Interferon-lambda contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses

Abstract

Virus-infected cells secrete a broad range of interferon (IFN) subtypes which in turn trigger the synthesis of antiviral factors that confer host resistance. IFN-alpha, IFN-beta and other type I IFNs signal through a common universally expressed cell surface receptor, whereas IFN-lambda uses a distinct receptor complex for signaling that is not present on all cell types. Since type I IFN receptor-deficient mice (IFNAR1(0/0)) exhibit greatly increased susceptibility to various viral diseases, it remained unclear to which degree IFN-lambda might contribute to innate immunity. To address this issue we performed influenza A virus infections of mice which carry functional alleles of the influenza virus resistance gene Mx1 and which, therefore, develop a more complete innate immune response to influenza viruses than standard laboratory mice. We demonstrate that intranasal administration of IFN-lambda readily induced the antiviral factor Mx1 in mouse lungs and efficiently protected IFNAR1(0/0) mice from lethal influenza virus infection. By contrast, intraperitoneal application of IFN-lambda failed to induce Mx1 in the liver of IFNAR1(0/0) mice and did not protect against hepatotropic virus infections. Mice lacking functional IFN-lambda receptors were only slightly more susceptible to influenza virus than wild-type mice. However, mice lacking functional receptors for both IFN-alpha/beta and IFN-lambda were hypersensitive and even failed to restrict usually non-pathogenic influenza virus mutants lacking the IFN-antagonistic factor NS1. Interestingly, the double-knockout mice were not more susceptible against hepatotropic viruses than IFNAR1(0/0) mice. From these results we conclude that IFN-lambda contributes to inborn resistance against viral pathogens infecting the lung but not the liver.

Figure 1. Induction of IFN-λ2 genes in virus-infected lung and liver of IFNAR1^0/0 mice.

Animals were either infected by the intranasal route with 10⁶ pfu of influenza A virus strain SC35M-ΔNS1 or PR8-ΔNS1, or else by the intraperitoneal route with 10 pfu of hepatotropic THOV-ΔML. Animals treated with plain buffer served as negative controls. At 17 hours post infection, the influenza virus-infected mice were killed and the lungs were removed for analysis. The liver of the THOV-infected mouse was harvested when the animal was severely diseased at 72 hours post infection. RNA samples from the organs were reverse transcribed and analyzed by PCR for transcripts of the indicated genes.

Figure 2. Exogenous IFN-λ protects IFNAR1^0/0 mice against intranasal challenge with influenza A virus but not against intraperitoneal challenge with THOV.

(A) Survival of mice intranasally treated for 10 hours with a mock preparation or 7,500 units of either IFN-λ2 or IFN-λ3 before challenge with 100 pfu (∼20 LD₅₀) of influenza A virus strain SC35M. (B) Survival of mice intraperitoneally treated for 10 hours with a mock preparation or 15,000 units of IFN-λ3 before infection with 100 pfu (∼20 LD₅₀) of THOV.

Figure 3. IFN-λ activates Mx1 gene expression in lung but not liver of IFNAR1^0/0 mice.

(A) Western blot analysis of Mx1 protein levels in lungs of mice at 20 hours post intranasal application of 3,500 units of IFN-λ3. Animals treated with a mock preparation served as control. Two animals of each group are shown. (B) Mx1 protein levels in the liver of IFNAR1^0/0 mice at 20 hours post intraperitoneal application of 15,000 units of IFN-λ3 or terminally ill at 72 hours post infection with hepatotropic THOV-ΔML or RVFV clone 13. Two animals for each group are shown. Liver extract from a wild-type mouse killed at 20 hours post intraperitoneal treatment with 100,000 units of human IFN-αB/D served as positive control.

Figure 4. Mice lacking functional receptors for IFN-λ show slightly reduced resistance to influenza A virus.

Wild-type and IL28Rα^0/0 mice were infected by the intranasal route with 5×10⁴ pfu of SC35M. (A) Survival and (B) virus titers in lungs at 72 hours post infection were recorded. Combined data of several independent experiments are shown. (**: p<0.01).

Figure 5. Mice lacking functional receptors for both IFN-α/β and IFN-λ exhibit enhanced susceptibility toward highly attenuated influenza A viruses but not toward two different attenuated hepatotropic viruses.

Survival of IFNAR1^0/0 (diamonds) and IFNAR1^0/0IL28Rα^0/0 double knockout mice (circles) after (A) intranasal infection with the indicated doses of SC35M-ΔNS1, (B) intranasal infection with 10⁶ pfu of PR8-ΔNS1, (C) intraperitoneal infection with the indicated doses of THOV-ΔML, and (D) intraperitoneal infection with the indicated doses of RVFV clone 13. Groups consisted of four to nine animals.

Figure 6. Inverse correlation of Mx1 protein levels and viral load in lungs of mice lacking functional receptors for IFN-α/β, IFN-λ or both.

Groups of mice were infected with 10⁵ pfu of SC35M-ΔNS1 and either killed at (A) 48 hours post infection to determine viral titers in the lung or at (B) 20 hours post infection to determine Mx1 protein levels by western blotting. Two animals of each group are shown. Actin-normalized Mx1 signal intensities are indicated. The calculated value of the wild-type mice was set to 100%. (*: p<0.05), ***: p<0.001).