Increased Toll-Like Receptor (TLR) 2 and TLR4 Expression in Monocytes from Patients with Type 1 Diabetes: Further Evidence of a Proinflammatory State

Article (PDF Available)inJournal of Clinical Endocrinology & Metabolism 93(2):578-83 · February 2008with204 Reads
DOI: 10.1210/jc.2007-2185 · Source: PubMed
Abstract
Type 1 diabetes (T1DM) is associated with increased cardiovascular mortality. It is a pro-inflammatory state as evidenced by increased circulating biomarkers and monocyte activity. The toll-like receptors (TLRs) are pattern recognition receptors, expressed abundantly on monocytes. TLR2 and TLR4 are important in atherosclerosis. However, there is a paucity of data examining TLR2 and TLR4 expression in T1DM and examining its contribution to the proinflammatory state. Thus, we examined TLR2 and TLR4 expression in monocytes from T1DM patients compared with controls (n = 31 per group). The study was performed at the University of California Davis Medical Center. Healthy controls (n = 31) and T1DM patients (n = 31) were included in the study. TLR2 and TLR4 surface expression and mRNA were significantly increased in T1DM monocytes compared with controls. Downstream targets of TLR, nuclear factor kappaB, myeloid differentiation factor 88, Trif, and phosphorylated IL-1 receptor-associated kinase were significantly up-regulated in T1DM. Finally, the release of IL-1beta and TNF-alpha was significantly increased in monocytes from T1DM compared with controls and correlated with TLR2 and TLR4 expression (P < 0.005). In addition, TLR2 and TLR4 expression was significantly correlated to glycosylated hemoglobin, carboxymethyllysine, and nuclear factor kappaB (P < 0.02). Thus, we make the novel observation that TLR2 and TLR4 expression and signaling are increased in T1DM and contribute to the proinflammatory state.
Increased Toll-Like Receptor (TLR) 2 and TLR4
Expression in Monocytes from Patients with Type 1
Diabetes: Further Evidence of a Proinflammatory
State
Sridevi Devaraj, Mohan R. Dasu, Jason Rockwood, William Winter, Steven C. Griffen, and
Ishwarlal Jialal
Laboratory for Atherosclerosis and Metabolic Research (S.D., M.R.D., J.R., I.J.), Department of Pathology and Internal Medicine (S.C.G.,
I.J.), University of California Davis Medical Center, Sacramento, California 95817; and Department of Pathology (W.W.), University of
Florida at Gainesville, Gainesville, Florida 32611
Context: Type 1 diabetes (T1DM) is associated with increased cardiovascular mortality. It is a pro-
inflammatory state as evidenced by increased circulating biomarkers and monocyte activity. The
toll-like receptors (TLRs) are pattern recognition receptors, expressed abundantly on monocytes.
TLR2 and TLR4 are important in atherosclerosis. However, there is a paucity of data examining TLR2
and TLR4 expression in T1DM and examining its contribution to the proinflammatory state.
Objective: Thus, we examined TLR2 and TLR4 expression in monocytes from T1DM patients com-
pared with controls (n 31 per group).
Setting: The study was performed at the University of California Davis Medical Center.
Patients: Healthy controls (n 31) and T1DM patients (n 31) were included in the study.
Results: TLR2 and TLR4 surface expression and mRNA were significantly increased in T1DM mono-
cytes compared with controls. Downstream targets of TLR, nuclear factor
B, myeloid differenti-
ation factor 88, Trif, and phosphorylated IL-1 receptor-associated kinase were significantly up-
regulated in T1DM. Finally, the release of IL-1
and TNF-
was significantly increased in monocytes
from T1DM compared with controls and correlated with TLR2 and TLR4 expression (P 0.005). In
addition, TLR2 and TLR4 expression was significantly correlated to glycosylated hemoglobin, car-
boxymethyllysine, and nuclear factor
B(P 0.02).
Conclusion: Thus, we make the novel observation that TLR2 and TLR4 expression and signaling are
increased in T1DM and contribute to the proinflammatory state. (J Clin Endocrinol Metab 93:
578–583, 2008)
T
ype 1 diabetes (T1DM) is associated with an increased risk
of coronary artery disease (1). Inflammation plays a pivotal
role in all stages of atherosclerosis. Recent studies have shown
that T1DM is a proinflammatory state (2–7). Schalkwijk et al. (4)
reported elevated C-reactive protein (CRP) levels in T1DM pa-
tients without macrovascular disease compared with controls. In
the EURODIAB study (2, 3), levels of CRP, plasma IL-6, and
TNF were significantly higher in T1DM subjects and, in a cross-
sectional study, correlated with the severity of diabetic vascular
disease. In addition, Targher et al. (6) reported increased CRP
levels in T1DM patients without complications. Furthermore,
we have demonstrated in two independent studies that patients
with T1DM exhibit increased inflammation as evidenced by in-
creased plasma CRP levels and increased monocyte activity, and
these are more pronounced in T1DM with microvascular com-
plications (5, 7).
Macrophages are the predominant participants in innate im-
mune responses in atherosclerosis via protein receptors. In par-
0021-972X/08/$15.00/0
Printed in U.S.A.
Copyright © 2008 by The Endocrine Society
doi: 10.1210/jc.2007-2185 Received September 28, 2007. Accepted November 13, 2007.
First Published Online November 20, 2007
Abbreviations: CML, Carboxymethyllysine; CRP, C-reactive protein; GAPDH, glyceralde-
hyde-3-phosphate dehydrogenase; HbA
1c
, glycosylated hemoglobin; IRAK, IL-1 receptor
associated kinase; LPS, lipopolysaccharide; MyD88, myeloid differentiation factor 88;
NFK
, nuclear factor
B; pIRAK, phosphorylated IL-1 receptor-associated kinase; T1DM,
type 1 diabetes; TLR, toll-like receptor.
ORIGINAL ARTICLE
Endocrine Research
578 jcem.endojournals.org J Clin Endocrinol Metab. February 2008, 93(2):578 –583
ticular, the members of the toll-like receptor (TLR) family play
a critical role in the inflammatory components of atherosclerosis.
TLRs are a family of pattern recognition receptors that are im-
portant in the regulation of immune function and inflammation
(8–13). Their activation by various ligands triggers a signaling
cascade leading to cytokine production and initiation of an adap-
tive immune response (13). TLRs are up-regulated in several
inflammatory disorders. However, there is a paucity of data on
TLR in diabetes, a cardiovascular risk equivalent (14). Among
the TLRs, TLR2 and TLR4 play an important role in athero-
sclerosis. TLRs 2 and 4 can recognize components of the bacte-
rial cell wall such as lipopolysaccharide (LPS) and peptidogly-
cans and lipopeptides. The activation of these receptors on cells
of the innate immune system leads to the production of cyto-
kines, chemokines, and the up-regulation of cell surface mole-
cules. TLRs are expressed in multiple tissues. The predominant
site of TLR expression is on cells of the innate immune system,
especially monocytes.
TLR2 and TLR4 expression is up-regulated in atherosclerotic
plaque macrophages and in animal models of atherosclerosis
(8–13). TLR4 binds to the LPS (endotoxin) of the outer mem-
brane of Gram-negative bacteria. TLR2 recognizes and signals
bacterial lipoproteins, peptidoglycans, and lipoteichoic acid
from Gram-positive bacterial cell walls. Knockout of TLR4 is
associated with reduction in lesion size, lipid content, and mac-
rophage infiltration in hypercholesterolemic apolipoprotein
E / mice (15). In addition, TLR2/low-density lipoprotein re-
ceptor-deficient /, and in a recent paper, TLR2/apolipopro-
tein E /, mice are protected from the development of athero-
sclerosis (16, 17). Furthermore, two groups have demonstrated
that deficiency of myeloid differentiation factor 88 (MyD88),
one of the downstream TLR intracellular signaling molecules,
results in reduction in plaque size, lipid content, expression of
proinflammatory genes, and systemic expression of proinflam-
matory cytokines, such as IL-1 and TNF (15, 18). There is a
paucity of data examining the role of TLR in diabetes. TLR4
mRNA expression is induced in adipose tissue of db/db mice
(19). Furthermore, Mohammad et al. (20) recently showed in-
creased TLR2 and TLR4 expression in type 1 diabetic nonobese
mice, and this correlated with increased nuclear factor
B
(NFK
) activation in response to the TLR4 ligand, LPS, resulting
in increased proinflammatory cytokines.
Although we have shown enhanced monocyte activity in
T1DM (5, 7), the contribution of TLR2 and TLR4 to the pro-
inflammatory state of T1DM has not been explored to date.
Thus, we studied TLR2 and TLR4 activation and downstream
signaling in monocytes isolated from patients with T1DM and
matched controls.
Subjects and Methods
Type 1 diabetic patients (n 31) (onset 20 yr and on insulin therapy since
diagnosis; present age 18 yr or older with duration of diabetes 1 yr or more)
were recruited from the Diabetes and Pediatric Clinics at University of Cal-
ifornia Davis Medical Center and advertisements in the local newspaper.
Subjects younger than 18 yr were not studied due to University of California
Davis Medical Center institutional review board restrictions. None of the
patients were on Glucophage (Bristol-Myers Squibb Co., Princeton, NJ),
and/or the thiazolidinediones, angiotensin converting enzyme inhibitors,
angiotensin receptor blockers, or statins.
Healthy controls (n 31), age older than 18 yr, were included if they
had normal complete blood count, no family history of diabetes or other
chronic diseases, normal kidney, liver, thyroid function, and fasting
plasma glucose less than 100 mg/dl. Healthy controls and T1DM pa-
tients were matched for age (within 10 yr), gender, and race. In addition,
56% of T1DM patients were positive for either glutamate decarboxylase
65 or tyrosine phosphatase 1A autoantibodies (measured using the Kro-
nos ELISA at the clinical laboratories in University of Gainesville,
Gainesville, FL).
Exclusion criteria were: mean glycosylated hemoglobin (HbA
1c
) over
the last year more than 10%; inflammatory disorders; microvascular and
macrovascular complications; abnormal liver, renal, or thyroid function;
malabsorption; steroid therapy, antiinflammatory, antihypertensive, or
hypolipidemic drugs; antioxidant supplements in the past 3 months;
pregnancy; smoking; abnormal complete blood count; alcohol consump-
tion more than 1 oz/d; consumption of N-3 polyunsaturated fatty acid
capsules (1 g/d); and chronic high intensity exercisers.
Informed consent was obtained from participants in the study, which
was approved by the institutional review board at University of Califor-
nia Davis. After history and physical examination, fasting blood (30 ml)
was obtained.
A complete blood count, plasma lipid and lipoprotein profile, creat-
inine, aspartate aminotransferase, alanine aminotransferase, glucose,
glycated hemoglobin, and TSH were assayed by standard laboratory
techniques in the Clinical Pathology Laboratory. Free fatty acid levels
were assayed using reagents from Wako Chemicals (Richmond, VA).
Monocyte isolation
Mononuclear cells were isolated from fasting heparinized blood by
Ficoll Hypaque centrifugation, followed by magnetic separation using
the depletion technique (Miltenyi Biotech, Auburn, CA), as described
previously (5, 7). Using this technique, more than 86% of cells were
identified as monocytes by CD14 staining. Isolated monocytes were stud-
ied before and after activation with LPS (from Escherichia coli 026:B6,
1
g/ml; Sigma Chemicals, St. Louis, MO) or Pam3CSK4 (200 ng/ml;
InvivoGen, San Diego, CA).
Surface expression of TLR2 and TLR4
Monocytes from control and T1DM were incubated with antihuman
TLR2 and TLR4 antibodies (InvivoGen) or isotype controls, and surface
expression of TLR2 and TLR4 was analyzed using BD FACSArray
(Franklin Lakes, NJ) after gating for CD14. Results were expressed as
mean fluorescence intensity of 10,000 cells. Because LPS is the ligand for
TLR4, surface expression of TLR4 was monitored in both resting and
LPS-activated monocytes from both groups. In addition, surface expres-
sion of TLR2 was examined in resting and Pam3CSK4 (TLR2 ligand)
activated monocytes from both groups. Intraassay and interassay coef-
ficient of variation for TLR2 and TLR4 expression was less than 5% and
less than 15%, respectively.
RT-PCR of TLR2 and TLR4 mRNA
RNA was extracted from monocytes using TRIZOL (Invitrogen
Corp., Carlsbad, CA). RT-PCR was performed using primers specific for
TLR2 and TLR4 (InvivoGen), with glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH) as control (R&D Systems, Minneapolis, MN).
Band intensities were determined using Imagequant software (GE
Healthcare, Piscataway, NJ). TLR2 and TLR4 mRNA was expressed as
a ratio to GAPDH.
Western blotting
Cell lysates were prepared, and Western blotting for TLR down-
stream signaling proteins, MyD88 (eBiosciences, San Diego, CA), IL-1
receptor-associated kinase (IRAK)-1 (Cell Signaling, Boston, MA), and
J Clin Endocrinol Metab, February 2008, 93(2):578 –583 jcem.endojournals.org 579
TIR-domain containing adapter inducing interferon
(Trif; eBio-
sciences) was performed using specific rabbit antibodies to the respective
(phospho)proteins (21).
-actin was used as loading and internal control
for MyD88 and Trif and IRAK was used for phosphorylated IRAK (pI-
RAK). Densitometric ratios were computed to examine differences in
expression between controls and T1DM.
NFK
activity was examined as a readout of TLR signaling. Briefly,
nuclear extracts of cells were prepared using NE-PER reagents from
Pierce (Rockford, IL) as described previously (21), and NFK
DNA
binding activity was determined in the nuclear extracts using TransAM
assay (Active Motif, Carlsbad, CA) according to the manufacturer’s in-
structions. Briefly, nuclear extracts were suspended in TransAM lysis
buffer; suspensions were then microcentrifuged at 14,000 g for 10 min
at 4 C. Nuclear proteins (5
g total protein) were incubated in 96-well
plates with immobilized oligonucleotides containing the NF-
B consen-
sus DNA-binding site (5-GGGACTTTCC-3)for1hatroom temper-
ature. Wells were then washed three times. To each well, 100
l p65
subunit monoclonal antibody (1:1000 dilutions) was added; they were
left for1hatroom temperature. Wells were washed three times. Then
100
l horseradish-peroxidase-conjugated secondary antibody (1:1000
dilutions) was added to each well for1hatroom temperature. The
absorbance at 450 nm was determined using a spectrophotometer using
a standard for NFK
. NFK
activity was expressed as nanograms of
NFK
p65 per milligram cell protein.
The release of cytokines, IL-1
, and TNF in the supernates of monocytes
isolated from controls and T1DM was also determined as a readout of
up-regulated TLR expression, using ELISA (R&D Systems). Release of cy-
tokines was expressed as picograms per milligram of cell protein as described
previously (5, 7, 21).
Statistical analyses were performed using SAS software (SAS Institute
Inc., Cary, NC). Data are expressed as mean SD for parametric data,
and as median and interquartile range for nonparametric data. Paramet-
ric data were analyzed using paired, two-tailed t tests and nonparametric
data using Wilcoxon signed rank tests. Level of significance was set at P
0.05. Spearman’s rank correlation was computed to assess association
between variables.
Results
Baseline subject characteristics are depicted in Table 1. There were
no significant differences in age, body mass index, and male to
female ratio between control and T1DM groups. In addition, there
were no significant differences in the lipid profile. As expected, lev-
els of glucose, HbA
1c
, and free fatty acids were significantly higher
in T1DM compared with controls (Table 1). Levels of the advanced
glycation endproduct, carboxymethyllysine (CML) were signifi-
cantly increased in T1DM compared with controls.
Monocyte surface expression of TLR2 and TLR4 was signif-
icantly up-regulated upon activation with Pam3CSK4 and en-
dotoxin, respectively. Furthermore, TLR2 expression was sig-
nificantly increased in T1DM compared with controls in resting
and Pam3CSK4 activated cells (Fig. 1A; P 0.05). In both rest-
ing and LPS-activated monocytes, TLR4 expression was signif-
icantly up-regulated in T1DM compared with controls (Fig. 1B;
P 0.05). There were no significant differences in TLR2 and
TLR4 expression between T1DM patients who were positive for
either glutamate decarboxylase 65 or tyrosine phosphatase au-
toantibodies vs. those who were negative.
TLR2 and TLR4 mRNA as quantitated by RT-PCR revealed
significant up-regulation of TLR2 (Fig. 1C; P 0.05) and TLR4
mRNA (P 0.05) in T1DM monocytes compared with controls.
Downstream signaling of TLR, i.e. NFK
activity and the
downstream proteins, MyD88, Trif, and IRAK-1 protein phos-
phorylation were examined. As shown in Fig. 2, NFK
activity
and MyD88, Trif and IRAK-1 phosphorylation were signifi-
cantly increased in monocytes from T1DM patients compared
with controls (P 0.05). In addition, there was a significant
correlation between TLR2 expression and NFK
activity (r
0.48; P 0.005), and TLR4 expression and NFK
activity,
respectively (r 0.74; P 0.001).
We examined downstream readouts of TLR, IL-1
, and TNF
from monocytes of controls and T1DM patients (Table 2). There
was a significant up-regulation of these cytokines in T1DM pa-
tients compared with controls in both basal state as well as after
activation.
Furthermore, there was a significant correlation between
HbA
1c
and TLR4 expression (r 0.38; P 0.02) and TLR2
expression (r 0.52; P 0.001), but no significant correlation
between TLR expression and free fatty acid levels. In addition,
CML levels significantly correlated with TLR2 expression (r
0.29; P 0.05) and TLR4 expression (r 0.35; P 0.05),
respectively. There was a significant correlation between TLR2
expression and IL-1
and TNF
, respectively, both in the resting
(r 0.55 and r 0.50; P 0.0001) and activated state (r 0.4
and r 0.53; P 0.005), and between TLR4 and IL-1
and
TNF
, respectively, both in the resting (r 0. 54 and r 0.52;
P 0.0001) and activated state (r 0.41 and r 0.38; P
0.005).
Discussion
T1DM is a proinflammatory state characterized by increased
levels of circulating biomarkers of inflammation and monocyte
activity. TLR2 and TLR4 play a critical role in atherosclerosis.
The increased inflammation in T1DM may be mediated in part
via activation of the innate immune pathway by the TLRs. How-
ever, there are no studies examining TLR expression in T1DM
and their contribution to the proinflammatory state of T1DM. In
this report, we provide novel data on up-regulated TLR2 and
TLR4 expression and signaling in monocytes of T1DM.
TABLE 1. Baseline subject characteristics
Controls
(n 31)
T1DM
(n 31)
Age (yr) 32 13 32 13
BMI (kg/m) 25 425 4
Male to female ratio 12:19 12:19
Glucose (mg/dl) 85 11 131 68
a
HbA
1c
(%)
5.4 0.3 7.8 1.1
a
CML (ng/ml) 3.8 2.0 5.0 1.9
a
CRP (mg/liter) 1.1 (0.6, 1.7) 1.7 (0.9, 2.1)
a
Free fatty acids (mM) 0.28 0.1 0.39 0.23
a
Total cholesterol (mg/dl) 175 26 180 37
Triglycerides (mg/dl) 80 39 81 47
LDL cholesterol (mg/dl) 112 20 111 28
HDL cholesterol (mg/dl) 47 15 52 17
Data are expressed as mean SD, and median and interquartile range for CRP.
BMI, Body mass index; HDL, high-density lipoprotein; LDL, low-density
lipoprotein.
a
P 0.05 compared with controls.
580 Devaraj et al. Increased TLR2 and TLR4 in T1DM J Clin Endocrinol Metab, February 2008, 93(2):578–583
There is a paucity of data examining the
role of TLR2 and TLR4 in hyperglycemia/
diabetes. TLR4 mRNA expression is in-
duced in adipose tissue of db/db mice (19).
Mohammad et al. (20) showed increased
TLR4 expression in type 1 diabetic nono-
bese mice, and this correlated with in-
creased NFK
activation in response to the
TLR4 ligand, LPS, resulting in increased
proinflammatory cytokines. Park et al.
(22) have shown significant differences in
TLR2 polymorphisms between T1DM and
controls, however, they failed to examine
TLR2 expression, and, thus, the relevance
of their findings to the proinflammatory
state of T1DM is unclear. In a limited re-
port (n 5 patients), Creely et al. (23) dem-
onstrated increased TLR2 but not TLR4
expression in adipocytes of subjects with
type 2 diabetes; however, they failed to ex-
amine any correlations with glycated he-
moglobin or downstream readouts, and
their sample size was very small. This may
have explained the failure to observe an in-
crease in TLR4 expression, despite an in-
creased endotoxin level, the ligand for
TLR4.
In addition to showing that TLR2 and
TLR4 surface expression is increased on
monocytes isolated from T1DM compared
with controls, we demonstrate increased
TLR2 and TLR4 mRNA in T1DM. Fur-
thermore, when incubated with the TLR4
ligand, LPS, the increase in TLR4 expres-
sion was more pronounced compared with
resting cells, indicating further activation
of the pathway. Similarly, when incubated
with the TLR2 ligand, Pam3CSK4, there
was increased TLR2 expression.
TLRs are characterized by an extra-
cellular ligand binding domain, single
transmembrane domain, and intracellu-
lar domain (8 –13). Upon ligand binding,
the TLR subunits associate, leading to the
formation of a complex of Toll-interact-
ing region domain containing adaptor
proteins of the MyD88 family. Subse-
quent downstream signal transduction
events lead to the activation of MAPKs
and NFK
, and transcription of proin-
flammatory chemokines such as mono-
cyte chemoattractant protein-1 and
cytokines such as IL-1, IL-6, and TNF
(8–12). Although TLR4 appears to signal
through a MyD88-dependent and a
MyD88-independent pathway, down-
stream adapter proteins that get acti-
Controls T1 DM
0
5
10
15
20
25
30
35
40
45
BA SA L
TLR2- Su rf ace Expr essi on (m fi )
Controls T1 DM
0
10
20
30
40
50
60
70
AC TI VA TE D
TLR2 Su rf ace Expr essi on (m fi )
*
#
A
B
C
Controls T1 DM
0
25
50
BA SA L
TLR4 Su rf ace Expr essi on (m fi )
Controls T1 DM
0
10
20
30
40
50
60
70
AC TI VA TE D
TLR4 Su rf ace Expr essi on (m fi )
*
#
TLR4
TLR2
GAPD H
Control Ty pe 1 Diabete s
*
*
0
0. 5
1
1. 5
2
2. 5
TL R2 TL R4
m RNA /G AP D H ra ti o
C ont ro
l
T1 DM
*
*
*
P<0.05 vs Control-Basa l
#
P<0.05 vs Control-Activate d
* P<0.05 vs Control-Basa l
#
P<0.05 vs Control-Activate d
P<0.05 vs control
FIG. 1. TLR2 and TLR4 expression in T1DM. Surface expression of TLR2 (resting and Pam3CSK4
activated) (A) and surface expression of TLR4 (resting and LPS activated) (B) on human monocytes
isolated from control and T1DM patients (n 31 per group) were assessed by flow cytometry as
described in Subjects and Methods. Data are expressed as mean fluorescence intensity units (mfi).
*, P 0.05 vs. control basal; #,
P 0.05 vs. control activated. C, TLR2 and TLR4 mRNA in T1DM.
Representative RT-PCR results for TLR2 and TLR mRNA from three different controls and three
T1DM patients were assessed as described in Subjects and Methods using GAPDH as control. Lower
panel, The TLR2 and TLR4 to GAPDH ratio, respectively, for all control and T1DM patients (n 31
per group). *, P 0.05 vs. control.
J Clin Endocrinol Metab, February 2008, 93(2):578 –583 jcem.endojournals.org 581
vated include Trif and pIRAK. MyD88 is also involved in
NFK
activation by every TLR identified so far except TLR3,
leading to increased expression of proinflammatory cyto-
kines, IL-1 and IL-6. In this study, in addition to demonstrat-
ing increased TLR2 and TLR4 mRNA and protein in T1DM
compared with controls, we show increased expression of the
downstream signaling as evidenced by increased NFK
DNA
binding activity, as well as increased expression of the adapter
proteins, i.e. MyD88, Trif, and IRAK, in monocytes from
patients with T1DM compared with controls, possibly con-
tributing to increased IL-1
and TNF
release. It also appears
that Trif activation is unique to the TLR4 pathway of proin-
flammatory cytokine activation; this will be explored in future
studies. We have previously shown increased expression of
monocyte IL-1 and IL-6 in T1DM compared with controls (5,
7). Furthermore, we report a significant correlation between
TLR2 and TLR4 expression and glycemic control, as well as
advanced glycation end-product (AGE) levels as denoted by
increased CML, NFK
, IL-1
, and TNF
release (a readout of
TLR activation). In this context, we have also shown in hy-
perglycemia that there is increased monocyte synthesis and
secretion of IL-1
, mediated via induction of p38MAPK, and
NFK
(24). Although Shi et al. (25) have demonstrated that
free fatty acids increase TLR4 expression in murine adipo-
cytes and macrophages, we failed to observe any correlation
of increased TLR2 and TLR4 expression with increased free
fatty acid levels in T1DM. In addition, it is important to note
that glycemia and AGE in T1DM may contribute to increased
TLR2 and TLR4 expression. Although we cannot directly
implicate that increased cytokine release in T1DM is due to
increased TLR2 and TLR4 activity, the significant correlation
warrants future studies in diabetic TLR knockout mice to
elucidate the role of TLR in the induction of cytokines/che-
mokines in diabetes. Thus, increased TLR2 and TLR4 expres-
sion in T1DM may contribute to the proinflammatory state of
T1DM. Although we cannot exclude the contribution of
TLR2 and TLR4 to the pathogenesis of T1DM, this was not
the intent of this study, and we purposely studied T1DM pa-
tients with more than 1-yr duration of diabetes to avoid the
autoimmune component of T1DM. To appreciate better the
role of TLR in the pathogenesis of T1DM, both TLR7 and
TLR9 need to be studied because they are related to autoim-
munity. We have previously shown that T1DM is a pro-
inflammatory state, and in this manuscript, we provide evi-
dence of increased TLR2 and TLR4 expression, which may
contribute to the proinflammatory state of diabetes. Previ-
ously, increased biomarkers of inflammation, including CRP,
have correlated to endothelial dysfunction, carotid athero-
sclerosis, and calcium score by electron beam computed to-
TABLE 2. Release of monocyte cytokines in control and T1DM patients
Controls (n 31) T1DM (n 31)
Resting Activated Resting Activated
IL-1
(pg/mg cell protein) 4.6 2.5 21.6 13.9 27 16
a
118 90
a
TNF
(pg/mg cell protein) 2.3 (1.6, 2.4) 4.1 (2.8, 5.3) 6.2 (3, 7.3)
a
9.8 (7.8, 14.2)
a
Data are expressed as mean SD or median and interquartile range.
a
P 0.001 compared with controls.
*
*p<0.001 compared to Controls
Controls T1DM
5
15
Monocyte Nuclear NFKbp65 Activity
(ng/mg cell protein)
MyD88
pIRAK-1
Beta actin
TRIF
C T1DM
* P<0.05 vs contro
0
0.5
1
1.5
2
2.5
MyD88/b-actin pIRAK-1/IRAK TRIF/b-actin
ratio
Control
T1DM
**
*
C
B
A
FIG. 2. TLR signaling proteins in T1DM. A, NFK
activity in control (C) and
T1DM monocytes was performed using TransAM reagents as described in
Subjects and Methods.*,P 0.001 compared with controls. B,
Representative Western blotting results of TLR downstream signaling
proteins MyD88, pIRAK-1, and Trif was performed using specific rabbit
antibodies to the respective (phospho)proteins as described in Subjects
and Methods using
-actin as loading and internal control for MyD88 and
Trif and IRAK for pIRAK-1. C, Control. Densitometric ratios. *, P 0.05 vs.
control. Controls and T1DM, n 31 per group.
582 Devaraj et al. Increased TLR2 and TLR4 in T1DM J Clin Endocrinol Metab, February 2008, 93(2):578–583
mography, thus linking inflammation to diabetic complica-
tions (26 –28). Thus, it is not unreasonable to speculate that
in T1DM, TLR2 and TLR4, by contributing to the proinflam-
matory state, promote atherogenesis.
In conclusion, this is the first demonstration of increased
TLR2 and TLR4 expression and activity in T1DM monocytes.
Future studies will examine molecular mechanisms for increased
TLR2 and TLR4 expression, and determine their contribution to
the proinflammatory state of diabetes using diabetic TLR knock-
out mice.
Acknowledgments
We thank Danielle Zak, B.S., for technical assistance.
Address all correspondence and requests for reprints to: I. Jialal,
M.D., Ph.D., Director, Laboratory for Atherosclerosis and Metabolic
Research, University of California Davis Medical Center, 4635 II Ave-
nue, Research I Building, Room 3000, Sacramento, California 95817.
E-mail: ijialal@ucdavis.edu.
This work was supported by Juvenile Diabetes Research Foundation
International Grant 2007-585 (to I.J.) and National Institutes of Health
Grants K24 AT 00596 (to I.J.) and DK69801 (to S.D.).
Disclosure Statement: The authors have nothing to disclose.
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    • "It is likely that other mediating factors apart from hypertriglyceridaemia and hyperglycaemia may be involved in mediating neuropathy development in type 2 diabetes. Potential candidates include increased circulating inflammatory markers including NF-κB, which is upregulated in sural nerves in patients and rodent models [41][42][43] and involved in altered ther- monociception [42]. Furthermore, the pro-inflammatory cytokine TNF-α has been implicated in mechanical and thermal hyperalgesia in addition to ectopic firing of sensory neurons [41, , 45], which occurs in a dose-dependent manner [41] . "
    [Show abstract] [Hide abstract] ABSTRACT: Objectives: Diabetic peripheral neuropathy (DPN) is a common and debilitating complication of diabetes mellitus. Treatment largely consists of symptom alleviation and there is a need to identify therapeutic targets for prevention and treatment of DPN. The objective of this study was to utilise novel neurophysiological techniques to investigate axonal function in patients with type 2 diabetes and to prospectively determine their relationship to serum lipids in type 2 diabetic patients. Methods: Seventy-one patients with type 2 diabetes were consecutively recruited and tested. All patients underwent thorough clinical neurological assessments including nerve conduction studies, and median motor axonal excitability studies. Studies were also undertaken in age matched normal control subjects(n = 42). Biochemical studies, including serum lipid levels were obtained in all patients. Patient excitability data was compared to control data and linear regression analysis was performed to determine the relationship between serum triglycerides and low density lipoproteins and excitability parameters typically abnormal in type 2 diabetic patients. Results: Patient mean age was 64.2±2.3 years, mean glycosylated haemoglobin (HbA1c%) was 7.8±0.3%, mean triglyceride concentration was 1.6±0.1 mmol/L and mean cholesterol concentration was 4.1±0.2mmol/L. Compared to age matched controls, median motor axonal excitability studies indicated axonal dysfunction in type 2 diabetic patients as a whole (T2DM) and in a subgroup of the patients without DPN (T2DM-NN). These included reduced percentage threshold change during threshold electrotonus at 10-20ms depolarising currents (TEd10-20ms)(controls 68.4±0.8, T2DM63.9±0.8, T2DM-NN64.8±1.6%,P<0.05) and superexcitability during the recovery cycle (controls-22.5±0.9, T2DM-17.5±0.8, T2DM-NN-17.3±1.6%,P<0.05). Linear regression analysis revealed no associations between changes in axonal function and either serum triglyceride or low density lipoprotein concentration when adjusted for renal function, a separate risk factor for neuropathy development. Our findings indicate that acutely, serum lipids do not exert an acute effect on axonal function in type 2 diabetic patients: TEd(10-20ms)(1.2(-1.4,3.8);P = 0.4) and superexcitability (2.4(-0.05, 4.8);P = 0.06). Conclusions: These findings suggest that serum triglyceride levels are not related to axonal function in type 2 diabetic patients. Additional pathogenic mechanisms may play a more substantial role in axonal dysfunction prior to DPN development.
    Full-text · Article · Apr 2016
    • "TLR expression is increased in a plethora of inflammatory disorders, including diabetes [18]. Enhanced expression of TLR4 has been shown in monocytes of diabetic patients with microvascular complications [19,20]. Due to the single nucleotide polymorphisms (SNPs) in the extracellular domain of TLR4 there is deregulation of the TLR4 signaling that may alter the ligand binding capacity. "
    Article · Jan 2016 · PLoS ONE
    • "Another essential element of the innate immune system is the Toll Like Receptor (TLR) system, which consists of a group of transmembrane proteins expressed in immune and in nonimmune cells, whose role is to recognize components of foreign pathogens. TLRs have been associated with T1D development in mouse and in human studies [35, 36], thus underlining the importance of innate immune system in diabetes etiology. Emerging data have identified miRNAs as important regulators of development and function of innate immune components in addition to the aforementioned adaptive immune cells. "
    [Show abstract] [Hide abstract] ABSTRACT: MicroRNAs are small noncoding RNA molecules that regulate gene expression in all cell types. Therefore, these tiny noncoding RNA molecules are involved in a wide range of biological processes, exerting functional effects at cellular, tissue, and organ level. In pancreatic islets of Langerhans, including beta-cells, microRNAs are involved in cell differentiation as well as in insulin secretion, while in immune cells they have been shown to play pivotal roles in development, activation, and response to antigens. Indeed, it is not surprising that microRNA alterations can lead to the development of several diseases, including type 1 diabetes (T1D). Type 1 diabetes is the result of a selective autoimmune destruction of insulin-producing beta-cells, characterized by islet inflammation (insulitis), which leads to chronic hyperglycemia. Given the growing importance of microRNA in the pathophysiology of T1D, the aim of this review is to summarize the most recent data on the potential involvement of microRNAs in autoimmune diabetes. Specifically, we will focus on three different aspects: (i) microRNAs as regulators of immune homeostasis in autoimmune diabetes; (ii) microRNA expression in pancreatic islet inflammation; (iii) microRNAs as players in the dialogue between the immune system and pancreatic endocrine cells.
    Full-text · Article · Sep 2015
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