Influenza A represents a significant cause of morbidity and mortality worldwide. Bacterial complications of influenza A confer the greatest risk to patients. TH17 pathway inhibition has been implicated as a mechanism by which influenza A alters bacterial host defense. Here we show that preceding influenza causes persistent Staphylococcus aureus infection and suppression of TH17 pathway activation in mice. Influenza does not inhibit S. aureus binding and uptake by phagocytic cells but instead attenuates S. aureus induced TH17 related antimicrobial peptides necessary for bacterial clearance in the lung. Importantly, exogenous lipocalin 2 rescued viral exacerbation of S. aureus infection and decreased free iron levels in the bronchoalveolar lavage from mice coinfected with S. aureus and influenza. These findings indicate a novel mechanism by which influenza A inhibits TH17 immunity and increases susceptibility to secondary bacterial pneumonia. Identification of new mechanisms in the pathogenesis of bacterial pneumonia could lead to future therapeutic targets.

Suppression of type 17 immunity by type I interferon (IFN) during influenza A infection has been shown to enhance susceptibility to secondary bacterial pneumonia. Although this mechanism has been described in coinfection with gram-positive bacteria, it is unclear whether similar mechanisms may impair lung defense against gram-negative infections. Furthermore, precise delineation of the duration of type I IFN-associated susceptibility to bacterial infection remains underexplored. Therefore, we investigated the effects of preceding influenza A virus infection on subsequent challenge with the gram-negative bacteria Escherichia coli or Pseudomonas aeruginosa and the temporal association between IFN expression with susceptibility to Staphylococcus aureus challenge in a mouse model of influenza and bacterial coinfection. Here we demonstrate that preceding influenza A virus led to increased lung E. coli and P. aeruginosa bacterial burden, which was associated with suppression of type 17 immunity and attenuation of antimicrobial peptide expression. Enhanced susceptibility to S. aureus coinfection ceased at day 14 of influenza infection, when influenza-associated type I IFN levels had returned to baseline levels, further suggesting a key role for type I IFN in coinfection pathogenesis. These findings further implicate type I IFN-associated suppression of type 17 immunity and antimicrobial peptide production as a conserved mechanism for enhanced susceptibility to both gram-positive and gram-negative bacterial coinfection during influenza infection.

Pseudomonas aeruginosa strains with loss-of-function mutations in the transcription factor LasR are frequently encountered in the clinic and the environment. Among the characteristics common to LasR-defective (LasR−) strains is increased activity of the transcription factor Anr, relative to their LasR+ counterparts, in low-oxygen conditions. One of the Anr-regulated genes found to be highly induced in LasR− strains was PA14_42860 (PA1673), which we named mhr for microoxic hemerythrin. Purified P. aeruginosa Mhr protein contained the predicted di-iron center and bound molecular oxygen with an apparent Kd of ∼1 µM. Both Anr and Mhr were necessary for fitness in lasR+ and lasR mutant strains in colony biofilms grown in microoxic conditions, and the effects were more striking in the lasR mutant. Among genes in the Anr regulon, mhr was most closely coregulated with the Anr-controlled high-affinity cytochrome c oxidase genes. In the absence of high-affinity cytochrome c oxidases, deletion of mhr no longer caused a fitness disadvantage, suggesting that Mhr works in concert with microoxic respiration. We demonstrate that Anr and Mhr contribute to LasR− strain fitness even in biofilms grown in normoxic conditions. Furthermore, metabolomics data indicate that, in a lasR mutant, expression of Anr-regulated mhr leads to differences in metabolism in cells grown on lysogeny broth or artificial sputum medium. We propose that increased Anr activity leads to higher levels of the oxygen-binding protein Mhr, which confers an advantage to lasR mutants in microoxic conditions.

Seasonal and pandemic influenza is a cause of morbidity and mortality worldwide. Most people infected with influenza virus display mild-to-moderate disease phenotypes and recover within a few weeks. Influenza is known to cause persistent alveolitis in animal models; however, little is known about the molecular pathways involved in this phenotype. We challenged C57BL/6 mice with influenza A/PR/8/34 and examined lung pathologic processes and inflammation, as well as transcriptomic and epigenetic changes at 21 to 60 days after infection. Influenza induced persistent parenchymal lung inflammation, alveolar epithelial metaplasia, and epithelial endoplasmic reticulum stress that were evident after the clearance of virus and resolution of morbidity. Influenza infection induced robust changes in the lung transcriptome, including a significant impact on inflammatory and extracellular matrix protein expression. Despite the robust changes in lung gene expression, preceding influenza (21 days) did not exacerbate secondary Staphylococcus aureus infection. Finally, we examined the impact of influenza on miRNA expression in the lung and found an increase in miR-155. miR-155 knockout mice recovered from influenza infection faster than controls and had decreased lung inflammation and endoplasmic reticulum stress. These data illuminate the dynamic molecular changes in the lung in the weeks after influenza infection and characterize the repair process, identifying a novel role for miR-155. (Am J Pathol 2017, 187: 851e863; http://dx

Influenza A is a highly contagious respiratory virus that causes seasonal epidemics and occasional worldwide pandemics. The primary cause of influenza-related mortality is bacterial super-infection. There are numerous mechanisms by which preceding influenza infection attenuates host defense, allowing for increased susceptibility to bacterial pneumonia. Herein, we demonstrate that influenza inhibits Staphylococcus aureus-induced production of IL-33. Restoration of IL-33 during influenza A and MRSA super-infection enhanced bacterial clearance and improved mortality. ILC2s and alternatively activated macrophages are not required for IL-33 mediated protection during super-infection. We show that IL-33 treatment resulted in neutrophil recruitment to the lung, associated with improved bacterial clearance. These findings identify a novel role for IL-33 in anti-bacterial host defense at the mucosal barrier.