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. 2016 Dec 9:7:578.
doi: 10.3389/fimmu.2016.00578. eCollection 2016.

Increased Toll-Like Receptors Activity and TLR Ligands in Patients with Autoimmune Thyroid Diseases

Shiqiao Peng  1 , Chenyan Li  1   2 , Xinyi Wang  1   3 , Xin Liu  1   4 , Cheng Han  1 , Ting Jin  1   5 , Shanshan Liu  1   6 , Xiaowen Zhang  1 , Hanyi Zhang  1 , Xue He  1 , Xiaochen Xie  1 , Xiaohui Yu  1   2 , Chuyuan Wang  1   2 , Ling Shan  1 , Chenling Fan  1 , Zhongyan Shan  1   2 , Weiping Teng  1   2
Affiliations

Increased Toll-Like Receptors Activity and TLR Ligands in Patients with Autoimmune Thyroid Diseases

Shiqiao Peng et al. Front Immunol. .

Erratum in

Abstract

Objective: Autoimmune thyroid disease (AITD) is an organ-specific disorder due to the interplay between environmental and genetic factors. Toll-like receptors (TLRs) are pattern recognition receptors expressed abundantly on monocytes. There is a paucity of data on TLR expression in AITD. The aim of this study was to examine TLR expression, activation, ligands, and downstream signaling adaptors in peripheral blood mononuclear cells (PBMCs) extracted from untreated AITD patients and healthy controls.

Method: We isolated PBMC of 30 healthy controls, 36 patients with untreated Hashimoto's thyroiditis, and 30 patients with newly onset Graves' disease. TLR mRNA, protein expression, TLR ligands, and TLR adaptor molecules were measured using real-time PCR, Western blot, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). PBMC was simulated with TLR agonists. The effects of TLR agonists on the viability of human PBMC were evaluated using the MTT assay. The supernatants of cell cultures were measured for the pro-inflammatory cytokines, interleukin (IL)-6, tumor necrosis factor alpha (TNF-α), and IL-10 by ELISA.

Results: TLR2, TLR3, TLR9, and TLR10 mRNA were significantly increased in AITD patients compared with controls. TLR2, TLR3, TLR9, high mobility group box 1 (HMGB1), and RAGE expression on monocytes was higher in patients than control at baseline and TLR agonists' stimulation. The release of TNF-α and IL-6 was significantly increased in PBMCs from AITD patients with TLR agonists, while IL-10 was significantly decreased. Downstream targets of TLR, myeloid differentiation factor 88 (MyD88), and myeloid toll/IL-1 receptor-domain containing adaptor-inducing interferon-β were significantly elevated in AITD patients. Levels of TLR2 ligands, HMGB1, and heat shock protein 60 were significantly elevated in AITD patients compared with those in controls and positively correlated with TgAb and TPOAb, while sRAGE concentration was significantly decreased in AITD patients.

Conclusion: This work is the first to show that TLR2, TLR3, and TLR9 expression and activation are elevated in the PBMCs of patients with AITD and TLRs may participate in the pathogenesis of AITD.

Keywords: autoimmune thyroid disease; innate immunity; pathogenesis; signaling; toll-like receptor.

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Figures

Figure 1
Figure 1
Toll-like receptor (TLR) mRNA expression in peripheral blood mononuclear cells (PBMCs). Bar graphs comparing TLR1–TLR10 mRNA expression levels in PBMCs from patient with Hashimoto’s thyroiditis, Graves’s disease, and healthy controls. TLRs expression was measured by real-time PCR using GAPDH as endogenous control. Results are shown as fold change regarding TLRs gene expression in AITD patients relative to healthy subjects. Each bar represents the 1 ± SEM of three groups. One-way ANOVA was used to compare the differences of expression between AITD patients and healthy controls. AITD, autoimmune thyroid disease; HT, Hashimoto’s thyroiditis; GD, Graves’ disease; HC, healthy controls. Level of significant is *P < 0.05 and **P < 0.01.
Figure 2
Figure 2
Cell viability in peripheral blood mononuclear cells after toll-like receptor (TLR) agonists stimulation. Bar graphs comparing of cell viability optical density between unstimulated and stimulated cells treated with either unstimulate or stimulate of TLR agonists in both AITD patients and controls by MTT assay. Each bar represents the mean ± SD of three groups. One-way ANOVA was used to compare the differences of expression between AITD patients and healthy controls. AITD, autoimmune thyroid disease; HT, Hashimoto’s thyroiditis; GD, Graves’ disease; HC, healthy controls. Pam3CSK4, Poly I:C, and CpG ODN were used as TLR2, TLR3, and TLR9 agonists, respectively. Level of significant is *P < 0.05 and **P < 0.01.
Figure 3
Figure 3
Time-dependent modulation of TLR2, TLR3, TLR9, high mobility group box 1 (HMGB1), and receptor for advanced glycation end products (RAGE) expression in peripheral blood mononuclear cells (PBMCs) by specific agonist activation. PBMCs from healthy controls were treated with a TLR2, TLR3, or TLR9 agonist for 0, 2, 6, 8, 12, 24, and 48 h stained with anti-CD14 and anti-TLR2, TLR3, TLR9, HMGB1, and RAGE mAbs for flow cytometry. Percentages of CD14+TLR2+ (A), CD14+TLR3+ (B), CD14+TLR9+ (C), CD14+HMGB1+ (D), CD14+RAGE+cells (E) from HC was measured by flow cytometry. Results are shown as mean ± SD of control group. Level of significant is *P < 0.05. Statistical analyses were conducted using one-way ANOVA. Pam3CSK4, Poly I:C, and CpG ODN were used as TLR2, TLR3, and TLR9 agonists, respectively.
Figure 4
Figure 4
TLR2, TLR3, and TLR9 surface protein expression in monocytes after toll-like receptor agonists stimulation. Peripheral blood mononuclear cells from AITD patients and healthy controls were treated with a TLR2, TLR3, or TLR9 agonist or PBS as basal for 24 h and stained with anti-CD14 and anti-TLR2, TLR3, and TLR9 mAbs for flow cytometry. We first selected singlets using forward scatter area Vs forward scatter height parameters. From the singlet population, we removed dead cells from total events by gating on LIVE/DEAD events. Then, we selected total monocytes from the living using the pan-monocyte marker CD14, then TLR2 positive, TLR3 positive, and TLR9 positive, respectively. (A) Flow cytometry dot plots from AITD and HC depicting TLR2 expression by CD14 enriched monocyte cells (left panels). The number in the right quadrants represents the percentage of CD14+TLR2+ between AITD and healthy controls. (B) Flow cytometry dot plots from AITD and HC depicting TLR3 expression by CD14 enriched monocyte cells (left panels). Percentages of CD14+TLR3+ cells were compared between HC and AITD patients (right panels). (C) Flow cytometry dot plots from AITD and HC depicting TLR9 expression by CD14 enriched monocyte cells (left panels). Percentages of CD14+TLR9+ cells were compared between HC and AITD patients (right panels). Each bar represents the mean ± SD of three groups. Level of significant is *P < 0.05 and **P < 0.01. AITD, autoimmune thyroid disease; HT, Hashimoto’s thyroiditis; GD, Graves’ disease; HC, healthy controls. Statistical analyses were conducted using one-way ANOVA. Pam3CSK4, Poly I:C, and CpG ODN were used as TLR2, TLR3, and TLR9 agonists, respectively.
Figure 5
Figure 5
High mobility group box 1 (HMGB1) and receptor for advanced glycation end products (RAGE) surface protein expression in monocytes after TLR9 agonist stimulation. Peripheral blood mononuclear cells from AITD patients and healthy controls were treated with a TLR9 agonist or PBS as basal for 24 h and stained with anti-CD14 and anti-HMGB1, and RAGE mAbs for flow cytometry. We first selected singlets using forward scatter area Vs forward scatter height parameters. From the singlet population, we removed dead cells from total events by gating on LIVE/DEAD events. Then, we selected total monocytes from the living using the pan-monocyte marker CD14, then HMGB1 positive and RAGE positive cells selected, respectively. (A) Flow cytometry dot plots from AITD and HC depicting HMGB1 or match isotype control expression by CD14 enriched monocyte cells (left panels). The number in the right quadrants represents the percentage of CD14+HMGB1+ between AITD and healthy controls. (B) Flow cytometry dot plots from AITD and HC depicting RAGE expression by CD14 enriched monocyte cells (left panels). Percentages of CD14+RAGE+ cells were compared between HC and AITD patients (right panels). Control cells were stained with the secondary antibodies without using the anti-RAGE primary antibody. Each bar represents the mean ± SD of three groups. Level of significant is *P < 0.05 and **P < 0.01. AITD, autoimmune thyroid disease; HT, Hashimoto’s thyroiditis; GD, Graves’ disease; HC, healthy controls. Statistical analyses were conducted using one-way ANOVA. CpG ODN was used as a TLR9 agonist.
Figure 6
Figure 6
Release of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-10 in the cultured supernatants from AITD patients and healthy controls. (A) Peripheral blood mononuclear cells (PBMCs) isolated from subjects were stimulated with Pam3CSK4, poly I:C, and CpG ODN for 24 h, TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). (B) PBMCs were treated as in (A) and the release of IL-6 was measured by ELISA. (C) PBMCs were treated as in (A) and the release of IL-10 was measured by ELISA. Level of significant is *P < 0.05 and **P < 0.01. Values were shown as mean ± SD and expressed as picograms per milliliter. AITD, autoimmune thyroid disease; HT, Hashimoto’s thyroiditis; GD, Graves’ disease; HC, healthy controls. Statistical analyses were conducted using one-way ANOVA. Pam3CSK4, poly I:C, and CpG ODN were used as TLR2, TLR3, and TLR9 agonists, respectively.
Figure 7
Figure 7
Toll-like receptor (TLR) signaling proteins in peripheral blood mononuclear cell from patients with Hashimoto’s thyroiditis, Graves’s disease, and healthy controls. (A) Representative Western blotting results of TLR downstream signaling proteins MyD88 and toll/IL-1 receptor-domain containing adaptor-inducing interferon-β (TRIF) was performed using specific antibodies to the respective proteins as described in methods. β-actin was used as loading and internal control for MyD88 and TRIF. (B) A ratio of TRIF/β-actin was determined to represent a mean net density. (C) A ratio of MyD88/β-actin was determined to represent a mean net density. Level of significant is *P < 0.05 and **P < 0.01. Values were shown as mean ± SD. HT, Hashimoto’s thyroiditis; GD, Graves’ disease; HC, healthy controls. One-way ANOVA was used to compare the differences of TRIF and MyD88 proteins of these three groups.
Figure 8
Figure 8
Dot plots showing serum levels of high mobility group box 1 (HMGB1), heat shock protein 60 (HSP60), and sRAGE in patients with Hashimoto’s thyroiditis, Graves’s disease, and healthy controls. Serum levels of HMGB1 (A), HSP60 (B), and sRAGE (C) were measured by using enzyme-linked immunosorbent assay. Results are shown in the graphic. HT, Hashimoto’s thyroiditis; GD, Graves’ disease; HC, healthy controls. Level of significant is *P < 0.05 and **P < 0.01. Values were shown as mean ± SD. Statistical analyses were conducted using one-way ANOVA.
Figure 9
Figure 9
Correlation analysis of laboratory parameters and serum levels of high mobility group box 1 (HMGB1) and heat shock protein 60 (HSP60) in HT patients. Relationship between serum expression of HMGB1 with TPOAb (A) and TgAb (B), HSP60 with TPOAb (C) and TgAb (D) are presented in scatter plots. Pearson’s and Spearman’s correlation and P-values are given in all cases, r = Pearson correlation coefficient. HT, Hashimoto’s thyroiditis.
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