Endosomal Toll-Like Receptor Status in Patients with Metabolic Syndrome

Abstract

Background:

Metabolic syndrome (MetS) predisposes to both diabetes and cardiovascular disease, and inflammation is pivotal in MetS. The Toll-like receptors (TLRs), TLR2 and TLR4, are implicated in both diabetes and atherosclerosis, are increased in MetS, and contribute to the inflammatory burden. Recent studies also suggest an evolving role of endosomal TLRs in diabetic complications. However, there is a paucity of data with regard to the expression of endosomal TLRs such as TLR3, 7–9 in MetS.

Aim:

Thus, in this short report, we examine expression of monocytes TLR3 and TLR9 in our cohort of subjects with nascent MetS compared to matched controls.

Subjects and Methods:

Monocytes were isolated from subjects with MetS (n = 45) and matched controls (n = 37), respectively, and TLR3 and TLR9 expression was assessed by intracellular flow cytometry and correlated with nuclear factor (NF)-κB expression.

Results:

We demonstrate increased endosomal TLR9 expression in MetS compared to controls that correlate with increased nuclear NF-κB expression in the monocytes of these subjects, with no change in TLR3 protein.

Conclusion:

Future studies are required to confirm these findings and determine the role of TLR9 in the increased cardiovascular risk in MetS.

Introduction

Metabolic syndrome (MetS) is a cardiometabolic cluster of abnormalities that predisposes to both diabetes and cardiovascular disease.¹ Inflammation appears to be pivotal in the pathogenesis of the MetS, diabetes, and cardiovascular diseases.² The pattern recognition receptors and Toll-like receptors (TLRs) appear to be crucial in metainflammation.^3
–5 The TLRs are classified into two distinct groups on the basis of their cellular distribution and ligand repertoire. External TLRs 1, 2, 4, 5, and 6 are located on the cell surface and recognize ligands of mainly bacterial and fungal origin. Internal members of the TLR family (TLR3, 7, 8, and 9) are localized to endosomes, where their primary function is to sense microbial and host-derived nucleic acids. It has been previously reported that cell surface TLRs, such as TLR2 and TLR4, are implicated in atherosclerosis and increased in diabetes and MetS and can contribute to the inflammatory burden in these states.^3
–5 Recently, endosomal TLRs have been shown to be potential contributors to the inflammation in different human studies and rodent models.⁶ Recent studies support an evolving role of endosomal TLRs in the development of secondary complications of T2DM because of their proinflammatory nature.⁶ However, there is a paucity of data with regard to the expression of endosomal TLRs, such as TLR3, 7–9 in MetS. TLRs signal either through a MyD88- or non-MyD88-dependent pathway.^3
–5 Since TLR3 does not signal like other TLRs through MyD88 and has a controversial role in atherosclerosis, we wanted to examine the expression of TLR3 in MetS. Furthermore, representative of the other MyD88 signaling pathway, endosomal TLRs, is TLR9, which has been implicated in atherosclerosis.^7,8 Thus, in this short report, we examine the expression of TLR3 and TLR9 in our cohort of monocytes of subjects with nascent MetS compared to matched controls.⁹

Subjects and Methods

All subjects were recruited from the Sacramento County, California, through fliers and advertisements in the newspaper, as described previously.⁹ The subjects (aged 21–70 years) with MetS (n = 45) and healthy control subjects (n = 37) were studied. MetS was defined using the modified criteria of the NCEP ATP III.⁹ Control subjects needed to have ≤2 features of MetS and not be on blood pressure medications with normal fasting glucose (<100 mg/dL) and triglycerides (<200 mg/dL).

Other exclusion criteria for both groups were diabetes, clinical atherosclerosis (coronary artery disease, peripheral arterial disease, cardiovascular disease [CVD], and so on), smoking, hypo- or hyperthyroidism, malabsorption, anticoagulant therapy, steroid therapy, anti-inflammatory drugs, statin and other hypolipidemic therapy, hypoglycemic agents, angiotensin receptor blockers, triglyceride > 400 mg/dL (for MetS subjects), oral contraceptives, use of antioxidant supplements in the past 6 months, pregnancy, abnormal complete blood count, alcohol consumption >1 oz/day, consumption of n-3 polyunsaturated fatty acid, postmenopausal women on estrogen replacement therapy, active wounds, recent surgery, inflammatory or malignant disease, C-reactive protein (CRP) > 10 mg/L, chronic high-intensity exercisers (exercise > 100 min/week), and any evidence of microalbuminuria. Diabetes was excluded by two fasting glucose levels (screening and day of monocyte isolation) <126 mg/dL and an HbA_1c <6.5%.

Informed consent was obtained from participants in the study, which was approved by the Institutional Review Board at the University of California, Davis. After history and physical examination, fasting blood was obtained. A complete blood count, plasma lipid and lipoprotein profile, glucose, and high-sensitivity CRP (hsCRP) were assayed by standard laboratory techniques in the Clinical Pathology Laboratory. Insulin levels were assayed by the enzyme-linked immunosorbent assay (Linco Biosystems), and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated from glucose and insulin levels, as previously described.⁹

Monocyte isolation

Mononuclear cells were isolated from fasting heparinized blood by Ficoll–Hypaque centrifugation followed by magnetic separation using the depletion technique (Miltenyi Biotech), as previously described.⁹

Surface expression of TLR3 and TLR9

Monocytes from control and MetS subjects were fixed at room temperature for 30 min with a fixation buffer and then incubated with anti-human TLR3 and TLR9 antibodies (InvivoGen) or isotype controls in permeabilization buffer for 30 min at room temperature, and endosomal expression of TLR3 and TLR9 was analyzed using the BD FACSArray, as previously described.⁹ Results were expressed as mean fluorescence intensity of 10,000 cells.

Cell signaling studies

Monocyte nuclear extracts were prepared as previously described,¹⁰ and the nuclear factor (NF)-κB p65 activity in the nuclear extracts was assessed using reagents from Bio-Rad using the Bioplex multiplex assays following the manufacturer's instructions. Plasma biomediators, including interleukin (IL)-1, IL-6, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factors (TNF), were quantified, as previously described.⁹

Statistical analysis

Data are expressed as mean ± SD or, for skewed variables, as median and interquartile range. Log transformations were applied to skewed data before parametric analyses. Comparisons between the control and MetS groups were made with two-sample t-tests for subject characteristics, and TLR3 and TLR9 were compared with analysis of covariance to control for age, waist circumference, and body mass index. Spearman rank correlation coefficients were computed to assess the association between metabolic risk factors and the monocyte activity. Data were analyzed using the SAS version 9.4 (SAS Institute).

Results

Table 1 shows salient baseline characteristics of the studied population. All features of MetS, including waist, blood pressure, glucose, and triglycerides, were higher, and high-density lipoprotein cholesterol was lower in MetS compared to controls. In addition, there was a significant increase in hsCRP levels and HOMA-IR, as shown previously.⁹

Table 1.

Salient Baseline Characteristics

Variable	Controls (n = 37)	MetS (n = 45)	P value (controls versus MetS)
Sex, F/M	30/7	36/9	1.0
Age (years)	48 ± 12	51 ± 10	0.25
Waist (cm)	93 ± 15	106 ± 12	<0.0001
BMI (kg/m²)	30.9 ± 6.8	34.1 ± 6.5	0.02
BP-systolic (mmHg)	117 ± 12	130 ± 11	<0.0001
BP-diastolic (mmHg)	73 ± 8	81 ± 9	<0.0001
Glucose (mg/dL)	89 ± 7	100 ± 12	<0.0001
Total cholesterol (mg/dL)	190 ± 30	201 ± 29	0.09
Triglycerides (mg/dL)	72 (60, 97)	150 (103, 174)	<0.0001
HDL cholesterol (mg/dL)	55 ± 14	41 ± 11	<0.0001
LDL cholesterol (mg/dL)	119 ± 24	129 ± 22	0.03
hsCRP (mg/L)	1.3 (0.5, 2.8)	4.1 (1.7, 5.6)	0.0002
HOMA-IR	1.4 (1.1, 2.9)	2.7 (1.9, 5.1)	<0.0001

Results are presented as mean ± standard deviation or median (25th percentile, 75th percentile).

BMI, body mass index; CRP, C-reactive protein; HDL, high-density lipoprotein; hsCRP, high-sensitivity CRP; HOMA-IR, homeostasis model assessment of insulin resistance; LDL, low-density lipoprotein; MetS, metabolic syndrome.

With regard to monocyte expression of endosomal TLRs, we report on TLR3 and TLR 9 expression in Fig. 1. While there was no change in TLR3, there was a significant upregulation in the TLR9 expression in monocytes of MetS compared to controls (P = 0.005), controlling for age, waist, and body mass index, which were greater in MetS patients compared to controls, suggesting it is a feature of the MetS independent of adiposity. TLR9 abundance did not correlate with any features of the MetS or increase with increasing features of MetS.

FIG. 1.

Endosomal TLR3 and TLR9 in monocytes of subjects with metabolic syndrome (MetS). Least squares geometric mean (TLR3), mean (TLR), and 95% confidence intervals, adjusted for age, body mass index, and waist circumference. MetS, metabolic syndrome; TLR, Toll-like receptor.

A pivotal downstream marker of activation of TLR9 is the transcription factor, NF-κB, and we have previously shown increased nuclear NF-κB binding and nuclear pp65/p65 ratio in monocytes of these MetS subjects compared to controls (P < 0.001). Thus, we examined correlation of TLR9 with NF-κb, and there was a significant correlation between nuclear pp65/p65 ratio and the TLR9 expression (r = 0.28, P = 0.02). Surprisingly, unlike with TLR4 and TLR2, there were no significant correlations with the biomediators, including IL-1, IL-6, and MCP-1.⁹

Discussion

Endosomal TLRs detect damage-associated molecular products (DAMPs) and, after activation, induce a strong inflammatory response mediated by NF-κB. We and others have previously reported increased cell surface TLRs, such as TLR2 and TLR4, in MetS in both monocytes and subcutaneous adipose tissue^4,11 associated with increased cellular and systemic inflammation. Hence, we hypothesized that inflammation in MetS may also be due to activation of endosomal TLRs such as TLR3 and TLR9. In this report, we demonstrated increased endosomal TLR9 expression in MetS compared to controls that correlate with increased nuclear NF-κb expression in the monocytes of these subjects. However, there was no significant change in the TLR3 protein.

Increased endosomal TLR signaling has been shown to be involved in some chronic inflammatory diseases such as systemic lupus erythematoses, T1DM, multiple sclerosis, and rheumatoid arthritis.⁶ Activation of TLR9 on dendritic cells suppresses the immunosuppressive anti-inflammatory Treg cells, thereby promoting inflammation.¹² Duramad et al. and Walker et al. also supported the inflammatory nature of TLR9 and suggested that it may be responsible for systemic inflammation in both mice and humans.^13,14 Previously, TLR9 has been shown to be upregulated in diabetic wounds and modulates the severity of diabetic wounds.¹⁵ Endosomal TLR9 appears to activate systemic inflammation by upregulating cytokines such as interferon gamma and TNF through an NF-κB-mediated pathway.⁶ We have previously shown increased NF-κB as well as increased monocyte derived and systemic increase in cytokines in MetS compared to controls.⁹ In this report, we show a significant correlation between increased endosomal TLR9 and the NF-κB activity corroborating the earlier findings. However, unlike TLR2 and TLR4, we failed to show a significant correlation with biomediators, suggesting that TLR9 might not be such a significant contributor to the increased inflammatory burden. In addition, it is possible that with activation of TLR9 with its classical ligand, CpG DNA motif, we would have seen a significant correlation with biomediators.

CpG ODN activates the TLR9-MyD88-ERK1/2 pathway, inducing foam cell formation.¹⁶ In addition, TLR9 agonists induce macrophage accumulation of lipids, especially triglycerides in vitro.⁸ Two studies have shown that activation of TLR9 facilitated the formation of foam cells in an NF-κB-dependent manner.¹⁷ TLR9 is present in human atheroma.⁷ In hypercholesterolemic apo E*3 Leiden mice, TLR9 was significantly increased following femoral artery cuff placement. Blocking TLR7/9 significantly reduced arterial wall inflammation and macrophage infiltration.¹⁸ Furthermore, inactivation of TLR9 results in decreased MyD88 and p-p65-NF-κB and alleviated atherosclerosis progression.¹⁹ In another study, obesity, either diet induced or due to leptin gene deficiency, resulted in increased TLRs 9, 11–13 in adipose tissue, concomitant with increased NF-κB and proinflammatory cytokines.²⁰ These data suggest that TLR9 is proinflammatory and promotes atherosclerosis. However, a single report suggested that TLR9 is antiatherogenic without a very clear explanation, but may have resulted from differences in gender and treatment time.²¹ Our data of increased TLR9 in human MetS support the general consensus that TLR9 could also contribute to the increased CVD risk of MetS by promoting inflammation through the activation of NF-κB.

In conclusion, in this preliminary report, we make the novel observation of upregulation of the endosomal TLR9 in monocytes of MetS patients compared with controls and, in conjunction with TLR2 and TLR4, may contribute to the chronic inflammation in these subjects. Further research is required to confirm these findings and test the effect of agonists/antagonists of these endosomal TLRs in MetS and animal models.

Footnotes

Author Disclosure Statement

No conflicting financial interests exist.

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