Samples

A total of 60 HT patients admitted to the Department of Endocrinology, the Second Affiliated Hospital of Dalian Medical University, and 20 healthy controls from the Physical Examination Center of the Second Affiliated Hospital of Dalian Medical University were enrolled in this study. Among them, 30 HT patients and 10 healthy controls were used for flow cytometry experiments and Western blotting. Ten HT patients were selected as the HT group, including 3 male patients and 7 female patients, with an average age of 40.05 ± 11.84 years, and 10 healthy controls were selected as the healthy control group (HC group), including 3 male patients and 7 female patients, with an average age of 40.90 ± 12.56 years (Table 1). Another 30 HT patients and 10 healthy controls were used for targeted gas chromatography-mass spectrometry (GC-MS) detection of medium-chain fatty acid content. Ten HT patients were selected as the HT group, including 3 male patients and 7 female patients, with an average age of 43.00 ± 8.69 years, and 10 healthy controls were selected as the healthy control group (HC group), including 3 male patients and 7 female patients, with an average age of 41.30 ± 10.29 years (Table 2). In general, all 80 samples were divided into four groups on average: Healthy control group (HC group), Hashimoto thyroiditis CD4+T cell inactive group (HT group), Hashimoto thyroiditis CD4+T cell activation (TCC group), Hashimoto thyroiditis CD4+T cell activation + Etomoxir group (TCC + ETO group) (Table 3).

Table 1 The clinical baseline data of flow cytometry and Western blotting in the control and HT groupTable 2 The clinical baseline data of targeted metabolomics in the control and HT groupTable 3 Experimental groupingDiagnostic criteria for Hashimoto’s thyroiditis

a.diffuse, uneven low-echo changes, nodular or uneven, solid goiter; b. positive TgAb and/or TPOAb and normal TSH (0.3–4.5 mIU/L), T3 (2.1–5.4 pmol/L), T4 (9–25 pmol/L); c. no history of thyroid surgery, radioactive iodine therapy; d. no medications affecting thyroid function or immune function;

Inclusion and exclusion criteria

Inclusion criteria for Hashimoto’s thyroiditis group: (a) patients meeting the above diagnostic criteria, (b) normal blood lipid levels (TC ≤ 5.20 mmol/L, TG 0.56 ~ 1.70 mmol/L). Exclusion criteria: (a) patients with other autoimmune diseases, such as type 1 diabetes (T1DM), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD), etc., (b) patients with acute and chronic infectious diseases, such as acute and chronic hepatitis, pneumonia, etc., (c) patients taking non-steroidal drugs, glucocorticoids, or antibiotics, (d) patients with malignant tumors or immune deficiencies, (e) pregnant or lactating women. Inclusion criteria for healthy control group: (a) FT3, FT4, TSH, TPOAb, TgAb were normal, (b) B-ultrasound showed normal thyroid, (c) no history of any autoimmune thyroid disease, (d) normal blood lipid levels (TC ≤ 5.20 mmol/L, TG 0.56 ~ 1.70 mmol/L). Exclusion criteria: (a) patients with other autoimmune diseases, such as T1DM, SLE, RA, MS, IBD, etc., (b) patients with acute and chronic infectious diseases, such as acute and chronic hepatitis, pneumonia, etc., (c) patients taking non-steroidal drugs, glucocorticoids or antibiotics, (d) patients with malignant tumors or immune deficiencies, (e) pregnant or lactating women.

Targeted metabolomics

The concentrations of medium and long-chain fatty acids in CD4+T cells were detected by targeted gas chromatography-mass spectrometry (GC-MS).

Instruments

The GC analysis was performed on trace a 1300 gas chromatograph (Thermo Fisher Scientific, USA). Mass spectrometric detection of metabolites was performed on ISQ 7000 (Thermo Fisher Scientific, USA).

Gas chromatography conditions: The GC analysis was performed on trace 1300 gas chromatograph (Thermo Fisher Scientific, USA). The GC was fitted with a capillary column Thermo TG-FAME (50 m*0.25 mm ID*0.20 μm) and helium was used as the carrier gas at 0.63 mL/min. Injection was made in split mode at 8:1 with an injection volume of 1 µL and an injector temperature of 250℃. The temperature of the ion source and transfer line were 300℃ and 280℃, respectively. The column temperature was programmed to increase from an initial temperature of 80℃, which was maintained for 1 min, followed by an increase to 160℃ at 20℃/min, which was maintained for 1.5 min, and increase to 196℃ at 3℃/min, which was maintained for 8.5 min, and finally to 250℃ at 20℃/min and kept at this temperature for 3 min.

Mass spectrum conditions

Mass spectrometric detection of metabolites was performed on ISQ 7000 (Thermo Fisher Scientific, USA) with electron impact ionization mode. Single ion monitoring (SIM) mode was used with an electron energy of 70 eV.

Animal model of EAT

The EAT-susceptible mouse model, female CBA/J mice (45, 4-week-old), SPF level, was purchased from Beijing Huafukang Biotechnology Experimental Animal Research Institute. The experimental mice were raised in the SPF Animal Experimental Center of Dalian Medical University. The nursing and experiments of laboratory animals were conducted by the guidelines of the Chinese Academy of Medical Sciences. This study was approved by the Medical Ethics Committee of Dalian Medical University(Ethics Approval Number: AEE22092). The in vivo experiment injected the drug concentration of 20 mg/kg Etomoxir (Sigma, E1905), and the drug intervention time was twice a week for 2 weeks. After adaptive feeding for a week, the mice were randomly divided into 3 groups: Con group (n = 15), mTg group (n = 15), and mTg + Etomoxir group (n = 15) (Table 3). Before the experiment, mTg was obtained and prepared from the frozen KM mouse thyroid. According to the previous preliminary experiments in the laboratory, the induction dose, frequency of immunization injection, modeling time and detection methods of EAT mice (namely mTg group) were determined: after a week of adaptive feeding, mTg was dissolved in Freund’s complete reagent (Sigma, F5881) (200 µg/mouse) and injected into multiple points subcutaneously after the neck for immunization at the age of 5 weeks; mTg was dissolved in Freund’s incomplete reagent (Sigma, F5506) (200 µg/mouse) and injected into multiple points subcutaneously after the neck for enhancing immunization at the age of 7 weeks. At the 11th week, two mice were randomly selected from each group to detect the modeling results: serum ELISA method was used to detect TgAb, TSH, T4, and thyroid HE staining. The successful modeling of EAT mice: compared with the Con group, the serum TgAb level in the mTg group was significantly increased, while the difference between TSH and T4 in serum was not obvious, and the thyroid lymphocyte infiltration in the Con group. From the 11th week to the 12th week, Etomoxir was dissolved in ultrapure water after high pressure and injected intraperitoneally into EAT mice (20 mg/kg). The control group was injected with the same amount of PBS at the same site through the same route. All mice were killed at the 13th week of the experiment, and the serum from the orbit and abdominal aorta, thyroid tissue, and spleen tissue were collected for subsequent experimental studies.

Determination of serum TgAb, TSH, and T4 levels by ELISA

Serum sample preparation: Collect the whole blood samples of mice in 1.5 ml EP tubes, place at room temperature for 1–2 h, centrifuge at 1000×g for 20 min, carefully collect the supernatant in a new EP tube, and store in a freezer at -80 °C for future use. TgAb kit sample preparation: To prevent the measured OD values from being beyond the range, dilute the serum of Con group by 1, mTg group by 4, and mTg + Etomoxir group by 2; TSH kit sample preparation: Dilute all groups of serum by 4; T4 kit samples do not need to be diluted. Then use TgAb (FineTest, EM1402), TSH (FineTest, EM1433), and T4 (FineTest, EU0402) ELISA kits to determine the corresponding serum levels. Methods are performed according to the manufacturer’s instructions.

Cell CultureHuman peripheral blood cell culture CD4+T cell sorting

Blood was mixed with the same amount of PBS, spread on the surface of the lymphocyte separation solution, and centrifuged at 2000 rpm/min for 20 min. After centrifugation, the cells in the cloud layer were gently absorbed into a new centrifuge tube, and washed with 5 ml PBS. After centrifugation at 1500 rpm/min for 5 min, 1 ~ 2 times, human peripheral blood mononuclear cells were obtained. The centrifuged peripheral blood mononuclear cell precipitation was resuspended with buffer solution and transferred into a 1.5 ml EP tube. Then 10 µl Isolation cocktail was added to the EP tube, mixed evenly, and left for 5 min. Under the condition of avoiding light, the Vortex RapidSpheresTM vortex was vibrated for 30 s, and then 10 µl was added to the sample EP tube and mixed evenly, and then 750 µl buffer was added and mixed evenly. The sample in the 1.5 ml EP tube was transferred into the frozen tube, and the frozen tube was placed in the magnet magnetic field of the Cell Separation Magnet. After 3 min, the frozen tube was upright and then upside down for 30 s. At this time, non-target cells and impurities were adsorbed on the surface of the frozen tube by the magnet, and the liquid in the tube contained sorted CD4+T cells. The liquid in the tube was poured into a new 1.5 ml EP tube, and the centrifuge was set at 1000 rpm/min for 10 min.

Culture, activation and intervention of CD4+T cells

CD4+T cells sorted by immune magnetic beads were added to the complete culture medium (RPMI-1640 + 10% fetal bovine serum + double antibody), gently blown and transferred into a culture flask, and placed in an incubator (37 °C, 5% CO2) for culture, which was the HT group. After sorting, CD4+T cells were inoculated into 24-well plates, and a 25ul activator was added to each 1 × 106 cells for 48 h of culture and activation. The successful activation was defined as a large number of cells forming a mass under the microscope, which was the TCC group. The activated TCC group cells were added with 50µM Etomoxir for 24 h of culture, which was the TCC + ETO group.

Mouse cell culture Preparation of spleen cell suspension

Take a fresh mouse spleen, remove the white membrane and excess tissue on the surface of the spleen tissue, and wash with PBS. Separate the spleen into several small pieces, add 2 ml complete medium, and put into the grinder for grinding. Gradually add 5 ~ 10 ml complete medium during the grinding process, grinding until there is no obvious spleen tissue in the grinder. Filter through a 200 mesh filter into a 15 ml centrifuge tube to obtain the spleen cell suspension. Centrifuge the prepared spleen cell suspension at 4 °C, 2000 rpm/min for 5 min, discard the supernatant, and blow the cell precipitation with 5 ml red blood cell lysate repeatedly for 20 times. Cleave in a refrigerator at 4 °C or on ice, add 5 ml PBS immediately after 5 min to stop the cleavage of red blood cells, and centrifuge at 4 °C, 2000 rpm/min for 5 min. Blow and re-suspend the spleen cell precipitation that removes the influence of red blood cells with PBS, and centrifuge at 4 °C, 2000 rpm/min for 5 min. Carefully absorb and discard the PBS, re-suspend the cell precipitation with complete medium transfer it into a petri dish or a culture flask, and place it in a constant temperature CO2 incubator at 37 °C for 6 ~ 8 h.

Separation, activation and culture of splenic CD4+T cells

The spleen cell suspension collected from the culture flask was centrifuged at 1000 rpm/min for 10 min at room temperature. The centrifuged medium was discarded, and the cells were resuspended by repeated blowing with 4 ml MojoSortTM Buffer. The cells were placed in an EP tube on ice for 1 min. After filtration through a 200 mesh filter, the cells were centrifuged at 300 g for 5 min. The resuspended cells were counted and the cell density was adjusted to 1 × 108/ml. Prepare a new 1.5 ml EP tube and number it. Add 100 µl of the cell suspension and 5 µl of Mouse TruStain FcXTM, and incubate at room temperature for 10 min. Add 10 µl of mouse CD4+T cell-labeled magnetic beads that were pre-vortexed 5 times, and incubate on ice for 15 min. Add 1 ml MojoSortTM Buffer, and place the EP tube in a Cell Separation Magnet for 5 min. At this point, our target cells were adsorbed on the tube wall, while non-target cells and impurities were in the solution. Pour out the solution, resuspend the cells adsorbed on the tube wall with 1 ml of complete medium, transfer to a 24-well plate, add 25 µl of activator per 1 × 106 cells, and cultured in a CO2 incubator.

Hematoxylin-eosin (HE) staining

After the mice were killed, the thyroid tissue was fixed in 4% paraformaldehyde (Solarbio, P1110) and dehydrated to be transparent. After wax immersion and paraffin embedding, the tissue was sliced by a paraffin sectioning machine and stained with eosin and hematoxylin.

Western blotting

RIPA: PMSF was added to the cell precipitation at a ratio of 100:1. Total proteins were extracted from each group of cells and protein samples were prepared. Proteins were isolated by 15% SDS/PAGE and transferred to PVDF membrane, which was blocked with a rapid blocking solution for 10 min. The membrane was cut and incubated with anti-mTOR, anti-ACC1 (1:1000 dilution, Abcam), anti-FASN, anti-CPT1A (1:1000, CST), anti-RORγt, anti-Foxp3 (1:1000, Bioss) and anti-β-actin, anti-GAPDH antibodies overnight at 4 °C. The membrane was bound in TBST for the third time, incubated with goat anti-rabbit IgG secondary antibody (1:20000 dilution, Abcam) at room temperature for 1 h, and washed three times with TBST. Finally, the protein bands were visualized using a chemiluminescence system (Bio-rad).

Flow cytometry

CD4+T cells from humans or mice were collected and stained with CD4 and CD25 antibodies in vitro, followed by staining with IL-17 antibody after membrane rupture on ice. The percentage of T cell subsets was analyzed using Agilent flow cytometry and software.

Spleen index

The body weight and spleen weight of mice were recorded, and the spleen index was calculated: spleen weight (mg)/body weight (g).

Data analysis

In this experiment, the protein bands obtained by Western blotting were analyzed by ImageJ software, and the results of flow cytometry were statistically analyzed by Agilent.SPSS 26.0 and GraphPad Prism 7.0 were used for statistical analysis of the experimental data. The mean ± standard deviation was used to represent the measurement data by the normal distribution. If the data between the two groups met the normal distribution, the independent sample t-test was used, and if not, the rank sum test was used. The variance analysis was used for the data of three groups or more, where p < 0.05 indicates that the difference in results is statistically significant, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Abbreviations: HT, Hashimoto’s thyroiditis; TPOAb, thyroglobulin antibody; TgAb, thyroid peroxidase antibody; Th, helper T; Th17, T helper cell 17; IL-17, interleukin-17; Treg, regulatory T; TGF-β, transforming growth factor-β; FAO, fatty acid oxidation; CPT1A, carnitine palmitoyltransferase 1 A; ETO, Etomoxir; ACC1, acetyl-COA carboxylase 1; FASN, Fatty acid synthase; mTOR, mammalian target of rapamycin; RORγt, Retinoic acid-related orphan receptor gamma t; Foxp3, forkhead box P3; T1DM, type 1 diabetes; SLE, systemic lupus erythematosus; RA, Rheumatoid arthritis; MS, multiple sclerosis; IBD, inflammatory bowel disease; SREBP1, Sterol-regulatory element binding protein 1.