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O020 Influenza a virus protein PB1-F2 activates the NLRP3 inflammasome to induce inflammation
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Abstract / Cytokine 59 (2012) 505–506
mutated TAK1, mutants in either activation loop (T184 and T187) or kinase domain (D156) leaded to inhibition of MAPKs. The activation of IKKs was slightly decreased in D156A mutant, but not T (184; 187) A. Furthermore, mutation in phosphorylation sites of TAK1 enhanced the K63-linked ubiquitination. By in vivo ubiquitination assay, we showed that mutation in either K158 or K209 of TAK1 was unable to abolish polyubiquitination completely, but we confirmed that K158 of TAK1 as the critical site in TLR4 pathway for IKK activation and TAK1 phosphorylation that, thereby, activates of MAPKs. The proteomic analysis for binding partners of TAK1 in different modification status was in line with activity assay. Furthermore, we also identified K562 of TAK1 as a novel ubiquitination site. Conclusion. We identified that ubiquitination in K209 of TAK1 regulates IKKs activation and the phosphorylation of TAK1 itself. The activation of TAK1 is able to transmit signal to activate MAPKs, and, in turn, block the K63-linked ubiquitination of TAK1 in feedback regulation. Our data suggested that TAK1-mediated activation of downstream effectors in TLR4 is carefully regulated by the crosstalk of phosphorylation and ubiquitination. Disclosure of interest: None declared.
Reference [1] Tseng PH, Matsuzawa A, Zhang W, Mino T, Vignali DA, Karin M. Nat Immunol 2010;11:70–5. http://dx.doi.org/10.1016/j.cyto.2012.06.050
O020 Influenza a virus protein PB1-F2 activates the NLRP3 inflammasome to induce inflammation A. Mansell 1, J. McAuley 1, M. Tate 2, E. Latz 3, 1 University of Melbourne, Australia, 2 Monash Institute of Medical Research, Melbourne, Australia, 3 University of Bonn, Bonn, Germany
symptoms and death. PB1-F2 is a non-structural IAV protein that has gained attention in recent years mainly attributable via its ability to increase pathogenicity by disrupting macrophages. Critical to human health, most avian influenza and human H3N2 viruses carry the information for full-length PB1-F2 protein, whereas human nonpandemic H1N1 IAV strains harbour a truncated version. Importantly, PB1-F2 peptides form protein aggregates and the formation of PB1-F2 amyloid-like fibres are observed in infected cells. There is increasing awareness to the important role of PB1-F2 in enhanced pathogenesis, although the underlying mechanism for its role in inflammation and macrophage dysfunction is not understood. Methods. We have elucidated the mechanism of PB1-F2 induced inflammation via the NRLP3-inflammasome using a series ofPB1-F2-deficient viral strains and PB1-F2 peptides combined with inflammasome-deficient macrophages. Results. Mice treated with IAV lacking expression of PB1-F2 display decreased inflammation, neutrophil, macrophage and DC infiltration to the lung. Conversely, mice inoculated with PB1-F2 peptide display increased inflammatory cell infiltration and inflammation. Following on from this, we have found that PB1-F2 peptide induces maturation of IL-1b in human PBMCs, which is reduced by inhibition of phagocytosis and Caspase-1. PB1-F2 also induced IL-1b secretion in murine macrophages that is phagocytosis-dependent. Importantly, cells deficient in caspase-1, ASC and NLRP3 display ablated IL-1b maturation upon challenge with PB1-F2 indicating PB1-F2 induces inflammation via a NLRP3 inflammasome. These findings suggest a critical role for PB1-F2 in inducing the hyperinflammatory state characterised in pandemic IAV infections and its possible role in macrophage dysfunction via inflammasomemediated pyroptosis. Conclusion. Our findings identify a previously unknown role for PB1-F2-induced inflammation, via recognition by the NLRP3-inflammasome, leading to maturation of IL-1b. Activation of the inflammasome may also explain the observation of PB1F2-mediated macrophage dysregulation, possibly via inflammasome-mediated pyroptosis. Elucidating mechanisms of the molecular basis for disease severity of emerging influenza viruses, especially those implicated in global outbreaks is essential to develop better treatment regimes to improve the clinical outcomes for patients with severe acute infections. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.051
Introduction. Characteristic of pandemic influenza A virus (IAV) strains is the hyperinflammatory response associated with infections which can lead to severe clinical
Abstract / Cytokine 59 (2012) 505–506
mutated TAK1, mutants in either activation loop (T184 and T187) or kinase domain (D156) leaded to inhibition of MAPKs. The activation of IKKs was slightly decreased in D156A mutant, but not T (184; 187) A. Furthermore, mutation in phosphorylation sites of TAK1 enhanced the K63-linked ubiquitination. By in vivo ubiquitination assay, we showed that mutation in either K158 or K209 of TAK1 was unable to abolish polyubiquitination completely, but we confirmed that K158 of TAK1 as the critical site in TLR4 pathway for IKK activation and TAK1 phosphorylation that, thereby, activates of MAPKs. The proteomic analysis for binding partners of TAK1 in different modification status was in line with activity assay. Furthermore, we also identified K562 of TAK1 as a novel ubiquitination site. Conclusion. We identified that ubiquitination in K209 of TAK1 regulates IKKs activation and the phosphorylation of TAK1 itself. The activation of TAK1 is able to transmit signal to activate MAPKs, and, in turn, block the K63-linked ubiquitination of TAK1 in feedback regulation. Our data suggested that TAK1-mediated activation of downstream effectors in TLR4 is carefully regulated by the crosstalk of phosphorylation and ubiquitination. Disclosure of interest: None declared.
Reference [1] Tseng PH, Matsuzawa A, Zhang W, Mino T, Vignali DA, Karin M. Nat Immunol 2010;11:70–5. http://dx.doi.org/10.1016/j.cyto.2012.06.050
O020 Influenza a virus protein PB1-F2 activates the NLRP3 inflammasome to induce inflammation A. Mansell 1, J. McAuley 1, M. Tate 2, E. Latz 3, 1 University of Melbourne, Australia, 2 Monash Institute of Medical Research, Melbourne, Australia, 3 University of Bonn, Bonn, Germany
symptoms and death. PB1-F2 is a non-structural IAV protein that has gained attention in recent years mainly attributable via its ability to increase pathogenicity by disrupting macrophages. Critical to human health, most avian influenza and human H3N2 viruses carry the information for full-length PB1-F2 protein, whereas human nonpandemic H1N1 IAV strains harbour a truncated version. Importantly, PB1-F2 peptides form protein aggregates and the formation of PB1-F2 amyloid-like fibres are observed in infected cells. There is increasing awareness to the important role of PB1-F2 in enhanced pathogenesis, although the underlying mechanism for its role in inflammation and macrophage dysfunction is not understood. Methods. We have elucidated the mechanism of PB1-F2 induced inflammation via the NRLP3-inflammasome using a series ofPB1-F2-deficient viral strains and PB1-F2 peptides combined with inflammasome-deficient macrophages. Results. Mice treated with IAV lacking expression of PB1-F2 display decreased inflammation, neutrophil, macrophage and DC infiltration to the lung. Conversely, mice inoculated with PB1-F2 peptide display increased inflammatory cell infiltration and inflammation. Following on from this, we have found that PB1-F2 peptide induces maturation of IL-1b in human PBMCs, which is reduced by inhibition of phagocytosis and Caspase-1. PB1-F2 also induced IL-1b secretion in murine macrophages that is phagocytosis-dependent. Importantly, cells deficient in caspase-1, ASC and NLRP3 display ablated IL-1b maturation upon challenge with PB1-F2 indicating PB1-F2 induces inflammation via a NLRP3 inflammasome. These findings suggest a critical role for PB1-F2 in inducing the hyperinflammatory state characterised in pandemic IAV infections and its possible role in macrophage dysfunction via inflammasomemediated pyroptosis. Conclusion. Our findings identify a previously unknown role for PB1-F2-induced inflammation, via recognition by the NLRP3-inflammasome, leading to maturation of IL-1b. Activation of the inflammasome may also explain the observation of PB1F2-mediated macrophage dysregulation, possibly via inflammasome-mediated pyroptosis. Elucidating mechanisms of the molecular basis for disease severity of emerging influenza viruses, especially those implicated in global outbreaks is essential to develop better treatment regimes to improve the clinical outcomes for patients with severe acute infections. Disclosure of interest: None declared. http://dx.doi.org/10.1016/j.cyto.2012.06.051
Introduction. Characteristic of pandemic influenza A virus (IAV) strains is the hyperinflammatory response associated with infections which can lead to severe clinical