Effects of high-fat diet on thyroid autoimmunity in the female rat

Abstract

Background: While contributions of dyslipidemia to autoimmune diseases have been described, its impact on thyroid autoimmunity (TA) is less clear. Programmed cell death 1(PD-1)/PD-ligand 1 (PD-L1) immune checkpoint is crucial in preventing autoimmune attack while its blockade exacerbates TA. We thus unveiled the effect of high-fat diet (HFD) on TA, focusing on the contribution of PD-1/PD-L1.

Methods: Female Sprague Dawley (SD) rats were randomly fed with a regular diet or HFD (60% calories from fat) for 24 weeks. Then, thyroid ultrasonography was performed and samples were collected for lipid and thyroid-related parameter measure.

Results: HFD rats exhibited hyperlipemia and abnormal biosynthesis of the unsaturated fatty acid in serum detected by lipidomics. These rats displayed a relatively lower echogenicity and increased inflammatory infiltration in thyroid accompanied by rising serum thyroid autoantibody levels and hypothyroidism, mimicking human Hashimoto's thyroiditis. These alterations were concurrent with decreased mRNA and immunostaining of intrathyroidal PD-1 and also serum PD-1 levels but not the PD-L1 expression, suggesting a role of a PD-1 pathway. Meanwhile, the infiltration of B and T cell, a key cellular event inhibited by the PD-1 signals, was enhanced in the thyroid of HFD rats, along with thyroid fibrosis and apoptosis.

Conclusions: Our data suggest that HFD triggers TA through a mechanism possibly involving downregulation of PD-1-related immunosuppression, providing a novel insight into the link between dyslipidemia and autoimmune toxicities.

Keywords: High-fat diet; Lymphocytic infiltration; PD-1; PD-L1; Thyroid autoimmunity.

Fig. 1

Building a HFD model in female rats. A Body weights. B Serum levels of CHO, TG, and LDL-c. Values were mean ± SEM, n = 10; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001

Fig. 2

HFD induced changes in serum lipidome. A Classification of the serum lipid by the biological process. B The OPLS-DA score plot derived from the LC-MS of serum obtained from the HFD (blue) and control (grey) groups. C Heatmap presentation of differential lipid metabolites. The relative values were presented by color differences, with columns representing samples and rows representing lipids. D-G The content in four main kinds of differential lipids (PC, MePC, ChE, and TG) were statistically analyzed (n = 6)

Fig. 3

HFD triggered the development of HT-like autoimmunity. A Representative thyroid images under ultrasound scanning (n = 5). Red arrows: thyroid gland. B Representative thyroid sections stained with H&E (n = 10; Magnification ×400). Red arrows: infiltrated monocytes. C Representative thyroid sections stained with Masson’s trichrome (n = 5; Magnification ×400). Red arrows: thyroid fibrosis. D Representative immunofluorescence staining images showing TUNEL-labeled (green) apoptotic cells counterstained with DAPI (blue) in thyroids (n = 5; Magnification×400). Red arrows: apoptotic cells. E Serum levels of thyroiditis-related parameters, including TPOAb, TgAb, T3, T4 and TSH (n = 10). F Quantitation of the degree of monocyte infiltration in thyroids using H&E. Values were mean ± SEM; ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001; ns, no statistical significance

Fig. 4

HFD disturbed the PD-1 signaling in thyroid and serum. A, B Representative images of PD-1 and PD-L1 staining in the thyroid (5 rats per group). Each right-hand panel (× 400) depicts a magnified image of the boxed area of the corresponding image in the left panel (× 100). C Relative mRNA expression of PD-1 and PD-L1 in the thyroid (n = 5). D Serum expression of sPD-1 and sPD-L1 (n = 10). Values were mean ± SEM; ^*p < 0.05, and ^***p < 0.001; ns, no statistical significance

Fig. 5

HFD increased lymphocyte infiltration in the thyroid. A Representative images of CD45 (mainly lymphocytic lineage), CD4 (CD4⁺ T cell), CD8 (CD8⁺ T cell) and CD19 (B cells) staining of thyroid sections. Each right panel (magnification ×2000) depicted a magnified image of the boxed area (red) of the corresponding image in the middle panel (magnification ×400). B Quantification of the data displayed in (A) revealed an increased infiltration of thyroid parenchyma by the CD45⁺ cells, CD4⁺ T cells, CD8⁺ T cells and B cells. Cells have been randomly counted in five nonoverlapping fields (magnification ×400) of tissue sections from 5 rats per group. Values were mean ± SEM; ^**p < 0.01