Conserved Nontypeable Haemophilus influenzae -Derived TLR2-Binding Lipopeptides Synergize with IFN-β to Increase Cytokine Production by Resident Murine and Human Alveolar Macrophages

Abstract

Nontypeable Haemophilus influenzae (NTHi) is strongly associated with exacerbations of chronic obstructive pulmonary disease, which often coincide with viral respiratory infections. TLR2 contributes importantly to innate immunity to NTHi, but whether this pathway is affected by simultaneous antiviral responses is unknown. To analyze potential interactions, resident murine and human alveolar macrophages (AMφ) were exposed, in the presence or absence of the appropriate rIFN-β, to synthetic lipopeptides corresponding to the triacylated N-terminal fragments of three outer membrane proteins (OMP) (PCP, P4, and P6) that are highly conserved among different NTHi strains. Synthetic OMP elicited strong release of IL-6, the principal inducer of airway mucin genes, and induced CCL5 and CXCL10 from murine AMφ only when IFN-β was also present. Surprisingly, combined stimulation by OMPs and IFN-β also markedly enhanced TNF-α release by murine AMφ. Stimulation with PCP plus IFN-β induced IFN-regulatory factor 1 expression and sustained STAT1 activation, but did not alter the activation of MAPKs or NF-κB. AMφ derived from STAT1-deficient mice did not demonstrate increased production of TNF-α in response to PCP plus IFN-β. Analysis of wild-type and STAT1-deficient AMφ using real-time PCR showed that increased TNF-α production depended on transcriptional up-regulation, but not on mRNA stabilization. The synergistic effect of synthetic OMP and IFN-β was conserved between murine AMφ and human AMφ for IL-6, but not for TNF-α. Thus, IFN-β, which is produced by virally infected respiratory epithelial cells, converts normally innocuous NTHi OMP into potent inflammatory stimulants, but does so via different mechanisms in mice and humans.