Effect of mTOR inhibitor on autoimmune thyroiditis

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Abstract

BACKGROUND: In autoimmune thyroiditis, hormone replacement therapy generally does not inhibit the activity of autoreactive T lymphocytes destroying the thyroid gland and promoting the antithyroid antibody response at the early stages of the disease.

AIM: This work aimed to find an approach aimed at suppressing the autoimmune reaction in autoimmune thyroiditis.

METHODS: Experimental autoimmune thyroiditis (EAT) was induced by double (at day 1 and day 14) thyroglobulin immunization of 15 weeks old C57BL/6 mice using complete and incomplete Freund’s adjuvant. Experimental animals were given a 0.05% NaI solution during feeding. The mice were divided into three groups; group 1 included intact mice (n = 5), group 2 included mice with induced EAT (n = 7), and group 3 included mice with induced EAT and subsequently administered sirolimus (n = 7). CD4+, CD8+, double-positive and double-negative T lymphocytes, and CD4+CD25+FoxP3+ T cell levels were determined using flow cytometry. Follicle destruction was measured by hematoxylin and eosin staining and the apoptosis was measured by staining with active caspase-3 antibodies. Thyroid antibodies were determined using enzyme-linked immunosorbent assay. Statistical processing was performed using one-way ANOVA and Student’s t-test (p <  0.05); data were presented as mean ± standard deviation (SD). Distribution normality was tested using the Shapiro–Wilk and Bartlett tests.

RESULTS: In the EAT group, the mTOR protein inhibitor (sirolimus) prevented thymus involution, increased the number of thymic regulatory T cells, and suppressed the secretion of IFN-γ and IL-17A cytokines. The inhibitor reduced lymphoid infiltration (D450 = 0.25) compared to mice with EAT (D450 = 2.4) and cellular apoptosis (D450 = 0.005 vs 0.0125 in the EAT group). This, in turn, resulted in lower levels of thyroid peroxidase autoantibodies (D450 = 3.0 compared to 19 in the experimental group). Therefore, sirolimus suppresses the autoimmune reaction by activating regulatory T immunity.

CONCLUSION: Sirolimus helps reduce the autoimmune aggression, improve the thyroid gland structure, and mitigate the severity of the disease.

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About the authors

Anastasia N. Tikhonova

Kazan (Volga Region) Federal University

Email: askatix@mail.ru
SPIN-code: 6842-5993

student, Depart. of Biochemistry, Biotechnology and Pharmacology

Russian Federation, Kazan

Anastasia V. Burtseva

Kazan (Volga Region) Federal University

Email: renardtriste00@gmail.com

Graduate student, Depart. of Biochemistry, Biotechnology and Pharmacology

Russian Federation, Kazan

Maria V. Tikhomirova

Kazan (Volga Region) Federal University

Email: MVTikhomirova@kpfu.ru
ORCID iD: 0000-0001-9357-3649
SPIN-code: 4208-9021

Cand. Sci. (Biology), Senior Lecturer, Depart. of Biochemistry, Biotechnology and Pharmacology

Russian Federation, Kazan

Zinaida I. Abramova

Kazan (Volga Region) Federal University

Author for correspondence.
Email: ziabramova@mail.ru
ORCID iD: 0000-0003-3749-3411
SPIN-code: 5293-9741

Dr. Sci. (Biology), Professor, Depart. of Biochemistry, Biotechnology and Pharmacology

Russian Federation, Kazan

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2. Fig. 1. Severity of autoimmune thyroiditis in mice: a, hematoxylin and eosin staining of thyroid tissue. The red arrow shows lymphocytic infiltration, the yellow arrow shows destroyed thyroid follicles; b, lymphocytic infiltration measured by the average absorbance (D450); c, plasma thyroid peroxidase antibodies; d, immunohistochemistry assay of active caspase-3 localization in thyroid tissue; e, average absorbance of caspase-3 in follicular cells of the thyroid gland. **** p < 0.0001.

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3. Fig. 2. Depletion of DP cells in the thymus of mice with experimental autoimmune thyroiditis: a, representative plots of anti-CD4/CD8 staining of thymocytes from control mice with experimental autoimmune thyroiditis and the sirolimus-treated group; b, T cell development stages determined by flow cytometry in mice with experimental autoimmune thyroiditis (red), the sirolimus-treated group (purple), and the control group (blue). * p < 0.05; ** p < 0.01.

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4. Fig. 3. Analysis of apoptosis in thymocytes by FCM analysis of annexin V/propidium iodide (PI) double staining: a, representative flow cytometry plots; b, T lymphocytes with signs of apoptosis (annexin V+) in the total cell population (in %). * p < 0.05; ** p < 0.01.

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5. Fig. 4. Evaluation of CD25 and FoxP3 expression in thymocytes: a, representative flow cytometry plots; b, CD25+ FoxP3+ cells in mice from the control group, the experimental autoimmune thyroiditis group, and the sirolimus-treated group. * p < 0.05; ** p < 0.01.

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6. Fig. 5. Flow cytometric staining of CD3+CD4+ T cells for intracellular cytokines: a, representative FACS staining; b, number of cells (in %) synthesizing IFN-γ, IL-4, or IL-17A. * p < 0.05; ** p < 0.01.

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