UMass Chan Medical School UMass Chan Medical School
eScholarship@UMassChan eScholarship@UMassChan
Open Access Publications by UMMS Authors
2017-01-01
HMGB1, IL-1alpha, IL-33 and S100 proteins: dual-function HMGB1, IL-1alpha, IL-33 and S100 proteins: dual-function
alarmins alarmins
Damien Bertheloot
University of Bonn
Et al.
Let us know how access to this document bene<ts you.
Follow this and additional works at: https://escholarship.umassmed.edu/oapubs
Part of the Cellular and Molecular Physiology Commons, and the Immunity Commons
Repository Citation Repository Citation
Bertheloot D, Latz E. (2017). HMGB1, IL-1alpha, IL-33 and S100 proteins: dual-function alarmins. Open
Access Publications by UMMS Authors.
https://doi.org/10.1038/cmi.2016.34. Retrieved from
https://escholarship.umassmed.edu/oapubs/3078
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.
This material is brought to you by eScholarship@UMassChan. It has been accepted for inclusion in Open Access
Publications by UMMS Authors by an authorized administrator of eScholarship@UMassChan. For more
information, please contact
Lisa.Palmer@umassmed.edu.
OPEN
REVIEW
HMGB1, IL-1α, IL-33 and S 100 proteins: dual-function
alarmins
Damien Bertheloot
1
and Eicke Latz
1,2,3
Our immune system is based on the close collaboration of the innate and adaptive immune systems for the rapid
detection of any threats to the host. Recognition of pathogen-derived molecules is entrusted to specific germline-
encoded signaling receptors. The same receptors have now also emerg ed as efficient detectors of misplaced or
altered self-molecules that signal tissue damage and cell death following, for example, disruption of the blood
supply and subsequent hypoxia. Many types of endogenous molecules have been shown to provoke such sterile
inflammatory states when released from dying cells. However, a group of proteins referred to as alarmins have both
intracellular and extracellular functions which have been the subject of intense research. Indeed, alarmins can
either exert beneficial cell housekeeping functions, leading to tissue repair, or provoke deleterious uncontrolled
inflammation. This group of proteins includes the high-mobility group box 1 protein (HMGB1), interleukin (IL)-1α,
IL-33 and the Ca
2+
-binding S100 proteins. These dual-function proteins share conserved regulatory mechanisms,
such as secretory routes, post-translational modifications and enzymatic processin g, that govern their extracellular
functions in time and space. Release of alarmins from mesenchymal cells is a highly relevant mechanism by which
immune cells can be alerted of tissue damage, and alarmins play a key role in the development of acute or chronic
inflammatory diseases and in cancer development.
Cellular & Molecular Immunology (2017) 14, 43–64; doi:10.1038/cmi.2016.34; published online 29 August 2016
Keywords: alarmin; HMGB1; IL-1α;IL-33;inflammation; S100 proteins
INTRODUCTION
The immune recognition of an infection and the subsequent
battle against the infecting pathogen is governed by the
concerted efforts of both the innate and the adaptive immune
systems. The first sensing of microbial invasion requires
germline-encoded signaling receptors, also known as pattern-
recognition receptors (PRRs), that evolved to acquire sensing
specificity for foreign signature molecules generally termed
pathogen-associated molecular patterns. This system was first
theorized by Charles Janeway
1
25 years ago, and has since been
extended to include the more recently recognized ability of the
innate immune system to also sense tissue damage by
recognizing mislocalized or altered endogenous molecules
termed damage-associated molecular patterns (DAMPs).
2,3
These molecules found in a foreign environment trigger
sterile inflammation and, in the best case, promote tissue repair
and the resolution of the inflammation. However, innate
immune responses can also fuel uncontrolled or chronic
inflammation. Since the introduction of the DAMP model by
Polly Matzinger,
2
many molecules that are released during
proinflammat ory cell death have been ascribed a DAMP
function. These include heat-shock proteins (HSPs), adenosine
triphosphate (ATP), nucleosomes, mitochondrial components
and several alarmins: dual-function proteins that have distinct
roles inside or outside the cells. The term alarmin describes
protein functions leading to a rapid inflammatory response upon
the release of these biologically active molecules. Notably,
alarmins are also found in the nucleus in the resting state and
likely exert biologically meaningful, yet understudied, functions.
For example, failure of interleukin (IL)-33 to translocate to the
nucleus results in a lethal inflammatory response, suggesting that
the nuclear localization of, in this case, IL-33 has a protective
function.
4
In this review, we summarize recent knowledge on
the most thoroughly studied dual-function proteins, the high-
mobility group box 1 (HMGB1) protein, IL-1α, IL-33 and S100
proteins, with a focus on their extracellular functions.
1
Institute of Innate Immunity, University Hospitals, University of Bonn, 53127 Bonn, Germany;
2
German Center for Neurodegenerative Diseases (DZNE),
53117 Bonn, Germany and
3
Department of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, MA 01605, USA
Correspondence: Professor Dr Med E Latz, Institute of Innate Immunity, University Hospital, Sigmund-Freud-Strasse 25, 53127 Bonn, Germany.
E-mail: eicke.latz@uni-bonn.de
Received: 22 February 2016; Revised: 16 May 2016; Accepted: 17 May 2016
Cellular & Molecular Immunology (2017) 14, 43–64
&
2017 CSI and USTC All rights reserved 2042-0226/17
www.nature.com/cmi
HIGH-MOBILITY GROUP BOX 1
HMGB1 is one of the most abundant nonhistone nuclear
proteins and is a member of the HMG protein family that
contributes to chromatin architecture and modulates gene
expression. Once released from stromal or immune cells, and
upon interaction with a large panel of cell surface receptors,
HMGB1 exerts a plethora of cell regulatory functions, from
maturation, proliferation and motility to inflammation, survi-
val and cell death (Figure 1).
HMGB1 expression, cellular localization and post-
translational modifications
In resting cells, HMGB1 is localized in the nucleus, where it
exerts an important function for chromatin structure and gene
expression (Figure 1).
5,6
HMGB1 is involved in chromosomal
DNA repair and contributes to nucleosome mobility by
promoting histone sliding along the DNA strand. Conse-
quently, Hmgb1 gene knockout is lethal, further arguing for
the importance of HMGB1. Structurally, HMGB1 is composed
of two DNA-binding HMG-box domains, namely Box A and
Box B, followed by a flexible and negatively charged C-terminal
tail. The C-terminal tail is believed to mediate a change in the
three-dimensional structure of HMGB1 from a collapsed to a
more linear conformation that most likely regulates HMGB1
binding to its ligands.
7
Interestingly, post-translational mod-
ifications, such as acetylation, methylation and phosphoryla-
tion, have been shown to govern HMGB1 cellular localization
(Figure 1).
8–11
Upon activation, monocytes and macrophages
were found to hyperacetylate HMGB1 at nuclear localization
sites, leading to its cytosolic relocalization. This process was
Figure 1 Role of HMGB1 in inflammation. Under resting conditions, HMGB1 is localized in the nucleus, where it plays an important role
in chromatin structure and gene expression. The translocation of HMGB1 to the cytoplasm is regulated by post-translational modifications
such as acetylation, methylation and phosphorylation (1). Because of the lack of a secretion signal, HMGB1 is actively secreted through a
caspase-1-dependent, noncanonical secretory vesicular pathway (2). HMGB1 can also be passively released from damaged cells either
alone or in complex with RNA, DNA or nucleosomes (3). Interestingly, during apoptotic cell death, ROS production induces the terminal
oxidation of HMGB1 that inhibits its proinflammatory function and switches HMGB1 function toward tolerogenicity (4). Once in the
extracellular space, HMGB1 binds to several receptors in either free or complexed form (5). HMGB1 receptors, including RAGE and TLR4,
bind free HMGB1 or HMGB1 in complex with DNA or LPS. Through its interaction with RAGE, the internalization of the HMGB1–DNA
complex increases the activation of TLR9 localized in the endosome. However, HMGB1 complex formation with nucleic acids and
potentially with other molecules can be inhibited by direct interaction with TIM-3. Other receptors, such as TLR2, IL-1R and CXCR4,
recruit HMGB1 in complex with nucleosomes, IL-1β or CXCL12, respectively. Thus, sensing of HMGB1 mediates mechanisms of
inflammation, cell migration, proliferation and differentiation (6). Furthermore, acting through the CXCL12/CXR4 axis, HMGB1 enhances
chemotaxis (7). HMGB1, high-mobility group box 1 protein; IL, interleukin; LPS, lipopolysaccharide; RAGE, receptor for advanced glycation
end-products; ROS, reactive oxygen species; TLR, Toll-like receptor.
Dual-function alarmins in the heart of inflammation and disease
D Bertheloot and E Latz
44
Cellular & Molecular Immunology
recently shown to be mediated by the activation of the
JAK/STAT1 (Janus kinase/signal transducer and activator of
transcription 1) pathway.
12
In addition, in monocytes, HMGB1
cytoplasmic localization can be regulated by the phosphoryla-
tion of its nuclear localization signal,
11
and this was found to
depend on protein kinase C activity.
9
Furthermore, in neu-
trophils, HMGB1 cytosolic translocation seems to depend on
the methylation of Lys42 that decreases HMGB1 binding
affinity to DNA and thus enables its translocation to the
cytosol.
8
These studies investigated the role of each modifica-
tion in a rather exclusive manner. It is therefore difficult to
conclude whether these mechanisms regulating HMGB1 cel-
lular localization function in parallel or are differentially
modulated in a cell type- or cell activation-dependent manner.
Because the nuclear localization of HMGB1 is likely to act as a
regulatory mechanism with regard to HMGB1 extracellular
function, a more integrative study of HMGB1 post-
translational modification will be necessary to better compre-
hend the regulation of HMGB1 cytosolic translocation before
its release.
Adding to the complexity of HMGB1 activity, the redox state
of HMGB1 is believed to orchestrate its extracellular
function.
13
HMGB1 possesses three cysteines at positions 23,
45 and 106. These enable three different oxidation states of
HMGB1 that license three mutually exclusive functions:
alarmin, chemoattraction or tolerance.
14–16
In fact, the forma-
tion of a disulfide bond between Cys23 and Cys45 was shown
to confer proinflammatory properties to HMGB1.
14,15
Reduced
HMGB1, with all cysteines in the thiol state, loses its alarmin
function and behaves as a chemoattractant.
15
In addition, upon
cellular stress or apoptotic cell death, reactive oxygen species
(ROS) production by mitochondria leads to the terminal
oxidation of HMGB1 (sulfonate cysteines), granting HMGB1
tolerogenic properties that seem to mainly depend on Cys106
(Figure 1).
16
Consequently, post-translational modifications orchestrate
HMGB1 activity from cell localization to extracellular function
and thus act as a crucial functional switch.
HMGB1 release and extracellular functions
HMGB1 can be released either passively by necrotic and
damaged cells or by active mechanisms triggered upon immune
cell activation. Once released in the extracellular space,
HMGB1 mediates inflammation, cell migration, proliferation
and differentiation (Figure 1).
17–19
In fact, extracellular
HMGB1 was shown to act as a chemoattractant for myeloid
cells,
20
smooth muscle cells (SMCs)
21
and mesoangioblasts,
thereby promoting muscle tissue repair.
22
HMGB1 is considered to be one of the most mobile nuclear
proteins and interacts only very transiently with chromosomal
DNA. This loose nuclear DNA binding enables the leakage of
HMGB1 upon cell damage or necrosis.
5,23
In contrast, during
apoptosis, HMGB1 was long believed to be trapped inside the
nucleus, where it binds strongly to hypoacetylated chromatin.
23
However, it was since demonstrated that oxidized HMGB1 can
be released from late-stage apoptotic cells, thereby promoting
tolerance in a caspase-1-dependent manner.
16,24,25
It was also
proposed that macrophages secrete HMGB1 following the
phagocytosis of apoptotic cells,
26
thus further challenging the
idea that apoptosis is a ‘silent’ death.
In addition to the cell death-dependent release of HMGB1,
which likely represents its most important role, immune cells
such as macrophages and monocytes are known to actively
secrete HMGB1 once stimulated by cytokines (interferon-γ
(IFNγ), tumor necrosis factor (TNF) and IL-1) or pathogen-
derived molecules (lipopolysaccharide (LPS)).
27–30
Moreover,
macrophages release HMGB1 following the activation of the
NLRP3 or NLRC4 inflammasomes.
27,28,31
Because HMGB1
lacks a leader sequence that would enable its transfer to the
endoplasmic reticulum (ER) and the Golgi, active secretion of
HMGB1 follows a nonclassical vesicular pathway.
32
Indeed,
several proteins, including not only HMGB1 but also IL-1β and
IL-18, have been shown to depend on autophagy for their
active secretion through the unconventional secretory
pathway.
33,34
Interestingly, following cell starvation, HMGB1
was proposed to enhance autophagy through its interaction
with Beclin1, and this was dependent on ROS production and
the HMGB1 redox state.
35
HMGB1 cytoplasmic translocation
both promoted autophagy and limited the apoptotic pathway.
Thus, HMGB1 cytoplasmic translocation not only enables
release but also can actively promote its own secretion.
Several receptors have been shown to trigger downstream
signaling upon binding to HMGB1 either directly or in
complex with other molecules (Figure 1). In macrophages
and dendritic cells, HMGB1 binds directly to Toll-like receptor
4 (TLR4) and induces the secretion of proinflammatory
cytokines.
36
Interestingly, binding of HMGB1 to TLR4 depends
on reduced Cys106.
14,36
Moreover, the activation of TLR4 by
proinflammatory HMGB1 (disulfide form) was recently shown
to require myeloid differentiation factor 2 (MD-2).
37
Upon
binding to TLR4-MD-2, HMGB1 triggers the MyD88-
dependent activation of nuclear factor (NF)-κB and the
subsequent release of proinflammatory cytokines (i.e., TNFα,
IL-1β and IL-6). Intriguingly, HMGB1 can also bind directly to
LPS, thereby strengthening its ability to activate TLR4 through
CD14.
38
In contrast, binding of HMGB1 to CD24 and the
further mobilization of Siglec-10 (or mouse Siglec-G) was
shown to antagonize HMGB1-induced TLR4 activation in
dendritic cells.
39
In this study, Chen et al.
39
found that the
effect of CD24/Siglec-10 was restricted to stimulation with
endogenous proteins (HMGB1, HSP70 and HSP90) and did
not hold upon the activation of cells with pathogen-derived
molecules. Hence, the authors proposed that the CD24/Siglec-
10 pathway is a self-regulatory mechanism, limiting deleterious
immune activation by endogenous damage-signaling
molecules.
Another important cell surface receptor for HMGB1 is the
receptor for advanced glycation end-products (RAGE). In fact,
HMGB1 binding to RAGE was demonstrated shortly after
RAGE discovery
40
and has since been characterized in many
settings. First, RAGE proved to mediate the previously
described role of HMGB1 in neurite outgrowth during the
Dual-function alarmins in the heart of inflammation and disease
D Bertheloot and E Latz
45
Cellular & Molecular Immunology
development of the nervous system.
40,41
In dendritic cells,
HMGB1 release and sensing by RAGE was shown to be critical
for homing to the lymph nodes and further cross-activation of
T lymphocytes.
42–44
In endothelial cells, HMGB1 was further
shown to promote the expression of RAGE and surface
adhesion proteins (intercellular adhesion molecule 1 (ICAM-1)
and vascular cell adhesion molecule 1 (VCAM-1)) and also to
induce RAGE-dependent cytokine production.
45–47
In addition to TLR4 and RAGE, HMGB1 interacts with
several more receptors once in complex with other molecules.
Indeed, extracellular HMGB1 can interact with IL-1β,
48
CXCL12,
49
and nucleosomes,
50
thereby promoting the activa-
tion of IL-1 receptor (IL-1R), CXCR4 and TLR2, respectively.
Furthermore, extracellular HMGB1, like other HMGB pro-
teins, is found in complex with either DNA or RNA, promot-
ing sensing by their putative receptors.
51–53
Interestingly, yet
another receptor of HMGB1, TIM-3, expressed at the surface
of tumor-associated dendritic cells, was recently shown to
compete with nucleic acids for binding to HMGB1, thereby
dampening the efficacy of antitumor DNA vaccines or
chemotherapy.
54
These studies revealed the importance of
controlling the purity of the recombinant HMGB1 protein
used in experiments. In fact, several publications have argued
that high-purity HMGB1 has a limited proinflammatory
activity on its own.
48,51,53,55
Yet, the propensity of HMGB1
to co-purify with innate immune stimulants is not the only
characteristic that can influence its biological function. One has
to take into account that different HMGB1 preparations can
vary in their amount of protein oxidation, thus influencing the
biological activity of recombinant HMGB1. In addition,
although HMGB1 redox status has now been described as a
major regulatory mechanism of HMGB1 extracellular function,
the role of other known post-translational modifications of
HMGB1 in the extracellular space remains unknown. Hence, in
the future, it would be beneficial to further analyze the
extracellular functions of HMGB1, paying special attention to
its purity and to the exact nature of its post-translational state.
HMGB1 in sepsis and sterile injury
HMGB1 was shown to mediate several acute effects in the
context of infection or sterile tissue damage following myo-
cardial infarction, stroke, acute lung injury or ischemia–
reperfusion injury often occurring after transplantation and
trauma. During sepsis, HMGB1 acts as a late mediator of
inflammation that can be sustained for several days and
correlates with an unfavorable prognosis.
29
In fact, upon
infection, the presence of large amounts of pathogen-derived
molecules, such as endotoxin, induces a biphasic secretion of
cytokines:
56
(1) the release of conventional proinflammatory
cytokines, such as TNFα and IL-1β (peak levels are found
within hours) and (2) the late release of HMGB1 that often
leads to lethality. Interestingly, treatment of animals with anti-
HMGB1 antibodies or the HMGB1-binding antagonist throm-
bomodulin provides protection even when administered several
hours after the peak secretion of the early cytokines.
45,57
In
accordance with these findings, conditional deletion of
HMGB1 in myeloid cells protected mice against LPS-induced
endotoxic shock.
58
Furthermore, a recent clinical study in
which septic patients were treated with a combination of
Polymyxin B and thrombomodulin found improved survival of
the treated cohort.
59
These data argue that HMGB1 may
represent a pharmacological target for the treatment of septic
shock. However, a study with larger groups and across different
microbes and infection routes will be necessary before large-
scale use in the clinic.
It is worth noting that the treatment of septic animals with
recombinant HMGB1 A box, an antagonist competing with
full-length HMGB1 for receptor binding, was also shown to
provide protection against sepsis.
60
In addition, the use of a
monoclonal anti-RAGE antibody after cecal ligation and
puncture (CLP) reduced lethality even when it was adminis-
tered 36 h after CLP.
61
This study followed the previous work
by Liliensiek et al.,
62
who first indicated a role for RAGE in the
development of acute inflammation during sepsis. In addition
to RAGE, the effects of HMGB1 in sepsis are believed to be
partly mediated through TLR4.
37,38
Further reinforcing this
hypothesis, treatment of CLP-elicited septic mice with a specific
inhibitor of HMGB1 binding to MD-2 (P5779) decreased
lethality.
37
However, as mentioned earlier, binding of HMGB1
to LPS strengthens TLR4 activation and thus could potentially
contribute to HMGB1-induced acute inflammation following
bacterial infection.
In contrast to its role during sepsis, in the context of sterile
tissue damage and cell death, HMGB1 mediates early inflam-
mation responses that only last hours. Indeed, HMGB1 levels
increase in the circulation following major events such as
stroke, myocardial infarction or hemorrhagic shock. These
conditions induce an ischemia–reperfusion injury in tissues
where HMGB1 is passively released.
63–69
In these settings, both
TLR4- and RAGE-dependent inflammatory pathways mediate
the early effect of HMGB1 release. Indeed, in the ischemic liver
and kidney, TLR4 was shown to trigger inflammation in
response to HMGB1 release.
64,65
Furthermore, RAGE
expressed at the surface of microglial cells was found to
mediate part of the deleterious function of extracellular
HMGB1 in the ischemic brain.
68
Strikingly, following brain
injury, released HMGB1 was found to signal through RAGE in
the lung, thereby mediating pulmonary dysfunction after lung
transplantation.
69,70
In accordance with this finding, HMGB1
release from gut epithelial cells was recently shown to mediate
lung injury following trauma and hemorrhagic shock.
71
These
studies illustrate the ability of HMGB1 to mediate its alarmin
function in a systemic manner across the entire body. Here
again, collaboration between RAGE and TLR4 is responsible
for the deleterious effects of HMGB1 in acute sterile inflam-
mation. Hence, direct targeting of HMGB1 with antibodies or
thrombomodulin will probably be the most appropriate treat-
ment strategy for transplanted patients or for those suffering
from a traumatic injury. In fact, anti-HMGB1 antibodies have
already shown some success in animal models of sterile
inflammation and could therefore be promising for use in
humans.
68,72,73
Dual-function alarmins in the heart of inflammation and disease
D Bertheloot and E Latz
46
Cellular & Molecular Immunology