The Role of the Inflammasome and Interleukin-1β in Inflammatory Disease
Creators: name: Pinar, Anita Anna
Publisher: name: Monash University
Year: 2016
Subjects: Medicine

description: <b>Background</b>: Influenza A virus (IAV) is the major cause of respiratory tract infections during seasonal epidemics with sporadic pathogenic pandemic outbreaks culminating<br>in high fatality. Understanding the molecular basis for disease severity is vital for predicting potential emerging IAVs. IAV PB1-F2 peptide strains from highly pathogenic<br>IAV outbreaks trigger severe inflammation whereas strains from seasonal IAVs do not. The ‘inflammatory residues’ and the predicted hydrophobic motif within PB1-F2 contribute to its virulence. PB1-F2 from the highly pathogenic mouse adapted laboratory PR8 strain contains the ‘inflammatory residues’ compared to the seasonal Wuhan influenza strain which lacks this motif.<br><b>Hypothesis</b>: Activation of the NLRP3 inflammasome by PB1-F2 during IAV infection is a critical contributor to pathogenic IAV pathophysiology. Targeting the inflammasome may therefore alleviate the excessive inflammation associated with these infections.<br><b>Aims</b>: This study implemented mutating amino acid residues in the Wuhan PB1-F2 strain for residues in PR8 (a previously shown NLRP3 inflammasome activating strain)<br>to identify the molecular signature motifs in PB1-F2 that activate the inflammasome and contribute to the inflammatory phenotype. This study also examined the<br>inflammatory potential of an emerging avian IAV strain and investigated a potential mechanism by which PB1-F2 induces inflammasome activation to contribute to the<br>inflammatory phenotype characteristic of pathogenic IAV outbreaks.<br><b>Methods and Results</b>: Stimulation of primed (LPS: 100ng/ml, 3h) wild-type murine bone marrow derived macrophages (WT BMMs) and human peripheral blood<br>mononuclear cells (hPBMCs) (LPS: 50pg/ml, 3h) with PB1-F2 peptides (10-100μg/ml) for 6h. Supernatants were assayed via ELISA for the levels of IL-1β protein expression<br>for inflammasome activation. LPS (200ng/ml, 3h) primed WT BMMs were stimulated with PB1-F2 peptides (200μg/ml) for 6h. Concentrated supernatants and lysates samples were harvested and analysed via immunoblotting for the detection of proteolytically cleaved caspase-1 and secreted IL-1β protein expression. Additionally, cerulean-tagged ASC inflammasome reporter iBMMs were stimulated with PB1-F2<br>peptides (100μg/ml) and imaged live for 4h for the formation of cerulean ASC specks indicative of inflammasome activation. Pre-treatment of LPS (100ng/ml, 3h) primed<br>WT BMMs and hPBMCs with the NLRP3 inflammasome inhibitor, MCC950, or with the mitochondrial ROS inhibitor, MitoTEMPO, significantly reduced the secretion of IL-1β and formation of ASC specks following challenge with PB1-F2 peptides of pathogenic IAVs.<br><b>Conclusions and significance</b>: This study demonstrated, for the first time, that the ‘inflammatory residues’ within PB1-F2s of pathogenic IAVs are required for activating<br>the inflammasome and contribute to the inflammatory phenotype characteristic of pathogenic IAVs. These findings uniquely identify and signify the PB1-F2 amino acid<br>sequence as a predictive tool and as a novel, reliable and rapid approach for identifying and predicting the pathogenicity of potential emerging influenzas. The further significance of these findings is that targeting the NLRP3 inflammasome with MCC950 and/or with MitoTEMPO presents these as viable anti-inflammatory agents for dampening inflammasome-mediated inflammation.<br>