Introduction: Aging is characterized by the deterioration of a wide range of functions in tissues and organs, and Alzheimer’s disease (AD) is a neurodegenerative disease characterized by cognitive impairment. Hypothyroidism occurs when there is insufficient production of thyroid hormones (THs) by the thyroid. The relationship between hypothyroidism and aging as well as AD is controversial at present. Methods: We established an animal model of AD (FAD4T) with mutations in the APP and PSEN1 genes, and we performed a thyroid function test and RNA sequencing (RNA-Seq) of the thyroid from FAD4T and naturally aging mice. We also studied gene perturbation correlation in the FAD4T mouse thyroid, bone marrow, and brain by further single-cell RNA sequencing (scRNA-seq) data of the bone marrow and brain. Results: In this study, we found alterations in THs in both AD and aging mice. RNA-seq data showed significant upregulation of T-cell infiltration- and cell proliferation-related genes in FAD4T mouse thyroid. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that upregulated genes were enriched in the functional gene modules of activation of immune cells. Downregulated energy metabolism-related genes were prominent in aging thyroids, which reflected the reduction in THs. GSEA showed a similar enrichment tendency in both mouse thyroids, suggesting their analogous inflammation state. In addition, the regulation of leukocyte activation and migration was a common signature between the thyroid, brain, and bone marrow of FAD4T mice. Conclusions: Our findings identified immune cell infiltration of the thyroid as the potential underlying mechanism of the alteration of THs in AD and aging.

In order to prove the relationship between alteration of thyroid hormones (THs) and Alzheimer’s disease (AD), as well as aging, we established animal models of AD and naturally aging and performed thyroid function test and bulk RNA sequencing. We also studied gene perturbation correlation in AD mouse thyroid, bone marrow, and brain by further single-cell RNA sequencing (scRNA-seq) data of the bone marrow and brain. Our findings identified immune cell infiltration of the thyroid as the potential underlying mechanism of the alteration of THs in AD and aging.

Aging can be defined as the increased risk of illness and ultimately death due to functional deterioration over time, which is an inevitable physiological process of humanity [1]. Currently, the incidence of age-related neurodegenerative diseases is increasing in the global aging population [2]. Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases, which is characterized by cognitive decline and dementia, and is the fifth leading cause of death. The pathological features are extracellular deposition of amyloid β (Aβ) peptide, neurofibrillary tangles caused by hyperphosphorylated tau, neuronal dysfunction, and synaptic damage [3].

Hypothyroidism can be classified into primary hypothyroidism, secondary hypothyroidism, and peripheral hypothyroidism. Primary hypothyroidism is characterized by the failure of the thyroid itself with elevated serum TSH levels and decreased free thyroxine (FT4) levels [4]. Secondary hypothyroidism, a consequence of imbalance of the HPT axis, is mostly attributed to deficient stimulation of the thyroid by TSH and thyroid stimulating hormone-releasing hormone, which originate from the pituitary and hypothalamus, respectively. The diagnosis of secondary hypothyroidism is based on low FT4 levels together with low to normal TSH concentrations [5].

THs are important for brain development and have critical effects on neurogenesis, myelinization, and cellular repair in the mature brain [6]. Moreover, THs are important for preserving essential brain functions, including neurotransmission and memory [7]. Epidemiological studies have demonstrated the correlation between hypo- and hyperthyroidism and dementia risk [8]. More recently, not only subclinical thyroid dysfunction but also variation in thyroid function within the normal range has been proved to be associated with the risk of AD [9, 10]. Lower TSH levels were associated with an increased risk of dementia [11], and higher TSH levels were closely related to a lower risk of dementia within the normal range of thyroid function [8]. Low TH levels in the central nervous system may accelerate AD by increasing amyloid precursor protein (APP) expression followed by the accumulation of Aβ [12], and hypothyroidism is considered to increase the risk of AD twofold [13]. Therefore, normal thyroid function is a vital factor in maintaining cognitive function in aging. On the other hand, brain disease could lead to thyroid dysfunction. It has been reported that the accumulation of Aβ is toxic to hypothalamic cells, leading to the disruption of the HPT axis followed by hypothyroidism [14]. AD patients exhibited clinical manifestations of hypothyroidism, such as depressed mood and apathy. However, the precise relationship and potential underlying mechanism between thyroid function and AD need further elucidation.

Here, a mouse model of AD (FAD4T) with mutations in the APP and PSEN1 genes was established, which has no influence on thyroid development. Interestingly, we found that this mouse model was characterized by the alteration of THs. We performed bulk RNA sequencing of the thyroid from 2-month-old FAD4T mice, 8-month-old FAD4T mice, 18-month-old naturally aging mice, 2-month-old wild-type (WT) mice, and 8-month-old WT mice to explore the potential underlying mechanism of the alteration of THs in AD and aging. Because the thyroid exhibited an inflammatory milieu and a close relationship between inflammatory cells and bone marrow, we performed scRNA-Seq of the bone marrow as well as the brain from AD mice and analyzed and compared the data from the above organs to investigate the origin of the inflammatory environment.

Animals

All mouse lines were generated and maintained on a C57BL/6J background, and they were all purchased from GemPharmatech Co., Ltd. and housed in the same environment. The APP gene with humanized Swedish and Indian mutations and the PSEN1 gene with humanized M146V and L286V mutations were transferred into the mouse model of FAD4T. The proposal for the study was approved by the Committee for the Ethics of Animal Experiments of the Sixth People’s Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, China (permit number: 2022-0592).

Study Population

Eighty-four patients over 18 years were divided into adults (22–46 years old) and elderly individuals (60–88 years old); they were admitted to the hospital for thyroid tumors requiring surgery and selected for the randomized trial. The age, sex, and preoperative fasting thyroid function of the patients were recorded. Thyroid function tests are routine tests for patients with thyroid tumors. Experienced operators were responsible for the determination of thyroid stimulating hormone (TSH), free tri-iodothyronine (FT3), and FT4 levels in all subjects in strict accordance with the correct procedure. Thyroid tumors were diagnosed by a skilled ultrasound technician. Patients with previous thyroid surgery or other thyroid diseases such as Hashimoto’s thyroiditis or taking medicine that affects thyroid function for nearly 2 weeks were excluded. The proposal for the study was approved by the Ethics Committee of Shanghai Sixth People’s Hospital (permit number: 2022-088). All participants provided written informed consent.

Enzyme-Linked Immunosorbent Assay

The mice were anesthetized by intraperitoneal injection with 4% chloral hydrate solution, and then blood was taken from the eye. Measurement of T4, T3, and TSH concentrations was performed using an enzyme-linked immunosorbent assay kit (mlbio, Shanghai, China) according to the manufacturer’s instructions. Excel 2019 was used for all data statistics, and Prism 9.0.2 was used for T test and Pearson correlation analysis.

Tissue Processing and Library Construction for Bulk RNA-Seq

A total of 2-month FAD4T (n = 3), 2-month WT (n = 3), 8-month FAD4T (n = 3), and 8-month WT (n = 3) female mice and the same number of male mice, as well as 2-month WT (n = 3) and 18-month WT (n = 3) male mice, were analyzed. The thyroids were dissected and frozen quickly in liquid nitrogen. Bulk RNA-Seq experiments were performed by LC-Bio Technology Co., Ltd., Hangzhou, China. Total RNA was extracted and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s procedure. After quality testing of the RNA, samples with a total RNA amount greater than 1 μg, a concentration greater than 50 ng/μL, a RIN value greater than 7.0, and an OD260/280 greater than 1.8 entered the downstream experiments. Poly (A) RNA was purified from 1 μg of the total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) using two rounds of purification and then fragmented into small pieces using the Magnesium RNA Fragmentation Module (NEB, cat. e6150, USA) at 94°C for 5–7 min. The fragments of RNA were reverse transcribed to create cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA). U-labeled second-stranded DNAs were then synthesized from cDNA along with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA), and dUTP solution (Thermo Fisher, cat.R0133, USA). By treating U-labeled second-stranded DNAs with the heat-labile UDG enzyme (NEB, cat. M0280, USA) and amplifying the products with PCR, we obtained a cDNA library with an average insert size of 300 ± 50 bp. Eventually, we performed 2 × 150 bp paired-end sequencing (PE150) on an Illumina NovaSeq™ 6000 (LC-Bio Technology Co., Ltd., Hangzhou, China) following the vendor’s recommended protocol.

Bulk RNA-Seq Data Analyses

Next, the original data were analyzed. Differentially expressed genes (DEGs) were determined according to the fold change in expression (fold change not less than 2) and statistical significance (p < 0.05). Finally, KEGG analysis and GO functional enrichment were employed to identify the roles of DEGs. Gene set enrichment analysis (GSEA) was conducted to analyze differentially expressed gene sets. Normalized enrichment score and false discovery rate were used to quantify enrichment magnitude and statistical significance, respectively.

ScRNA-Seq Data Analysis

External scRNA-seq data from previously published studies were retrieved from the Gene Expression Omnibus: FAD4T bone marrow and brain (GSE214917). Generally, a processed digital gene expression matrix was loaded using Seurat [15] in R (version 3.6.3). We filtered cells with fewer than 500 expressed genes and high mitochondrial reads (>50%). After principal component analysis and clustering using default settings of the external scRNA-seq data, we identified DEGs from FAD4T bone marrow and brain (FAD4T vs. WT) using the “FindMarkers” function in Seurat with logfc.threshold = 0, min.pct = 0. We filtered and calculated the number of DEGs in each cell type with a threshold of absolute log fold change >0.5 and adjusted p value <0.05. Gene set expression scores of overlapping genes between scRNA-seq and bulk RNA-seq were calculated using the Seurat function “AddModuleScore.” The gene set expression score was visualized using a violin plot. GO functional enrichments were performed using Metascape [16].

RNA Extraction and Quantitative RT-PCR

Returned samples from bulk RNA-seq were used for validation by quantitative RT-PCR, and insufficient returned samples were supplemented with mice of the same genotype. In addition, total RNA was extracted from normal human thyroid tissue adjacent to tumors. Five hundred nanograms of total RNA were reverse transcribed into cDNA using an EZBioscience® Color Reverse Transcription Kit (with gDNA remover) (A0010CGQ). Quantitative (q)RT-PCR was performed by using the SYBR Green PCR system on the ABI HT7900 platform (Applied Biosystems). Relative gene expression levels of each RT-PCR product were analyzed using the threshold cycle (2–ΔΔCT) method and normalized to the expression of the housekeeping gene ACTB.

Statistical Analysis

All data were analyzed using GraphPad Prism (v9.0.2) software for statistical significance. The p value was determined by Student’s t test for each pair of groups.

Alteration of Thyroid Hormones in FAD4T and Aging Mice

We performed an integrated analysis of the preoperative thyroid function of patients, and the results showed that FT4 and FT3 levels were significantly decreased in aged men. However, there was no significant difference in aged women (Fig. 1a). Therefore, we speculated that a reduction in THs occurs with age, and this phenomenon was more common in aged men. To reduce the interference of external conditions, naturally aging and FAD4T mouse models were used for subsequent experiments. Dissection and exposure of the thyroid showed that there was no significant difference in the appearance of the aging thyroid compared with the young thyroid except being slightly larger, and the difference in the thyroid between FAD4T and WT mice of the same age was barely noticeable to the naked eye. Both naturally aging mice and 2-month-old FAD4T mice showed significantly decreased levels of T4 (Fig. 1b, c). However, TSH decreased in only 8-month-old FAD4T female mice (Fig. 1d).

Fig. 1.

Thyroid function in aging and FAD4T model and experimental grouping and process. a Comparison of thyroid function between aged and young humans. FT3, FT4, and TSH (n = 30 males and n = 12 females). b Comparison of thyroid function between 2-month-old (2 M) and 18-month-old (18 M) male mice. Tri-iodothyronine (T3) (n = 8), thyroxine (T4) (n = 11), and TSH (n = 9). c Comparison of thyroid function between 2-month-old wild-type (2 M W) and 2-month-old FAD4T (2 M T) mice (n = 3 per group). d Comparison of thyroid function between 8-month-old wild-type (8 M W) and 8-month-old FAD4T (8 M T) mice (n = 3 per group).

Fig. 1.

Thyroid function in aging and FAD4T model and experimental grouping and process. a Comparison of thyroid function between aged and young humans. FT3, FT4, and TSH (n = 30 males and n = 12 females). b Comparison of thyroid function between 2-month-old (2 M) and 18-month-old (18 M) male mice. Tri-iodothyronine (T3) (n = 8), thyroxine (T4) (n = 11), and TSH (n = 9). c Comparison of thyroid function between 2-month-old wild-type (2 M W) and 2-month-old FAD4T (2 M T) mice (n = 3 per group). d Comparison of thyroid function between 8-month-old wild-type (8 M W) and 8-month-old FAD4T (8 M T) mice (n = 3 per group).

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Gene Expression Analysis in 2-Month-Old FAD4T Mouse Thyroid

To explore the potential underlying mechanism of the alteration of THs in AD and aging, we generated triplicate libraries from 2-month-old and 8-month-old FAD4T and WT control mouse thyroids, respectively. We also generated RNA-seq datasets of young WT and 18-month-old naturally aging mouse thyroids (Fig. 2a). Samples were divided into five groups according to age (2-month-old FAD4T, 8-month-old FAD4T, and naturally aging) and sex (Fig. 2a). FAD4T mice exhibited early stage AD pathological features, such as cognitive disorders, in the 2-month-old mouse model. We analyzed DEGs in 2-month-old FAD4T thyroids from male and female mice. Gene expression patterns in both male and female FAD4T mouse thyroids demonstrated significant upregulation of T-cell infiltration signatures. Upregulated genes in the FAD4T mouse thyroid included the cytotoxic T-cell marker Cd8a and the T helper (Th) cell marker Cd4 [17] (Fig. 2b, left: female, right: male). The expression of the T-cell transcription factor Tcf7 [18] was also enriched in FAD4T mice. Other upregulated lymphocyte signature genes included the RAG complex genes (Rag1 and Rag2) [19], Trbc2 [20], Cd28 [21], and chemokine-related genes (Ccr9 and Ccl17). In addition to common T-cell infiltration events in the FAD4T mouse thyroid, we observed specific upregulation of cell proliferation marker genes in female FAD4T mice, such as Mki67, Cdk1, and Top2a [22], suggesting a potential inflammatory environment in female FAD4T mouse thyroid, similar to Hashimoto’s disease [23]. GO and KEGG pathway analyses of the DEGs were performed to identify potential biological processes associated with FAD4T perturbation (Fig. 2e, f). The functions of the upregulated genes were mainly involved in lymphocyte differentiation, T-cell differentiation, and mononuclear cell differentiation. We observed two distinct functional gene modules in the upregulated genes (Fig. 2c). Leukocyte activation and regulation of T-cell activation were related to positive regulation of immune responses such as inflammation. The other module included the mitotic cell cycle and cell cycle phase transition of infiltrated lymphocytes. Downregulated genes were enriched in muscle cell differentiation. The most critical gene modules of downregulated genes included the regulation of ion transport (especially calcium ions), blood circulation, and metal ion transport, which could affect muscle tissue development. These terms may be associated with the decline of the parathyroid gland (hypoparathyroidism) [24, 25]. Another module represented the decline in metabolism, such as the alpha-amino acid metabolic process and carboxylic acid biosynthetic process (Fig. 2d). The KEGG pathway enrichment analysis of upregulated genes also indicated the activation of lymphocytes. Enriched pathways included Th17-cell differentiation, Th1- and Th2-cell differentiation, natural killer cell-mediated cytotoxicity, and leukocyte transendothelial migration, while downregulated pathways were glycine, serine, and threonine metabolism, adrenergic signaling in cardiomyocytes, and biosynthesis of amino acids. Thus, a certain degree of hypoparathyroidism and reduction in THs may result from lymphocyte infiltration in the 2-month-old FAD4T mouse thyroid [26]. We identified the gene expression overlap between 2-month-old FAD4T and WT aging mouse thyroids of different sexes (online suppl. SFig. 1a; for all online suppl. material, see https://doi.org/10.1159/000536089). Overlapping genes indicated immune cell infiltration in the senescence-associated secretory phenotype [27]. Protein-protein interaction (PPI) analysis of upregulated genes showed interaction of T-cell receptor (TCR) and Ras protein signal transduction (online suppl. SFig. 1b). The PPI network of downregulated genes mapped an additional ion transmembrane transport module, transmembrane receptor protein serine/threonine kinase signaling pathway, and metabolism function in the thyroid (online suppl. SFig. 1c).

Fig. 2.

Gene expression analysis in 2-month-old FAD4T mouse thyroid. a Flow chart of experimental grouping. b DEGs in thyroids from 2-month-old FAD4T male and female mice (left: female, right: male) (n = 3 per group). c Two distinct functional gene modules in upregulated genes (leukocyte activation and regulation of T-cell activation, mitotic cell cycle, and cell cycle phase transition). d Two distinct functional gene modules in downregulated genes (muscle cell differentiation and decline of metabolism). e GO and KEGG pathway analysis of the upregulated genes. f GO and KEGG pathway analysis of the downregulated genes.

Fig. 2.

Gene expression analysis in 2-month-old FAD4T mouse thyroid. a Flow chart of experimental grouping. b DEGs in thyroids from 2-month-old FAD4T male and female mice (left: female, right: male) (n = 3 per group). c Two distinct functional gene modules in upregulated genes (leukocyte activation and regulation of T-cell activation, mitotic cell cycle, and cell cycle phase transition). d Two distinct functional gene modules in downregulated genes (muscle cell differentiation and decline of metabolism). e GO and KEGG pathway analysis of the upregulated genes. f GO and KEGG pathway analysis of the downregulated genes.

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Gene Expression Analysis in 8-Month-Old FAD4T Mouse Thyroid

We further checked whether similar gene expression perturbation events occurred in the 8-month-old FAD4T mouse thyroid. At this stage, astroglial activation could be observed in the brain. A volcano plot showed weak gene expression upregulation changes in both female and male mice (Fig. 3a). However, we could still identify GO terms of lymphocyte differentiation, T-cell differentiation, and mononuclear cell differentiation with low gene counts, suggesting a reduction in the degree of lymphocyte infiltration (Fig. 3b, d). Of note, a large number of downregulated genes were enriched in muscle system processes. The KEGG pathways in which the downregulated genes were enriched included the calcium signaling pathway and cGMP-PKG signaling pathway (Fig. 3c, e). However, few overlapping genes were found between 8-month-old FAD4T and WT aging mouse thyroids (number of overlapping DEGs <10). The PPI network of upregulated genes was mainly enriched in leukocyte chemotaxis and GPCR ligand binding (online suppl. SFig. 2a). The PPI network of downregulated genes was strongly enriched in class I MHC-mediated antigen processing and presentation, potassium ion transmembrane transport, and multiple metabolic processes (online suppl. SFig. 2b).

Fig. 3.

Gene expression analysis in 8-month-old FAD4T mouse thyroid. a DEGs in thyroids from 8-month-old FAD4T male and female mice (left: female, right: male) (n = 3 per group). Functional gene modules in upregulated genes (b) and downregulated genes (c). d GO and KEGG pathway analysis of the upregulated genes. e GO and KEGG pathway analysis of the downregulated genes.

Fig. 3.

Gene expression analysis in 8-month-old FAD4T mouse thyroid. a DEGs in thyroids from 8-month-old FAD4T male and female mice (left: female, right: male) (n = 3 per group). Functional gene modules in upregulated genes (b) and downregulated genes (c). d GO and KEGG pathway analysis of the upregulated genes. e GO and KEGG pathway analysis of the downregulated genes.

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Gene Expression Signatures in Aging Thyroid

The WT aging mouse thyroid exhibited a large number of DEGs (online suppl. SFig. 3a). A series of oxygen deprivation-induced upregulated genes, including Hyou1, were upregulated in aging thyroids [28]. We observed significant enrichment of the unfolded protein reaction process and endoplasmic reticulum stress (online suppl. SFig. 3b). These processes are associated with apoptosis and autophagy in thyroid cells [29, 30]. The downregulated genes in the aging thyroid were related to basic cell energy metabolism, such as oxidative phosphorylation, respiratory electron transport chain, ATP metabolic process, and NADH dehydrogenase complex assembly (online suppl. SFig. 3c). Downregulation of energy metabolism could also reflect the reduction of THs [31]. Interestingly, the KEGG pathways in which downregulated genes were enriched included multiple neurodegenerative disorders (online suppl. SFig. 3d).

GSEA Enrichment of Aging-Related Gene Set Functions in the FAD4T Mouse Thyroid

Next, we performed GSEA of all genes in three groups (2-month-old, 8-month-old, and aging) to validate the gene expression changes in DEGs. In the 2-month-old group, GSEA suggested that the gene sets were mainly involved in the TCR signaling pathway, chemokine signaling pathway, and primary immunodeficiency (Fig. 4a). In the 8-month-old group, oxidative phosphorylation, cellular tight junction, and ribosome functions were inhibited (Fig. 4b). The aging group demonstrated a similar enrichment tendency as the 2-month-old and 8-month-old groups (Fig. 4c). Thus, gene expression in FAD4T mice could reflect aging perturbations in the inflammatory state and a reduction in THs.

Fig. 4.

GSEA of all genes in the three groups. a 2-month-old group. b 8-month-old group. c Aging group.

Fig. 4.

GSEA of all genes in the three groups. a 2-month-old group. b 8-month-old group. c Aging group.

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Gene Perturbation Correlation between the Thyroid, Bone Marrow, and Brain in FAD4T Mice

Due to the occurrence of immune cell infiltration events in FAD4T mice, we explored whether similar changes occurred in the bone marrow. To link the perturbation events between the FAD4T mouse thyroid, brain, and bone marrow, we generated scRNA-Seq datasets of brain from 2-month-old WT and FAD4T female mice and bone marrow from 2-month-old WT and FAD4T female mice as well as male mice (Fig. 2a). We compared DEGs from the three tissues. DEGs from the brain and bone marrow were generated using scRNA-seq data to locate the specific cell types. We checked the expression score of the thyroid DEG module in bone marrow immune cell subsets (online suppl. SFig. 4a, left: female, right: male). NKT cells and granulocytes harbored high expression scores across all immune cells. Although few overlapping genes were observed between thyroid and bone marrow NKT cell DEGs (online suppl. SFig. 4b), most critical gene functions, such as leukocyte transendothelial migration and gamma-delta T-cell differentiation, were enriched in female and male samples (online suppl. SFig. 4c). DEGs between the sexes (female vs. male) in the FAD4T mouse thyroid showed a high module score in bone marrow B cells (online suppl. SFig. 4d). Overlapping genes (Pou2f2, E2f2, Ighm) represented plasma cells and B cell function (online suppl. SFig. 4e, f). A relatively high degree of B cell infiltration pattern could also be observed in female papillary thyroid carcinoma patients [32]. In the brain, NKT cells and neutrophils showed a high expression score of upregulated FAD4T mouse thyroid DEGs, which included Trbc2, Ccr7, Csf3r, and Cd52 (online suppl. SFig. 4g–i). Synchronous gene perturbations showed that the regulation of leukocyte activation and migration was a common signature between the thyroid, brain, and bone marrow of FAD4T mice.

Validation of Gene Expression Patterns in FAD4T Mouse, Naturally Aging Mouse, and Aging Human Thyroids

We observed T-cell infiltration and cell proliferation events in FAD4T mice, especially in the 2-month-old FAD4T mouse thyroid; in contrast, the lymphocyte infiltration degree decreased in the 8-month-old FAD4T mouse thyroid. Although the changes in unfolded protein reaction and metabolism were more prominent, genes involved in inflammatory infiltration were also upregulated in the natural aging mouse thyroid. Therefore, we verified the gene expression patterns in FAD4T and naturally aging mouse thyroids, including Rasgrp1, CD3g, CD8a, Lat, Themis, and Tcf7, which are associated with T-cell infiltration, and Mki67, Rrm2, Pclaf, and Top2a, which are associated with cell proliferation. The expression of several T-cell infiltration- and cell proliferation-related genes was upregulated in the 2-month-old FAD4T mouse thyroid regardless of sex (Fig. 5a, b). Interestingly, the number of upregulated genes decreased in the 8-month-old FAD4T mouse thyroid (Fig. 5c, d). This result seems to be in rough agreement with the transcriptome sequencing results. The expression of Rasgrp1 and CD3g was upregulated in the naturally aging mouse thyroid, which indicated the existence of inflammatory infiltration in the aging group (Fig. 5e). Furthermore, no age-related difference in the expression of lymphocyte signature genes was found in adjacent noncancerous tissue in men (Fig. 5f). This may have been because our sample size was too small and the heterogeneity was too high.

Fig. 5.

Validation of gene expression patterns in FAD4T mice, naturally aging mice, and aging human thyroids. Validation of T-cell infiltration- and cell proliferation-related genes in 2-month-old female (a) and male (b) mouse thyroids and 8-month-old female (c) and male (d) mouse thyroids (n = 3 per group). Validation of T-cell infiltration-related genes in aging mice (e; n = 3 in the 2-month-old group and n = 4 in the 18-month-old group) and male thyroids (f; n = 5 in the young male group and n = 3 in the aged male group).

Fig. 5.

Validation of gene expression patterns in FAD4T mice, naturally aging mice, and aging human thyroids. Validation of T-cell infiltration- and cell proliferation-related genes in 2-month-old female (a) and male (b) mouse thyroids and 8-month-old female (c) and male (d) mouse thyroids (n = 3 per group). Validation of T-cell infiltration-related genes in aging mice (e; n = 3 in the 2-month-old group and n = 4 in the 18-month-old group) and male thyroids (f; n = 5 in the young male group and n = 3 in the aged male group).

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This study presented the potential underlying mechanism of the alteration of THs in AD and aging. According to the results of bulk RNA-seq, both 2-month-old and 8-month-old FAD4T mice demonstrated immune cell infiltration in the thyroid. We speculated that the reason for the reduction in THs in aging and FAD4T mice was closely associated with immune cell infiltration. The pathological manifestations, including inflammatory infiltration and thyroid dysfunction, are also observed in another disease called Hashimoto’s thyroiditis, in which CD8+ T cells attack thyrocytes and CD4+ T cells promote B cell transition into plasmocytes followed by secretion of antibodies, both of which accelerate apoptosis of the thyroid and eventually result in hypothyroidism [33]. Therefore, we suppose that the chronic and lasting attack of inflammatory cells causes the thyroid to decompensate and causes a reduction in THs in FAD4T and aging mice, similar to Hashimoto’s thyroiditis. According to the results of scRNA-Seq, the activation and migration of immune cells are common signatures among the thyroid, brain, and bone marrow of FAD4T mice, which provides evidence that bone marrow serves as a provider of immune cells to participate in the pathogenesis of TH reduction in AD and aging. RasGRP1, one of the coaugmented genes in aged thyroid and FAD4T thyroid, is a member of the RasGRP family and is highly expressed mainly in T cells [34]. RasGRP1 has been proven to participate in many inflammatory diseases, the deficiency of which hinders the proliferation, activation, and motility of immunocytes [35]. CD3g is another coaugmented gene in aged thyroid and FAD4T thyroid. It plays a significant role in the adaptive immune process. As part of the TCR complex, it delivers TCR-mediated signals followed by the activation of downstream signaling pathways [36]. Therefore, immune cell infiltration of the thyroid is a potential underlying mechanism of the alteration of THs in AD and aging.

Moreover, imbalance of the HPT axis may play a role in the alteration of THs in FAD4T mice, although the evidence is not sufficient. Aβ deposits and neurofibrillary tangles lead to the degeneration and apoptosis of neurons followed by brain dysfunction. The hypothalamus is the most significant structure responsible for the regulation of neuroendocrine functions in the central nervous system. As a direct target of pathology in AD, the hypothalamus was also found to contain Aβ deposits and neurofibrillary tangles, similar to the cortex and hippocampus [37]. We speculate that hypothalamic dysfunction destroys the HPT axis and decreases the secretion of thyroid stimulating hormone-releasing hormone, which suppresses TSH secretion from the pituitary and eventually causes a reduction in THs.

In conclusion, immune cell infiltration of the thyroid is a potential underlying mechanism of the alteration of THs in AD and aging. However, our study has some limitations, and we only explained the alteration of THs from the level of the potential underlying mechanism. We propose that immune cell infiltration and destruction of the HPT axis might just be the tip of the iceberg. Because of the deficiency of verification in the population, whether the conclusion can be extended to humans remains in doubt. Nevertheless, our results still provide a firm foundation for further research on the relationship among AD, aging, and the thyroid in the future. In addition, as a promising target for the treatment of AD, the thyroid may provide new ideas for delaying the progression of AD in the future.

We thank Dr. Youben Fan, Dr. Minggao Guo, and Dr. Zhili Yang from the Department of General Surgery, Shanghai Jiao Tong University Affiliated Shanghai Sixth People’s Hospital, for discussions and suggestions.

The proposal for the study was approved by the Committee for the Ethics of Animal Experiments of the Sixth People’s Hospital, affiliated with Shanghai Jiao Tong University School of Medicine, China (permit number: 2022-0592) and the Ethics Committee of Shanghai Sixth People’s Hospital (permit number: 2022-088). All participants provided written informed consent.

The authors declare no competing interests.

This study was performed with the support of the National Natural Science Foundation of China (81972543), Zhejiang Medical Science and Technology Program (2021KY329, 2023KY1151), Ningbo Science and Technology Program (Ningbo Natural Science Foundation [202003N4243, 2021J022]), and Ningbo Yinzhou Science and Technology Program (2020AS0073).

Yijun Wu, Jun Pan, Bo Wu, and Jun Xu conceived and designed the study. Siyuan Zhu, Yidan Pang, Xiangwei Zhang, and Chunying Yang performed the experiment and analyzed the data. Junjie Gao, Ping Fang, Yaohui Zhang, Yunjin Yao, Fangyu Ju, Fang Ye, Hongyi Zhu, Peng Liao Lufeng Yao, and Lulu Dai assisted with the animal experiments and provided suggestions. Siyuan Zhu and Yidan Pang wrote the manuscript. Yijun Wu, Junjie Gao, Jun Pan, Bo Wu, and Jun Xu reviewed and edited the manuscript.

Additional Information

Siyuan Zhu, Yidan Pang, Xiangwei Zhang, and Chunying Yang have contributed equally to this work and shared the first authorship.

scRNA-Seq data have been deposited into the GEO repository with the accession code GSE214917. Bulk RNA-Seq data have also been deposited into the GEO repository with the accession code GSE224575. Additional data that support the findings of this study are available from the corresponding author upon request. Source data are provided with this paper. Further inquiries can be directed to the corresponding author.

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