Analyzing genetic and environmental molecular mechanisms causing autoimmune thyroid diseases

Albert Einstein College Of Medicine, Bronx NY

Investigators

Linked publications, trials & patents

Abstract

The autoimmune thyroid diseases (AITD), Graves’ disease (GD) and Hashimoto’s thyroiditis (HT), are the most common autoimmune diseases. Because their mechanisms are not fully understood, AITD are treated symptomatically (hormone replacement in HT or hormone suppression in GD) and as a result, patients are often difficult to manage. Therefore, new therapies are needed that target the autoimmune mechanisms causing AITD. AITD are complex diseases caused by interactions between susceptibility genes and environmental triggers. We and others have mapped & confirmed several AITD genes, including thyroglobulin (Tg). We have also shown that interferon alpha (IFNα), produced during viral infections, is the key cytokine triggering AITD. The current proposal aims to dissect the genetic and environmental mechanisms causing AITD in order to target them with novel therapies. Our focus is on genetic mechanisms predisposing to AITD (Aim 1), environmental triggers of AITD (Aim 2), and medication triggers of AITD (Aim 3). In Aim 1 we will test the hypothesis that missense SNPs in Tg that are associated with AITD predispose to AITD by increasing Tg misfolding and enhancing its degradation into immunogenic peptides. We will use a novel muse model of AITD developed by us, in which autoimmune thyroiditis is induced by immunization with human Tg cDNA in a non-replicating Adenovirus vector. This model enables us to test the effects of different Tg SNPs shown to be susceptible or protective for AITD, and to assess in vivo mechanisms by which these Tg variants trigger AITD. In Aim 2 we will test the hypothesis that IFNα triggers AITD by engaging the autophagic degradation of Tg into immunogenic peptides. We will use cell lines and a mouse model with thyroid over-expression of IFNα to define the autophagy-lysosomal pathways of Tg degradation; we will also test the immunogenicity of the generated Tg peptides in mouse models and PBMC’s from AITD patients. This aim will define a new unifying mechanism for triggering autoimmunity by IFNα-mediated autoantigen degradation. In Aim 3 we will test the hypothesis that Programmed death-ligand 1 (PD-L1) expressed on thyrocytes has intrinsic activity protecting them from intracellular stress during inflammation, and that Immune Checkpoint Inhibitors (ICI’s) trigger thyroiditis by blocking these intrinsic protective effects of PD-L1. We will use a new mouse model of ICI-thyroiditis we developed to dissect the mechanisms by which PD-L1 blockade triggers thyroiditis in vivo. This aim will define the mechanisms of ICI-induced thyroiditis as well as the role of thyroidal PD-L1 in AITD. Collectively, the studies in this proposal will help advance our long-term goal, to design targeted mechanism-based therapies for autoimmune thyroid diseases. REVISED SPECIFIC AIMS Our long-term goal is to design mechanism-based therapies for autoimmune thyroid diseases (AITD). Currently AITD are treated symptomatically since their mechanisms are not fully understood and as a result, patients are often difficult to manage [1]. Therefore, new therapies targeting the mechanisms causing AITD are needed. AITD are complex diseases caused by interactions between susceptibility genes & environmental triggers [2]. We and others mapped & confirmed several AITD genes, including thyroglobulin (Tg) [3]. We also showed that interferon alpha (IFNα), produced during viral infections, is the key cytokine triggering AITD [4-6]. The current proposal aims to dissect the genetic & environmental mechanisms causing AITD in order to target them with novel therapies. We propose to study: (1) Genetic mechanisms focusing on Tg (Aim 1); (2) Environmental viral triggers of AITD via IFNα (Aim 2); and (3) Medication triggers of AITD (Aim 3). Our hypothesis is that complex interactions between susceptibility genes & environmental factors including medications lead to cellular changes in thyroid cells that facilitate the immune attack on them. Our specific aims are: Specific Aim 1: To test the hypothesis that Tg missense SNPs predispose to AITD by promoting misfolding of Tg and enhancing its degradation into immunogenic peptides (e.g., Tg.2098). We will use a new mouse model we developed in which experimental autoimmune thyroiditis (EAT) is induced by Tg-cDNA immunization [7]. Testing if missense Tg SNP variants accelerate AITD in vivo - We will mutate the Tg-cDNA to create different missense SNP variants associated with AITD [8] and test their contribution to the induction of EAT. Testing if misfolded Tg accelerates AITD - We will mutate the Tg-cDNA used for immunization to induce Tg misfolding, and test if misfolded Tg accelerates EAT in our new mouse model. Preventing AITD through mutation of Tg.2098 - We showed that Tg.2098 is a major Tg T-cell epitope in AITD [9, 10]. We will mutate Tg-cDNA to delete Tg.2098 & examine if this can prevent EAT development. Specific Aim 2: To test the hypothesis that IFNα triggers AITD by activating the unfolded protein response (UPR) and autophagy/lysosomal pathways causing Tg degradation into immunogenic peptides (e.g., Tg.2098). Analyzing molecular interactions that mediate Tg degradation - We found that IFNα triggers Tg degradation through engagement of autophagy/lysosomal pathways in thyrocytes [11]. We will assess Tg degradation mechanisms by: (i) co-immunoprecipitation (co-IP) of Tg with autophagy/lysosomal markers and electron microscopy analysis of immunogold labeled Tg; (ii) identifying Tg LC3-interacting regions (LIR) that facilitate Tg autophagic degradation, then mutating the LIRs & testing if this blocks Tg autophagic degradation. Testing IFNα-induced Tg degradation in vivo - We developed a new mouse model with thyroidal IFNα expression resulting in decreased Tg levels, upregulation of ER-stress & autophagic markers and increased cathepsin S & L levels in their thyroids. We will examine mechanisms of Tg degradation and test if blocking autophagy by deleting Atg7 or by blocking lysosomal cathepsins S & L will prevent Tg degradation in vivo. Testing if IFNα induces post-translational modifications in Tg - This will be done using Mass Spectrometry (MS) analysis of Tg obtained from thyroid cells treated or not treated with IFNα. Testing immunogenicity of IFNα-induced Tg peptides - Ex vivo, we will compare the reactivities of sera & T-cells from AITD patients to “IFNα-processed” (degraded) Tg vs. native Tg. In vivo, we will compare the immunogenicity of “IFNα-processed” Tg vs. native Tg by using them to induce EAT, and testing if preventing Tg degradation, by blocking autophagy, can prevent/treat the EAT phenotype induced by IFNα-processed Tg. Specific Aim 3: To test the hypothesis that programmed death ligand 1 (PD-L1) expressed by thyrocytes has intrinsic activity protecting them from intracellular stress and apoptosis during inflammation, and that therapies with Immune Checkpoint Inhibitors (ICI’s) trigger thyroiditis by blocking the intrinsic protective effects of PD-L1. We found that PD-L1 suppression predisposes thyroid cells to the toxic effects of interferons (IFN) leading to apoptosis, while upregulation of PD-L1 induces pro-survival mechanisms. We recently developed a new mouse model for anti-PD-L1 (αPD-L1) induced thyroiditis. We will use this new mouse model in which mice are treated with αPD-L1 to test the role of PD-L1 in thyroid cell response to inflammation. In summary, we propose to analyze 3 key mechanisms causing AITD: (1) Genetic mechanisms focusing on the Tg gene; (2) Viral mechanisms via IFNα; and (3) PD-L1 related mechanisms. We will use novel mouse models, cell lines, and techniques we have recently developed that for the first time make these analyses possible. Our multidisciplinary team has the capacity, experience, and expertise to achieve the aims of our proposal, and hopefully our mechanistic studies will lead to novel therapeutic targets in AITD. Our long-term goal is to translate the mechanisms we identify into novel therapies to benefit AITD patients.