Iodine activates NLRP3 inflammasomes in PBMCs of patients with autoimmune thyroiditis and regulates Th1 and Th17 cell differentiation

Abstract

Objectives: Inflammasomes are associated with various autoimmune diseases. Herein, we aimed to study the occurrence of inflammasomes in peripheral blood mononuclear cells (PBMCs) from patients with autoimmune thyroiditis (AIT), and the relationship between their abundance and the inflammatory response index of AIT. Furthermore, we examined the effect of iodine on inflammasomes containing NLR family pyrin domain-containing 3 (NLRP3) and inflammasome activation of helper T (Th) cell differentiation regulation in cultured PBMCs.

Methods: We collected PBMCs and serum samples from 50 patients with AIT with normal thyroid function and 50 controls matched for age and sex. In PBMCs, the mRNA and protein expressions of certain inflammasome constituents (e.g., NLRP1, NLRP3, absent in melanoma 2 (AIM2) and caspase-1), interleukin (IL)-1β and IL-18 were assessed using qRT-PCR and western blotting. Enzyme-linked immunosorbent assays (ELISAs) assessed the serum levels of IL-1β and IL-18. Flow cytometry was employed to examine NLRP3 expression on CD14+ monocytes and Th1 and Th17 cell percentages in the groups. AIT- or healthy control-derived PBMCs were stimulated using sodium iodide, with or without lipopolysaccharide (LPS) for 72 h.

Results: PBMCs from patients with AIT had significantly higher levels of pro-IL-18, pro-IL-1β and NLRP3 than did the PBMCs from the healthy controls (P < 0.05). Compared with those from the controls, AIT-derived PBMCs had enhanced levels of active IL-18 and active caspase-1 p20 (P < 0.05), whereas their abundance of active IL-1β was similar (P > 0.05). In serum, the AIT group had enhanced levels of IL-18 compared with the healthy controls (P < 0.05) but had similar levels of IL-1β (P > 0.05). NLRP3 expression on CD14+ monocytes from AIT patients was significantly augmented compared with the healthy controls (P< 0.01). Significantly increased percentages of Th1 and Th17 cells were detected in AIT patients compared with those in the healthy participants (P < 0.001). Sodium iodide treatment upregulated NLRP3 expression in PBMCs during 72 h of culture (P < 0.001). The percentage of Th1 and Th17 cells in AIT patients increased in an iodine-dependent manner (P < 0.01). Iodine had no significant effect on the number of these cells in the healthy control group (P > 0.05).

Conclusion: AIT-derived PBMC NLRP3 activity and expression increased. Iodine might regulate the immune and inflammatory response of patients with AIT by activating NLRP3 and promoting Th1 and Th17 cell differentiation.

Keywords: T cell subsets; autoimmune thyroiditis; inflammasome; iodine; peripheral blood mononuclear cells.

Figure 1

PBMCs from patients with AIT had increased inflammasome component levels. (A) NLRP1, NLRP3, AIM2 and CASP1 mRNA levels in AIT patients and control thyroid tissues (n = 50 per group). Representative immunoreactive protein bands (B) and related quantitative determination (C) of the protein levels of NLRP1, NLRP3, AIM2, caspase-1 and caspase-1 p20 in PBMCs from the AIT and HC groups (n = 20 per group). GAPDH expression was used for normalization. The columns represent the mean ± SD values. The patient and control data were compared using Student’s t-test or Mann–Whitney U test. *P < 0.05; **P < 0.01. AIT, autoimmune thyroiditis; HC, healthy control; PMBC, peripheral blood mononuclear cell.

Figure 2

Increased levels and maturation of IL-1β and IL-18 PBMCs from patients with AIT. (A) IL-1β and IL-18 mRNA levels in PBMCs from patients with AIT and HCs (n = 50 per group). Representative immunoreactive protein bands (B) and related quantitative determination (C) of pro-IL-18, active IL-18, pro-IL-1β and active IL-1β levels in patient and control PBMCs (n = 20 per group). GAPDH expression was used for normalization. The columns represent the mean ± SD values. Patient and control data were compared using Student’s t-test or Mann–Whitney U test. *P < 0.05; **P < 0.01. AIT, autoimmune thyroiditis; HC, healthy control; PMBC, peripheral blood mononuclear cell.

Figure 3

IL-18 and IL-1β levels in PBMCs from patients with AIT. (A) Serum levels of IL-1β in PBMCs from patients with AIT and controls. (B) Serum levels of IL-18 in patient and control PBMCs. The columns represent the mean ± SD values. Mann–Whitney U-tests and Student’s t-tests were employed to compare the data between the AIT and HC groups. *P < 0.05; **P < 0.01. AIT, autoimmune thyroiditis; HC, healthy control; PMBC, peripheral blood mononuclear cell.

Figure 4

Correlation analysis of inflammasome component mRNA levels in PBMCs and serum autoantibody levels in patients with autoimmune thyroiditis (AIT). Correlation analysis of NLRP3, IL-18 and IL-1β mRNA levels in PBMCs and the serum levels of thyroid peroxidase antibody (TPOAb)/thyroglobulin antibody (TgAb) levels (A, B, C, D, E, F). The Spearman rank test was used to carry out bivariate correlation analysis (r = the Spearman correlation coefficient). PMBC, peripheral blood mononuclear cell.

Figure 5

NLRP3 expression on monocytes of HC and AIT groups. (A) HC group, (B) AIT group and (C) the corresponding quantitative analysis (n = 30 per group). The columns represent the mean ± SD values. Mann–Whitney U-tests and Student’s t-tests compared the data between the patients and HCs. *P < 0.05; **P < 0.01; ***P < 0.001. AIT, autoimmune thyroiditis; HC, healthy control.

Figure 6

Iodine’s effects on the relative mRNA and protein levels of NLRP3 and its components in PBMCs from the AIT group and the HC group. (A) NLRP3 mRNA relative expression; (B) CASP1 mRNA relative expression; (C), (D) representative immunoreactive protein bands in the HC and AIT groups; (E) the relative protein level of NLRP3; (F) caspase-1 relative protein level; (G) the relative protein level of caspase-1 P20 (*P < 0.05; **P < 0.01; ***P < 0.001: in comparison with the lipopolysaccharide (LPS) group. ^#P < 0.05; ^##P < 0.01; ^###P < 0.001: AIT group vs HC group). Different concentrations of iodine: iodide 1: 5 × 10⁻⁵ mmol/L, iodide 2: 2 × 10⁻⁴ mmol/L, iodide 3: 1 × 10⁻³ mmol/L and iodide 4: 1 × 10⁻² mmol/L. AIT, autoimmune thyroiditis; HC, healthy control.

Figure 7

NLRP3 levels in monocytes of the healthy control (HC) group and the autoimmune thyroiditis (AIT) group after lipopolysaccharide (LPS) and sodium iodide stimulation, as determined using flow cytometry. HC group: (A) only culture medium without a stimulant; (B) 2 μg/mL LPS; (C, D, E, F) different concentrations of iodine were added; (G) statistical chart of NLRP3 expression in the monocytes of the six groups (*P < 0.05; **P < 0.01; ***P < 0.001). AIT group: (H) only culture medium without a stimulant; (I) 2 μg/mL LPS; (J, K, L, M) different concentrations of iodine were added; (N) statistical chart of NLRP3 expression in monocytes of the six groups (*P < 0.05; **P < 0.01; ***P < 0.001). Different concentrations of iodine: iodide 1: 5 × 10⁻⁵ mmol/L, iodide 2: 2 × 10⁻⁴ mmol/L, iodide 3: 1 × 10⁻³ mmol/L and iodide 4: 1 × 10⁻² mmol/L.

Figure 8

Flow cytometry analysis of the changes in Th1 and Th17 cell differentiation percentage in autoimmune thyroiditis (AIT) monocytes after lipopolysaccharide (LPS) and sodium iodide stimulation. Th1 cell: (A) only culture medium without a stimulant; (B) 2 μg/mL LPS; (C, D, E, F) different concentrations of iodine were added. Th17 cell: (A) only culture medium without a stimulant; (B) 2 μg/mL LPS; (C, D, E, F) different concentrations of iodine were added; (N) statistical chart of Th1 cell differentiation percentage changes in the six groups; (M) statistical chart of the change in the percentage of Th17 cell differentiation in the six groups (*P < 0.05; **P < 0.01; ***P < 0.001). Different concentrations of iodine: iodide 1: 5 × 10⁻⁵ mmol/L, iodide 2: 2 × 10⁻⁴ mmol/L, iodide 3: 1 × 10⁻³ mmol/L and iodide 4: 1 × 10⁻² mmol/L.