The Four-and-a-Half LIM Domain Protein 2 Supports Influenza A Virus-Induced Lung Inflammation by Restricting the Host Adaptive Immune Response.

Four-and-a-half LIM domain protein 2 (FHL2) is a multifunctional adaptor protein with fine-tuning adjustment properties. It acts as a regulator of signaling cascades but also as a cofactor of transcription and controls several anti-inflammatory immune responses. Recently, we described FHL2 as a novel regulator of influenza A virus propagation. We have shown that in vitro FHL2 restricts viral replication by accelerating the interferon regulatory factor 3-dependent transcription of the Ifnb1 gene. In this work, we unraveled an ambiguous role of FHL2 during influenza A virus infection in vivo. Although FHL2 restrained viral replication during the first 24 hours of infection, it significantly delayed viral clearance afterward. Comparison of lung immune status of wild-type and FHL2 knockout mice during influenza virus infection did not acknowledge significant differences in the innate host immune response but revealed an improved migration of dendritic cells from infected lungs into draining lymph nodes as well as increased levels of activated CD8+ T lymphocytes accumulated in the lungs of FHL2 knockout mice.

Phosphorylation of influenza A virus NS1 protein at threonine 49 suppresses its interferon antagonistic activity.

Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed.

Inhibition of p38 mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal H5N1 infection.

Highly pathogenic avian influenza viruses (HPAIV) induce severe inflammation in poultry and men. One characteristic of HPAIV infections is the induction of a cytokine burst that strongly contributes to viral pathogenicity. This cell-intrinsic hypercytokinemia seems to involve hyperinduction of p38 mitogen-activated protein kinase. Here we investigate the role of p38 MAPK signaling in the antiviral response against HPAIV in mice as well as in human endothelial cells, the latter being a primary source of cytokines during systemic infections. Global gene expression profiling of HPAIV-infected endothelial cells in the presence of the p38-specific inhibitor SB 202190 revealed that inhibition of p38 MAPK leads to reduced expression of IFNß and other cytokines after H5N1 and H7N7 infection. More than 90% of all virus-induced genes were either partially or fully dependent on p38 signaling. Moreover, promoter analysis confirmed a direct impact of p38 on the IFNß promoter activity. Furthermore, upon treatment with IFN or conditioned media from HPAIV-infected cells, p38 controls interferon-stimulated gene expression by coregulating STAT1 by phosphorylation at serine 727. In vivo inhibition of p38 MAPK greatly diminishes virus-induced cytokine expression concomitant with reduced viral titers, thereby protecting mice from lethal infection. These observations show that p38 MAPK acts on two levels of the antiviral IFN response. Initially the kinase regulates IFN induction and, at a later stage, p38 controls IFN signaling and thereby expression of IFN-stimulated genes. Thus, inhibition of MAP kinase p38 may be an antiviral strategy that protects mice from lethal influenza by suppressing excessive cytokine expression.

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.

ß-catenin promotes the type I IFN synthesis and the IFN-dependent signaling response but is suppressed by influenza A virus-induced RIG-I/NF-κB signaling.

BACKGROUND: The replication cycle of most pathogens, including influenza viruses, is perfectly adapted to the metabolism and signal transduction pathways of host cells. After infection, influenza viruses activate several cellular signaling cascades that support their propagation but suppress those that interfere with viral replication. Accumulation of viral RNA plays thereby a central role. Its sensing by the pattern recognition receptors of the host cells leads to the activation of several signal transduction waves that result in induction of genes, responsible for the cellular innate immune response. Type I interferon (IFN) genes and interferon-stimulated genes (ISG) coding for antiviral-acting proteins, such as MxA, OAS-1 or PKR, are primary targets of these signaling cascades. ß- and γ-catenin are closely related armadillo repeat-containing proteins with dual roles. At the cell membrane they serve as adapter molecules linking cell-cell contacts to microfilaments. In the cytosol and nucleus, the proteins form a transcriptional complex with the lymphoid enhancer factor/T-cell factor (LEF/TCF), regulating the transcription of many genes, thereby controlling different cellular functions such as cell cycle progression and differentiation. RESULTS: In this study, we demonstrate that ß- and γ-catenin are important regulators of the innate cellular immune response to influenza A virus (IAV) infections. They inhibit viral replication in lung epithelial cells by enhancing the virus-dependent induction of the IFNB1 gene and interferon-stimulated genes. Simultaneously, the prolonged infection counteracts the antiviral effect of ß- and γ-catenin. Influenza viruses suppress ß-catenin-dependent transcription by misusing the RIG-I/NF-κB signaling cascade that is induced in the course of infection by viral RNA. CONCLUSION: We identified ß- and γ-catenin as novel antiviral-acting proteins. While these factors support the induction of common target genes of the cellular innate immune response, their functional activity is suppressed by pathogen evasion.

A single point mutation (Y89F) within the non-structural protein 1 of influenza A viruses limits epithelial cell tropism and virulence in mice.

The nonstructural protein 1 (A/NS1) of influenza A viruses (IAV) harbors several src homology (SH)-binding motifs (bm) that mediate interactions with cellular proteins. In contrast to the sequence variability of the second SH3bm, tyrosine 89, within the SH2bm is a highly conserved residue among IAV strains. This prompted us to evaluate the necessity of this SH2bm for IAV virulence. In an in vivo mouse model, we observed drastic reductions in weight loss, mortality, and virus titers in lung and bronchoalveolar lavage fluid after infection with the mutant virus PR8 A/NS1-Y89F (PR8 Y89F) when compared with wild-type virus (PR8 wt). Concomitantly, we observed decreased inflammation and less severe pathologic changes, reflecting reduced levels of virus titers. At histologic analysis, lungs infected with PR8 wt virus showed widespread destruction of the bronchiolar epithelium, with extensive distribution of virus antigen within tracheal, bronchial, bronchiolar, and alveolar epithelium. In marked contrast, the bronchiolar epithelium after infection with the mutant PR8 Y89F virus was entirely intact, and the severity and extent of viral infection was reduced and strongly restricted to alveoli. These findings demonstrate that change of a single residue of the highly conserved SH2bm within the A/NS1 results in restricted virus spread in mouse lung and strongly reduced virulence, which illustrates the necessity of the SH2bm for IAV-induced pathogenicity.

The adaptor protein FHL2 enhances the cellular innate immune response to influenza A virus infection.

The innate immune response of influenza A virus-infected cells is predominantly mediated by type I interferon-induced proteins. Expression of the interferon ß (IFNß) itself is initiated by accumulating viral RNA and is transmitted by different signalling cascades that feed into activation of the three transcriptional elements located in the IFNß promoter, AP-1, IRF-3 and NF-κB. FHL2 (four-and-a-half LIM domain protein 2) is an adaptor molecule that shuttles between membrane and nucleus regulating signalling cascades and gene transcription. Here we describe FHL2 as a novel regulator of influenza A virus propagation. Using mouse FHL2 wild-type, knockout and rescued cells and human epithelial cells with different expression levels of FHL2 we showed that FHL2 decreases influenza A virus propagation by regulating the intrinsic cellular antiviral immune response. On virus infection FHL2 translocates into the nucleus, potentiating the IRF-3-dependent transcription of the IFNß gene.

The influenza virus PB1-F2 protein has interferon antagonistic activity.

PB1-F2 is a nonstructural protein of influenza viruses encoded by the PB1 gene segment from a +1 open reading frame. It has been shown that PB1-F2 contributes to viral pathogenicity, although the underlying mechanisms are still unclear. Induction of type I interferon (IFN) and the innate immune response are the first line of defense against viral infection. Here we show that influenza A viruses (IAVs) lacking the PB1-F2 protein induce an enhanced expression of IFN-ß and IFN-stimulated genes in infected epithelial cells. Studying molecular mechanisms underlying the PB1-F2-mediated IFN antagonistic activity showed that PB1-F2 interferes with the RIG-I/MAVS protein complex thereby inhibiting the activation of the downstream transcription factor IFN regulatory factor 3. These findings were also reflected in in vivo studies demonstrating that infection with PR8 wild-type (wt) virus resulted in higher lung titers and a more severe onset of disease compared with infection with its PB1-F2-deficient counterpart. Accordingly, a much more pronounced infiltration of lungs with immune cells was detected in mice infected with the PB1-F2 wt virus. In summary, we demonstrate that the PB1-F2 protein of IAVs exhibits a type I IFN-antagonistic function by interfering with the RIG-I/MAVS complex, which contributes to an enhanced pathogenicity in vivo.

Interleukin-27 displays interferon-gamma-like functions in human hepatoma cells and hepatocytes.

UNLABELLED: Interleukin-27 (IL-27) is a cytokine belonging to the IL-6/IL-12 cytokine family. It is secreted by antigen-presenting cells, strongly acts on T cells, and also stimulates innate immune cells. In most studies, the effects of IL-27 on T cells were investigated; however, not much is known about possible effects of IL-27 on other cell types. IL-27 signals via the common IL-6-type cytokine receptor chain gp130 and the IL-27-specific chain WSX-1. Given the importance of gp130 in regulating liver responses such as the acute phase response or liver regeneration, we investigated whether IL-27 could also have a function in liver cells. We find that IL-27 stimulates hepatoma cells and hepatocytes by inducing a sustained signal transducer and activator of transcription (STAT)1 and STAT3 activation. Whereas the STAT3 mediated responses to IL-27 (gamma-fibrinogen and hepcidin induction) are not detectable, we observe an interferon-gamma (IFN-gamma)-like STAT1 response leading to the induction of interferon-regulated proteins such as STAT1, STAT2, interferon response factor (IRF)-1, IRF-9, myxovirus resistance A and guanylate binding protein 2. CONCLUSION: Our study provides evidence for a function of IL-27 in hepatoma cells and hepatocytes and shows that IL-27 responses are not restricted to the classical immune cells. Our results suggest that IL-27 exerts IFN-like functions in liver cells and that it can contribute to the antiviral response in these cells.

Phosphorylation of TRIM28 Enhances the Expression of IFN-ß and Proinflammatory Cytokines During HPAIV Infection of Human Lung Epithelial Cells.

Human infection with highly pathogenic avian influenza viruses (HPAIV) is often associated with severe tissue damage due to hyperinduction of interferons and proinflammatory cytokines. The reasons for this excessive cytokine expression are still incompletely understood, which has hampered the development of efficient immunomodulatory treatment options. The host protein TRIM28 associates to the promoter regions of over 13,000 genes and is recognized as a genomic corepressor and negative immune regulator. TRIM28 corepressor activity is regulated by post-translational modifications, specifically phosphorylation of S473, which modulates binding of TRIM28 to the heterochromatin-binding protein HP1. Here, we identified TRIM28 as a key immune regulator leading to increased IFN-ß and proinflammatory cytokine levels during infection with HPAIV. Using influenza A virus strains of the subtype H1N1 as well as HPAIV of subtypes H7N7, H7N9, and H5N1, we could demonstrate that strain-specific phosphorylation of TRIM28 S473 is induced by a signaling cascade constituted of PKR, p38 MAPK, and MSK1 in response to RIG-I independent sensing of viral RNA. Furthermore, using chemical inhibitors as well as knockout cell lines, our results suggest that phosphorylation of S473 facilitates a functional switch leading to increased levels of IFN-ß, IL-6, and IL-8. In summary, we have identified TRIM28 as a critical factor controlling excessive expression of type I IFNs as well as proinflammatory cytokines during infection with H5N1, H7N7, and H7N9 HPAIV. In addition, our data indicate a novel mechanism of PKR-mediated IFN-ß expression, which could lay the ground for novel treatment options aiming at rebalancing dysregulated immune responses during severe HPAIV infection.

The LIM-only protein FHL2 attenuates lung inflammation during bleomycin-induced fibrosis.

Fibrogenesis is usually initiated when regenerative processes have failed and/or chronic inflammation occurs. It is characterised by the activation of tissue fibroblasts and dysregulated synthesis of extracellular matrix proteins. FHL2 (four-and-a-half LIM domain protein 2) is a scaffolding protein that interacts with numerous cellular proteins, regulating signalling cascades and gene transcription. It is involved in tissue remodelling and tumour progression. Recent data suggest that FHL2 might support fibrogenesis by maintaining the transcriptional expression of alpha smooth muscle actin and the excessive synthesis and assembly of matrix proteins in activated fibroblasts. Here, we present evidence that FHL2 does not promote bleomycin-induced lung fibrosis, but rather suppresses this process by attenuating lung inflammation. Loss of FHL2 results in increased expression of the pro-inflammatory matrix protein tenascin C and downregulation of the macrophage activating C-type lectin receptor DC-SIGN. Consequently, FHL2 knockout mice developed a severe and long-lasting lung pathology following bleomycin administration due to enhanced expression of tenascin C and impaired activation of inflammation-resolving macrophages.