MDA5 and MAVS mediate type I interferon responses to coxsackie B virus

Abstract

Coxsackie B viruses (CVB) are enteroviruses that have been associated with a variety of human diseases, including myocarditis. In the present study, we found that MDA5 and its adaptor molecule MAVS are critical for type I interferon responses to CVB, since the absence of either MAVS or MDA5 leads to deficient type I interferon production and early mortality in mice infected with CVB. Pancreatic and hepatic necrosis were observed on histopathological examination of MAVS and MDA5 knockout mice infected with CVB. Inflammatory cytokine production in response to systemic CVB infection was independent of MAVS. Surprisingly, virus titers were not elevated in MAVS-deficient mice, despite significant reductions in type I interferon levels. These data highlight the importance of type I interferon in host defense and provide insight on the mechanisms of innate immune responses following coxsackievirus infection.

FIG. 1.

IFN-α/βR-deficient mice show increased susceptibility to lethal infection with CVB3. (A) IFN-α/βR^−/− mice (n = 6) and age- and sex-matched wild-type C57BL/6 (n = 6) were injected with 10⁴ PFU of CVB3 and monitored for survival. The median survival was 2 days for IFN-α/βR^−/− mice (solid line) versus 4 days for wild-type C57BL/6 mice (dashed line, P = 0.0009). (B) Histopathology of livers and pancreata from a wild-type C57BL/6 mouse and an IFN-α/βR^−/− mouse 48 h after infection with CVB3. Both pancreata demonstrate severe necrosis of the acinar cells, but only the IFN-α/βR^−/− mouse liver shows severe necrosis without associated inflammatory cellular infiltration. Magnification, ×400.

FIG. 2.

Absence of MAVS is associated with high mortality in CVB3-infected mice. (A) MAVS^−/− mice (n = 10) and littermate controls, including MAVS^+/+ mice (n = 6) and MAVS^+/− mice (n = 21), were injected i.p. with 10⁴ PFU of CVB3 and then monitored for survival. Median survival times were 3 days for MAVS^−/− mice versus 7 days for MAVS^+/+ mice (P < 0.001) and 6.5 days for MAVS^+/− mice (P = 0.021). The graph shows data combined from two independent experiments. (B) Histopathology of a liver from a MAVS^−/− mouse 2 days postinfection reveals severe necrosis without inflammatory cells. Magnification, ×400.

FIG. 3.

MDA5-deficient mice have increased susceptibility to lethal infection with CVB3. (A) MDA5^−/− mice (n = 6) and age- and sex-matched wild-type controls on a pure 129 SvJ background (n = 7) were injected i.p. with 10⁴ PFU of CVB3 and then monitored for survival. A total of 50% of MDA5^−/− mice died, whereas all of the wild-type mice survived more than 14 days (P = 0.03). (B) Histopathology of a liver from a MDA5^−/− mouse at 4 days postinfection reveals severe hepatic necrosis. Necrotic hepatocytes stain bright red. Brown granules overlying necrotic cells represent acid hematin. Magnification, ×400.

FIG. 4.

Pancreatic IFN-α levels are significantly reduced in MAVS^−/− and MDA5^−/−mice infected with CVB3. Age- and sex-matched wild-type (C57BL/6 or B6.129 or 129 SvJ), IFN-α/βR^−/−, MAVS^−/−, or MDA5^−/− mice were inoculated with 10⁴ PFU of CVB3 i.p. Organs were harvested 48 h after infection, and the cytokine levels of serum and pancreata (normalized to organ weight) were determined by ELISA. Statistical significance was calculated by using the Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The data are shown from four to five animals per group. Dashed line indicates limit of detection of the assay.

FIG. 5.

Virus titers were significantly elevated in IFN-α/βR^−/− but not MAVS^−/− or MDA5^−/− mouse sera and livers after infection with CVB3. Wild-type (C57BL/6, B6.129, or 129 SvJ), IFN-α/βR^−/−, MAVS^−/−, or MDA5^−/− mice were inoculated with 10⁴ PFU of CVB3 i.p. Organs were harvested 48 h after infection, and the virus titers of serum (A), pancreas (B), and liver (C) were determined by plaque assay on HeLa cells. Statistical significance was calculated by using the Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The data are shown from four to five animals per group. (D) In a separate experiment, wild-type B6.129 or MAVS^−/− mice were similarly inoculated with CVB3 and tail bled daily. Serum virus titers were determined by plaque assay on HeLa cells. The data are shown from four animals per group. The dashed line indicates limit of detection of the assay.

FIG. 6.

Viral RNA recognition pathways for CVB and their impact on type I IFN induction, viral replication, and mortality in mice in vivo. The dsRNA replicative intermediate of CVB interacts with the helicase-binding domain of MDA5 in the cell cytoplasm. Activation of the adaptor MAVS triggers downstream IRFs 3 and 7. Production of IFN-α and IFN-β leads to the engagement of the IFN-α/βR. Absence of the IFN-α/βR leads to decreased survival and increased virus titers after CVB challenge. In contrast, absence of MAVS or MDA5 leads to decreased serum levels of type I IFN and decreased survival but does not lead to increased virus titers in serum after challenge with CVB.