NOD.H-2^h4 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA.). Mice were bred and raised under specific pathogen-free conditions in the animal facility at China Medical University. NOD.H-2^h4 mice at 4 weeks of age with a mean weight of 20 g were randomly divided into the control and spontaneous autoimmune thyroiditis (SAT) groups. Mice in the control group (16 mice, 8 per time-point) were given sterile water. SAT group (16 mice, 8 per time-point) were given 0.005% mg/L NaI in their drinking water for 8 or 16 weeks. Each group of mice was killed at 8 and 16 weeks after grouping. All of the animal study procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were performed according to the institutional ethical guidelines. Thyroid tissues were used for HE staining (32 mice, 8 per group), RT-PCR (32 mice, 8 mice per group) and Western blotting (32 mice, 8 per group).

This study was carried out in accordance with the recommendations of “the regional Ethics Committee guidelines and institutional policies, the Ethics Committee of China Medical University.” The protocol was approved by “The Ethics Committee of China Medical University (the protocol number was [2014]94).” All subjects gave written informed consent in accordance with the Declaration of Helsinki.

Thyroid tissues were obtained from 23 patients with HT who were undergoing thyroidectomies for treatment. Non-HT thyroid tissues collected from 24 paracancerous tissues from patients without HT as controls. Freshly harvested thyroid tissues were used for immunohistochemical staining (12 patients, 6 per group), RT-PCR (47 patients, 24 patients with HT and 23 patients without HT), and Western blotting (16 patients, 8 per group).

Serum was obtained from 35 patients with HT and 21 healthy controls. Patients included in the HT group were positive for at least one autoantibody. Serum samples were stored at −80°C for subsequent analysis.

Thyroid tissues were obtained from six patients with HT for primary thyrocyte culture, and all of these subjects were euthyroid. The tissue was processed as previously described (8). Fresh thyroid tissue was scissors into small pieces of about 1 mm³ and incubated with Collagenase IV (1.25 mg/ml; Sigma, C1889) in Hank's solution at 37°C for 4 h. Cells suspension was filtered through a nylon cell strainer with 70 μm. The filtrate was centrifuged at 1,000 × g for 5 min. Cells were resuspended in RPMI-1640 containing 10% fetal bovine serum (FBS). Then, thyroid cells were plated in 6-well plates (1 × 10⁶ cells per well). After 24 h the supernatant containing no adherent cells was removed.

Primary thyrocytes and Nthy-ori 3-1 (a human thyroid follicular epithelial cell line from the Health Protection Agency Culture Collections, UK) were cultured in RPMI-1640, 10% FBS and Pen/Strep in 5% CO₂ at 37 °C. Different doses of rhIL-34 (from 0 to 50 ng/mL; R&D Systems, Minneapolis, MN, USA) were used at indicated times as stimulators of thyrocytes. Primary thyrocytes were treated with rh-IL-34 (50 ng/mL) alone or plus CSF-1R inhibitor (25 μM; Tocris Cookson, Bristol, UK) for 24 h. Nthy-ori 3-1 were treated with increasing doses of the IFN-γ (from 0 to 1000 IU/mL; R&D Systems, Minneapolis, MN, USA) for 24 h.

Western blotting was performed as previously described (8). Anti-IL-34 and STAT3 antibodies (1:1,000; Abcam, Cambridge, MA, UK), anti-CSF-1R and p-STAT3 antibodies (1:1,000; Cell Signaling), anti-Bax and Bcl-2 antibodies (1:1,000; BioLegend, San Diego, CA, USA), and anti-GAPDH antibodies (1:1,000; Santa Cruz, Dallas, TX, USA) were used in our study.

Immunohistochemistry (IHC) was performed as previously described (8). Rabbit anti-human antibody IL-34 antibodies (1:500; Biorbyt LLC, San Francisco, CA, USA) were used at 1:500 dilutions to observe the expression of IL-34 in thyroid tissues. The staining intensity was measured by the Image-Pro Plus 5.0 software (Media Cybernetics, Inc. Silver Spring, MD USA). The mean densitometry of the image is designated as the levels of IL-34 expression.

Serum IL-34 levels were measured using an ELISA kit according to the manufacturer's directions (BioLegend, San Diego, CA, USA). Each sample was measured three times.

We used RT-PCR to detect mRNA expression, as previously described (8).

The following primers were used: human IL-34, F: 5′-TTGACGCAGAATGAGGAGTG-3′; R: 5′-CCCTCGTAAGGCACACTGAT-3′; Mouse IL-34, F: 5′- ATCTACAGGAGGTTCAGACATTGC-3′; R: 5′-AACAGTACAGCAGTTCCATGACC-3′; Human Bcl-2, F: 5′-ACATCCATTATAAGCTGTCGCAGAGG-3′; R: 5′- TGCAGCGGCGAGGTCCTG-3′; Human Bax: F: 5′-GATGCGTCCACCAAGAAGCTGAG-3′; R: 5′-CACGGCGGCAATCATCCTCTG-3′. Human GAPDH: F: 5′-CAGGAGGCATTGCTGATGAT-3′; R: 5′-GAAGGCTGGGGCTCATTT-3′;

Mouse GAPDH: F: 5′-GGTTGTCTCCTGCGACTTCA-3′; R: 5′- TGGTCCAGGGTTTCTTACTCC-3′. The results are shown as the mean ratio of the test molecule/GAPDH±SD.

Apoptotic thyrocytes were detected using an Annexin V FITC Apoptosis Detection Kit (Dojindo Molecular Technologies, Rockville, MD) according to the manufacturer's directions. Thyrocyte fluorescence was analyzed by a FACS Calibur (BD Biosciences, USA) cytometer with CellQuest software.

All data are presented as the mean ± SD or median. The nonparametric Mann-Whitney test and t-test were performed to compare the variables between groups. Associations were analyzed by using the Spearman correlation test. All graphs were performed using GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). All western blot analysis be scanned and the abundance assessed quantitatively using Image J (NIH Image, Bethesda, MD, USA). All quantitative data analyses were performed using SPSS version 20.0 (IBM, Armonk, New York, USA), and P < 0.05 was considered significant.

Thyroid follicles in the control group had a uniform morphology and moderate size. In the SAT groups, thyroid follicles were significantly enlarged, exhibited different morphologies, and contained a large amount of lymphocyte infiltration. We further examined the levels of the IL-34 in control group and SAT group by western blotting and RT-PCR. IL-34 expression was significantly lower in the SAT groups than in the control groups, but there was no difference between the groups treated with NaI water for 16 and 8 weeks (Figure 1).

IL-34 is expressed in human thyroid follicular epithelial cells. We performed IHC analysis and measured the expression of IL-34 in human thyroid tissue. The result suggests a lower level of IL-34 expression in patients with HT compared to in patients without HT. We detected IL-34 expression in follicular epithelial cells in patients without HT. In contrast, IL-34 expressed in thyroid follicular epithelial cells and infiltrating lymphocytes in the thyroid tissue of patients with HT (Figure 2).

The clinical features of patients with HT (n = 24) and without HT (n = 23) are shown in Table 1. The level of autoantibodies in the HT group was significantly higher than that in the non-HT group (p < 0.001). There was no significant difference in the levels of TSH, FT4, and FT3 between the HT and non-HT groups. Then, we analyzed the correlation between the mRNA expression of IL-34 in thyroid tissue and thyroid function parameters. IL-34 protein expression in the thyroid tissue of the HT group was significantly lower than that of the non-HT group (Figure 3A), Similarly, IL-34 mRNA expression in the thyroid tissue of the HT group was significantly lower than that of the non-HT group (Figure 3B) and a negative correlation was observed between the mRNA expression of IL-34 and both TPOAb (r = −0.368, p = 0.015) and TgAb (r = −0.356, p = 0.019) (Figure 3C). IL-34 mRNA expression in thyroid tissue has no correlation with TSH (p = 0.151), FT3 (p = 0.630), and FT4 (p = 0.409).

The clinical features of patients with HT (n = 21) and healthy controls (n = 35) are shown in Table 1. The level of autoantibodies in the HT group was significantly higher than that in the healthy control group (p < 0.001). As before, we analyzed the correlation between serum IL-34 levels and thyroid function. Serum IL-34 levels in the HT group were significantly lower than those in the healthy control group (p < 0.05) (Figure 4A), and a significant negative correlation was observed between IL-34 and TgAb (r = −0.502, p = 0.021) (Figure 4B). Serum IL-34 levels has no correlation with TPOAb (p = 0.407), TSH (p = 0.07), FT3 (p = 0.268) and FT4 (p = 0.409).

As a result of IL-34 treatment, increased CSF1R expression was found in thyrocytes, and p-STAT3 expression was evidently increased in a dose-dependent manner upon exposure IL-34. These findings suggested that IL-34 stimulation mediates STAT3 activation by binding to CSF1R (Figures 5, 6B).

As shown in Figure 6A, flow cytometric analysis demonstrated that treatment with IL-34 significantly decreased thyrocyte apoptosis compared with control (Figure 6A). Following IL-34 treatment, the protein expression of Bcl-2 increased, and the expression of Bax was decreased, resulting in an increased Bcl-2/Bax ratio (Figure 6B). A similar tendency was discovered in the mRNA expression of the Bcl-2/Bax ratio (Figure 6C). Decreased protein expression levels, including p-STAT3/STAT3and Bcl-2/Bax, were observed following treatment with CSF1R inhibitor (Figure 6B), suggesting that the IL-34/CSF-1R/STAT3 pathway is involved in the apoptosis resistance of thyrocytes in HT.

IL-34 expression decreased after Nthy-ori 3-1 cells were treated with IFN-γ, and IL-34 expression was significantly lower in the IFN-γ-treated group (treated with an IFN-γ concentration of 1000 UI/mL) than in the untreated group (p < 0.05) (Figure 7). Reduction of IL-34 expression in Nthy-ori 3-1 cells can be observed after Nthy-ori 3-1 cells damage induced by high doses of IFN-γ. We conclude that IL-34 may be associated with damage to thyrocytes.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.