TYK2 signaling promotes the development of autoreactive CD8+ cytotoxic T lymphocytes and type 1 diabetes.

Tyrosine kinase 2 (TYK2), a member of the JAK family, has attracted attention as a potential therapeutic target for autoimmune diseases. However, the role of TYK2 in CD8+ T cells and autoimmune type 1 diabetes (T1D) is poorly understood. In this study, we generate Tyk2 gene knockout non-obese diabetes (NOD) mice and demonstrate that the loss of Tyk2 inhibits the development of autoreactive CD8+ T-BET+ cytotoxic T lymphocytes (CTLs) by impairing IL-12 signaling in CD8+ T cells and the CD8+ resident dendritic cell-driven cross-priming of CTLs in the pancreatic lymph node (PLN). Tyk2-deficient CTLs display reduced cytotoxicity. Increased inflammatory responses in ß-cells with aging are dampened by Tyk2 deficiency. Furthermore, treatment with BMS-986165, a selective TYK2 inhibitor, inhibits the expansion of T-BET+ CTLs, inflammation in ß-cells and the onset of autoimmune T1D in NOD mice. Thus, our study reveals the diverse roles of TYK2 in driving the pathogenesis of T1D.

HSPA8 Single-Nucleotide Polymorphism Is Associated with Serum HSC70 Concentration and Carotid Artery Atherosclerosis in Nonalcoholic Fatty Liver Disease.

There is an association between nonalcoholic fatty liver disease (NAFLD) and atherosclerosis, but the genetic risk of atherosclerosis in NAFLD remains unclear. Here, a single-nucleotide polymorphism (SNP) of the heat shock 70 kDa protein 8 (HSPA8) gene was analyzed in 123 NAFLD patients who had been diagnosed using a liver biopsy, and the NAFLD phenotype including the maximum intima-media thickness (Max-IMT) of the carotid artery was investigated. Patients with the minor allele (A/G or G/G) of rs2236659 showed a lower serum heat shock cognate 71 kDa protein concentration than those with the major A/A allele. Compared with the patients with the major allele, those with the minor allele showed a higher prevalence of hypertension and higher Max-IMT in men. No significant associations between the HSPA8 genotype and hepatic pathological findings were identified. In decision-tree analysis, age, sex, liver fibrosis, and HSPA8 genotype were individually associated with severe carotid artery atherosclerosis (Max-IMT ≥ 1.5 mm). Noncirrhotic men aged ≥ 65 years were most significantly affected by the minor allele of HSPA8. To predict the risk of atherosclerosis and cardiovascular disease, HSPA8 SNP genotyping might be useful, particularly for older male NAFLD patients.

Immunohistochemical detection of enteroviruses in pancreatic tissues of patients with type 1 diabetes using a polyclonal antibody against 2A protease of Coxsackievirus.

AIMS/INTRODUCTION: The need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde-fixed and paraffin-embedded samples is increasing. The gold standard monoclonal antibody (clone 5D8/1) against EV-envelope protein (VP1) was proven to cross-react with other proteins. Another candidate marker of EV proteins is 2A protease (2Apro ), which is encoded by the EV gene and translated by the host cells during EV replication, and participates processing proproteins to viral capsid proteins. MATERIALS AND METHODS: We raised polyclonal antiserum by immunizing a rabbit with an 18-mer peptide of Coxsackievirus B1 (CVB1)-2Apro , and examined the specificity and sensitivity for EV on formaldehyde-fixed and paraffin-embedded tissue samples. RESULTS: Enzyme-linked immunosorbent assay study showed a high titer of antibody for 18-mer peptide of CVB1-2Apro , cross-reacting with CVB3-2Apro peptide. IHC showed that antiserum against 2Apro reacted with CVB1-infected and VP1-positive Vero cells. Confocal laser scanning microscopy showed that antigen stained by the 2Apro antibody located in the same cell with VP1 stained by 5D8/1. IHC using 2Apro antiserum showed dense staining in the islets of EV-associated fulminant type 1 diabetes pancreas and that located in the same cell stained positive for VP1 (5D8/1). Specificity of 2Apro antiserum by IHC staining was confirmed by negative 2Apro in 14 VP1-negative non-diabetes control pancreases. CONCLUSIONS: Our study provides a new polyclonal antiserum against CVB1-2Apro , which might be useful for IHC of EV-infected human tissues stored as archive of formaldehyde-fixed and paraffin-embedded tissue samples.

Regulation of Viral Infection in Diabetes.

Though there is no 'Diabetes Virus', multiple agents such as mumps virus, rubella virus, influenza virus, type A hepatitis virus, enterovirus, rotavirus, cytomegalovirus, varicella-zoster virus, human herpesvirus 6, Epstein-Barr virus, and also SARS-CoV-2 have been reported to be associated to diabetes [...].

TYK2 Promoter Variant Is Associated with Impaired Insulin Secretion and Lower Insulin Resistance in Japanese Type 2 Diabetes Patients.

Accumulating evidence has suggested that viral infection causes type 1 diabetes due to direct ß-cell damage and the triggering of autoimmune reactivity to ß cells. Here, we elucidated that the tyrosine kinase 2 (Tyk2) gene, encoding an interferon receptor signaling molecule, is responsible for virus-induced diabetes in mice, and its promoter variant confers a risk of type 1 diabetes in humans. This study investigated the relationship between a TYK2 promoter variant (TYK2PV) and insulin secretion in type 2 diabetes patients. TYK2PV status was determined using direct DNA sequencing and its associations with fasting insulin, C-peptide, and homeostatic model assessment of insulin resistance (HOMA-IR) were evaluated in type 2 diabetes patients without sulfonylurea or insulin medication. Of the 172 patients assessed, 18 (10.5%) showed TYK2PV-positivity. Their body mass index (BMI) was significantly lower than in those without the variant (23.4 vs. 25.4 kg/m2, p = 0.025). Fasting insulin (3.9 vs. 6.2 µIU/mL, p = 0.007), C-peptide (1.37 vs. 1.76 ng/mL, p = 0.008), and HOMA-IR (1.39 vs. 2.05, p = 0.006) were lower in those with than in those without the variant. Multivariable analysis identified that TYK2PV was associated with fasting insulin ≤ 5 µIU/mL (odds ratio (OR) 3.63, p = 0.025) and C-peptide ≤ 1.0 ng/mL (OR 3.61, p = 0.028), and also lower insulin resistance (HOMA-IR ≤ 2.5; OR 8.60, p = 0.042). TYK2PV is associated with impaired insulin secretion and low insulin resistance in type 2 diabetes. Type 2 diabetes patients with TYK2PV should be carefully followed in order to receive the appropriate treatment including insulin injections.

SARS-CoV-2 Infection and Pancreatic ß Cell Failure.

SARS-CoV-2 infection primarily causes pulmonary symptoms; however, accumulating reports indicate that some patients with COVID-19 have multiple organ dysfunction or failure. Although diabetes is considered a risk factor for severe COVID-19, SARS-CoV-2 infection may also be a causal factor for diabetes mellitus in patients with COVID-19. According to the research reviewed in this paper, the pancreas and pancreatic ß cells appear to be targets of SARS-CoV-2 and are damaged by direct or indirect effects of the infection. However, controversial results have been reported between study groups, mainly due to the limited number of cases with diabetes precipitated by COVID-19. In this review, we comprehensively discuss the published findings on the potential association between SARS-CoV-2 infection or COVID-19 and pancreatic ß-cell damage leading to diabetes onset. These findings will further contribute to our understanding of the pathogenesis of diabetes mellitus.

Genetic Susceptibility of the Host in Virus-Induced Diabetes.

Enteroviruses, especially Coxsackie B viruses, are among the candidate environmental factors causative of type 1 diabetes. Host genetic factors have an impact on the development of virus-induced diabetes (VID). Host background, in terms of whether the host is prone to autoimmunity, should also be considered when analyzing the role of target genes in VID. In this review, we describe the genetic susceptibility of the host based on studies in humans and VID animal models. Understanding the host genetic factors should contribute not only to revealing the mechanisms of VID development, but also in taking measures to prevent VID.

Combined effect of canagliflozin and exercise training on high-fat diet-fed mice.

Sodium-glucose cotransporter 2 inhibitors (SGLT2is) have been reported to improve obesity, diabetes, and nonalcoholic fatty liver disease (NAFLD) in addition to exercise training, whereas the combined effects remain to be elucidated fully. We investigated the effect of the combination of the SGLT2i canagliflozin (CAN) and exercise training in high-fat diet-induced obese mice. High-fat diet-fed mice were housed in normal cages (sedentary; Sed) or wheel cages (WCR) with or without CAN (0.03% of diet) for 4 wk. The effects on obesity, glucose metabolism, and hepatic steatosis were evaluated in four groups (Control/Sed, Control/WCR, CAN/Sed, and CAN/WCR). Numerically additive improvements were found in body weight, body fat mass, blood glucose, glucose intolerance, insulin resistance, and the fatty liver of the CAN/WCR group, whereas CAN increased food intake and reduced running distance. Exercise training alone, CAN alone, or both did not change the weight of skeletal muscle, but microarray analysis showed that each resulted in a characteristic change of gene expression in gastrocnemius muscle. In particular, in the CAN/WCR group, there was acceleration of the angiogenesis pathway and suppression of the adipogenesis pathway compared with the CAN/Sed group. In conclusion, the combination of an SGLT2i and exercise training improves obesity, insulin resistance, and NAFLD in an additive manner. Changes of gene expression in skeletal muscle may contribute, at least in part, to the improvement of obesity and insulin sensitivity.

Impaired upregulation of Stat2 gene restrictive to pancreatic ß-cells is responsible for virus-induced diabetes in DBA/2 mice.

Viral infection is a putative causal factor for the development of type 1 diabetes, but the exact pathogenic mechanism of virus-induced diabetes (VID) remains unclear. Here, to identify the critical factors that regulate VID, we analyzed encephalomyocarditis D (EMC-D) VID-sensitive DBA/2 mice in comparison with resistant B6 mice. EMC-D virus-induced cell death occurred more frequently in DBA/2 ß-cells than in B6 ß-cells with 100U/ml IFN-ß priming in vitro. We therefore purified ß-cells using flow cytometry from mice two days after EMC-D virus infection and subjected them to microarray analysis. As a results, innate immune response pathway was found to be enriched in B6 ß-cells. The signal transducer and activator of transcription 2 (Stat2) gene interacted with genes in the pathway. Stat2 gene expression levels were lower in DBA/2 mice than in B6 mice, restrictive to ß-cells. Moreover, administration of IFN-ß failed to upregulate Stat2 gene in DBA/2 ß-cells than in those of B6 in vivo. The viral titer significantly increased only in the DBA/2 pancreas. Thus, these provided data suggest that impaired upregulation of Stat2 gene restrictive to ß-cells at the early stage of infection is responsible for VID development in DBA/2 mice.

Subtyping of Type 1 Diabetes as Classified by Anti-GAD Antibody, IgE Levels, and Tyrosine kinase 2 (TYK2) Promoter Variant in the Japanese.

OBJECTIVE: Type 1 diabetes (T1D) is known to be caused by Th1 cell-dependent autoimmunity. Recently, we reported that TYK2 promoter variant serves as a putative virus-induced diabetes susceptibility gene associated with deteriorated interferon-dependent antiviral response. TYK2 is also related to HIES, that is, Th2 cell-dependent. Therefore, TYK2 promoter variant may be also associated with the pathogenesis of T1D, modulating Th1/Th2 balance. RESEARCH DESIGN AND METHODS: We assessed the association between anti- GAD Ab, IgE levels, and TYK2 promoter variant among 313 T1D patients, 184 T2D patients, and 264 YH controls in the Japanese. RESULTS: T1D patients had elevated IgE (median, 56.7U/ml; p<0.0001) compared with T2D patients (22.5U/ml) and controls (43.3U/ml). Contrary to our expectations, there was no correlation between TYK2 promoter variant and IgE levels. We found that T1D could be subtyped as four groups based on anti-GAD Ab and IgE profile: Subtype 1, anti-GAD Ab positive and non-elevated IgE (47.0%); Subtype 2, anti-GAD Ab negative and non-elevated IgE (35.1%); Subtype 3, anti-GAD Ab positive and elevated IgE (10.9%); and Subtype 4, anti-GAD Ab negative and elevated IgE (7.0%). In Subtype 2, a significantly higher incidence was observed in T1D cases carrying the TYK2 promoter variant (OR, 2.60; 95%CI, 1.03-6.97; p=0.032), and also showing a flu-like syndrome at diabetes onset (OR, 2.34; 95%CI, 1.27-4.35; p=0.003). INTERPRETATION: Anti-GAD Ab and IgE profiling helps classifying T1D into four groups that recognize variable pathogenic bases of T1D.

Adjusting the 17ß-Estradiol-to-Androgen Ratio Ameliorates Diabetic Nephropathy.

Diabetes is manifested predominantly in males in experimental models, and compelling evidence suggests that 17ß-estradiol (E2) supplementation improves hyperglycemia in humans. We previously generated a severely diabetic transgenic (Tg) mouse model by ß-cell­specific overexpression of inducible cAMP early repressor (ICER) and found that male but not female ICER-Tg mice exhibit sustained hyperglycemia and develop major clinical and pathologic features of human diabetic nephropathy (DN). Thus, we hypothesized that differences in circulating hormone levels have a key role in determining susceptibility to diabetes. Here, we examined whether DN in male ICER-Tg mice is rescued by adjusting the androgen-to-E2 ratio to approximate that in normoglycemic female ICER-Tg mice. We treated hyperglycemic male ICER-Tg mice with orchiectomy (ORX), E2 pellet implantation, or both. E2 pellet implantation at an early stage of DN with or without ORX caused a rapid drop in blood glucose and a dramatic increase in ß-cell number, and it markedly inhibited DN progression [namely, E2 reduced glomerulosclerosis, collagen 4 deposition and albuminuria, and prevented hyperfiltration]. Furthermore, E2 pellet implantation was more effective than ORX alone and induced a remarkable improvement, even when initiated at advanced-stage DN. In contrast, induction of normoglycemia by islet transplant in ICER-Tg mice eliminated albuminuria but was less effective than E2 + ORX in reducing glomerulosclerosis, collagen 4 deposition, and hyperfiltration. These findings indicate that E2 treatment is effective, even after establishment of DN, whereas glucose normalization alone does not improve sclerotic lesions. We propose that E2 intervention is a potential therapeutic option for DN.

TYK2 Promoter Variant and Diabetes Mellitus in the Japanese.

BACKGROUND: Recently, natural mutation of Tyrosine kinase 2 (Tyk2) gene has been shown to determine susceptibility to murine virus-induced diabetes. In addition, a previous human genome-wide study suggested the type 1 diabetes (T1D) susceptibility region to be 19p13, where the human TYK2 gene is located (19p13.2). METHODS: Polymorphisms of TYK2 gene at the promoter region and exons were studied among 331 healthy controls, and 302 patients with T1D and 314 with type 2 diabetes (T2D) in the Japanese. FINDINGS: A TYK2 promoter haplotype with multiple genetic polymorphisms, which are in complete linkage disequilibrium, named TYK2 promoter variant, presenting decreased promoter activity, is associated with an increased risk of not only T1D (odds ratio (OR), 2.4; 95% confidence interval (CI), 1.2 to 4.6; P = 0.01), but also T2D (OR, 2.1; 95% CI, 1.1 to 4.1; P = 0.03). The risk is high in patients with T1D associated with flu-like syndrome at diabetes onset and also those without anti-glutamic acid decarboxylase autoantibody. INTERPRETATION: The TYK2 promoter variant is associated with an overall risk for diabetes, serving a good candidate as a virus-induced diabetes susceptibility gene in humans. FUNDING: Ministry of Education, Culture, Sports, Science and Technology and of Health, Labor and Welfare of Japan.

Reduced Tyk2 gene expression in ß-cells due to natural mutation determines susceptibility to virus-induced diabetes.

Accumulating evidence suggests that viruses play an important role in the development of diabetes. Although the diabetogenic encephalomyocarditis strain D virus induces diabetes in restricted lines of inbred mice, the susceptibility genes to virus-induced diabetes have not been identified. We report here that novel Tyrosine kinase 2 (Tyk2) gene mutations are present in virus-induced diabetes-sensitive SJL and SWR mice. Mice carrying the mutant Tyk2 gene on the virus-resistant C57BL/6 background are highly sensitive to virus-induced diabetes. Tyk2 gene expression is strongly reduced in Tyk2-mutant mice, associated with low Tyk2 promoter activity, and leads to decreased expression of interferon-inducible genes, resulting in significantly compromised antiviral response. Tyk2-mutant pancreatic ß-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by ß-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic ß-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.

Suppression of Th1-mediated autoimmunity by embryonic stem cell-derived dendritic cells.

We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs) using two models of autoimmune disease, namely non-obese diabetic (NOD) mice and experimental autoimmune encephalomyelitis (EAE). Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases.

Regulation of human autoimmune regulator (AIRE) gene translation by miR-220b.

Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4(+) T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein-Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3'UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3'UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3'UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3'UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation.

Autoimmune gastro-pancreatitis with anti-protein disulfide isomerase-associated 2 autoantibody in Aire-deficient BALB/cAnN mice.

Although the autoimmune regulator (Aire) knockout (KO) mouse model has been reported to present various organ-specific autoimmune diseases depending on genetic background, autoimmune pancreatitis in mice of BALB/c background has not yet been reported. Here, we report that Aire KO mice with BALB/cAnN background showed significant lymphoid cell infiltration in the pancreas and stomach. To examine whether the phenotype in the pancreas and stomach is due to autoimmune reaction associated with autoantibody production, indirect immunofluorescence staining followed by Western blot analysis was performed. Consequently, the autoantibody against pancreas and stomach was detected in the sera of Aire KO mice, and the target antigen of the autoantibody was identified as protein disulfide isomerase-associated 2 (Pdia2), which was reported to be expressed preferentially in the pancreas and stomach. Thus, Aire KO mice of BALB/cAnN background can serve as a useful animal model for autoimmune gastro-pancreatitis with anti-Pdia2 autoantibody production.