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. 2011 Apr;121(4):1497-507.
doi: 10.1172/JCI44005. Epub 2011 Mar 14.

RNA Sensor-Induced Type I IFN Prevents Diabetes Caused by a β Cell-Tropic Virus in Mice

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Free PMC article

RNA Sensor-Induced Type I IFN Prevents Diabetes Caused by a β Cell-Tropic Virus in Mice

Stephen A McCartney et al. J Clin Invest. .
Free PMC article

Abstract

Viral infections have been linked to the onset of type I diabetes (T1D), with viruses postulated to induce disease directly by causing β cell injury and subsequent release of autoantigens and indirectly via the host type I interferon (IFN-I) response triggered by the virus. Consistent with this, resistance to T1D is associated with polymorphisms that impair the function of melanoma differentiation associated gene-5 (MDA5), a sensor of viral RNA that elicits IFN-I responses. In animal models, triggering of another viral sensor, TLR3, has been implicated in diabetes. Here, we found that MDA5 and TLR3 are both required to prevent diabetes in mice infected with encephalomyocarditis virus strain D (EMCV-D), which has tropism for the insulin-producing β cells of the pancreas. Infection of Tlr3-/- mice caused diabetes due to impaired IFN-I responses and virus-induced β cell damage rather than T cell-mediated autoimmunity. Mice lacking just 1 copy of Mda5 developed transient hyperglycemia when infected with EMCV-D, whereas homozygous Mda5-/- mice developed severe cardiac pathology. TLR3 and MDA5 controlled EMCV-D infection and diabetes by acting in hematopoietic and stromal cells, respectively, inducing IFN-I responses at kinetically distinct time points. We therefore conclude that optimal functioning of viral sensors and prompt IFN-I responses are required to prevent diabetes when caused by a virus that infects and damages the β cells of the pancreas.

Figures

Figure 1
Figure 1. Both MDA5 and TLR3 protect from EMCV-D infection.
WT, Mda5–/–, Tlr3–/–, and DKO mice were infected with 103 PFU of EMCV-D i.p. and monitored for survival (n = 20) in 2 independent experiments. WT mice survived infection, Mda5–/– mice succumbed on day 5, Tlr3–/– succumbed on day 22 with 30% surviving infection, and DKO mice succumbed on day 2.
Figure 2
Figure 2. MDA5 controls EMCV-D infection in the heart.
WT and KO mice were infected with 103 PFU of EMCV-D. Serum and heart (n ≥ 6 per time point, 3 independent experiments) were harvested at days 2, 4, and 7 from surviving mice and were evaluated for (A) troponin levels by ELISA and (B) virus titer by plaque assay or fixed in formalin and paraffin embedded for histology. Tissue sections were stained for EMCVpol antigen by immunohistochemistry (C) (left column: original magnification, ×100 magnification; scale bars: 200 microns; right column: original magnification, ×400; scale bars: 50 micron) or stained by H&E and evaluated for pathology (D) (original magnification, ×100; scale bar: 200 micron). In C, strong and diffuse EMCVpol reactivity is particularly evident in the myocardium of Mda5–/– and DKO; in contrast, heart from WT and Tlr3–/– animals showed only mild (WT) or focal (Tlr3–/–) staining, which is shown in detail in the right panel. In D, areas of myocarditis are observed in WT and Tlr3–/– on day 7 PI as well as in Mda5–/– day 4 PI; only mild inflammation is observed in Tlr3–/– day 4 PI. Statistical significance was calculated by 2-tailed Student’s t test and is indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
Figure 3. TLR3 and MDA5 protect pancreatic islets from EMCV-D infection.
WT and KO mice were infected with 103 PFU EMCV-D. Serum samples were evaluated for (A) blood glucose (n ≥ 8) or (B) amylase and lipase (n ≥ 4) at the indicated times after infection in 2 independent experiments. The pancreas was harvested at the indicated times and viral titers were determined by plaque assay (C) (n = 6 per time point, 3 independent experiments) or fixed in formalin and paraffin embedded for histology. Tissue sections were stained using anti-EMCVpol (D) by immunohistochemistry (n > 3) to visualize virus infiltration (original magnification, ×100; scale bars: 200 micron) or by H&E and evaluated for pathology (E) (original magnification, ×200; scale bars: 100 micron). To further examine the role of MDA5 for protection in the pancreas, Mda5+/– animals on the C57BL/6 background were infected with EMCV-D in parallel with WT and Mda5–/– animals and were evaluated for blood glucose (F) and pancreatic viral titers (G) (n = 5). In addition, Mda5–/– animals on the 129/SvJ background were evaluated for hyperglycemia (H) (n = 12) after infection with EMCV-D. Statistical significance was calculated by 2-tailed Student’s t test and is indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 4
Figure 4. Stromal MDA5 and hematopoietic cell TLR3 protect against EMCV-D infection.
(A and B) BM chimeras were generated between WT and Mda5–/– (n = 10 each) and WT and Tlr3–/– animals (n = 12 each) in 2 independent experiments. Chimeras were infected with 103 PFU of EMCV-D and evaluated for (A) survival and (B) blood glucose. Mda5–/–→WT and WT→Tlr3–/– chimeras survived infection, WT→Mda5–/– chimeras succumbed on day 6, and Tlr3–/–→WT chimeras succumbed on day 14 with 67% surviving infection. (C) MDA5 expression in the pancreas (original magnification, ×200; scale bars: 100 micron). Fixed tissue sections were made from Mda5–/– and WT pancreas on days 0, 2, and 4 after EMCV infection and stained with anti-MDA5 (n = 3). MDA5 is expressed in islets before infection and induced in both islets and exocrine pancreas after infection. (D) TLR3 expression in the pancreas. Frozen tissue sections were made from WT and Tlr3–/– pancreas from uninfected animals (first, second, and fourth panels) or from WT pancreas 12 hours after EMCV infection (third panel). Sections were stained with anti-TLR3 (brown) or costained with anti-TLR3 (brown) and synaptophysin (blue) to visualize expression in the islets (third panel) and are shown at different magnifications as indicated (original magnification, ×200, scale bars: 100 micron; original magnification, ×400, scale bars: 50 micron; original magnification, ×600, scale bars: 33 micron) (n = 3). TLR3 expression is found in the islets as well as in duct epithelial cells, vascular endothelial cells and interstitial stromal cells (arrowheads indicate TLR3+ interstitial cells).
Figure 5
Figure 5. CD11c+ DC control EMCV-D infection and development of diabetes.
(A) Identification of CD11c+ DCs in the islets. Serial frozen sections were made from the pancreas of uninfected WT animals and stained with anti-CD11c (left panel) and anti-TLR3 (right panel) (original magnification, ×400; scale bar 50 micron) (n = 3). Arrowheads indicate CD11c+ cells that are also TLR3+. (BE) CD11c+ DC are required for protection from EMCV-D–induced diabetes. CD11c-DTR mice were treated with PBS (n = 8) or DT (n = 8), then monitored for (B) blood glucose, (C) survival, (D) organ viral titers in the pancreas, heart, and spleen, and (E) serum IFN-I after EMCV-D infection. (F) Frozen sections were prepared from WT and CD11c-DTR PBS and DT-treated mice 48 hours after treatment. Immunohistochemical analysis of CD11c+ cells in the pancreas and spleens of these animals revealed depletion primarily in the spleen after DT treatment (original magnification, ×200; scale bars: 100 micron) (n = 3). Arrowheads indicate CD11c+ cells in the pancreas. (G) Frozen sections of spleens from WT and Tlr3–/– mice were used to perform immunohistochemical evaluation of TLR3 expression in the marginal and periarteriolar zones of the spleen (original magnification, ×200, scale bars: 100 micron) (n = 3). Statistical significance was calculated by 2-tailed Student’s t test and is indicated as follows: *P < 0.05; **P < 0.01.
Figure 6
Figure 6. MDA5 and TLR3 induce type I IFN responses that are distinct in timing and magnitude.
(A) Serum samples from WT, Mda5–/–, Tlr3–/–, and DKO mice were harvested at specified time points after EMCV-D infection and evaluated for type I IFN production by bioassay (n = 6 samples per time point performed in duplicate; results from 3 independent experiments). Irf3–/– (n = 8; 2 independent experiments) and Ifnb–/– (n = 6) mice were infected with EMCV-D and monitored for (B) blood glucose levels and (C) survival. BM chimeras with Irf3–/– or Ifnb–/– BM into WT hosts (n = 6 for each) were infected with EMCV-D and monitored for (D) blood glucose and (E) survival. Statistical significance was calculated by 2-tailed Student’s t test and is indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 7
Figure 7. EMCV-D–induced diabetes in Tlr3–/– mice is characterized by islet infection, apoptosis, and myeloid cell infiltrate.
Pancreas tissue samples from WT or Tlr3–/– mice were harvested at days 0, 2, 4, or 7 PI as indicated, fixed in formalin, and paraffin embedded. (A) Sections were costained with anti-EMCVpol (brown) and anti-synaptophysin (red) (top left panel); active caspase-3 (brown) and anti-synaptophysin (blue) (top right panel); anti–Iba-1 (brown) and anti-synaptophysin (blue) with H&E-stained insert (bottom left panel); or anti-EMCVpol (brown) and anti–Iba-1 (red) (bottom right panel) (left panels: original magnification, ×200, scale bars: 100 micron, right panels: original magnification, ×400, scale bars: 50 micron). By morphology, Iba-1+ cells correspond to large cells with irregular nuclei resembling macrophages (A, bottom left panel insert). As demonstrated by double immunohistochemistry, these islets contain numerous apoptotic cells and Iba1+ cells that react with antibodies against EMCVpol. (B) To visualize myeloid cell infiltrates, sections from WT and Tlr3–/– pancreas were stained with anti–Iba-1 (original magnification, ×200; scale bars: 100 micron). In WT pancreas, Iba-1+ cells are found at the periphery of the islets, whereas In Tlr3–/–, Iba-1+ cells infiltrate the islet.

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