Background: Influenza virus is the major
cause of respiratory tract infections resulting in severe immunopathologies.
Understanding the molecular basis for disease severity is vital for predicting potential
emerging influenzas. Influenza PB1-F2 peptide strains from pandemic influenzas
trigger severe hyper-inflammation and activate the NLRP3 inflammasome whereas
strains from mild seasonal influenzas do not. The ‘4 inflammatory residues’ at
the bioactive C-terminus of PB1-F2 correlate with virulence. Further, a sequence
of hydrophobic residues may also influence secondary structure and enhance the inflammatory
phenotype. The 'hyper-inflammatory
PR8’ strain contains ‘the 4 inflammatory residues’
whereas the
'non-inflammatory Wuhan' strain does not. This study has
implemented sequence swapping to identify the residues necessary to convert the
Wuhan strain into the PR8 strain to characterise their ability to activate the NLRP3
inflammasome and predict potential emerging influenzas.
Aim: To examine whether differences between hyper-inflammatory and non-inflammatory PB1-F2 strains influence the degree of inflammasome activation and induce influenza pathophysiology.
Methods: Immortalised wild-type bone marrow macrophages (iBMMs) were primed with LPS (100 ng/ml) for 3 h and stimulated with PB1-F2 strains (10 µg/ml) for 6 h. Supernatants were assayed via ELISA for levels of IL-1β protein expression for inflammasome activation. Cerulean-tagged ASC iBMMs were also stimulated with PB1-F2 (100 µg/ml) strains and imaged live for 4 h for the formation of cerulean ASC specks indicative of inflammasome activation.
Results: Stimulation of iBMMs with hyper-inflammatory PB1-F2 strains (10 µg/ml) increased inflammasome activation compared to non-inflammatory PB1-F2 strains. Stimulation of cerulean-tagged ASC iBMMs with hyper-inflammatory PB1-F2 strains (100 µg/ml) increased ASC speck formation compared to non-inflammatory strains.
Conclusion: Hyper-inflammatory PB1-F2 strains potently activate the inflammasome in iBMMs compared to non-inflammatory strains. Sequence swapping of residues within PB1-F2 identified the combination necessary for inflammasome activation and will be a novel and vital tool for predicting potential emerging influenzas.