Review
IL-6 pathway in the liver: From physiopathology to therapy
Dirk Schmidt-Arras, Stefan Rose-John⇑
Interleukin 6 (IL-6) is a pleiotropic four-helix-bundle cytokine that exerts multiple functions in the body. In the liver, IL-6 is an important inducer of the acute phase response
and infection defense. IL-6 is furthermore crucial for hepatocyte homeostasis and is a
potent hepatocyte mitogen. It is not only implicated in liver regeneration, but also in
metabolic function of the liver. However, persistent activation of the IL-6 signaling pathway is detrimental to the liver and might ultimately result in the development of liver
tumors. On target cells IL-6 can bind to the signal transducing subunit gp130 either in
complex with the membrane-bound or with the soluble IL-6 receptor to induce intracellular signaling. In this review we describe how these different pathways are involved in
the physiology and pathophyiology of the liver. We furthermore discuss how IL-6 pathways can be selectively inhibited and therapeutically exploited for the treatment of liver
pathologies.
Ó 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All
rights reserved.
Keywords: Interleukin-6; Interleukin-6receptor; Trans-signaling; Inflammation; Cancer; Liver regeneration.
Received 16 December 2015; received in
revised form 15 January 2016; accepted 3
February 2016
Abbreviations: EGF, epidermal growth
factor; EGFR, epidermal growth factor
receptor; Fc, constant portion of an IgG
antibody; gp130, glycoprotein 130 kDa;
IL, interleukin; JAK1, Janus kinase 1;
JNK1, Jun amino-terminal kinase 1;
LPS, lipopolysaccharide; R, receptor; s,
soluble; SOCS3, suppressor of cytokine
signaling 3; STAT, signal transducer and
activator of transcription; TLR, Toll-like
receptor; TNF-a, tumor necrosis factor a.
Introduction
Interleukin-6 and gp130 signal transduction
Interleukin-6 (IL-6) is a four-helical cytokine of
184 amino acids [1]. The protein is synthesized
by fibroblasts, monocytes, macrophages, T cells
and endothelial cells. IL-6 synthesis and secretion is induced during inflammatory conditions
such as upon stimulation of Toll-like receptor
(TLR)-4 by lipopolysaccharide or upon stimulation of cells by IL-1 or tumor necrosis factor
(TNF)-a [1]. When the IL-6 cDNA was molecularly cloned as B cell stimulating factor 2, it
turned out that IL-6 was identical to the 26 kDa
protein and to hybridoma growth factor [2].
On target cells, IL-6 binds to the 80 kDa
Interleukin-6 receptor (IL-6R), which is not signaling competent. Signaling is initiated upon
association of the IL-6/IL-6R complex with a second receptor protein, glycoprotein (gp) 130.
Gp130 dimerization leads to the activation of
the tyrosine kinase JAK1, which is constitutively
bound to the cytoplasmic portion of gp130 [3].
After autophosphorylation, JAK1 phosphorylates
five tyrosine residues within the cytoplasmic
portion of gp130. This leads to the activation of
several intracellular signaling pathways including the MAP kinase and PI3 kinase pathway and
the signal transducer and activator of transcrip-
tion (STAT) 1 and STAT3 pathway. Subsequently,
STAT3 is able to upregulate suppressor of
cytokine signaling (SOCS) 3, which leads to a
downregulation of gp130 signals and thereby
represents a negative feed-back loop [3,4].
IL-6 trans-signaling
Importantly, it was shown that IL-6 has only a
measurable affinity for the IL-6R but not for
gp130. Likewise, the IL-6R has no measurable
affinity for gp130 [3]. Only when the IL-6/IL-6R
complex is formed, binding to gp130 can be
demonstrated [3]. This has important consequences. Whereas gp130 is expressed on all cells
of the body, the IL-6R is only expressed on few
cell types such as hepatocytes, some leukocytes
and some epithelial (e.g. biliary epithelial cells)
and non-epithelial cells (e.g. hepatic stellate
cells) (Fig. 1A). Therefore, only IL-6R expressing
cells can directly respond to the cytokine IL-6 [3].
Interestingly, the IL-6R was shown to be
cleaved at the cell surface by the metalloprotease
a disintegrin and metalloprotease (ADAM) 17, a
process called shedding [5]. The shed soluble
IL-6R (sIL-6R) could still bind its ligand IL-6.
The complex of IL-6 and sIL-6R was able to
associate with gp130 and initiate intracellular
Institute of Biochemistry, ChristianAlbrechts-University Kiel, Olshausenstrasse 40, Kiel, Germany
Key point
On target cells, IL-6 can signal
via the IL-6 classic or IL-6
trans-signaling pathway.
⇑ Corresponding author. Address: Institute of Biochemistry, Christian-AlbrechtsUniversity Kiel, Olshausenstrasse 40,
D-24098 Kiel, Germany. Tel.: +49 431
880 3336; fax: +49 431 880 5007.
E-mail address: rosejohn@biochem.
uni-kiel.de (S. Rose-John).
Journal of Hepatology 2016 vol. xxx j xxx–xxx
Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
Review
Summary
Review
Review
signaling [6]. Strikingly, this could also be
demonstrated to occur on cells, which did not
express the membrane-bound IL-6R [6]. This process has been named IL-6 trans-signaling
(Fig. 1B). Therefore, IL-6 trans-signaling has the
potential to largely increase the spectrum of
target cells of IL-6 [7].
Hyper-IL-6 is an artificial fusion protein of
IL-6 coupled to the sIL-6R via a flexible peptide
linker [8] (Fig. 1C). It has been shown to be
100–1000 times more potent than the separate
proteins IL-6 and sIL-6R. Hyper-IL-6 has been used
to analyze the responsiveness of cells to IL-6 or to
the trans-signaling complex IL-6/sIL-6R. It turned
out that many cell types including neurons [9],
glial cells [10], endothelial cells [11], smooth
muscle cells [12], hematopoietic stem cells [13]
and embryonic stem cells [14] do not express
IL-6R and are therefore dependent on transsignaling in their response to IL-6.
A fusion protein of the entire extracellular
portion of gp130 coupled to the Fc portion of a
human IgG1 antibody (sgp130Fc) turned out to
be a selective inhibitor of IL-6 trans-signaling
[15] (Fig. 1D). IL-6 signaling via the membranebound IL-6R was not affected by this protein.
This surprising effect could be explained by the
fact that neither IL-6 nor IL-6R alone showed a
measurable affinity for gp130 [15].
Under normal conditions, IL-6 levels in the
blood are extremely low (1–5 pg/ml). Surprisingly, sIL-6R in the blood has been found at concentrations of 40–70 ng/ml. And a soluble form
of gp130 (sgp130), which is generated by alternative splicing, is found at concentrations of
around 400 ng/ml [16]. We have argued that
the sIL-6R and sgp130 constitute a buffer in the
blood since IL-6, once secreted will bind to the
sIL-6R with an affinity of 1 nM. Thereupon,
the complex of IL-6/sIL-6R binds to sgp130 with
a hundred times higher affinity (10 pM) and is
neutralized. Only when IL-6 levels exceed the
sIL-6R concentration, IL-6 can bind to
membrane-bound IL-6R on target cells [17,18].
This concept might, however, not be fully valid
in case of paracrine activity of IL-6 e.g. in the liver
where activated Kupffer cells secrete IL-6 and
neighboring hepatocytes respond to a strong
local increase in IL-6.
(CRP), serum amyloid A (SAA), haptoglobin and
fibrinogen. Functionally, some acute phase proteins are components of the complement system
and of the coagulation cascade. Other acute
phase proteins are protease inhibitors, transport
proteins or participants in inflammatory
responses, such as secreted phospholipase A2
[20,21]. As already mentioned, the major inducer
of the hepatic acute phase proteins is the cytokine IL-6, which is secreted by neutrophils,
monocytes and macrophages upon TLR stimulation by e.g. lipopolysaccharide [20,21]. Activated
myeloid cells in addition release the inflammatory cytokines IL-1 and TNF-a, which can lead
to massive production and secretion of IL-6 from
other cells such as endothelial cells and fibroblasts thereby functioning as a positive feedforward loop [22]. Interestingly, in IL-6 knockout
mice, the acute phase response is largely inhibited upon injection of turpentine, whereas it is
almost normal upon injection of lipopolysaccharide [23]. This might be due to compensation by
other IL-6 type cytokines such as Interleukin-11,
leukemia inhibitory factor, oncostatin M, ciliary
neurotrophic factor, and cardiotrophin 1, which
have been demonstrated to induce the synthesis
of acute phase proteins in hepatocytes or hepatoma cells. It is therefore believed that members
of this cytokine family are actors of liver physiopathology [24,25].
Although not all functions of the acute phase
proteins are known, their induced expression is
thought to be beneficial for the response of the
body to infectious insults and inflammation
[21]. Clinically, the extent of the acute phase protein levels such as CRP is used to measure the
extent of the inflammatory condition. 80–85%
of patients who show CRP levels of more than
100 mg per liter are diagnosed with bacterial
infections [26].
The duration of the acute phase response is
normally 24–48 h, after which the organism
returns to normal liver function. Under severe
conditions, such as advanced cancer and the
acquired immunodeficiency syndrome, the acute
phase can convert to a chronic state of inflammation although the molecular mechanisms of such
a chronicity are not completely understood
[20,21].
IL-6 and the acute phase response
IL-6 and liver regeneration
More than 25 years ago, it was found that
Interleukin-6 was also identical with hepatocyte
stimulating factor 2. Under inflammatory conditions, this factor was known to induce the liver
to synthesize a group of proteins called acute
phase proteins [19].
In humans the acute phase proteins, which
are most induced, include C-reactive protein
The most important reaction of the liver to injury
is liver regeneration [27,28] and it became clear
from experiments with parabiotic animals that
soluble extrahepatic factors provide the stimulus
for hepatocyte proliferation [29]. Only 2 h after
hepatectomy, the level of TNF-a increased followed by a dramatic upregulation of IL-6 levels
in the liver vein (Fig. 2A) [30]. After hepatectomy
Key point
While IL-6 classic signaling is
crucial for the induction of the
acute phase response, IL-6
trans-signaling mediates strong
mitogenic signals during liver
regeneration.
2
Journal of Hepatology 2016 vol. xxx j xxx–xxx
Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
JOURNAL OF HEPATOLOGY
A
B
HC
C
IL-6R
D
ADAM17
sIL-6R
BEC
IL-6
IL-6
sIL-6R
gp130
HSC
gp130 ECD
Flexible
peptide
linker
EC
KC
}
IgG1 Fc
IL-6R
IL-6 transsignaling
Fig. 1. Interleukin 6 (IL-6) signals via two distinct pathways on target cells. (A) Expression of IL-6R and gp130 on different cell types of the liver. HC: hepatocyte, BEC:
biliary epithelial cell, HSC: hepatic stellate cell, KC: Kupffer cell, EC: endothelial cell (B) In the liver, IL-6 can directly bind to its cognate membrane-bound IL-6 receptor
(mIL-6R) on hepatocytes (HC), leukocytes like KC and stellate cells (HSC). The complex of IL6/IL-6R subsequently recruits the signaling b-subunit gp130 and induces
downstream signaling. This process is termed ‘‘IL-6 classic signaling”. Cells that do not express the mIL-6R, like endothelial cells (EC) can react to IL-6 via ‘‘IL-6 transsignaling”: a soluble form of the IL-6R (sIL-6R) is proteolytically released by a disintegrin and metalloprotease 17 (ADAM17) from cells expressing the mIL-6R. The complex
of IL-6/sIL-6R can bind to gp130 and induce downstream signaling. (C) Hyper-IL-6 is a fusion protein of IL-6 and the soluble IL-6 receptor. Hyper-IL-6 is a potent stimulator
of gp130. D. sgp130Fc is a fusion protein of the extracellular portion of gp130 to the Fc part of human IgG1. sgp130Fc selectively inhibits IL-6 trans-signaling without
interfering with IL-6 classic signaling (also see Fig. 6).
or liver damage, gut-derived factors like
lipopolysaccharide (LPS) activate liver-resident
Kupffer cells resulting in a TNF-a-dependent
secretion of IL-6 (Fig. 2B) [31].
Consistently, IL-6 knockout mice show
impaired liver regeneration [32]. Also under cholestatic conditions, the number of regenerative
liver progenitor cells is reduced if IL-6 signaling
is blunted [33]. These experimental data pointed
to an important role of IL-6 in liver regeneration.
Mice transgenic for the human sIL-6R showed
that this protein acts as a serum binding protein
for IL-6 and prolongs the half-life of IL-6 [34] and
double transgenic mice expressing human IL-6
and human sIL-6R exhibited permanent hepatocyte proliferation [35,36]. We concluded from
these results that IL-6 in the presence of sIL-6R
was a potent stimulus of liver regeneration
[36]. Therefore, the potential of Hyper-IL-6
(Fig. 1C) to accelerate liver regeneration was
tested.
When mice were treated with recombinant
IL-6 or Hyper-IL-6 after 50% hepatectomy, it
turned out that only Hyper-IL-6 significantly
accelerated liver weight gain and led to a 36 h
earlier peak of mitosis in hepatocytes (Fig. 2C)
[37]. Likewise, Hyper-IL-6, but not IL-6, was
shown to reverse D-galactosamine mediated
liver toxicity and to significantly improve the
survival rate of the animals [38]. Interestingly,
it was shown that Hyper-IL-6 when genetically
delivered via a recombinant adenovirus led to
the survival of more than 90% of the mice
whereas only 13% of the mock-treated animals
survived the D-galactosamine treatment. Treatment with an adenovirus coding for IL-6 resulted
only in survival of 21% of the animals [39]. These
experiments demonstrated that stimulation of
IL-6 trans-signaling via the sIL-6R dramatically
accelerated and improved liver regeneration suggesting a physiologic role of the sIL-6R in this
process [4,40].
Since hepatocytes express much more gp130
than IL-6R the presence of IL-6 and sIL-6R results
in more gp130 activation and therefore to a
higher amplitude of the IL-6 signal (Fig. 2D). Furthermore, it was shown that the complex of IL-6
and sIL-6R was internalized much less efficiently
than IL-6 leading to longer duration of the IL-6
signal when mediated by trans-signaling [41].
This explains why, in the absence of any liver
insult, hepatocytes permanently proliferated in
IL-6/sIL-6R double transgenic mice, while hepatocytes did not show any mitogenic response in
IL-6 single transgenic mice. The signal induced
via the membrane-bound IL-6R on hepatocytes
was not sufficient to induce a proliferative
response [35,36].
Since all IL-6 type cytokines use the gp130
receptor for signaling it is interesting to ask
whether there is potential competition of members of this cytokine family for gp130 (see
above). As depicted in Fig. 2D, hepatocytes have
been shown to express far more gp130 than IL6R making it unlikely that gp130 will be the limiting factor in the response to other members of
the IL-6 type cytokine family [42].
These experiments demonstrated the potential of exogenous stimulation of IL-6 transsignaling in the induction of liver regeneration
but they did not directly show that IL-6 transsignaling actually occurred in vivo. Therefore,
experiments were conducted with the recombinant sgp130Fc protein, which specifically blocks
Journal of Hepatology 2016 vol. xxx j xxx–xxx
3
Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
Review
IL-6 classic
signaling
Review
B
Proliferation
Liver vein cytokine level
IL-6
STAT3
P P
STAT3
HC
sIL-6R/gp130
IL-6R/gp130
TNF-α
IL-6
2h
24 h
KC
TNFR
C
Liver mass regeneration BrdU incorporation
A
Hyper-IL-6
IL-6
36 h
72 h
Hyper-IL-6
IL-6
72 h
120 h
sIL-6R
Review
TNF-α
IL-6
D
gp130
gp130
IL-6R
Fig. 2. IL-6 is a major driver of liver regeneration. (A, B) Early after hepatocyte damage, Kupffer cells secrete TNF-a, which induces massive expression of IL-6 in an
autocrine manner. IL-6 then stimulates hepatocyte proliferation. (C) IL-6 trans-signaling enhances liver regeneration. Mice injected with Hyper-IL-6 show accelerated
hepatocyte proliferation and liver regeneration after partial hepatectomy in comparison to IL-6. Modified from [37] and [31]. (D) IL-6 trans-signaling can boost IL-6
signaling on hepatocytes. IL-6 engages IL-6 receptor molecules but not all gp130 receptor molecules on the surface of hepatocytes. The presence of the soluble IL-6R
enhances the possibility to engage gp130 receptor molecules on the cell surface. This leads to an amplification and prolongation of gp130 signals.
Key point
There is a growing body of evidence that IL-6 is needed for
the proper control of metabolic
functions.
IL-6 trans-signaling without affecting signaling
via the membrane-bound IL-6R (Fig. 1D). Alternatively, transgenic mice were generated, which
overexpress the sgp130Fc protein [43]. In these
mice, trans-signaling is abrogated whereas IL-6
signaling via the membrane-bound IL-6R is
intact.
In these sgp130Fc transgenic mice, the
response to D-galactosamine induced liver damage was shown to be compromised. Interestingly,
the liver damage-induced glycogen consumption
in the liver of the transgenic mice was strongly
reduced indicating that glycogen consumption
depended on IL-6 trans-signaling [44]. Upon
acute CCl4 damage, blockade of IL-6 transsignaling led to higher liver damage and to
reduced refilling of hepatocyte glycogen stores
[45]. In the concanavalin A hepatitis model it
was shown that IL-6 classic signaling via the
membrane-bound IL-6R rather than IL-6 transsignaling was responsible for the observed
neutrophil accumulation and the induced liver
damage [46]. Collectively, these experiments
demonstrate that IL-6 trans-signaling in the liver
plays an important role in the regenerative
response of this organ to injury [47].
Interestingly, IL-6 triggered the activation of
YAP and Notch independently of the downstream
effector STAT3 in primary hepatocytes and in
mouse liver upon partial hepatectomy, suggesting an inflammation induced participation of
the YAP and Notch pathways during liver regeneration [48].
IL-6 in obesity and insulin resistance
The role of IL-6 signaling on hepatic metabolism,
obesity and insulin resistance is discussed
controversially.
The observation that serum levels of IL-6 correlate with the degree of obesity [49] and the
development of type 2 diabetes [50] suggested
that IL-6 is causally linked to metabolic disease
development. Proteomics analysis of TNF-astimulated adipocytes identified the adipokine
progranulin (PGRN) as an inducer of IL-6 expression in adipose tissue during obesity [51]. Furthermore, activation of JNK1 in adipose tissue
of mice fed a high-fat diet (HFD) resulted in insulin resistance in the liver in an IL-6 dependent
manner [52]. Consistently, acute infusions of IL6 in mice impaired insulin action on the liver
and skeletal muscle in hyperinsulinemiceuglycemic clamp analysis [53]. In the liver and
skeletal muscle, IL-6 signaling leads to a STAT3dependent upregulation of SOCS3 (Fig. 3A)
which, in turn, impairs insulin-mediated
Journal of Hepatology 2016 vol. xxx j xxx–xxx
Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
JOURNAL OF HEPATOLOGY
IL-6 action on the central nervous system stimulates energy expenditure [57], again underlining
a beneficial role of IL-6 in metabolism. Remarkably, we could recently show that most if not
all IL-6 signaling in the brain is mediated by
trans-signaling [67].
Most of the studies so far used total ablation
of IL-6 or tissue-specific inactivation of the IL6R. However, future studies have to consider that
IL-6 classic and IL-6 trans-signaling might differentially regulate metabolism. And indeed, a very
recent report demonstrated that blockade of IL-6
trans-signaling impaired the recruitment of
inflammatory macrophages to white adipose tissue under a HFD, while it did not alter insulin
resistance in that model [68]. Furthermore,
glycogen consumption and synthesis in hepatocytes was regulated by IL-6 trans-signaling in
two different models of liver damage [44,45].
Review
phosporylation of insulin receptor substrates 1/2
(IRS-1/2) and subsequent protein kinase B (PKB/
AKT) activation [54–56].
However, the role of IL-6 in control of metabolism seems to be more complex. The first hint
that IL-6 might also exert beneficial roles on the
metabolism came from the observation that mice
deficient for IL-6 (IL-6/) developed matureonset diabetes with increased leptin and insulin
levels, liver inflammation and hepatosteatosis,
in particular when fed a HFD [57,58]. Mice with
a hepatocyte-specific deletion of the IL-6R also
displayed insulin resistance in liver, skeletal
muscle and white adipose tissue and exaggerated
diet-induced liver inflammation, indicating a
protective role of IL-6 on hepatocytes. Interestingly, glucose homeostasis in these mice could
be restored after TNF-a-inhibition or Kupffer
cell-depletion [59]. Consistently, hepatocytespecific ablation of STAT3 or gp130 also led to
insulin resistance and predisposed to dietinduced liver steatosis (Fig. 3B) [60,61]. Hepatic
glucose production and release to the periphery,
in particular the expression of the gluconeogenic
enzyme glucose-6 phosphatase, is negatively
regulated by IL-6 in a STAT3-dependent manner
[59,60,62]. In addition, increase in insulin sensitivity through upregulation of IRS-2 by adiponectin, a well-recognized anti-diabetic cytokine has
been shown to be IL-6 mediated (Fig. 3A) [63].
These data show that IL-6 in the liver not only
regulates glucose metabolism but is also necessary to maintain liver tissue homeostasis for
proper control of metabolic functions. One of
the hallmarks of obesity is the development of
a chronic low-grade inflammatory state with
increased infiltration of T lymphocytes and
macrophages to adipose tissue. Interestingly,
mice with a myeloid-specific ablation of the IL6R also developed insulin resistance and
increased inflammation in the liver under a
HFD. The authors of that study could demonstrate that IL-6 mediates polarization of proinflammatory M1 macrophages to M2 macrophages in adipose tissue through upregulation
of the IL-4 receptor (IL-4R) [64]. The IL-4R is an
integral component of the receptor complexes
for the cytokines IL-4 and IL-13, which are
needed for M2 macrophage differentiation.
Therefore, in the context of obesity, elevated IL6 serum levels rather seem to counterbalance
obesity-associated hyperglycemia. Interestingly,
also during physical exercise, IL-6 is produced
by muscle cells, leading to an up to 100-fold
increase in IL-6 plasma levels [65].
One further has to consider that IL-6 plays an
important role in the brain-liver axis. Insulin signaling in agouti-related peptide-expressing neurons induces hepatic IL-6 expression and
concomitant downregulation of hepatic gluconeogenic enzymes (Fig. 3A) [66]. Furthermore,
IL-6 during liver tumorigenesis
IL-6 is crucial for the development of hepatocellular
carcinoma
Key point
Diabetes, obesity and male gender are associated
with an increased risk to develop hepatocellular
carcinoma (HCC). Additionally, high serum levels
of IL-6 have been reported in several liver
pathologies that predispose to the development
of HCC, including acute and viral hepatitis [69],
alcoholic cirrhosis [70] and primary biliary cirrhosis [71]. In a very recent prospective study,
elevated IL-6 serum levels correlated with an
increased risk to develop HCC [72]. And in
patients suffering from HCC, elevated serum
levels of IL-6 and the sIL-6R have been detected
[73].
Extensive work on hepatocarcinogenesis has
been performed using the murine diethylnitrosamine (DEN)-model of HCC. The cytotoxic
effects of DEN are dependent on its metabolic
activation by cytochrome P450 2E1 in hepatocytes. Once activated, DEN forms DNA adducts
leading to hepatocyte damage. In this early phase
of hepatocarcinogenesis, Kupffer cells become
activated in a TLR- and EGFR-dependent manner
to secrete TNF-a and IL-6 (Fig. 4) [74–76]. IL-6
induces a compensatory proliferation of hepatocytes, which accumulate DNA damages due to
DEN. Consistently, IL-6 deficient mice, as well
as mice with liver-specific loss of gp130 show a
lower incidence of HCC tumors and prolonged
survival in the DEN model [75,77]. In mulitdrug
resistance 2-deficient mice, cholestasis leads to
bile acid induced liver injury, inflammation and
fibrosis and at a later stage to hepatocyte dysplasia and carcinoma formation [78,79]. Interestingly, in these mice STAT3 or IL-6 ablation
aggravated hepatocyte damage through impaired
Journal of Hepatology 2016 vol. xxx j xxx–xxx
IL-6 is a major driver of hepatocellular carcinogenesis.
5
Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
Review
Local
hepatocyte
homeostasis
sIL-6R
IL-6R/
gp130
InsR
B
InsR
IL-6
NC
Adiponectin
SOCS3
IRS-2
?
X
A
SOCS3 IRS-2
Local
inflammation,
steatosis
TNF-α
KC
STAT3
P P
STAT3
AC
Glycogen
Review
Systemic
muscle,
WAT:
insulin
resistance
ER-α
NCOA5
G6Pase
Glcc
IL-6
Glc
KC
G6Pase
Glycogen
HC
HC
EC
EC
Glc
Glc
Fig. 3. Multiple effects of IL-6 on metabolism. (A) Engagement of the insulin receptor (InsR) on agouti-related peptide (AgRP)-expressing neurons leads to the expression
of IL-6 in the liver. Expression of IL-6 by Kupffer cells is negatively regulated by estrogen receptor a (ER-a) and nuclear receptor coactivator 5 (NCOA5). On hepatocytes IL-6
induces expression of insulin receptor substrate 2 (IRS-2), thereby enhancing insulin signaling. IL-6 dependent suppression of glucose-6 phosphatase (G6Pase) lowers
peripheral blood glucose levels and increases glycogen stores in hepatocytes. Upregulation of suppressor of cytokine signaling 3 (SOCS-3) by IL-6 impairs gp130 and InsR
signaling. (B) Loss of hepatic IL-6 signaling leads to hepatosteatosis, local inflammation, enhanced blood glucose levels and peripheral insulin resistance mediated by TNF-a.
HC: hepatocyte, EC: endothelial cell, KC: Kupffer cell, NC: neuronal cell, AC: adipocyte.
EGFR signaling and enhanced bile acid synthesis
leading to enhanced liver fibrosis, predisposing
to increased tumor formation [79].
IL-6 expression is a major hallmark of the
senescence-associated secretory phenotype.
Senescent hepatocytes in chronic liver disease
[80,81] or senescent cholangiocytes during primary sclerosing cholangitis [82] therefore also
contribute to increased IL-6 expression in the
liver under diseased conditions.
Interestingly, the incidence of HCC development is reduced in female mice as estrogen signaling via the estrogen receptor a, suppresses
expression of IL-6 (Fig. 4) [75], which correlates
well with the human situation. Very recently
nuclear receptor coactivator 5 (NCOA5) has been
shown to be another transcriptional repressor of
IL-6 expression. Predisposition of NCOA5haploinsufficient mice to insulin resistance, type
2 diabetes and HCC correlated with elevated IL-6
levels [83]. HCC development in the DEN model
was aggravated in mice with genetically induced
or dietary-mediated obesity, which could be
linked to elevated IL-6 levels [84]. A very recent
report suggested, however, that obesityenhanced HCC formation was independent of
the IL-6R on hepatocytes, suggesting that HCC
formation might be independent of IL-6 or rely
on IL-6 trans-signaling [85]. Indeed, work from
our laboratory shows that abrogation of IL-6
trans-signaling decreases tumor burden in the
DEN model to a similar degree as in IL-6/ mice,
indicating that IL-6 trans-signaling significantly
contributed to tumor formation in this model
(personal communication).
6
In the inflamed liver, IL-6 promotes the polarization of macrophages to a M2 phenotype
through induction of the IL-4 receptor a chain
[64]. In human HCC, the amount of infiltrating
tumor associated macrophages inversely correlates with prognosis in HCC patients and it could
be shown that indeed, IL-6 secreted from tumor
associated macrophages promoted the expansion
of HCC progenitor cells (HcPCs) [86]. Interestingly, at an early stage of hepatocarcinogenesis,
HcPCs appeared in ectopic lymphoid structures
containing M2 macrophages, B- and T lymphocytes creating a growth-promoting microenvironment [87]. Albeit HcPCs have a liver
progenitor cell phenotype, they originate
through dedifferentiation from hepatocytes
[88,89]. At a later stage, HcPCs aquire an IL-6
autocrine loop where expression of IL-6 is dependent on LIN28B-mediated degradation of the
miRNA Let-7, a negative regulator of IL-6 expression (Fig. 4) [90]. A similar epigenetic switch has
been shown to be involved in the generation of
breast cancer stem cells [91]. However, the autocrine IL-6 loop alone is not sufficient to drive HCC
formation, as HcPCs only formed tumors when
transplanted to mice with a fibrotic liver [90].
During the course of HCC development, HcPCs
then egress from ectopic lymphoid structures to
form HCC nodules in the liver and metastasis to
distant organs.
Taken together, while IL-6 secreted from
myeloid cells is important during an early phase
of HCC development, pre-neoplastic lesions are
fed by an autocrine IL-6 loop at a later stage
of tumorigenesis, but are still dependent on
Journal of Hepatology 2016 vol. xxx j xxx–xxx
Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
JOURNAL OF HEPATOLOGY
HCC nodules
Metastasis
Tumor promoting
niche
HCC - initial phase
Proliferation
HcPC
CD44
STAT3
P
P
STAT3
IL-6R
Transformation
STAT3
P P
IL-6
STAT3
let7
Lin28B
LC
gp130
gp130
IL-6
sIL-6R
gp130
Review
IL-6
M2
Polarization
TLR
EGFR
M1
Fig. 4. IL-6 promotes multiple steps of hepatocarcinogenesis. Kupffer cells are activated by damaged hepatocytes in a TLRand EGFR-dependent manner and secrete IL-6 and the soluble IL-6R, leading to a compensatory hepatocyte proliferation.
Within a tumor-promoting microenvironment, hepatocytes transform into HCC progenitor cells (HcPCs) that aquire an
autocrine IL-6 loop through upregulation of LIN28B. Hepatocyte transformation is facilitated by M2-type macrophages that
also secrete IL-6. Polarization of M1 to M2 macrophages is promoted by IL-6. HcPCs egress from the tumor-promoting niche to
form HCC nodules and eventually metastasis to distant organs.
additional signals from the tumor microenvironment to develop full-blown HCC.
Activation of the IL-6/JAK/STAT3 pathway in
inflammatory hepatocellular adenoma
Inflammatory hepatocellular adenomas (IHCA)
are rare benign tumors of the liver predominantly found in women and frequently associated with oral contraception, obesity and
alcohol abuse. They are characterized by constitutive activation of the acute phase genes in hepatocytes and highly polymorphous inflammatory
infiltrates in the liver [92].
The discovery of activating mutations in
gp130 and downstream signaling molecules,
including JAK1, STAT3, Fyn-related kinase (FRK)
and G-protein G(s) subunit alpha (GNAS)
(Fig. 5) in IHCA highlighted the importance of
the IL-6 pathway for the pathogenesis of benign
liver tumors. While novel activating STAT3 mutations are found in 12% of IHCA cases [93], small
in-frame deletions within the coding region of
gp130 are present in 60% of IHCA cases [92].
These deletions vary in length but always cluster
within a loop of the extracellular domain D2
which represents an IL-6 contact site. As a consequence stabilizing hydrophobic interactions
between domain D2 and D3 are lost and the
extracellular domain adopts an active conformation, resulting in constitutive downstream
signaling [94]. Persistent activation of IL-6
trans-signaling has already previously been
shown to induce hepatocellular adenoma formation in IL-6/sIL-6R double transgenic but not in
IL-6 single transgenic mice [95]. These findings
suggest that enhanced gp130 activation by IL-6
trans-signaling is needed to induce oncogenic
transformation of hepatocytes. Interestingly, the
described in-frame deletion mutants of gp130
display a delayed biosynthesis and are therefore
predominantly localized within the endoplasmic
reticulum, potentially resulting in altered signal
transduction [96] contributing to oncogenic
transformation (Fig. 5).
Albeit IL-6 has been shown to be critically
involved in the development of hepatocellular
carcinoma, only 1–2% of HCC cases harbor
gp130 deletion mutations [92]. These findings
support the notion that hepatocellular adenomas
only very rarely progress to HCC and that persistent activation of the IL-6 pathway alone is not
sufficient to trigger malignant transformation of
hepatocytes [92].
IL-6 directed therapy in liver pathologies
Acceleration of liver regeneration
As described above, different preclinical rodent
models of liver regeneration demonstrated that
Hyper-IL-6 strongly enhances liver regeneration.
Transient infusions of Hyper-IL-6 might therefore
Journal of Hepatology 2016 vol. xxx j xxx–xxx
7
Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
Review
be beneficial to patients after partial liver resection to boost hepatocyte-mediated regeneration.
However, these patients very often underwent
tumor resection and up to now it is not clear if
transient IL-6/Hyper-IL-6 therapy could cause
recurrence of tumor growth.
A more elegant way would be to enhance
ex vivo expansion of bi-potential liver progenitor
cells (LPC) by IL-6 or Hyper-IL-6 prior to its infusion to patients. Indeed IL-6 and even more
Hyper-IL-6 were able to enhance proliferation
of LPCs in vivo and in vitro and infusion of
expanded LPCs were able to regenerate impaired
liver function in vivo [33,97].
Review
Neutralization of IL-6 and IL-6R
IL-6 has been previously recognized as growth
factor in multiple myeloma and use of a neutralizing anti-IL-6 antibodies (Fig. 6) was effective in
gp130del
multiple myeloma patients [98]. In a very recent
clinical phase I/II study, however, monotherapy
with the anti-IL6 antibody siltuximab did not
show clinical activity against a series of solid
tumors [99]. Furthermore, it turned out that
anti-IL-6 antibody treatment led to the formation
of high molecular weight antibody-IL-6 complexes and thereby prevented IL-6 clearance
from the circulation leading to massive systemic
IL-6 elevations [100].
This led to the development of tocilizumab, a
humanized anti-IL-6R antibody (Fig. 6). Tocilizumab binds to the IL-6 binding site of the IL-6R
and thereby prevents IL-6 binding and also high
molecular weight antibody-IL-6 complex formation. Tocilizumab blocks IL-6 classic- and transsignaling. A series of clinical studies have shown
its therapeutic benefit in rheumatoid arhtritis
[101], juvenile idiopathic arthritis [102] and
Castleman’s disease [103]. A phase I/II clinical
Constitutive active
gp130
deletion mutants
(60%)
Plasma membrane
FRK
Ras
FRK mutations
(10%)
Src
α
γβ
GNAS (Gα S)
mutations (5%)
Endosome
gp130WT
FRK
JAK1
mutations (1%)
gp130del
JAK1
STAT3
STAT3
JAK1
Constitutive active
STAT3 mutants
(5%)
gp130del
STAT3
PP
Endoplasmic reticulum
Nucleus
Fig. 5. The IL-6/gp130 pathway is consitutively activated in inflammatory hepatocellular adenoma (IHCA). Activating
deletion mutations in gp130 are the most frequently occurring mutations in IHCA. Constitutively active gp130 can induce
STAT3 phosphorylation from the endoplasmic reticulum and endosomes, while Ras-activation occurs at the plasma
membrane. Activating mutations are also found in STAT3, the Fyn-related kinase (FRK) and G-protein G(s) subunit alpha
(GNAS).
8
j xxx–xxx
JournalofofHepatology
Hepatology2016
2016vol.
vol.xxx
xxxj xxx–xxx
Journal
Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
JOURNAL OF HEPATOLOGY
A
B
Anti-IL-6
IL-6
C
Anti-IL-6R
sgp130Fc
D
JAK inhibitors
E
STAT3 inhibitors
sIL-6R
gp130WT
JAK1
IL-6R
STAT3
acute phase
inflammation acute phase inflammation
infection defense
infection defense
metabolism
metabolism
acute phase inflammation acute phase inflammation
tissue regeneration
tissue regeneration
infection defense
infection defense
metabolism
metabolism
tissue regeneration
tissue regeneration
Fig. 6. Strategies to specifically block IL-6 signaling. (A) Neutralizing antibodies to IL-6 block both, IL-6 classic and IL-6 trans-signaling. (B) Antibodies directed against the
IL-6R inhibit binding of IL-6 to IL-6R and block both, IL-6 classic and IL-6 trans-signaling. (C) Recombinant sgp130Fc selectively blocks IL-6 trans-signaling by sequestration
of the IL-6/sIL-6R complex. Therefore, the beneficial effects of IL-6 like acute phase response and infection defense are not impaired by the sgp130Fc protein. (D) Small
molecule inhibitors of JAK kinases prevent phosphorylation of gp130 and downstream molecules like STAT3 of both IL-6 classic and IL-6 trans-signaling. (E) Small molecule
inhibitors of STAT3 prevent either dimerization of STAT3 through binding to its SH2 domain or prevent binding of STAT3 to DNA. STAT3-dependent effects of both IL-6
classic and IL-6 trans-signaling are blocked. However, STAT3-independent effects of IL-6 like activation of the gp130-YAP-Notch module during liver regeneration are not
altered.
trial also showed that tocilizumab might be also
benefical to prevent graft-versus host disease
after bone marrow transplantation [104].
Preclinical studies using murine xenograft
models showed that anti-IL-6R therapy suppresses tumor angiogenesis and tumor growth
in colon cancer and oral squamous cell carcinoma [105,106]. Also, in an HCC xenograft
model, tocilizumab reduced HCC growth by
blunting IL-6 signaling from tumor associated
macrophages to HCC cells and therefore preventing the formation of cancer stem cells [86]. The
fact that expansion of HcPCs is dependent on
IL-6 [90], that IL-6 promotes M2 macrophage
polarization [64] and that growth of HCC seems
to be promoted by M2 macrophages [107] suggest that HCC patients might benefit from an
IL-6 directed therapy. Beside its direct effect on
tumor growth, anti-IL-6R therapy has been considered for the treatment of tumor cachexia. In
preclinical models of cancer cachexia, genetic
loss of IL-6 or the use of anti-IL-6 antibodies prevented white adipose tissue browning and therefore counterbalanced cancer cachexia in these
models [108]. Consequently, in a study with a
single cancer patient, tocilizumab was able to
reverse cancer cachexia symptoms [109].
As outlined above, classic IL-6 receptor signaling is believed to control some crucial homeostatic mechanisms such as the hepatic acute
phase response [19–21,110]. In contrast, IL-6
trans-signaling seems to play a role in overshooting immunological reactions resulting in autoimmune diseases such as rheumatoid arthritis and
inflammatory bowel disease [111].
One therefore has to consider several caveats
when using anti-IL-6R antibody therapy. Anti-IL6R antibodies prevent both, classic IL-6 and IL-6
trans-signaling (Fig. 6). Classic IL-6 signaling
however is important for the induction of the
acute phase response as a first-line defense to
infection and as a prevention of sepsis
[112,113]. Classic IL-6 signaling indeed has been
shown to be important for the control of bacterial and viral, in particular hepatitis B virus
(HBV) infections [110,114]. Also a significant
reduction in the number of peripheral neutrophils [115] and an increase in body weight
and triglyceride levels has been observed as a
side effect of tocilizumab treatment [103]. These
parameters therefore have to be tightly monitored under anti-IL-6R treatment.
Given the fact that sgp130Fc transgenic mice
do not show a negative metabolic phenotype,
while possessing anti-inflammatory properties
in various preclinical models [68], sgp130Fc
treatment might represent a more advantageous
therapy option in diverse disease settings. A
recent phase I study has shown that sgp130Fc
is well tolerated in humans [116]. Further studies
have to reveal if sgp130Fc therapy is an option
for the treatment of HCC and other liver pathologies. A phase IIa clinical trial in patients with IL-6
related inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis is
planned for 2016.
Inhibition of IL-6 downstream signaling
As described above, IL-6 binding to IL-6R and
gp130 results in the activation of cytoplasmic
tyrosine kinases of the JAK and Src kinase family
and subsequent phosphorylation of STAT3. Inhibtion of JAK and Src kinases would therefore blunt
intracellular IL-6 signaling. Ruxolitinib and tasocitinib are small molecule inhibitors of JAK
kinases (Fig. 6D) and have been clinically
approved for the treatment of myeloproliferative
Journal of Hepatology 2016 vol. xxx j xxx–xxx
Key point
Patients with liver pathologies
might benefit from selective
inhibition of IL-6 pathways.
9
Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
Review
acute phase inflammation
infection defense
metabolism
tissue
regeneration
Review
Review
neoplasms and rheumatoid arthritis [117]. Other
compounds are currently under development
and in clinical trials for hematological malignancies, solid tumors and rheumatoid arthritis
[117,118]. Activated STAT3 can also be directly
targeted by small molecule inhibitors binding
to its SH2 or DNA binding domain (Fig. 6E). These
compounds however haven’t entered yet clinical
trials [119].
There are preclinical data available that both
JAK and STAT3 inhibition abrogates the proliferation of HCC cell lines and the growth of orthotopic HCC tumors [119–121]. Consequently, the
use of JAK inhibitors for the treatment of IHCA
has been suggested [122] and its use for the
treatment of HCC might be considered. However,
JAK kinase inhibition would not only abrogate
both, IL-6 classic and IL-6 trans-signaling, but
also interfere with the downstream signaling of
several cytokines. One therefore would need to
carefully monitor potential side effects.
hepatic pathologies. These experiments would
also help to unravel which cells of the liver provide the soluble IL-6R to induce IL-6 transsignaling. Another elegant way to dissect the different effects of IL-6 signaling in the liver would
be the use of mice with an inducible, cellautonomous and ligand-independent activation
of gp130.
Albeit both, classic and trans-signaling pathways lead to activation of the signaling subunit
gp130, the effects on intracellular signaling, but
also biological effects seem to differ between
IL-6 classic and IL-6 trans-signaling. We therefore need a more detailed spatial and temporal
cell biological analysis to better explain these
effects and to be able to identify cells in complex
tissues that underwent IL-6 trans-signaling.
There is now a growing body of evidence that
IL-6 trans-signaling is also implicated in liver
pathologies. Selective inhibition of IL-6 transsignaling rather than complete blockade of both
IL-6 signaling pathway might therefore be more
effective in the treatment of liver pathologies.
Conclusions and perspectives
IL-6 is a cytokine with pleiotropic functions in
the body. Under physiological conditions it is
essential for proper hepatic tissue homeostasis,
liver regeneration, infection defense and fine
tuning of metabolic functions. Persistent activation of the IL-6 pathway however seems to be
detrimental and can even lead to the development of liver cancer.
Although much progress has been made,
there are still many open questions concerning
the implication of IL-6 in physiology and pathology of the liver. In order to efficiently target only
the detrimental effects of IL-6 on the liver, we
need to better dissect the effects of IL-6 on different cell types of the liver.
Most of the murine models used so far investigated effects in the complete absence of IL-6.
However, in order to better understand the complex nature of IL-6 in the liver and other tissues, a
cell type-specific analysis of IL-6 signals is warranted. Deletion of the IL-6R from selected cell
types would be informative to analyze the effect
of IL-6 classic signaling on different cell types in
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10
Financial support
The work described in this review was supported
by grants from the Deutsche Forschungsgemeinschaft Bonn, Germany (SFB 841, projects C1, SFB
877, project A1 and the Cluster of Excellence
‘Inflammation at Interfaces’).
Conflict of interest
S. R-J is an inventor of patents owned by CONARIS Research Institute, which develops the
sgp130Fc protein together with Ferring Pharmaceuticals and he has stock ownership in CONARIS. No conflicts of interest, financial or
otherwise, is declared by D. S-A.
Authors’ contributions
Both authors contributed equally to this article.
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dx.doi.org/10.1016/j.jhep.2016.02.004
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Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
Review
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Review
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Please cite this article in press as: Schmidt-Arras D, Rose-John S. IL-6 pathway in the liver: From physiopathology to therapy. J Hepatol (2016), http://
dx.doi.org/10.1016/j.jhep.2016.02.004
Review
JOURNAL OF HEPATOLOGY