The Role of the Inflammasome and Interleukin-1β in Inflammatory Disease

Abstract

Background: Influenza A virus (IAV) is the major cause of respiratory tract infections during seasonal epidemics with sporadic pathogenic pandemic outbreaks culminatingin high fatality. Understanding the molecular basis for disease severity is vital for predicting potential emerging IAVs. IAV PB1-F2 peptide strains from highly pathogenicIAV 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.Hypothesis: 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.Aims: This study implemented mutating amino acid residues in the Wuhan PB1-F2 strain for residues in PR8 (a previously shown NLRP3 inflammasome activating strain)to identify the molecular signature motifs in PB1-F2 that activate the inflammasome and contribute to the inflammatory phenotype. This study also examined theinflammatory potential of an emerging avian IAV strain and investigated a potential mechanism by which PB1-F2 induces inflammasome activation to contribute to theinflammatory phenotype characteristic of pathogenic IAV outbreaks.Methods and Results: Stimulation of primed (LPS: 100ng/ml, 3h) wild-type murine bone marrow derived macrophages (WT BMMs) and human peripheral bloodmononuclear 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 expressionfor 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-F2peptides (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) primedWT 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.Conclusions and significance: This study demonstrated, for the first time, that the ‘inflammatory residues’ within PB1-F2s of pathogenic IAVs are required for activatingthe inflammasome and contribute to the inflammatory phenotype characteristic of pathogenic IAVs. These findings uniquely identify and signify the PB1-F2 amino acidsequence 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.