Type 1 diabetes is a proinflammatory state characterised by increased levels of circulating biomarkers of inflammation and monocyte activity. We have shown increased Toll-like receptor 2 (TLR2) and TLR4 expression and signalling in monocytes from type 1 diabetic patients. Several endogenous ligands of TLR2 and TLR4 have been identified; however, there is a paucity of data on levels of these endogenous ligands in diabetes. Thus, the aim of this study was to examine circulating levels of exogenous/endogenous ligands of TLR2 and TLR4 in type 1 diabetic patients and to compare these with the levels in matched healthy controls.
Healthy controls (n = 37) and type 1 diabetic patients (n = 34) were recruited, and a fasting blood sample was obtained. Circulating levels of endotoxin, heat-shock protein 60 (Hsp60), high-mobility group box 1 (HMGB1) and growth arrest-specific 6 (GAS6) proteins were assessed by ELISA, and TLR2 and TLR4 expression was determined by flow cytometry.
Levels of the classical TLR4 ligand, endotoxin, were significantly elevated in type 1 diabetic patients compared with those in matched controls. Hsp60 and HMGB1 concentrations were also significantly increased in the patients (p &lt; 0.01 and p &lt; 0.001, respectively). No significant differences were observed in GAS6.
We report the novel observation that levels of ligands of TLR2 and TLR4 are significantly elevated in type 1 diabetes, and this, in concert with hyperglycaemia, accounts for the increase in TLR2 and TLR4 activity, underscoring the proinflammatory state of type 1 diabetes

A Osterloh

AE Mullick

H Ghanim

H Wagner

I. Jialal

JS Park

K Nagai

M. R. Dasu

P Libby

S Devaraj

S. Devaraj

S. H. Park

SJ Creely

English

PubMed

Crossref

Springer - Publisher Connector

Increased levels of ligands of Toll-like receptors 2 and 4 in type 1 diabetes

SHORT COMMUNICATION
Increased levels of ligands of Toll-like receptors 2 and 4
in type 1 diabetes
S. Devaraj & M. R. Dasu & S. H. Park & I. Jialal
Received: 17 March 2009 /Accepted: 24 April 2009 /Published online: 20 May 2009
# The Author(s) 2009. This article is published with open access at Springerlink.com
Abstract
Aims/hypothesis Type 1 diabetes is a proinflammatory state
characterised by increased levels of circulating biomarkers
of inflammation and monocyte activity. We have shown
increased Toll-like receptor 2 (TLR2) and TLR4 expression
and signalling in monocytes from type 1 diabetic patients.
Several endogenous ligands of TLR2 and TLR4 have been
identified; however, there is a paucity of data on levels of
these endogenous ligands in diabetes. Thus, the aim of this
study was to examine circulating levels of exogenous/
endogenous ligands of TLR2 and TLR4 in type 1 diabetic
patients and to compare these with the levels in matched
healthy controls.
Methods Healthy controls (n=37) and type 1 diabetic
patients (n=34) were recruited, and a fasting blood sample
was obtained. Circulating levels of endotoxin, heat-shock
protein 60 (Hsp60), high-mobility group box 1 (HMGB1)
and growth arrest-specific 6 (GAS6) proteins were assessed
by ELISA, and TLR2 and TLR4 expression was deter-
mined by flow cytometry.
Results Levels of the classical TLR4 ligand, endotoxin,
were significantly elevated in type 1 diabetic patients
compared with those in matched controls. Hsp60 and
HMGB1 concentrations were also significantly increased
in the patients (p<0.01 and p<0.001, respectively). No
significant differences were observed in GAS6.
Conclusions/interpretation We report the novel observation
that levels of ligands of TLR2 and TLR4 are significantly
elevated in type 1 diabetes, and this, in concert with
hyperglycaemia, accounts for the increase in TLR2 and
TLR4 activity, underscoring the proinflammatory state of
type 1 diabetes.
Keywords Endotoxin . HMGB1 . Hsp60 . Inflammation .
Ligand . Toll-like receptor
Abbreviations
GAS6 Growth arrest-specific 6
HMGB1 High-mobility group box 1
hsp Heat shock protein
IFNβ Interferon-β
mfi Mean fluorescence intensity of 10,000 cells
MyD88 Myeloid differentiation primary response 88
NF-κB Nuclear factor κB
RAGE Receptor for advanced glycation end-products
TLR Toll-like receptor
Introduction
Type 1 diabetes is associated with an increased risk of
micro- and macrovascular complications [1]. Recent studies
have shown that type 1 diabetes is a proinflammatory state
[2]. Members of the Toll-like receptor (TLR) family play a
critical role in innate immunity [3]. The TLRs, TLR2 and
TLR4 also play an important role in atherosclerosis [3]. We
recently demonstrated increased levels of TLR2 and TLR4
expression, activity and signalling in monocytes of type 1
Diabetologia (2009) 52:1665–1668
DOI 10.1007/s00125-009-1394-8
S. Devaraj :M. R. Dasu : S. H. Park : I. Jialal (*)
Laboratory for Atherosclerosis and Metabolic Research,
UC Davis Medical Center,
4635 II Ave, Res. 1 Bldg, Rm 3000,
Sacramento, CA 95817, USA
e-mail: ishwarlal.jialal@ucdmc.ucdavis.edu
S. Devaraj : I. Jialal
VA Medical Center,
Mather, CA, USA
diabetic patients compared with those in matched controls
[4].
A number of endogenous molecules may also be potent
activators of the innate immune system [5] and are capable
of inducing the release of proinflammatory cytokines from
monocytes/macrophages via the TLR signal-transduction
pathways. However, there are limited data on levels of
endogenous ligands of TLR2 and TLR4 in type 1 diabetes.
The major ligand involved in TLR4 activation is endotoxin
[5]. The activation of these TLR2 and TLR4 on monocytes
leads to the production of cytokines and chemokines. Heat
shock proteins (hsps) are endogenous ligands of TLR2 and
TLR4 [6]. Using macrophages from C3H/HeJ mice with a
Tlr4 point mutation, the cytokine effects of recombinant
human Hsp60 were shown to be dependent on TLR4,
suggesting that Hsp60 may be a TLR4 ligand [6]. High-
mobility group box 1 (HMGB1) protein has been reported
to be a ‘late’ proinflammatory mediator in sepsis. HMGB1
can induce activation of intracellular signalling pathways
via activation of TLR2, TLR4 and the receptor for
advanced glycation end-products (RAGE) [7]. Extracellular
HMGB1 thus acts as an alarmin and can activate the innate
system and function in a synergistic fashion with other
proinflammatory mediators to induce responses. Growth
arrest-specific 6 (GAS6) protein is another endogenous
ligand of TLR2 and TLR4 that is induced in experimental
models of diabetic nephropathy [8]. Thus, in this study,
since type 1 diabetes has previously been shown to be a
proinflammatory state involving increased TLR2 and TLR4
expression, we determined the levels of endogenous
(Hsp60, HMGB1 and GAS6) and exogenous (endotoxin)
ligands of TLR2 and TLR4 in patients with type 1 diabetes
and compared these with the levels in matched healthy
controls. In a previous report [4], we examined myeloid
differentiation response 88 (MyD88)-dependent TLR acti-
vation, and we follow up this work in the present study by
examining levels of interferon-β (IFNβ), a biological
indicator of non-MyD88-dependent activation of TLRs.
Methods
Type 1 diabetic patients (n=34) (aged<20 years at onset
and on insulin therapy since diagnosis) with a duration of
diabetes of ≥1 year were recruited from the diabetes/
pediatric clinics at UC Davis Medical Center and advertise-
ments. All patients were aged ≥18 years at recruitment and
none was receiving metformin and/or thiazolidinediones, or
any anti-inflammatory therapy.
Healthy controls (n=37) aged >18 years were included if
they had a normal complete blood count, no family history
of diabetes or other chronic diseases, normal kidney, liver,
thyroid function and fasting plasma glucose <5.5 mmol/l.
Selection criteria were as described previously [4]. In-
formed consent was obtained from participants in the study,
which was approved by the Institutional Review Board at
UC Davis Medical Center. For each participant, a medical
history was obtained, a physical examination was under-
taken, and then a fasting blood sample (30 ml) was
collected. Routine laboratory tests were performed in the
clinical pathology laboratory.
Mononuclear cells were isolated from the heparinised
blood samples by Ficoll–Hypaque centrifugation followed
by negative magnetic separation using the depletion
technique (Miltenyi Biotech, Auburn, CA, USA) [4]. The
purity of the isolated mononuclear cells was 88%. Mono-
cytes from control and type 1 diabetic patients were
incubated with anti-human TLR2 and TLR4 antibodies
(Invivogen, San Diego, CA, USA) or isotype controls, and
surface expression of TLR2 and TLR4 was analysed using
BD FACSArray (Becton-Dickinson, Fullerton, CA, USA)
[4]. Results were expressed as mean fluorescence intensity
of 10,000 cells (mfi). The intra- and inter-assay CVs for
TLR2 and TLR4 expression were both found to be <5%
and <15%, respectively. Endotoxin levels were measured in
serum samples of controls and type 1 diabetic patients by
the Limulus amoebocyte lysate assay (Invivogen). Sand-
wich ELISA was used to assess levels of IFNβ (R&D,
Minneapolis, MN, USA), HMGB1 (Shino Test, Tokyo,
Japan), Hsp60 (Assay Designs, Ann Arbor, MI, USA) and
GAS6 (R&D, Minneapolis, MN, USA); the intra-assay CV
for each of these assays was <10%.
Statistical analyses were performed using SAS (SAS
Institute, Cary, NC, USA). Data are expressed as the mean±
SD for parametric data and as the median and interquartile
range for non-parametric data. Parametric data were analysed
using paired, two-tailed t tests; non-parametric data were
analysed using Wilcoxon signed rank tests. A p value of less
than 0.05 was considered significant. Spearman’s rank
correlation was computed to assess associations between
variables.
Results
There were no significant differences between the control
and type 1 diabetic groups in terms of age, BMI, male/
female ratio and lipid profile. Levels of glucose and HbA1c
were significantly higher in type 1 diabetic patients than in
controls (Table 1).
Levels of the major exogenous ligand for TLR4,
endotoxin, were significantly increased in type 1 diabetic
patients compared with those in the controls (Table 2).
Levels of Hsp60 and HMGB1 were also significantly
elevated (p<0.01 and p<0.001, respectively). There was
no significant difference between the two groups with
1666 Diabetologia (2009) 52:1665–1668
respect to GAS6 levels. IFNβ concentrations were signif-
icantly higher in type 1 diabetic patients compared with
controls (43.9±10.3 vs 26.7±3.2 nmol/l, respectively,
p<0.05).
Across the study sample as a whole, there was a
significant correlation between TLR2 and HMGB1 levels
(r=0.42, p<0.05), a trend towards a positive correlation
between TLR2 and Hsp60 levels (though this did not reach
statistical significance; r=0.33, p<0.09), and no correlation
between TLR2 and endotoxin (r=0.34, p>0.2). TLR4 was
significantly correlated with endotoxin (r=0.56, p<0.01)
and Hsp60 (r=0.61, p<0.01), and showed a trend towards a
positive correlation with HMGB1 (though this did not reach
statistical significance; r=0.39, p<0.1). The only ligand to
be significantly correlated with HbA1c was endotoxin
(r=0.64, p<0.01).
Discussion
In addition to regulating inflammation, both TLR2 and
TLR4 are relevant to atherosclerosis [3]. The expression and
activity of TLR2 and TLR4 are increased in monocytes from
type 1 diabetic patients [4]. Downstream targets of TLR,
nuclear factor-κB (NF-κB), MyD88, TIR-domain-containing
adapter-inducing IFN-β (TRIF) and phosphorylated IL-1
receptor-associated kinase (pIRAK), were significantly upre-
gulated in type 1 diabetes [4]. The release of IL-1β and
TNF-α from monocytes was significantly increased in type 1
diabetic patients compared with controls and was correlated
with TLR2 and TLR4 expression [4]. Also, high glucose
levels upregulate, and insulin downregulates, TLR2 and
TLR4 expression [9].
Given the importance of TLR2 and TLR4 in atheroscle-
rosis and their emerging role in the proinflammatory state of
type 1 diabetes, in this study we examined circulating levels
of several different ligands of TLR2 and TLR4 in patients
with type 1 diabetes and compared these with the levels in
matched controls. Creely et al. [10] have previously shown
increased levels of endotoxin in type 2 diabetes but not
increased TLR4 expression in adipose tissue. Furthermore,
they demonstrated that circulating endotoxin levels are
associated with atherosclerosis [10]. In this report, we
provide evidence of increased endotoxin levels, which could
contribute to the proinflammatory burden by activation of
TLR4 and downstream signalling pathways.
The hsps such as Hsp60 induce the production of
proinflammatory cytokines via activation of TLR2 and
TLR4 [6]. In this paper we show that type 1 diabetic
patients also have high circulating levels of Hsp60, which
could trigger the activation of TLR2 and TLR4, leading to a
proinflammatory state.
HMGB1 induces the activation of intracellular signalling
pathways via interaction with at least three pattern-
recognition receptors: TLR2, TLR4 and RAGE. Further-
more, HMGB1 stimulates the transcription of genes
encoding proteins involved in the proinflammatory state
[7]. In the present study, we show that type 1 diabetic
patients have high circulating levels of HMGB1, which
could trigger TLR2 and TLR4 activation, leading to a
proinflammatory state.
TLRs mainly signal through the adapter protein MyD88
via activation of NF-κB and mitogen-activated protein kinase,
resulting in the increased transcription of inflammation-
related genes, such as those encoding IL-1, TNF-α, MCP-1
[3]. In addition, a MyD88-independent pathway involving
Table 2 Levels of ligands of TLR2 and TLR4
Controls (n=37) Type 1 diabetes (n=34)
Endotoxin (nmol/l) 2.53±0.67 3.32±0.82**
HMGB1 (nmol/l) 0.13±0.04 0.20±0.06**
Hsp60 (pmol/l) 18.5±5.0 23.2±6.7**
GAS6 (nmol/l) 11.2±6.9 10.2±5.9
Data are expressed as mean±SD
**p<0.01 vs controls
Controls (n=37) Type 1 diabetes (n=34)
Age (years) 34±11 32±11
BMI (kg/m2) 24±4 25±4
Male/female ratio 19:18 15:19
Glucose (mmol/l) 4.9±0.5 7.2±3.8*
HbA1c (%) 5.4±0.4 7.9±1.4*
Total cholesterol (mmol/l) 4.5±0.8 4.7±0.8
Triacylglycerol (mmol/l) 0.9±0.4 0.9±0.5
LDL-cholesterol (mmol/l) 2.9±0.6 2.9±0.6
HDL-cholesterol (mmol/l) 1.1±0.4 1.3±0.4
Table 1 Characteristics of the
participants at baseline
Data are expressed as mean±SD
*p<0.05 vs controls
Diabetologia (2009) 52:1665–1668 1667
Trif is essential to TLR3 and TLR4 signalling and induces
IFNβ [3]. In the previous study [4], we showed that
activation of the MyD88 pathway is increased in type 1
diabetic patients. In the present study, we show that type 1
diabetic patients have high circulating levels of IFNβ
(downstream indicator of non-MyD88 pathway activation).
In summary, in this paper we report the novel observa-
tion that there is significant elevation of endogenous
ligands of TLR2 and TLR4 in type 1 diabetes, which, in
concert with hyperglycaemia, contributes to the increase in
TLR2 and TLR4 signalling that results in the proinflam-
matory state of type 1 diabetes. In future studies, we will
test levels of endogenous ligands and TLR2 and TLR4
expression and signalling in type 2 diabetes, which is also a
proinflammatory state.
Acknowledgements Grant support from the Juvenile Diabetes
Research Foundation (JDRF) (no. 1-2007-585 to I. Jialal) and the
National Institutes of Health (no. DK69801 to S. Devaraj; no.
K24AT00596 to I. Jialal). Technical support from C. Duncan-Staley.
Duality of interest The authors declare that there is no duality of
interest associated with this manuscript.
Open Access This article is distributed under the terms of the
Creative Commons Attribution Noncommercial License which per-
mits any noncommercial use, distribution, and reproduction in any
medium, provided the original author(s) and source are credited.
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1668 Diabetologia (2009) 52:1665–1668


Devaraj, S.

Dasu, M. R.

Park, S. H.

Jialal, I.

eScholarship - University of California

Acute modulation of toll-like receptors by insulin. Diabetes Care 31:1827–1831

Growth arrest-specific gene 6 is involved in glomerular hypertrophy in the early stage of diabetic nephropathy.

Heat shock proteins: linking danger and pathogen recognition.

High mobility group box 1 protein interacts with multiple Toll-like receptors.

Increased monocytic activity and biomarkers of inflammation in patients with type 1 diabetes.

Increased toll-like receptor (TLR) 2 and TLR4 expression in monocytes from patients with type 1 diabetes: further evidence of a proinflammatory state.

Lipopolysaccharide activates an innate immune system response in human adipose tissue in obesity and type 2 diabetes.

Toll-like receptors and atherosclerosis: key contributors in disease and health?

https://link.springer.com/content/pdf/10.1007%2Fs00125-009-1394-8.pdf

Increased levels of ligands of Toll-like receptors 2 and 4 in type 1 diabetes

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