Influence of thyroid hormone in the expression of the marker pro-apoptosis BID, in spite of the predominance of anti-apoptosis activation in intratiroidal lymphocytic infiltration in Hashimoto’s thyroiditis
Introduction
Hashimoto's thyroiditis (HT) is an autoimmune disease characterized by intrathyroid lymphocytic infiltrate and the presence of antibodies against thyroid antigens such as thyroglobulin, thyroperoxidase and thyroid stimulating hormone receptor (TSH-R) (Weetman and DeGroot, 2016; Weetman, 2001). The mechanisms proposed to be responsible for cell destruction are the autoreactivity against thyrocytes by antibodies, the release of lytic granules with perforin and granzyme B by cytotoxic T lymphocytes (CTLs), the induction of cell apoptosis and, finally, the inadequate presence of death receptors and its ligands that activate apoptotic pathways (Stassi and De Maria, 2002).
Apoptosis is a controlled mechanism of cell death involved in the regulation of homeostasis. Deregulation of apoptosis can lead to thyroid autoimmunity, allowing the survival of autoreactive lymphocytes and changing the susceptibility of the target organ (Salmaso et al., 2002; Rossi and Gaidano, 2003). Death receptors and their ligands, such as tumor necrosis factor (TNF), Fas, its ligand (FasL) and TRAIL (TNF-related apoptosis-inducing ligand), are inactive in normal thyroid tissue, however, in pro-inflammatory conditions, greater activation of pro-apoptotic molecules and less expression of anti-apoptotic molecules occurs (Mikoś et al., 2014; Wang and Baker, 2007). The death receptor Fas, also called CD95, is a member of the TNF receptor (TNF-R) superfamily. This receptor has the ability to induce apoptosis in a large number of cell types. In contrast, the expression of its ligand, Fas Ligand (FasL), is restricted to selective groups of cells, such as activated-T lymphocytes and natural killer cells. HT gland presents a higher rate of apoptosis probably due to abnormal expression of Fas and FasL on the surface of neighboring thyrocytes and low FasL levels in infiltrating lymphocytes, allowing that survival (Kaufmann et al., 2012; Giordano et al., 2001).
On the other hand, BCL2 (B cell lymphoma/leukemia 2) is an anti-apoptotic molecule that gives its name to a family of proteins related to apoptosis. In addition, other molecules of the family participate in the control of apoptosis, such as the anti-apoptotic myeloid cell leukemia sequence (MCL1) and pro-apoptotic BID (BH3-only BCL-2 family member) molecules. Kaczmarek et al. observed that BCL2 expression in HT was lower than in colloid goiter without inflammatory infiltrate (Wang and Baker, 2007; Davids and Letai, 2012; Hardwick and Soane, 2013; Kaczmarek et al., 2011). MCL1, the main protein related BCL-2 is found in the outer mitochondrial membrane, linked to the proteins Bak (Bcl-2 homologous antagonist/killer) and Bax, being relevant for the survival of cell lines, such as lymphocytes, stem cells, neutrophils and neurons. Thyroid hormone stimulates MCL1 expression, avoiding the stabilization of the mitochondrial membrane and the formation of channels that release mitochondrial cytochrome C and induce apoptosis. Pietrzak et al. studied the molecular mechanism by which triiodothyronine (T3) regulates the expression of MCL1, concluded that MCL1 is activated by T3 and propose that this is one of the mechanisms by which T3 regulates apoptosis (Perciavalle and Opferman, 2013; Pietrzak and Puzianowska-Kuznicka, 2008). In this sense, studies using T4 inhibitor (tetrac/Nanotetrac) demonstrated suppression of the anti-apoptosis genes MCL-1 and XIAP (X-linked inhibitor of apoptosis) and induction of the expression of CASP2 and BCL2L14 pro-apoptosis genes. Thus, the pro-apoptotic action of the T4 inhibitor corroborates the anti-apoptotic activity demonstrated by thyroxine (T4) (Davis et al., 2011, 2015). BID or “BH 3 interaction domain” has lower expression in normal thyroid cells. But, when thyroid cells are exposed to inflammatory cytokines, interleukin-1 and TNF-alpha, they remain more sensitive to apoptosis through BID overexpression. In addition, Wang et al. observed that in mice with BID overexpression, the risk of developing autoimmune thyroiditis induced by pathogenic factors known as iodine was higher than in controls (Davids and Letai, 2012; Mezosi et al., 2004; Wang et al., 2014).
Autoimmune thyroid diseases and their clinical characteristics are distinguished by the presence of proteins related to apoptosis and an imbalance between the degree of apoptosis and that of cell proliferation. In HT, apoptosis predominates in relation to the thyroid's ability to regenerate (Troncone et al., 2003; Morshed et al., 2013). The Ki-67 and p27Kip1 proteins are markers related to cell proliferation in thyroid cells. Ki-67 is a nuclear protein produced in dividing cells, used as a marker to identify active dividing cells. Doganay et al. evaluated the follicular epithelium in chronic lymphocytic thyroiditis and found the main expression of Ki-67 in the follicles adjacent to the lymphocytic infiltrate associated with regenerative hyperplasia (Sobecki et al., 2016; Doganay et al., 2005; Domoslawski et al., 2013). The p27Kip1 protein is a cell cycle inhibitor that protects tissues from diseases related to excessive cell proliferation, such as inflammatory diseases and neoplasms. Troncone et al. found that p27Kip1 downregulation occurs in HT and may play a role in HT pathogenesis. Fang et al. observed that TGF-beta promotes and interferon-gamma inhibits thyroid epithelial cell proliferation in their study done with IFN -/- NOD.H-2h4 (non-obese diabetic) mice that develop an autoimmune disease with the proliferation of thyroid epithelial cells. They found that cell proliferation occurs by downregulating the antiproliferative molecules p21 and p27 (Troncone et al., 2003; Fang et al., 2012). The need for clarification regarding the development of HT justifies studies on the mechanisms of cell protection and death of thyrocytes and lymphocytes that infiltrate the thyroid. This study aimed to evaluate anti, pro-apoptotic and cell proliferation markers in HT in histopathological samples of thyroidectomy products.
Section snippets
Study design
Paraffin blocks of total thyroidectomy products with anatomopathological and clinical diagnosis of Hashimoto's thyroiditis and normal thyroid were selected. New sections were prepared for the immunohistochemical analysis of pro-apoptotic (Fas, Ligand Fas, BID), anti-apoptotic (BCL-2, MCL-1), cell proliferation (Ki-67) and antiproliferation (p27Kip1) markers in cells of the thyroid and intrathyroid lymphocytic infiltrate. These data were correlated with the clinical and laboratory parameters of
Results
Of the 18 HT patients studied, all were female, with a mean age of 49.7 ± 13.5 (median 54.0; 22.0–67.0 years), 8 (44.4%) were in use levothyroxine and the majority (n = 15, 83.3%) underwent total thyroidectomy due to suspected neoplasia. The average concentration of FT4 was 1.20 ± 0.25 ng/mL (median 1.16; 0.80–1.67); TSH 3.88 ± 4.11 μIU/mL (median 2.58; 0.75–15.78). The volume of the thyroid gland was on average 37.3 ± 44.5 cm3 (median 22.2; 4.6–168.4). The lymphocytic infiltrate was classified
Discussion
The pathophysiology of autoimmune thyroid diseases involves dysregulation of apoptosis and lymphocytic infiltration. The clinical disease results from the balance between the pro and anti-apoptotic factors and the proliferative and antiproliferative factors in the thyroid cells and in the lymphocytes that infiltrate the thyroid (Troncone et al., 2003).
Thus, our study showed that the thyroid lymphocytes infiltration in patients with HT exhibited high expression of the cell proliferation marker,
Funding
The study was funded by the Foundation for Research Support of the State of São Paulo (FAPESP). DEZW has a research grant from CNPq (National Council of Research) proc 302827/2018-8.
Conflicts of interest
The authors declare that they have no conflict of interest/Competing interests.
CRediT authorship contribution statement
Jessica Castro de Vasconcelos: Formal analysis, Project administration, Methodology, Visualization, Writing – original draft. Icleia Barreto de Siqueira: Data curation, Formal analysis, Methodology, Supervision, Visualization. Frederico Fernandes Ribeiro Maia: Project administration, Methodology. Maria Cândida Ribeiro Parisi: Project administration, Methodology. Denise Engelbrecht Zantut-Wittmann: Conceptualization, Data curation, Methodology, Funding acquisition, Supervision, Writing – review
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2022, International ImmunopharmacologyCitation Excerpt :In addition, BCL-2 is an apoptosis inhibitory proteins during the pathogenesis of HT. During the development of HT, increased caspase proteins and decreased BCL-2 expression have been observed, which favor the thyrocytes apoptosis and thyroid gland destruction [21]. In this study, we demonstrated that AMIO enhanced endoplasmic reticulum stress and promoted thyroid epithelial follicular cell apoptosis, which clearly explained the pathogenesis of AMIO-induced HT.