding of the mechanisms underlying obesity associated adipose
inflammation has been further expanded, as more cells and molecules were discovered to
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participate in this process. In the lean state, immune cells in adipose tissues (primarily resident
M2-like macrophages together with T regulatory (Treg) cells and eosinophils) synthesize IL-10,
IL-4, and IL-13 to maintain an anti-inflammatory environment, through which adipose tissue
maintains insulin sensitivity (Samad and Ruf 2013). In contrast, obesity drives a shift in the
number and phenotype of immune cells (Olefsky and Glass 2010; Osborn and Olefsky
2012). Specifically, monocytes are recruited from the blood into the obese adipose tissue, where
they polarize to M1 macrophages and secret proinflammatory cytokines (e.g., TNF-α, HMGB1,

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and IL-6) contributing to insulin resistance. Other changes accounting for the proinflammatory
state include reduced numbers of eosinophils and Tregs, and elevated numbers of neutrophils, B
cells, mast cells, and interferon γ–producing Th1 and CD8+ T cells (Wagner 2014) (Fig. 1). The

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proinflammatory cytokines and chemokines promote inflammation and insulin resistance acting in
autocrine, paracrine, and endocrine manners in adipose and other target tissues.

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Similar as the classical inflammatory responses, the initial recruitment of immune cells during

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obesity arises from tissue derived signals in the white adipose tissue (WAT). Pathogen-associated
molecular patterns (PAMPs) are infectious and foreign “danger signals”, whereas
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damage-associated molecular patterns (DAMPs), for example, HMGB1, are non-infectious, yet
potentially harmful stimuli. Tissue resident immune cells express PAMP and DAMP receptors and,
upon their stimulation, initiate an inflammatory response characterized by the recruitment of more
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immune cells in order to eradicate the stimuli by engulfing the agent or infected cells (Janeway
and Medzhitov 2002). The immune cells then initiate tissue repair and remodeling, notably by
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clearing dead cells. The development of inflammation and dysfunction in WAT is most likely due
to a combination of endogenous and exogenous stress signals (Lumeng 2013). Indeed, obese
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WAT undergoes structural remodeling associated with an altered biochemical environment

(Kammoun et al. 2014). In the next several sections, we discuss with focus for the implication of
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HMGB1 in the pathogenesis of obesity and insulin resistance.

3. HMGB1 structural and functional properties

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HMGB1, which is abundantly expressed in almost all eukaryotic cells, was identified about 40
years ago as a nonhistone chromatin-binding protein with high electrophoretic mobility
(Andersson and Tracey 2011; Javaherian et al. 1978). The major structural features of HMGB1 are
its two DNA-binding domains termed as A and B box, and a C-terminal domain composed of
aspartic and glutamic acids (Andersson and Tracey 2011; Chen et al. 2014). HMGB1 contains two
nuclear localization sequences (NLS), which are recognized by the nuclear import complexes to
prevent its cytoplasm re-entry under physiological condition (Tang et al. 2016; Bonaldi et al. 2003;
Evankovich et al. 2010; Zhang et al. 2008; Youn and Shin 2006). Early studies demonstrated that
HMGB1 binds to the minor groove of DNA without showing sequence specificity, by which it
contributes to chromatin structure and gene expression (Liaw et al. 2016). Mice deficient in
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Hmgb1 die shortly after birth in part as a consequence of hypoglycemia (Calogero et al. 1999).
However, other than its nuclear role, HMGB1 has been noted to be released either passively from
damaged cells, or actively secreted by cells following cellular signal transduction (Wang et al.
1999; Tsung et al. 2007; Scaffidi et al. 2002; Gardella et al. 2002) (Fig. 2). Once in the
extracellular milieu and upon interaction with its receptors, HMGB1 exerts a plethora of functions,
including regulation of cell maturation, proliferation, migration, survival and death (Venereau et al.
2015). In addition, extracellular HMGB1 was shown to act as a chemoattractant for myeloid cells

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(Abraham et al. 2000), smooth muscle cells (SMCs) and meso-angioblasts, thereby promoting
muscle tissue repair (Bertheloot and Latz 2017). Interestingly, as the first critical step for its

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secretion from activated immune cells, its cytoplasmic translocation can be regulated by
post-translational modifications such as acetylation (Bonaldi et al. 2003), methylation and
phosphorylation (Ito et al. 2007; Oh et al. 2009; Youn and Shin 2006). For example, HMGB1 is

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hyperacetylated at its nuclear localization sites in monocytes and macrophages, leading to its
cytosolic relocalization. Cytosolic HMGB1 is then concentrated by default into secretory

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lysosomes, and secreted when monocytes receive an appropriate second signal (Bonaldi et al.
2003). This process was recently recognized to be abrogated by pharmacological inhibition of the
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JAK/STAT1 pathway (Lu et al. 2014). However, those studies on its post-translational
modifications were conducted in a rather exclusive manner. It would be, therefore, necessary to
clarify whether its cellular localization related post-translational modification mechanisms are in
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parallel or differentially modulated in a cell type- or cell activation-dependent manner (Bertheloot

and Latz 2017).
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According to in vitro and in vivo studies, the immune function of HMGB1 depends on the redox
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forms of key cysteine residues at positions 23, 45, and 106 (Magna and Pisetsky 2016). There are
three redox forms of HMGB1, including fully reduced HMGB1, disulfide HMGB1 and sulfonyl
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HMGB1 (Antoine et al. 2014). Fully reduced HMGB1 without posttranslational modifications is
the predominant form in the nucleus and cytoplasm under normal conditions, and it forms a
heterocomplex with CXCL12 that interacts with receptor CXCR4 to promote cell migration
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(Venereau et al. 2013; Lian et al. 2017). Disulfide HMGB1 with a disulfide bond connecting C23
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and C45 interacts with receptor TLR4 to induce cytokine and chemokine transcription and
secretion (Lian et al. 2017). Sulfonyl HMGB1 forms after exposure to ROS and has no activity for
cell migration or cytokine induction (Venereau et al. 2012). However, the investigation of specific
HMGB1 redox forms and their targeted pathways in mediating inflammation is just at its very
beginning, especially in the situation of obesity. Current and upcoming projects in this field will
be paving the way for future translational approaches targeting at the center of obesity-induced
inflammation.

4. Adipocyte necrosis and HMGB1 passive release

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Electron microscopy analysis revealed that in all instances, obesity-associated adipocyte death
exhibits features of necrosis, including ruptured plasma membranes, dilated endoplasmic
reticulum, cell debris in the extracellular space, and the appearance of small lipid droplets in the
cytoplasm (Ziegler and Groscurth 2004; Leist and Jaattela 2001; Imai et al. 2004; Cinti et al.
2005). These necrotic adipocytes lose their membrane integrity and release the intracellular
contents such as HMGB1 into the extracellular milieu (Wagner 2014; Rockenfeller et al. 2010),
where they act as alarmins to recruit inflammatory cells and activate resident immune cells. In

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sharp contrast, none of the classical ultrastructural features of apoptosis such as chromatin
condensation, plasma membrane blebbing (zeiosis), or membrane-bound apoptotic bodies with

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nuclear fragments, were found in obese db/db mice (Ziegler and Groscurth 2004; Leist and
Jaattela 2001; Cinti et al. 2005), suggesting that necrosis is likely to be the dominant form of
adipocyte death in obesity.

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Intra-abdominal pressure in obese subjects may serve as a non-pathological factor contributing to

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adipocyte necrosis. Under physical condition, adipocytes in the abdominal cavity are generally
subject to sudden varying pressures, as occurs for example, during cough, abdominal crunch or
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diverse physical exercises, and in particular, obese subjects are manifested by the higher
intra-abdominal pressure (Malbrain et al. 2004; Lambert et al. 2005; Cobb et al. 2005). A visceral
adipocyte in the abdominal cavity is, thus, much more commonly exposed to rupturing forces than
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its siblings in subcutaneous adipose tissue (Monteiro et al. 2006). As a matter of fact, adipocytes
death occurs more frequently in visceral compared with subcutaneous fat depots (Kammoun et al.
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2014).
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Hypoxia in adipose tissue may serve as another driver of adipocyte death in the setting of obesity
(Kammoun et al. 2014). Hypoxia can activate stress pathways such as endoplasmic reticulum (ER)
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and oxidative stress with direct effects on adipocytes (Koumenis et al. 2002). Large adipocytes are
often presence in obese individuals, and their sizes in terms of diameters can reach up to 180µm,
while the diffusion distance of oxygen is only 100–200µm (Ye et al. 2007; Goossens 2008;
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Helmlinger et al. 1997). More importantly, by employing in situ “hypoxyprobe” marker systems
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(such as pimonidazole) and biomarkers (e.g., leptin, plasminogen activator inhibitor type-1, matrix
metalloproteinase-2, and adrenomedullin) to measure WAT hypoxia in obese mice confirmed that
the hypoxic environment leads to devoid of nutrients, which prevents adipocytes to undergo
energy dependent apoptosis instead of necrosis (Rausch et al. 2008; Hosogai et al. 2007; Greijer
and van der Wall 2004). Furthermore, elevated concentration of free fatty acid (FFA) may serve as
a critical factor to induce adipocyte death (Rockenfeller et al. 2010; Wagner 2014). Indeed,
excessive FFA leads to mitochondria dysfunction along with accumulation of reactive oxygen
species (ROS), which then induces adipocyte necrosis. Similarly, inflammatory cytokines such as
TNF-α also induce adipocyte necrosis by increasing the production of ROS (Ventura et al. 2004).

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5. Active secretion of HMGB1 in WAT

Adipose tissue is vital for the life of mammals. Originally, adipose tissue was considered as an
inert depository for excess calories in the form of lipids. It has now been well recognized that
WAT also serves as an endocrine organ (Kammoun et al. 2014). In 1994, one of the first
adipocytes derived hormones, leptin, was cloned and found to be involved in the regulation of
energy homeostasis (Zhang et al. 1994). From then on, the term adipokine was used to describe

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any secreted substance from adipose tissue. An ever expanding number of adipokines have been
discovered including more than 50 cytokines, chemokines, hormone-like factors and other

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mediators (Wozniak et al. 2009; Lago et al. 2009). More recently, HMGB1 was proposed to be an
adipokine that acts as an innate pro-inflammatory mediator in WAT of patients with obesity,
which may be associated with insulin resistance and metabolic syndrome (Shimizu et al. 2016).

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Gunasekaran et al. demonstrated that HMGB1 was expressed 2-fold more in adipose tissue from
obese compared to normo-weight individuals, and LPS increased HMGB1 secretion by

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adipose-derived stem cells (ASCs), but not by adipocytes isolated from human adipose tissue
(Gunasekaran et al. 2013). The human preadipocyte cell line SW872, can actively secrete HMGB1
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(Nativel et al. 2013). Very low level of HMGB1 release in the media could be detected in
undifferentiated 3T3-L1 preadipocytes (Shimizu et al. 2016). However, from day 4 of
differentiation, HMGB1 level in the media of 3T3-L1 adipocytes rapidly increased without any
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stimulation. Moreover, HMGB1 is abundantly localized in the cytoplasm of 3T3-L1 adipocytes

and adipocytes of visceral adipose tissue from the obese patient (Guzman-Ruiz et al. 2014).
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Overall, these findings indicate that HMGB1 is secreted by 3T3-L1 adipocytes, and also by the
lipid-laden adipocytes in WAT of patients with obesity. However, it is yet to be well defined by
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which signaling pathway HMGB1 is released by adipocytes. Inactivation of JNK in adipose tissue
decreased the production of HMGB1 by 30 percent, suggesting that apart from JNK, other
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signaling molecules are also involved in HMGB1 secretion from 3T3-L1 adipocytes (Shimizu et
al. 2016).
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Other than adipocytes, a variety of other cells such as endothelial cells, preadipocytes, fibroblasts,
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and macrophages, are also critical components of white adipose tissue (Gesta et al. 2007; Cancello
and Clement 2006; Subramanian and Ferrante 2009). Gunasekaran et al. reported that HMGB1
production in adipose tissue mainly come from adipose-derived stromal cells (ASCs), which were
able to respond to inflammation by increasing HMGB1 secretion in a time-dependant manner
(Gunasekaran et al. 2013). Similarly, activation of endothelial inflammasomes due to increased
free fatty acids produces HMGB1, which disrupts inter-endothelial junctions and increases
paracellular permeability of endothelium contributing to early onset of endothelial injury during
obesity (Wang et al. 2016). Visfatin is another kind of adipokine, the instigation of endothelial
Nlrp3 inflammasome by visfatin and its subsequent caspase-1 activation trigger the release of
HMGB1 by endothelial cells, which acts in an autocrine or paracrine fashion to promote
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inter-endothelial junction disruption (Chen et al. 2015). Collectively, these results indicate that
obesity is clearly a state of low-grade chronic inflammation, and HMGB1 is definitely implicated
in this pathological process.

6. The direct effect of HMGB1 on macrophages, DCs and adipocytes

Among the immune cells that infiltrate obese adipose tissue, macrophages are functionally and

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numerically dominant (Ferrante 2007; Odegaard and Chawla 2011). The percentage of
macrophages in adipose tissue ranges from under 10% in lean mice and humans to over 50% in

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extremely obese, leptin-deficient mice and nearly 40% in obese humans, indicating that obesity
substantially alters the ratio of macrophages to adipocytes (Weisberg et al. 2003). In general,
macrophages with manifestation of a F4/80+CD11c+CD206− profile are called classically activated

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macrophages (M1), which can be activated either by IFN-γ or LPS to produce pro-inflammatory
cytokines (Sica and Mantovani 2012; Yao et al. 2016). While alternatively activated macrophages
(M2) are F4/80+CD11c−CD206+ and characterized by anti-inflammatory properties, and are

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activated by IL-4 or IL-13 (Rhee 2016). It has been well established that M2 macrophages
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predominate in insulin-sensitive adipose tissue in lean subjects, whereas M1 macrophages
accumulate commensurate with adipose tissue mass in the obese state (Lumeng et al. 2007;
Kosteli et al. 2010; Weisberg et al. 2003). Of note, HMGB1 plays a critical role in polarizing
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adipose-resident macrophages from M2 to an M1 phenotype (Ghosh et al. 2016). In lean subjects,

macrophages are observed interspersed among adipocytes in adipose tissue, whereas in obese
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mice and humans, they form crown-like structures around dead or dying cells. It is noteworthy
that majority of macrophages surround necrotic adipocytes in obese mice and humans suggesting
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that HMGB1 released from those dysfunctional adipocytes may directly activate macrophages
(Andersson and Tracey 2011; Strissel et al. 2007; Cinti et al. 2005). In turn, adipose tissue
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macrophages themselves may contribute to a feed forward mechanism of adipocyte death by

releasing TNF-α and ROS (Wagner 2014).
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Despite its importance as a professional antigen presenting cell, little is known regarding the role
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of dendritic cell (DC) in obese mice or humans (Cildir et al. 2013). One possible reason is that DC
and macrophage present some similar cell surface markers often leading to confusion. For
example, DC expresses CD11c which is also recognized as a marker of M1 pro-inflammatory
macrophages (Kammoun et al. 2014). However, in a recent study, Bertola et al. showed for the
first time the presence and accumulation of specific DCs in adipose tissue and that their numbers
are significantly elevated during obesity (Bertola et al. 2012). It was noted that adipose
tissue-derived HMGB1 activates TLR9 in the adipose recruited plasmacytoid DCs (pDCs) by
transporting extracellular DNA via RAGE (Ghosh et al. 2016). Extracellular HMGB1 is also
highly potent to stimulate DC activation (Messmer et al. 2004; Telusma et al. 2006; Dumitriu et al.
2005; Han et al. 2008), which then induces the expansion of CD4+ T cells as well as Th1 cells in
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adipose tissue, although the antigens responsible for T cell expansion are not clearly defined
(Winer et al. 2009). Th1 cells are suggested to be one of the important cells responsible for
adipose tissue inflammation and insulin resistance (Cildir et al. 2013). Consistent with this notion,
decreased Th1 cells and IFN-γ level in adipose tissue have shown to cause lower adipose tissue
inflammation and increase insulin sensitivity (Winer et al. 2009; Feuerer et al. 2009).

Unlike its effect on macrophages and DCs, the direct impact of HMGB1 on adipocyte viability

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and functionality is yet to be fully addressed. Generally, necrotic adipocytes lose their membrane
integrity and release HMGB1 into extracellular milieu to further impair adipocyte function,

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leading to obesity-associated complications such as insulin resistance and type 2 diabetes (Wagner
2014). While those discoveries are confirmative, controversial results were also reported. Feng
and colleagues reported that HMGB1 regulates autophagy in 3T3-L1 preadipocytes, and increased

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HMGB1 along with activated autophagy in adipose tissue of obese mice may be a protective
mechanism under stressed state induced by high-fat diet (HFD) (Feng et al. 2016). Similarly,

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Rocío et al. reported a positive role of HMGB1 in the regulation of β-cell secretory activity
(Guzman-Ruiz et al. 2014), but this effect could turn into detrimental in response to continuous
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high HMGB1 levels, as occurs in obesity. Overall, additional studies would be necessary to fully
address the above observations and to dissect the underlying mechanisms.
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7. HMGB1 signaling pathways

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Currently, three putative signaling pathways (i.e., RAGE, TLR2 and TLR4) have been recognized
to initiate HMGB1-mediated inflammatory responses (Park et al. 2004; He et al. 2016) (Fig. 3).
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RAGE, a member of the immunoglobulin superfamily of cell surface molecules, has ability to
bind to and transduce the biological effects of advanced glycation endproducts (AGEs), which
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accumulate in settings such as hyperglycemia, aging, inflammation, renal failure, and oxidative
stress (Lopez-Diez et al. 2016; Shekhtman et al. 2017). Except for AGEs, RAGE is also a signal
transduction receptor for non-AGE molecules such as HMGB1 (Schmidt 2017). In adipose tissue
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of obese subjects, it mainly triggers nuclear factor κB (NFκB) activation, a classical inflammatory
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mediator (van Beijnum et al. 2008). Of importantly note, HMGB1 binding to RAGE can
upregulate RAGE signaling, which amplifies inflammation by creating a positive feedback loop
(Yan et al. 1994). Extracellular HMGB1 also contributes to IL-6 secretion through RAGE, which
is one of the mediators that link obesity-derived chronic inflammation with insulin resistance
(Nativel et al. 2013). Particularly, adipose tissue contributes up to 35% of the circulating IL-6
(Nativel et al. 2013), which promotes low-grade inflammation to impair insulin signaling and
weaken insulin sensitivity in visceral fat (Wang et al. 2015). As a result, administration of soluble
RAGE or blocking antibody can induce a protective effect against inflammation and diabetes
(Nativel et al. 2013).

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Other than RAGE, TLR2 and 4 have also been characterized to be receptors for HMGB1. A great
deal of studies have consistently demonstrated that Tlr4 deficiency decreases inflammation and
insulin resistance in adipose tissue (Orr et al. 2012; Poggi et al. 2007; Tsukumo et al. 2016; Saberi
et al. 2009; Tsukumo et al. 2007; Suganami et al. 2007). Consistently, loss of Tlr4 reduces
HFD-induced hepatic steatosis and inflammation along with improved hepatic insulin sensitivity
(Orr et al. 2012; Poggi et al. 2007; Saberi et al. 2009; Tsukumo et al. 2016), and blockade of
TLR4 signaling confers protection against lipid-induced insulin resistance in skeletal muscle

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(Holland et al. 2011; Tsukumo et al. 2016). Similarly, TLR2 mediates FFA-induced
proinflammatory response in cultured adipocytes, macrophages, and myotubes (Konner and

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Bruning 2011; Nguyen et al. 2007; Senn 2006). Ablating TLR2 signaling reduces HFD-induced
lipid accumulation and inflammation in the liver and adipose tissue and generally improves
peripheral insulin sensitivity (Jin et al. 2013). Similar as RAGE signaling, ligation of HMGB1 to

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TLR2 or TLR4 activates NFκB, which then transcribes numerous mediators such as TNF-α, IL-6
and IL-1β to promote inflammatory response in adipose tissue (Park et al. 2003; Gunasekaran et al.

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2016). AN
8. The feasibility of HMGB1 as an early diagnostic biomarker for obesity and therapeutic
target
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Biomarkers are of great importance to provide valuable information for the prediction, diagnosis,
and observation of the therapeutic success of common complex multi-factorial metabolic diseases
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such as obesity and type 2 diabetes (T2D) (Muller 2012). Based on currently available knowledge,
biomarkers used in obesity include body parameters, plasma lipids, cytokines and chemokines
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(Jialal et al. 2014). Diagnosis based on these traditional biomarkers, however, usually takes place
at a late stage of disease development with limited information on disease etiology (Parrizas and
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Novials 2016). As obesity is an important risk factor for many metabolic diseases, knowledge
about early markers may facilitate to refine prevention strategies and to provide a useful tool for
the diagnosis and treatment of obesity and its complications. Therefore, only those biomarkers
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readily identified from easily accessible sources (e.g., blood) and associated with disease etiology
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could meet the demand. In this regard, serum level of HMGB1 emerges as an untapped promise.

Indeed, clinical studies demonstrated a positive correlation between HMGB1 and obesity in
pregnant women, children, and Chinese subjects (Giacobbe et al. 2015; Arrigo et al. 2013; Wang
et al. 2015). Giacobbe and colleagues evaluated serum HMGB1 levels in obese pregnant women,
and identified a direct and statistically significant correlation between BMI of the study group and
HMGB1 levels (Giacobbe et al. 2015). Similarly, Arrigo et al. reported a positive correlation
between serum HMGB1 and obesity in children (Arrigo et al. 2013). Independently, circulating
HMGB1 was noted to be significantly increased not only in obese individuals, but also in T2D
patients (Wang et al. 2015). However, these studies are manifested by some limitations such as
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small example size and the lack of different ethnicities. Therefore, larger sample size coupled with
different populations should be considered in the future study to further confirm the feasibility of
HMGB1 as a viable biomarker for early diagnosis of obesity.

Since HMGB1 plays a critical role in obesity-induced inflammation, antagonizing HMGB1

inflammatory pathway could bring on new therapeutic targets to counteract obesity-associated
pathologies. For example, neutralization of HMGB1 appears to reduce weight gain and liver

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inflammation in mice fed an obesogenic diet (Montes et al. 2015). Treating apoE-deficient mice
with anti-HMGB1 neutralizing antibody results in decreased lesions, as well as a reduction in
many inflammatory cell types such as dendritic cells, macrophages and CD4+ T cells in

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atherosclerotic lesions (Kanellakis et al. 2011). Metformin, a biguanide derivative, is the first-line
drug in the treatment of type 2 diabetes (Foretz et al. 2014). While recently, Horiuchi and
colleagues, identified HMGB1 as a novel metformin-binding protein and demonstrated that

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metformin inhibited cytokine activity of HMGB1 in vitro and in vivo (Horiuchi et al. 2017). This
is the first report of an extracellular function of metformin that regulates inflammation induced by
HMGB1, which suggests possible new ways to manage HMGB1-induced inflammation in obesity.

9. Conclusion remarks
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Obesity has emerged as an imminent global public health concern over the past several decades. It
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is featured by the persistent and low-grade inflammation, and serves as a causative factor for many
secondary diseases including T2D, cardiovascular disease, high cholesterol, and infertility. As an
innate alarmin once released from necrotic adipocytes, HMGB1 stimulates the activation of
D

resident immune cells in the adipose tissue. Upon activation, those resident immune cells actively
TE

secrete additional HMGB1, which then activates/recruits additional immune cells, and acts on
adipocytes in autocrine, paracrine or systemic fashion. Therefore, HMGB1 plays a critical role in
the initiation and maintenance of chronic inflammatory state in adipose tissue of obese subjects.
EP

There is also feasible evidence that HMGB1 could be a viable biomarker for early diagnosis of
obesity and therapeutic target for prevention and intervention of obesity-induced inflammation.
However, additional studies would be necessary to warrant its application in clinical settings.
C
AC

Acknowledgements
Our work was supported by the National Natural Science Foundation of China (81530024,
81471046, 81470988, 81500637 and 81670729), the Program Project for Type 1 Diabetes from
the Ministry of Science and Technology (2016YFC1305002), the Integrated Innovative Team for
Major Human Disease Programs of Tongji Medical College, Huazhong University of Science &
Technology, and the Innovative Funding for Translational Research from Tongji Hospital.

11
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Figure legends

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Fig. 1. The composition of immune cells in lean versus obese adipose tissue. In the lean state, the
major immune cells in adipose tissue are eosinophils, Th2 cells, Treg cells, and M2-like
macrophages. Eosinophils and Th2 cells promote an anti-inflammatory milieu through the
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secretion of IL-4, IL-10 and IL-13, supporting an M2-like phenotype of macrophages. In the obese
adipose tissue, adipocytes secret chemokines such as MCP-1, in response to proinflammatory
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signals (e.g., TNF-α), to recruit circulating monocytes into adipose tissue, which then differentiate
into adipose tissue macrophages with an M1-like phenotype. Other than macrophages, neutrophils
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are also recruited from circulation probably through an inflammatory chemokine-dependent

manner. Obesity also promotes a shift in T cell populations with a decrease in Treg population and
an increase in CD4+ and CD8+ effector T cells. B cells release pathogenic IgG2c type antibodies
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and are also found in adipose tissue of obesity individuals. Adipocytes, mast cells, and T cells
contribute to inflammation by releasing specific adipokines and cytokines. M1: M1 macrophages;
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M2: M2 macrophages; Treg: T regulatory cells; NEU: neutrophils; MC: mast cells; and EOS:
eosinophils.
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Fig. 2. An overview of HMGB1 in the induction and maintenance of chronic inflammation in

adipose tissue of obese subjects. In the state of obesity, elevated HMGB1 can either passively
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released from necrotic adipocytes or actively secreted by living adipocytes, activated

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immunocytes, and adipose-derived stromal cells such as endothelial cells. HMGB1 activates DCs
in adipose tissue, which stimulate the expansion of inflammatory Th1 cells. Upon activation, the
recruited pDCs induce the production of type I interferons to promote macrophage M1 program.
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Macrophages, which play an essential role in obesity-induced inflammation, can sense the
increased HMGB1 and produce ROS, TNF-α as well as many other proinflammatory cytokines to
further impair the function of adipocyes.
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Fig. 3. A summary of HMGB1 signaling pathways in adipose tissue. During the development of
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obesity-induced inflammation, RAGE and TLRs can be activated by the extracellular HMGB1
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(DAMP) as well as free nucleic acid molecules from necrotic adipocytes. HMGB1 is well known
to mediate RAGE, TLR2 and TLR4 activation. In addition, accumulated HMGB1 and DNA
owing to increased adipocytes death and adipose tissue remodeling can be sensed by TLR9. Upon
activation in this case, they mainly trigger NFκB activation to initiate the transcription of various
proinflammatory cytokines including TNF-α, IL-6 and IL-1β to promote inflammation in obesity.

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Highlights:

Obesity is characterized by a chronic, low-grade inflammation in local and systemic sites.

Both necrotic adipocytes and activated immune cells in adipose tissue account for the
increased HMGB1 level in obese subjects.
HMGB1 initiates and maintains chronic inflammatory state in adipose tissue through RAGE,
TLR2 and TLR4 signaling pathways.
HMGB1 could be a viable biomarker for early diagnosis of obesity and therapeutic target for

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prevention and intervention of obesity-induced inflammation.

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