MAPKAP kinase 3 suppresses Ifng gene expression and attenuates NK cell cytotoxicity and Th1 CD4 T-cell development upon influenza A virus infection.

MK2 and MK3 are downstream targets of p38 and ERK1/2. They control the mRNA stability of several inflammatory cytokines, including TNF-α and IL-10. Whereas MK2 is expressed ubiquitously, the expression of MK3 is restricted to muscle, liver, and heart tissues and T and NK cells. Using Mk-deficient and wild-type (WT) mice, we demonstrated an inhibitory effect of MK3, but not of MK2, on interferon (IFN)-γ expression in T and NK lymphocytes. The results provided evidence that the inhibitory effect of MK3 is based on negative feedback phosphorylation of p38 and ERK1/2, which causes decreased binding of Stat4 to the IFN-γ promoter and reduced expression of IFN-γ mRNA and protein. Consequently, all Mk3(-/-) mice challenged with the Th1-inducing influenza A virus (IAV) survived the WT LD50 virus dose. The reduced disease severity in the Mk3(-/-) mice was accompanied by a >10-fold reduction in viral lung titer and an increase in the number of activated NK cells and enhanced Th1 activation of CD4 T cells. Thus, our data describe the protein kinase MK3 as a novel regulator of the innate and adaptive immune responses.-Köther, K., Nordhoff, C., Masemann, D., Varga, G., Bream, J. H., Gaestel, M., Wixler, V., Ludwig, S. MAPKAP kinase 3 suppresses Ifng gene expression and attenuates NK cell cytotoxicity and Th1 CD4 T-cell development upon influenza A virus infection.

MAP kinase-activated protein kinases 2 and 3 are required for influenza A virus propagation and act via inhibition of PKR.

Influenza viruses have to overcome the type I interferon induced antiviral response to successfully propagate in target cells. A major antiviral factor induced by interferons is the protein kinase R (PKR) that is further activated by dsRNA and phosphorylates the eukaryotic initiation factor 2 (eIF2α). This results in inhibition of protein translation thereby limiting viral replication. Here we describe a novel mechanism by which influenza A viruses escape the antiviral action of PKR. We demonstrate that the mitogen-activated protein kinase-activated protein kinases (MAPKAPKs) MK2 and MK3 are activated on virus infection and, in their active form, directly interact with the repressor of the inhibitor of PKR p88(rIPK). This leads to recruitment of a tetrameric protein complex consisting of p88(rIPK), the inhibitor of PKR p58(IPK) and PKR itself, and finally results in inhibition of the kinase. The importance of MKs for influenza virus propagation was further underscored by demonstrating reduced viral progeny in cells genetically deficient in MK2 or MK3 genes as well as in highly proliferating tumor cells, in which expression of MKs was diminished by specific small interfering RNA. Accordingly, knockdown of MKs resulted in enhanced phosphorylation of PKR and its substrate eIF2α.

Oncolytic influenza virus infection restores immunocompetence of lung tumor-associated alveolar macrophages.

Non-small-cell lung cancer (NSCLC) is the most frequent type of lung cancer and demonstrates high resistance to radiation and chemotherapy. These tumors evade immune system detection by promoting an immunosuppressive tumor microenvironment. Genetic analysis has revealed oncogenic activation of the Ras/Raf/MEK/ERK signaling pathway to be a hallmark of NSCLCs, which promotes influenza A virus (IAV) infection and replication in these cells. Thus, we aimed to unravel the oncolytic properties of IAV infection against NSCLCs in an immunocompetent model in vivo. Using Raf-BxB transgenic mice that spontaneously develop NSCLCs, we demonstrated that infection with low-pathogenic IAV leads to rapid and efficient oncolysis, eliminating 70% of the initial tumor mass. Interestingly, IAV infection of Raf-BxB mice caused a functional reversion of immunosuppressed tumor-associated lung macrophages into a M1-like pro-inflammatory active phenotype that additionally supported virus-induced oncolysis of cancer cells. Altogether, our data demonstrate for the first time in an immunocompetent in vivo model that oncolytic IAV infection is capable of restoring and redirecting immune cell functions within the tumor microenvironment of NSCLCs.