The immunosenescence and lipotoxicity in thyrotoxicosis mice


 COVID-19 is a worldwide outbreak now, and it is found to be age-related. Immunosenescence may be a predisposing and severe factor for COVID-19. Besides, many infectious diseases in clinic are age-related, and elderly patients have longer hospitalization and worse prognosis. Therefore, finding suitable aging models is of great significance for fighting aging related diseases and promoting the prognosis of elderly patients. In this study, the relationship between thyrotoxicosis and aging was investigated by routine detection and serum metabonomics in mice. The results of routine blood test and flow cytometry showed significant decrease in neutrophils, lymphocytes, CD4+/CD8+ and CD4+IFN-γ + lymphocytes in thyrotoxicosis mice. Biochemical examination combined with serum metabolomics analysis showed that serious disorder of lipid metabolism may be one of the causes of immunosenescence, including lower cholesterol levels, lower levels of VD and bile acids, high level of glucocorticoids, triglycerides, free fatty acids, Sphingolipids and decrease of Docosanoids, especially DPA. This study proves that thyrotoxicosis mice are an accelerated aging model. In present study, the main performance is immunosenescence, which may be due to lipotoxicity, suggesting that the immunosenescence state can be adjusted by improving lipotoxicity, whether anti thyroxine or not. However, there are other manifestations of thyroid toxicity mouse model simulating aging, such as organ aging, which need to continue to be studied by means of system biology to provide more comprehensive evidence.

to the decrease of CD4+T cell count and the increase of CD8+T cell count, which was associated with poor immunity, increased morbidity and increased mortality 9 . Immunosenescence not only occurs in the elderly, but also is a marker of AIDS immune damage, and is related to the prognosis of AIDS 10 . Additionally, it is of great signi cance to evaluate the host immune function by combining the number and function of lymphocytes for the diagnosis, treatment and prognosis of diseases 11 . The ability to produce IFN-γ can be used as lymphocyte function 12 . However, the relationship between hyperthyroidism or thyrotoxicosis with immunosenescence has not been given special attention, the material basis of the impact on the immune system also needs to be clari ed.
Selecting appropriate aging models and detection indicators are very important for studying aging and exploring antiaging strategy. The existing aging animal models can re ect the aging characteristics or related diseases in one or some aspects, but cannot comprehensively re ect or detect the physiological changes related to aging 13 , so it is urgent to screen or develop animal models that can fully simulate the characteristics of human aging. Accelerated metabolism by thyroid hormones assumed to accelerated aging. Studies have found that elevated thyroxine levels are associated with aging and shortened lifespan, and the longevity of vertebrate species is usually positively correlated with low metabolic rate and low TH level 14,15 . Despite the length of life is judged in these models, the internal changes of thyroxine affecting aging are rarely mentioned, especially the aging related markers to predict the aging status and the effect of aging intervention. Thyrotoxicosis refers to excessive thyroid hormone in blood circulation caused by any reason, resulting in hyperthyroidism 16 , so exogenous administration of excessive thyroxine can cause thyrotoxicosis. Although many studies have used this model to study the mechanism of hyperthyroidism and the development of hyperthyroidism protective drugs, there is rare discussion on the model simulating aging. We speculate that thyrotoxicosis model is an ideal accelerated aging model, but a deeper understanding of the association between thyrotoxicosis and aging is needed. Our study design allows assessment of the effects of excessive thyroid hormones on immune system and metabolism without autoimmune factors or treatment side effects, especially focus on the in uence of metabolites on immune system. On the basis of routine biochemical and hematological indicators, metabonomics was used to analyze the changes of serum endogenous metabolites and fecal metabolites, the mechanisms behind the relationship of thyrotoxicosis and aging will be well explored.

Changes of differential blood count and biochemical index
Blood routine test results showed that the total number of white blood cells (WBC), neutrophils (NEUT), lymphocytes (LYMPH), red blood cells (RBC), hemoglobin (HGB), Hematocrit (HCT) and platelets (PLT) were all decreased, which suggested that pancytopenia was induced by excessive thyroxine in mice (Table 1).
Biochemical analysis showed that the levels of Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) and Total triglyceride (TG) were increased signi cantly after 20 days of continuous intake of overdose thyroxine. The levels of Low-density lipoprotein cholesterol (LDC-C), High-density lipoprotein cholesterol (HDL-C), Total cholesterol (TC), UREA and Creatine (CREA) decreased. Changes of subsets and function of lymphocytes As shown in Fig. 1, compared with the normal control group, the percentage of CD3+CD4+ in thyrotoxic mice decreased signi cantly, while the percentage of CD3+CD8+ increased signi cantly, so the percentage of CD4+/CD8+ decreased signi cantly. The percentage of CD3 + CD4 + IFN-r + was signi cantly decreased in thyrotoxicosis mice.

Results of serum LC-MS metabolomics analysis
There were signi cant differences in OPLS-DA score between thyrotoxicosis group and normal group (Fig. 2a). Based on Human Metabolome Database (HMDB), LC-MS-based metabolomics identi ed and quanti ed 821 metabolites in serum, of which 157 were differentially expressed metabolites (DEMs), including 81 upregulated and 76 downregulated (Fig.  2b). KEGG pathway analysis showed that these DEMs were enriched in the pathway of Steroid hormone biosynthesis, Primary bile acid biosynthesis, Bile secretion, Valine, leucine and isoleucine biosynthesis, Arachidonic acid metabolism, Sphingolipid signaling pathway and so on (Fig. 2c). The classi cation of DEMs was displayed in Fig. 2d. "Hydroxyl steroid" corresponded to "Steroid hormone biosynthesis", "Phosphosphingolipids" corresponded to "Sphingolipid signaling pathway", the DEMs in these two classes were all upregulated. "Bile acids, alcohols and derivatives" corresponding to "Primary bile acid biosynthesis" and "Bile secretion", there are 11 DEMs in this class, of them 7 were downregulated. "Eicosanoids' corresponded to "Arachidonic acid metabolism", in this class 9 of 11 were downregulated. The DEMs in "Fatty acids and conjugates" and "Lineolic acids and derivativities" classes had ups and down. 7 of 8 DEMs in "Arylsulfates" class were downregulated. The DEMs of "Glycerophosphocholines (GPC)", "Hydroxycinnamic acids and derivatives", "Pregnane steroids", and "Purines and purine derivatives" were all down regulated.

Results of serum GC-MS metabolomics analysis
There were signi cant differences in OPLS-DA score between thyrotoxicosis group and normal group (Fig. 3a). GC-MSbased metabolomics identi ed and quanti ed 885 metabolites in serum, of which 78 were differentially expressed metabolites (DEMs) (Fig. 3b

Discussion
Low immune status and defense ability of thyrotoxicosis In present study, the syndrome and mechanism of thyrotoxicosis mice were studied by routine detection combined with metabolomics. Blood routine examination showed that excessive thyroxine led to a decrease in the total number of leukocytes, mainly neutrophils and lymphocytes. It also led to anemia and thrombocytopenia. The ow cytometry test showed the decrease of CD4/CD8 ratio and the number of IFN-γ + CD4+T cells in thyrotoxicosis mice. The above evidences indicated that the immune function of thyrotoxicosis mice was damaged seriously in present study.
As reported, hyperthyroidism patients may present with single-cell lineage hematological abnormalities such as leucopenia, anemia, thrombocytopenia. The activity of hyperthyroidism (measured at T3 or T4 level) is signi cantly correlated with anemia and cytopenia 17 . But hyperthyroidism can also lead to pancytopenia 18 . Leukopenia, especially agranulocytosis, may be a complication of hyperthyroidism, which usually leads to severe illness, as well as severe secondary in ammation 19 . The decrease of CD4 / CD8 ratio is one of the markers of T cell "immunosenescence" 9 . The ability to produce IFN-γ can be used as lymphocyte function 12 . Th1 CD4 effector T cells can produce large amounts of IFN-γ to ght the infection of intracellular pathogens, thereby stimulating and maintaining effective cellular immune responses 20 . In present study, the decrease of CD4/CD8 ratio and the number of IFN-γ + CD4+T cells represent the decline of host immune function in thyrotoxicosis mice. Although there is a lack of information on the prevalence and determinants of thyroid function and COVID-19, the British Thyroid Association and the Society for Endocrinology issued a statement emphasizing that patients with thyroid diseases (especially thyrotoxicosis) may have a higher risk of complications, and the American Thyroid Association also recommended that patients with thyroid diseases maintain social distance and limit their exposure to COVID-19 21 . Actually, autoimmune factors mask the relationship between thyroxine and immunity in human, so the relationship between hyperthyroidism and COVID-19 appears very complex 22 .
Here, present study suggested that thyrotoxicosis related immunosenescence might be the cause of aggravation of infection disease, including COVID-19, which also explain the susceptibility and severity of the elderly to respiratory infections caused by decreased resistance. Biochemical test and metabonomics of serum revealed the changes of metabolites partly contributed to immunosenescence.

Abnormal cholesterol synthesis and metabolism in thyrotoxicosis mice
Biochemical test results and serum metabolomics results together displayed that impairment of lipid metabolism in thyrotoxicosis mice, which re ected by the reduced levels of cholesterol, apolipoproteins and elevated lipid metabolites in serum.
Cholesterol is the basic component of cell membrane, its biosynthetic and regulatory pathways are ubiquitous in various cells, including immune cells, which play an important regulatory role in innate and adaptive immune activities 23 .
Although high TC has been advertised as adverse to health, especially in atherosclerosis, studies have shown that low cholesterol is also harmful, hospitalized elderly patients are prone to acquired hypocholesterolemia, which is characterized by low concentrations of all lipoproteins (VLDL, LDL and HDL), which is related to poor prognosis as hypoproteinemia 24 . Plasma cholesterol is a negative acute phase reactant, total cholesterol decreases after surgery and under various pathological conditions, including trauma, sepsis, burn and liver dysfunction, and hypocholesterolemia is associated with in-hospital mortality 25,26,27 . The reason might be cholesterol and apolipoprotein played an important role in pathogen toxin clearance and regulation of in ammatory response 28 . Biochemical test results showed the TC, LDC-C and HDL-C level were decreased signi cantly in thyrotoxicosis mice. Although the decrease of serum cholesterol level is a known nding in hyperthyroidism 29 , the reason is still unknown. The metabolomic results of present study might give the answer.
Lanosterol is an intermediate product of cholesterol synthesis 31 . In present study, the endogenous cholesterol synthesis, intestinal cholesterol absorption and bile cholesterol absorption, and bile acid synthesis and excretion were all decreased, the evidences were the downregulation of Lanosterol, Campesterol and bile acids. In addition to Cholesterol, Dihydrocholesterol and 20a,22b-Dihydroxycholesterol were also found downregulated. Present study proved that the decrease level of cholesterol attributed to the decrease of synthesis and absorption. And the decrease level of bile acids in present study suggested that the decrease level of Cholesterol was not due to the increasing conversion of cholesterol to bile acids which was accordance with previous study 29 .
Bile acids are cholesterol derived metabolites, which play a recognized role in the digestion and absorption of dietary fat 32 . Bile acids are a physiological factor required for nutrient absorption, distribution, metabolism and excretion. They are also nutrient sensor and metabolic regulator 33 . Bile acids also have important immunomodulatory effects 34 . The primary bile acids produced by the liver are metabolized into secondary bile acids under the action of intestinal microbes, they play a role in maintaining intestinal barrier and preventing intestinal pathogens from colonization. These primary and secondary bile acids play a bene cial role in maintaining innate immunity by acting on their receptors at the interface of the host immune system 35 . Taurochenodeoxycholic acid has been found to enhance immunity by increasing CD4+/CD8+ value in peripheral blood in mice 36 . Chenodeoxycholic acid can inhibit the lipotoxicity of cardiomyopathy 37 . Previous study showed that the T3 dose dependently decreased the formation of cholic acid and chenodeoxycholic acid by inhibiting the expression of CYP7A1 and Cyp8b1 human liver cell lines 38 . In present study, several of bile acids were downregulated, including 3a,7a-Dihydroxy-5b-cholestan-26-al, Chenodeoxycholic acid, Chenodeoxycholic acid 3-glucuronide, Alpha-Muricholic acid, Deoxycholic acid, Taurochenodesoxycholic acid, Dihomodeoxycholic acid and Lithocholic acid. Especially Chenodeoxycholic acid and Deoxycholic acid reduced to undetectable levels. The downregulation of these bile acids seriously affects the metabolism of the lipids and drugs 39 .
Vitamin D is another product of cholesterol metabolism. In addition to the classical effects related to mineral homeostasis, vitamin D plays a new role in cell proliferation and differentiation, regulation of innate and adaptive immune system, prevention of cardiovascular and neurodegenerative diseases, and even anti-aging 40 . A recent study found that the serum 25-OHVit D levels in hyperthyroidism patient with hypercalcemia were lower than the normal range, and Vitamin D3 adjuvant therapy can improve thyroid related antibody level, thyroid function and bone metabolism in patients with hyperthyroidism complicated with hypercalcemia 41 . Early study showed that aging could reduce the ability of skin to produce previtamin D3 more than two times 42  The decrease level of bile acids proved that the decrease level of Cholesterol was not due to the increasing conversion of cholesterol to bile acids. Alternatively, it might due to the increasing conversion to glucocorticoids. Conversely to the downregulation of bile acids, another cholesterol metabolites glucocorticoids were upregulated in thyrotoxicosis mice, which might partly contribute to the decline of Cholesterol. They were all Hydroxysteroids, including Tetrahydrocorticosterone, 11b,17a,21-Trihydroxypreg-nenolone, Cortolone, 3a,21-Dihydroxy-5b-pregnane-11,20-dione, 18-Hydroxycorticosterone, Cortisol, 11-Dehydrocorticosterone, Dihydrocortisol, Corticosterone and Cortol (Table 3). Their upregulations indicated the increase of endogenic glucocorticoids (eGCs) in plasma which was in line with previous study 46 . As reported, increased hypothalamic-pituitary-adrenal (HPA) axis activity associated with cushing's syndrome, hyperthyroidism and aging 47 . GC is the most common cause of secondary osteoporosis and the main cause of nontraumatic osteonecrosis 48 . It is well known that GCs have immunosuppressive effect, which inhibits phagocytosis of macrophages and causes lymphocytic lysis, especially the decrease of helper T cells (Th) 49 . And CD4 + T cells are highly sensitive to GC induced apoptosis 50 . eGCs levels increase with age and can accelerate aging processes in vertebrate species 51,52 . Research also indicated that stress-induced GCs might play causal role for aging and agerelated disorders 53 . Aging and chronic stress together lead to abnormal HPA axis activation, leading to the increase of peripheral GC level, consequently accelerate cell aging and premature immunosenescence, including the reduction of primitive T cells, poor immune response to neoantigens, cell-mediated immune reduction, and thymus degeneration, resulting in an increase in the incidence of diseases related to immunosenescence, including tumors and COVID-19 54,55 . So, the increase of GCs is both the result and the cause of aging. And in present study the upregulated eGCs in thyrotoxicosis mice are not only the reason for the decline of immunity, but also the evidence that the model simulates aging.
In general, the changes in cholesterol synthesis and metabolism were mainly manifested by the decreasing of synthesis and bile acids mediated absorption, as well as the increasing conversion to glucocorticoids. The decrease of cholesterol, bile acids and VD, as well as excessive glucocorticoids resulting in immune decline, which are closely related to the aging. The cholesterol related differential metabolites were integrated into Table 3.

Lipotoxicity in thyrotoxicosis mice
Early study found that T3 or T4 may directly stimulate the lipolysis which is characterized by plasma high fatty acids 56 , however, there is no discussion about the relationship between high plasma fatty acids and lipotoxicity in hyperthyroidism. In present study, the lipids DEMs such triglycerides (TGs), Fatty acids and Sphingolipids were upregulated, which indicated lipotoxicity in thyrotoxicosis mice.
It is generally believed that the increase in blood lipids is due to the use of antithyroid drugs 57 . However, this study found that excessive thyroxine itself also leads to the increase of TGs level in serum. Combined with the upregulation of Monoradylglycerols, Diradylglycerols and large number of upregulated Fatty acids, a complete map of TGs metabolism in thyrotoxicosis model was displayed, that is the increase of fatty acids attributed to thyroxine mobilized the decomposition of adipose tissue, resulting in the increase of free fatty acids level in blood, with the increase of Monoradylglycerols (MGs), Diradylglycerols (DGs) led to the increase of TGs synthesis, at the same time, because of the decrease of lipoprotein, TG couldn't be transported in time, leading to the high level of TGs in serum 58 .
As well known, TGs are highly correlated with aging and age-related physiological dysfunction 59 . Various intermediates of fatty acid metabolism have been shown to cause cell stress and toxicity (lipotoxicity) in adipocytes and other related cell types (including cardiomyocytes, hepatocytes and immune cells), such as Sphingolipid, Ceramide and Diacylglycerol, which were also upregulated in present study 60 . Cells treated with sphingosine can rapidly induce mitochondrial membrane potential loss, mitochondria release cytochrome c and apoptotic cell death 61 . Sphingolipids implicated in the pathophysiology of cardiovascular disease, and inhibition of sphingolipid synthesis could attenuate cardiomyopathic symptoms 62 . Sphingolipids, including ceramide, play a role in aging and are also markers of aging. Many studies have shown that lowering ceramide concentration may delay or improve the symptoms of aging human aging 59 . Here, the upregulated Sphingolipids are another evidence that thyrotoxicosis mimics aging.
It was found that plasma long-chain free fatty acids are inversely correlated with longevity 63 . And long-chain fatty acids also affect immune cells. For example, it was found that a ω-6 polyunsaturated fatty acids (PUFAs) Linoleic acid (18:2) destroyed mitochondrial function, caused more oxidative damage than other free fatty acids (such as Palmitic acid), which mediated the selective loss (death) of CD4 (+) T lymphocytes in the liver, accelerated the occurrence of cancer 64 .
Linoleic acid could also decrease the function of immune cells by inhibited IFN-γ production in CD4 + T cells 65 . The severity of ICU patients with COVID-19 was associated with high levels of free fatty acid 66 . Mice given linoleic acid (LA) (C18:2) developed leukopenia, lymphopenia, lymphocyte damage, relative thrombocytopenia, hypercytokinemia, elevated alanine aminotransferase levels, hypoalbuminemia, hypocalcemia, shock, and renal failure, resembling lethal COVID-19 67 . In present study, Linoleic acid was also upregulated which might be the cause of loss and dysfunction of CD4 T cells.
On the contrary, ω-3 fatty acids have potential use in COVID-19 treatment because they have their antioxidant and antiin ammatory effects, as well as the ability of regulating platelet homeostasis and the risk of thrombosis 68 . A pilot study in 100 patients suggested that ω-3 fatty acids have a tendency to reduce the incidence rate and mortality of COVID-19 infection 69 . As we all know, intake ω-3 fatty acids can have bene cial health effects on many biological processes, such as improving immune status, protecting against infection and allegies, enhancing cognitive ability, optimizing neuromuscular function and reducing muscle loss 70,71 . ω-3 fatty acids regulate lipid metabolism, contribute to fatty acid oxidation and inhibit fat production, and lead to good lipid distribution and adipocyte metabolism 72 . ω-3 fatty acids improve body composition by reducing cortisol levels 73 , which suggested that ω-3 fatty acids could resist the adverse effects of cortisol. Coincidentally, the level of cortisol were upregulated, while the ω-3 fatty acids and their metabolites were found downregulated in thyrotoxicosis mice in present study, including Docosapentaenoic acid (22n-3) (DPA), Maresin 1 and 17,18-EpETE. So, the less of ω-3 fatty acids might also be the cause of harmful effect of eGCs in present study. The effects of ω-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been widely studied. DPA is the metabolic intermediate of EPA and DHA, less is known about DPA, however, available evidence suggests that DPA may also be superior to the health bene ts of EPA and DHA 74 . DPA has anti-in ammatory effects and can improve cardiovascular and metabolic diseases, and plasma DPA level was inversely associated with total mortality in older people 75 . In addition, DPA is particularly bene cial to the neuroprotection and early life development of the elderly 76 . DPA is also a precursor of docosanoids, such as Maresin 1, which was found downregulated in thyrotoxicosis mice in present study. Marensin 1 plays an important role in the remission of acute in ammation and organ protection by enhancing the phagocytosis of macrophages, which is bene cial to maintain host defense ability, homeostasis and wound healing 77 . In addition, 17,18-epoxyeicosatetraenoic acid (17,18-EpETE) was also found downregulated, it is a lipid metabolite endogenously generated from EPA which exhibits anti-allergic and anti-in ammatory properties 78 .
The upregulations of Glycerolipids, Sphingolipids, Fatty Acids and the downregulation of Docosanoids in present study were listed in Table 4. In this study, routine examination combined with metabonomics technology were used to make a comprehensive research report on thyrotoxicosis. Bioinformatics combined with a large number of literature research was used to deeply analyze the serum metabonomics. A variety of test results were mutually veri ed, and the relationship between thyrotoxicosis and aging was well established. The results were summarized in Fig. 4.
Serum metabolomics analysis showed that the metabolism of thyrotoxicosis mice was disordered, especially the synthesis and metabolism of cholesterol and lipids. The decrease in cholesterol synthesis and absorption led to a decrease in serum cholesterol, which consequently resulted in a decline in the levels of cholesterol derived VD and bile acids. The lack of VD led to osteoporosis and the decline of immunity. The decrease in bile acids not only caused disorder of lipid metabolism, but also contributed to decline in innate immunity. In contrast, another type of cholesterol derivative GCs increased signi cantly, which can cause osteoporosis, immunosenescence. Despite that VD and bile acids were downregulated while the GCs were upregulated, the results caused by their changes are indeed consistent, which are harmful to the body and relate to aging.
This study also showed that thyrotoxicosis model might be an appropriate model to study lipotoxicity, because a variety of lipotoxicity related metabolites were up-regulated, including Glycerolipids, Sphingolipids and Fatty Acids. Lipotoxicity is mainly manifested in its damage to the mitochondrial membrane, acting on immune cells leading to immunosenescence, and acting on organs causing organ senescence. Furthermore, GCs are also Lipids and lipid-like molecules, and the harmful effects of excessive GCs can also be classi ed as lipotoxicity. On the contrary, the Docosanoids were downregulated, but correspondingly, their decrease also led to the decline of homeostasis regulation ability and defense ability.
In summary, as shown in Fig. 4, the increase of harmful lipids metabolites and the decrease of protective lipids metabolites me eventually lead to the aging feather of thyrotoxicosis mice. In present study, the symptoms of aging were manifested as immunosenescence. Present study provided evidence that thyrotoxicosis model is an aging model, which can be used to study the mechanism of aging and anti-aging drugs, even to explore disease prevention measures, like COVID-19. Studies have con rmed that COVID-19 is an acute aging disease 4 , age and age-related diseases are the main risk factors leading to severe disease and COVID-19 death, but the reason for this age dependence is not clear 79 . Present study provides some clues, the decline of CD4/ CD8 ratio and IFN-γ production capacity, granulocytopenia indicated that the immune function and the defense ability of thyrotoxicosis mice was damaged seriously, the high levels of free fatty acid and eGCs were indicated lipotoxicity in thyrotoxicosis mic, all above factors was related with the severity of COVID-19 patients. Candidates that can improve lipotoxicity of thyrotoxicosis may have potential value in the development of anti-aging drugs, whether they have anti-thyroxine function or not.
The combination of metabonomics and conventional detection methods to study the toxic effects of excess thyroxine provide more information for the thyrotoxicosis model to simulate aging, mainly manifested as immunosenescence in present study, whereas the differential metabolites are also rooted in the generated organs or tissues, and also represent the aging lesions of organs. The organ changes might provide more informations for exploring the similarity of thyrotoxicosis and aging, and the proteomics of serum may provide more convenient markers for judgement of aging. The subsequent use of system biology to study serum and organ changes will make the evidence more complete for thyrotoxicosis simulating aging.

Methods
Animals and reagents Thyrotoxicosis model 24 mice were randomly divided into 2 groups (n = 12 per group): control group (C), model group (T). Intragastric administration of thyroxine tablets suspension (320mg / kg), once a day, for 20 days. The body weight was measured before and at the end of the model. At the end of the experiment, fasting the night before dissection, blood was taken from the main abdominal vein after anesthesia with 60mg·kg -1 pentobarbital sodium, and 6 mice in each group were for routine blood test and ow cytometry. 6 mice in each group were for biochemical analysis and metabonomics detection. About 1ml blood was injected to gel separation tube and the serum was separated after centrifugation of 1000g. 100ul serum was taken out and diluted 4 times for biochemical detection. The remaining serum was cryopreserved at -80 ℃ for proteomics and metabonomics detection.
Routine blood tests EDTAK2 anticoagulant whole blood was taken for routine blood test. Routine blood tests were carried by Sysmex XN-1000v [B1] automated hematology analyzer (SYSMEX Co., Ltd., Japan).

Detection of CD4 + , CD8 + T lymphocytes
The positive rates of CD4 + , CD8 + T lymphocyte subsets were analyzed by ow cytometry. Brie y, 3 mL of red blood cell lysis buffer was added to each sample to completely lyse red blood cells. Then the samples were washed and resuspended with DMEM to 1 × 10 6 cells/mL. Cells were transferred to 48-well plates with 100 μL volume of each well, 2 μL of cell activation cocktail (BD biosciences) and 1 μL of BrefeldinA (BD biosciences) were added to each sample and incubated at 37 °C for 6 h. After washed and re-suspended with PBS, samples were incubated with speci c uorescent antibodies (BD biosciences) of PC5.5 conjugated anti-mouse CD3 antibody (0.5 μl/sample), FITC conjugated anti-mouse CD4 antibody (0.2 μl/sample), and APC-A750 conjugated anti-mouse CD8 antibody (

Data and Statistical Analysis
All experimental data obtained from rats were expressed as mean ± SD. A one-way repeated measure analysis of variance (ANOVA) and a log-rank test were used to determine the signi cance of the differences in differential blood count, biochemical index and subsets and function of lymphocytes, respectively.
For LC-MS and GC-MS, differential expressed metabolites (DEMs) were selected on the basis of the combination of a statistically signi cant threshold of variable in uence on projection (VIP) values obtained from the (orthogonal) partial least-squares-discriminant analysis (OPLS-DA) model and p values from a two tailed Student's t test on the normalized peak areas from different groups, where metabolites with VIP values larger than 1.0 and p values less than 0.05 were considered as differential metabolites.
79. Santesmasses, D., et al. COVID-19 is an emergent disease of aging. Aging cell 19, e13230 (2020). Figure 1 Changes of subsets and function of lymphocytes. Compared with the normal control group, the percentage of CD3+CD4+ in thyrotoxicosis mice decreased signi cantly, while the percentage of CD3+CD8+ increased signi cantly, so the percentage of CD4+/CD8+ decreased signi cantly. The percentage of CD3 + CD4 + IFN-r + was signi cantly decreased in thyrotoxicosis mice. C: normal control group; T: thyrotoxicosis group.   The relationship of disordered lipid metabolism with immunosenescence in thyrotoxicosis mice Serum metabolomics analysis showed that the metabolism of thyrotoxicosis mice was disordered, especially the synthesis and metabolism of cholesterol and lipids. The decrease in cholesterol synthesis and absorption led to a decrease in serum cholesterol, which consequently resulted in a decline in the levels of cholesterol derived VD and bile acids. The lack of VD and bile acids led to the decline of immunity. In contrast, another type of cholesterol derivative GCs increased signi cantly, which can cause immunosenescence. A variety of lipotoxicity related metabolites were up-regulated, including Glycerolipids, Sphingolipids and Fatty Acids. Lipotoxicity is mainly manifested in its damage to the mitochondrial membrane, acting on immune cells leading to immunosenescence. Furthermore, GCs are also Lipids and lipid-like molecules, and the excessive GCs can also be classi ed as lipotoxicity factors. On the contrary, protective Docosanoids were downregulated, but correspondingly, their decrease weakens their ability to resist lipotoxicity, leading to the decline of defense ability. In conclusion, present study showed that immunosenescence was found in thyrotoxicosis mice, and lipotoxicity may be one cause, which provides a basis for simulating aging in the thyrotoxicosis mice model.