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SUMMARY Clinical severity of Staphylococcus aureus respiratory infection correlates with alpha toxin (AT) expression. AT activates the NLRP3 inflammasome; deletion of Nlrp3, or AT neutralization, protects mice from lethal S. aureus pneumonia. We tested the hypothesis that this protection is not due to a reduction in inflammasome-dependent cytokines (IL-1β/IL-18) but increased bactericidal function of macrophages. In vivo, neutralization of AT or NLRP3 improved bacterial clearance and survival, while blocking IL-1β/IL-18 did not. Primary human monocytes were used in vitro to determine the mechanism through which NLRP3 alters bacterial killing. In cells treated with small interfering RNA (siRNA) targeting NLRP3 or infected with AT-null S. aureus, mitochondria co-localize with bacterial-containing phagosomes. Mitochondrial engagement activates caspase-1, a process dependent on complex II of the electron transport chain, near the phagosome, promoting its acidification. These data demonstrate a mechanism utilized by S. aureus to sequester itself from antimicrobial processes within the cell., Graphical Abstract, In Brief In the lung, alpha toxin (AT) is a primary virulence factor used by S. aureus to evade innate immune responses. Cohen et al. demonstrate that AT activation of the NLRP3 inflammasome uncouples key components of the phagocytic killing machinery, namely, mitochondria dissociate from internalized bacteria. Without close association of mitochondria with internalized bacteria, macrophages are unable to effectively kill S. aureus.

The innate immune system possesses a multitude of pathways to sense and respond to microbial pathogens. One such family are the interferons (IFNs), a family of cytokines that are involved in several cellular functions. Type I IFNs are appreciated to be important in several viral and bacterial diseases, while the recently identified type III IFNs (IFNL1, IFNL2, IFNL3, IFNL4) have been studied primarily in the context of viral infection. Viral and bacterial infections however are not mutually exclusive, and often the presence of a viral pathogen increases the pathogenesis of bacterial infection. The role of type III IFN in bacterial and viral-bacterial co-infections has just begun to be explored. In this mini review we discuss type III IFN signaling and its role in microbial pathogenesis with an emphasis on the work that has been conducted with bacterial pathogens.

The balance between pro and anti-inflammatory signaling in innate immune responses to bacterial infection is especially critical in the lung. PDCD4, known primarily for its anti-tumor functions, influences inflammatory cytokine production and is negatively regulated by miR-21 (transcription) and p70S6K (phosphorylation), which are both targets of interferon (IFN) signaling. We postulated that PDCD4 has a central role in activating cytokine signaling, and functions as a target for IFN λ . To confirm the relationship between PDCD4 and inflammatory cytokines in the context of P. aeruginosa PAK infection, 16HBEs were treated with siRNA against PDCD4, stimulated with PAK (MOI 10, 4 h) and IL-8 was measured by RT-PCR. IL-8 induction was decreased in siRNA treated cells. Conversely, inhibiting miR-21 increased IL-8 induction in response to PAK. In response to PAK (MOI 10, 4 h), IFN λ mRNA was increased >10-fold in dendritic cells. IFN λ treatment of 16HBEs resulted in a STAT3 dependent decrease in miR-21 and increase in PDCD4 expression. IFN λ also reduced phosphorylation of p70S6K. The in vivo relevance of these pathways was determined using wt and IL-28R-/- (IFN λ receptor) mice. Similar levels of PDCD4 and miR-21 were found in the lungs of uninfected mice, however constitutive phosphorylation of p70S6K and PDCD4 was observed in knockout mice. IL-28R-/- mice had significantly improved clearance of PAK from the lung ( p p p = 0.0018) and PDCD4 mRNA significantly lower ( p = 0.0323) in IL-28R-/- compared to wt. Phosphorylation of p70S6K and PDCD4 in response to PAK was observed in WT and KO mice. These results indicate that PDCD4 is a central regulator of inflammatory cytokine production during P. aeruginosa pneumonia.

Introduction The balance between pro and anti-inflammatory signaling in innate immune responses to bacterial infection is especially critical in the lung. Airway epithelial cells, in addition to resident and recruited cells of immune origin, participate in what must be coordinated proinfammatory signaling in response to inhaled pathogens. The type III interferons are especially important in pulmonary infection, produced in response to viral infection and bacterial PAMPs. IFN-λ is activated by and induces NF-κB signaling and promotes expression of Th1 cytokines. We postulated that common respiratory pathogens, Staphylococcus aureus and Pseudomonas aeruginosa , would stimulate an IFN-λ response and that this would have an important effect on bacterial clearance from the airway. Methods To establish that bacterial components can stimulate IFN-λ, we measured the induction of IFN-λ by RT-PCR in murine BMDCs. Biological significance of IFN-λ induction was determined by comparing the ability of wt C57Bl/6 and IL-28R−/− mice (lacking the IFN-λ receptor) to handle an intranasal inoculation of 10 7 cfu of either PAK or USA300. Results In response to either P. aeruginosa PAK or USA300 MRSA on BMDCs there was a 10-fold increase in IFN-λ transcript by 4 h post infection which persisted for up to 8 h; in contrast to the 1000-fold induction of IFN-β by both organisms. The IL-28R−/− mice had significantly improved clearance of either pathogen from the airway and lung tissue ( P - BAL ( P Conclusion These results indicate that like IFN-β, bacterial PAMPs also activate IFN-λ signaling which may contribute to airway inflammation without augmenting pathogen clearance. Further dissection of the components of IFN-λ regulation may provide targets to diminish the pathology associated with airway inflammation without compromising the ability to clear pathogens.