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

Abstract

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 β-cells are unresponsive even to high dose of Type I interferon. Reversal of virus-induced diabetes could be achieved by β-cell-specific Tyk2 gene expression. Thus, reduced Tyk2 gene expression in pancreatic β-cells due to natural mutation is responsible for susceptibility to virus-induced diabetes.

Figure 1. Type 1 interferon (IFN-α/β) signalling pathway.

Tyk2 and Jak1 are reciprocal IFN receptor-associated molecules, mediating the downstream signal to induce ISGs to resist against viral infection (modified from ref. , Fig. 5.3).

Figure 2. Development of diabetes in EMC-D virus-infected Tyk2 KO mice.

(a) Blood glucose levels in WT (Tyk2+/+) and Tyk2 KO (Tyk2−/−) mice with B6 genetic background, which was used throughout the study except indicated, infected with EMC-D or EMC-B virus. (b) Insulin content of the pancreas from Tyk2+/+ and Tyk2−/− mice infected with EMC-D virus. (c) Virus titres in the pancreas from Tyk2+/+ and Tyk2−/− mice infected with EMC-D virus. (d) Blood glucose levels of lethally irradiated Tyk2+/+ or Tyk2−/− mice reconstituted with Tyk2+/+ or Tyk2−/− spleen cells infected with EMC-D virus. The irradiated Tyk2 WT recipient mice that were transferred spleen cells derived from either WT donor mice or Tyk2 KO mice (Tyk2+/+≪Tyk2+/+, or Tyk2+/+≪Tyk2−/−), or the irradiated KO recipient mice received spleen cells derived from either WT donor mice or Tyk2 KO mice (Tyk2−/−≪Tyk2+/+ or Tyk2−/−≪Tyk2−/−). †died by 10 days after infection, ‡died by 14 days after infection. (e) Blood glucose levels in MIP-Tyk2 Tg Tyk2−/− (Tyk2−/−+MIP-Tyk2) mice infected with EMC-D virus. (f) Histopathology of pancreatic islets from Tyk2−/−, Tyk2+/+ and MIP-Tyk2 Tg Tyk2−/− (Tyk2−/−+MIP-Tyk2) mice infected with EMC-D virus. Scale bar, 50 μm. In a,d,e, animals with blood glucose levels exceeding 14 mmol l⁻¹ were diagnosed as diabetic. All data represent the mean±s.e.m. Statistical significance (*P<0.05, **P<0.01) in a,d,e was determined by Kruskal–Wallis test with Bonferroni's post hoc analysis; in b,c by two-tailed Student's t-test in comparisons with Tyk2+/+, respectively.

Figure 3. Lack of biological importance of produced IFN-α in Tyk2 KO mice.

(a) IFN-α level in the serum. (b) IFN-α level in the pancreas. IFN-α produced in Tyk2 KO (Tyk2−/−) and Tyk2 WT (Tyk2+/+) mice on B6 background, infected with EMC-D virus were measured using ELISA. (c) Blood glucose levels after IFN-α treatment in Tyk2−/− and Tyk2+/+ mice without infection. Animals with blood glucose levels exceeding 14 mmol l⁻¹ were diagnosed as diabetic. (d) Blood glucose levels following intraperitoneal injection of high dose IFN-α (5,000 IU) for five consecutive days, after EMC-D virus infection. All data represent the mean±s.e.m. Statistical significance (*P<0.05 or **P<0.01) in a,b was determined by two-tailed Student's t-test; in c,d by Kruskal–Wallis test with Bonferroni's post hoc analysis.

Figure 4. Significance of the mutated Tyk2 gene in congenic mice.

(a) Blood glucose levels of B6 mice with Tyk2+/+, heterozygous mutated Tyk2 (Tyk2+/mt) and homozygous mutated Tyk2 gene (Tyk2mt/mt) infected with EMC-D virus. (b) Histopathology of pancreatic islets from Tyk2+/+ or Tyk2mt/mt mice with B6 background infected with EMC-D virus. Scale bar, 50 μm. (c) Blood glucose levels of in Tyk2+/+, Tyk2+/mt, Tyk2mt/mt gene carrying congenic mice, with SJL genetic background, infected with EMC-D virus. In a,c, animals with blood glucose levels exceeding 14 mmol l⁻¹ were diagnosed as diabetic. All data represent the mean±s.e.m. Statistical significance (*P<0.05, **P<0.01) in a,c was determined by Kruskal–Wallis test with Dunnett's post hoc analysis.

Figure 5. Expression of Tyk2 protein and gene in Tyk2 gene-mutated mice.

(a) Relative expression levels of the Tyk2 gene were measured by qRT–PCR (see Methods). Pancreatic β-cells, splenocytes and MEF cells were isolated from B6 mice with genotypes as indicated in the figure. (b) Luciferase assay using genes possessing Tyk2+/+ or Tyk2mt/mt promoter region. All data represent the mean±s.e.m. Statistical significance (*P<0.05, **P<0.01) was determined by two-tailed Student's t-test in comparisons with Tyk2+/+, respectively.

Figure 6. Expression of IFN-stimulated genes and antiviral effect in IFN-treated cells.

(a) Relative expression levels of the Tyk2 and Jak1 genes measured using qRT–PCR (See Methods) in IFN-β-stimulated pancreatic β-cells, splenocytes and MEF cells, isolated from various genotypes of mice with B6 background as indicated in the figure. (b) Relative expression levels of ISGs (PKR, 2–5AS, Mx1) measured by qRT–PCR in IFN-β-stimulated β-cells, splenocytes and MEF cells, isolated from B6 mice with genotypes as indicated in the figure. (c) IFN-dependent inhibitory activity against EMC-D virus-induced cell death in MEF or β-cells derived from mice with genotypes as indicated. (d) Blood glucose levels in MIP-Tyk2 Tg Tyk2mt/mt (Tyk2mt/mt+MIP-Tyk2) mice infected with EMC-D virus. In d, animals with blood glucose levels exceeding 14 mmol l⁻¹ were diagnosed as diabetic. In a,b, relative activity of each gene expression before and after IFN-β stimulation was shown. All data represent the mean±s.e.m. Statistical significance (*P<0.05, **P<0.01) in a,b were determined by two-tailed Student's t-test between IFN-β-stimulated each sample and that of Tyk2+/+; in c by Kruskal–Wallis test with Dunnett's post hoc analysis; in d by Kruskal–Wallis test with one-way analysis of variance.