Experimental evidence for alpha enolase as one potential autoantigen in the pathogenesis of both autoimmune thyroiditis and its related encephalopathy

https://doi.org/10.1016/j.intimp.2020.106563Get rights and content

Highlights

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    ENO1Ab can cause thyrocyte damage through ADCC effect via CD16+ NK cells.

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    ENO1Ab can cause cognitive dysfunction by disrupting the blood-brain barrier.

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    ENO1Ab may be the pathogenic antibody during AIT and its related encephalopathy.

Abstract

Alpha-enolase (ENO1) is a ubiquitous protein. Patients with autoimmune thyroiditis-associated encephalopathy have high serum ENO1Ab titers. We aimed to explore whether ENO1Ab was the pathogenic antibody in the thyroid and brain. The serum ENO1Ab titers were significantly increased in the mice immunized with Thyroglobulin (Tg). And in the mice immunized with ENO1, serum levels of both TgAb and thyroid-stimulating hormone (TSH) were significantly increased. Obvious CD16+ cell infiltration, IgG deposit and cleaved caspase-3 were observed in the thyroid of ENO1-immunized mice. Spatial learning and memory abilities and synaptic functions were impaired in ENO1-immunized mice. Furthermore, the expression levels of Iba-1, GFAP, interlukin-6, CDK5, and phosphorylated tau were increased, and endothelial tight junction proteins were decreased in the brain of ENO1-immunized mice. These results suggest that ENO1Ab can cause thyrocyte damage via ADCC effect and impair cerebral function by disrupting the blood–brain barrier.

Introduction

Autoimmune thyroiditis (AIT) is a multifactorial disorder, which is usually characterized by high expression of antithyroid antibodies and local lymphocyte infiltration. AIT-associated encephalopathy, also called Hashimoto encephalopathy (HE), is an important extrathyroidal autoimmune component involved in central nervous system damages and dysfunction. The most common manifestations include cognitive impairment (shown in 65% of patients with HE), seizures (59.2%), behavioral disorder (43%), and myoclonus (42.3%) [1]. Although patients with HE usually show good responses to corticosteroid therapy, the pathogenesis of HE is almost unknown [2]. Brain vasculitis and autoimmune responses induced by common autoantigens distributed between the brain and thyroid tissues have been hypothesized to be the most potential pathogenic mechanisms [3].

ENO1 is a ubiquitous and multifunctional protein. Anti-ENO1 antibody (ENO1Ab) is involved in many infectious and autoimmune diseases and plays a vital role in aggravating the progression of these diseases [4]. High ENO1Ab level has been observed in more than half of the patients with HE [5], suggesting that ENO1-specific autoimmune responses may be involved in the development of HE. However, no further direct evidence has supported it yet. Moreover, pathogenic mechanisms related with ENO1Ab in thyroid and brain disorders have not been reported either. Thus, this study was designed to explore whether ENO1 was a common autoantigen distributed between thyroid and brain tissues, and how the autoimmune responses against ENO1 affected the functions of the thyroid and brain using the mouse models with experimental autoimmune thyroiditis (EAT) and high serum ENO1Ab level (H-ENO1Ab). The latter was established for better understanding the in vivo pathogenicity of ENO1Ab.

Section snippets

Animals

CBA/J female mice were purchased from Beijing HFK Bioscience (Beijing, China). They were housed under specific pathogen free (SPF) conditions, including automatic 12-h light/dark cycles, regular mice diet, and constant temperature and humidity in the Laboratory Animal Science of China Medical University. This study was approved by the Institutional Animal Care and Use Committee of China Medical University.

Animal model establishment

A few 8-week-old mice were not given any treatment and used as normal intact mice after

ENO1 expression in normal thyroid and brain tissues

Western blot and immunohistochemical staining were used to examine the expression of ENO1 protein in thyroid and brain tissues from normal intact mice, as described in Fig. 1A. ENO1 was present in thyroid follicular cell and brain cells in the cerebral cortex and hippocampus (Fig. 2A and B). IF staining was used to further localize ENO1 protein in brain. The representative images showed ENO1 was expressed in neurons (NeuN+ cells), astrocytes (GFAP+ cells) and microglia (Iba-1+ cells, Fig. 2C).

Discussion

Enolase include three isoforms with α-enolase (ENO1) found in most of tissues, β-enolase predominantly in muscles, and γ-enolase only in neuron and neuroendocrine tissues. One early study has reported that thyroid follicular cells, connective tissue, and vessels did not express γ-isoform [25]. ENO1 protein expression has been found in a few cell types, such as epithelial vascular, endothelial, and neuronal cells [4]. It is a multifunctional protein located in the cytoplasm and nucleus as well

Conclusion

In summary, ENO1 can become a common autoantigen between thyroid and brain tissues, which is different from those classical thyroid-specific antigens. ENO1Ab may cause the injury of thyroid epithelial cells via ADCC, initiate or promote the autoimmune inflammatory responses against thyroid tissue, and result in insufficient production of thyroid hormones. At the same time, ENO1-specific IgG may damage brain microvascular endothelial cells through antibody-dependent cell-mediated injury and/or

Acknowledgements

We would like to thank Univ-bio for cytokine microarray technical support.

Funding

This work was supported by the National Key Research and Development Program of China (No. 2017YFC0907403); the General Program of National Nature Science Foundation of China (No. 81771741 and 81273296); Distinguished Professor at Educational Department of Liaoning Province (No. [2014]187).

Declaration of Competing Interest

The authors have no conflicts of interest in this manuscript.

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    • 1

    Yihan Lu and Juan Qin contributed equally to this work and share first authorship.

    2

    Present address: Department of Endocrinology and Metabolism, Shenyang the Fourth Hospital of People, Shenyang 110001, PR China.

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