Dane Parker

Innate Immunity in the Respiratory Epithelium

The airway epithelium represents the first point of contact for inhaled foreign organisms. The protective arsenal of the airway epithelium is provided in the form of physical barriers and a vast array of receptors and antimicrobial compounds that constitute the innate immune system. Many of the known innate immune receptors, including the Toll-like receptors and nucleotide oligomerization domain-like receptors, are expressed by the airway epithelium, which leads to the production of proinflammatory cytokines and chemokines that affect microorganisms directly and recruit immune cells, such as neutrophils and T cells, to the site of infection. The airway epithelium also produces a number of resident antimicrobial proteins, such as lysozyme, lactoferrin, and mucins, as well as a swathe of cationic proteins. Dysregulation of the airway epithelial innate immune system is associated with a number of medical conditions that can result in compromised immunity and chronic inflammation of the lung. This review focuses on the innate immune capabilities of the airway epithelium and its role in protecting the lung from infection as well as the outcomes when its function is compromised.

The NanA Neuraminidase of Streptococcus pneumoniae Is Involved in Biofilm Formation

ABSTRACT Streptococcus pneumoniae remains a major cause of bacteremia, pneumonia, and otitis media despite vaccines and effective antibiotics. The neuraminidase of S. pneumoniae, which catalyzes the release of terminal sialic acid residues from glycoconjugates, is involved in host colonization in animal models of infection and may provide a novel target for preventing pneumococcal infection. We demonstrate that the S. pneumoniae neuraminidase (NanA) cleaves sialic acid and show that it is involved in biofilm formation, suggesting an additional role in pathogenesis, and that it shares this property with the neuraminidase of Pseudomonas aeruginosa even though we show that the two enzymes are phylogenetically divergent. Using an in vitro model of biofilm formation incorporating human airway epithelial cells, we demonstrate that small-molecule inhibitors of NanA block biofilm formation and may provide a novel target for preventative therapy. This work highlights the role played by the neuraminidase in pathogenesis and represents an important step in drug development for prevention of colonization of the respiratory tract by this important pathogen.

Streptococcus pneumoniae DNA Initiates Type I Interferon Signaling in the Respiratory Tract

ABSTRACT The mucosal epithelium is the initial target for respiratory pathogens of all types. While type I interferon (IFN) signaling is traditionally associated with antiviral immunity, we demonstrate that the extracellular bacterial pathogen Streptococcus pneumoniae activates the type I IFN cascade in airway epithelial and dendritic cells. This response is dependent upon the pore-forming toxin pneumolysin. Pneumococcal DNA activates IFN-β expression through a DAI/STING/TBK1/IRF3 cascade. Tlr4−/−, Myd88−/−, Trif−/−, and Nod2−/− mutant mice had no impairment of type I IFN signaling. Induction of type I IFN signaling contributes to the eradication of pneumococcal carriage, as IFN-α/β receptor null mice had significantly increased nasal colonization with S. pneumoniae compared with that of wild-type mice. These studies suggest that the type I IFN cascade is a central component of the mucosal response to airway bacterial pathogens and is responsive to bacterial pathogen-associated molecular patterns that are capable of accessing intracellular receptors. IMPORTANCE The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen. The bacterium Streptococcus pneumoniae is a leading cause of bacterial pneumonia, leading to upwards of one million deaths a year worldwide and significant economic burden. Although it is known that antibody is critical for efficient phagocytosis, it is not known how this pathogen is sensed by the mucosal epithelium. We demonstrate that this extracellular pathogen activates mucosal signaling typically activated by viral pathogens via the pneumolysin pore to activate intracellular receptors and the type I interferon (IFN) cascade. Mice lacking the receptor to type I IFNs have a reduced ability to clear S. pneumoniae, suggesting that the type I IFN cascade is central to the mucosal clearance of this important pathogen.

Toxin-Induced Necroptosis Is a Major Mechanism of Staphylococcus aureus Lung Damage

Staphylococcus aureus USA300 strains cause a highly inflammatory necrotizing pneumonia. The virulence of this strain has been attributed to its expression of multiple toxins that have diverse targets including ADAM10, NLRP3 and CD11b. We demonstrate that induction of necroptosis through RIP1/RIP3/MLKL signaling is a major consequence of S. aureus toxin production. Cytotoxicity could be prevented by inhibiting either RIP1 or MLKL signaling and S. aureus mutants lacking agr, hla or Hla pore formation, lukAB or psms were deficient in inducing cell death in human and murine immune cells. Toxin-associated pore formation was essential, as cell death was blocked by exogenous K+ or dextran. MLKL inhibition also blocked caspase-1 and IL-1β production, suggesting a link to the inflammasome. Rip3 -/- mice exhibited significantly improved staphylococcal clearance and retained an alveolar macrophage population with CD200R and CD206 markers in the setting of acute infection, suggesting increased susceptibility of these leukocytes to necroptosis. The importance of this anti-inflammatory signaling was indicated by the correlation between improved outcome and significantly decreased expression of KC, IL-6, TNF, IL-1α and IL-1β in infected mice. These findings indicate that toxin-induced necroptosis is a major cause of lung pathology in S. aureus pneumonia and suggest the possibility of targeting components of this signaling pathway as a therapeutic strategy.

Staphylococcus aureus induces type I IFN signaling in dendritic cells via TLR9.

The importance of type I IFN signaling in the innate immune response to viral and intracellular pathogens is well established, with an increasing literature implicating extracellular bacterial pathogens, including Staphylococcus aureus, in this signaling pathway. Airway epithelial cells and especially dendritic cells (DCs) contribute to the production of type I IFNs in the lung. We were interested in establishing how S. aureus activates the type I IFN cascade in DCs. In vitro studies confirmed the rapid uptake of S. aureus by DCs followed promptly by STAT1 phosphorylation and expression of IFN-β. Signaling occurred using heat-killed organisms and in the absence of PVL and α-toxin. Consistent with the participation of endosomal and not cytosolic receptors, signaling was predominantly mediated by MyD88, TLR9, and IRF1 and blocked by cytochalasin D, dynasore, and chloroquine. To determine the role of TLR9 signaling in the pathogenesis of S. aureus pneumonia, we infected WT and Tlr9−/− mice with MRSA USA300. Tlr9−/− mice had significantly improved clearance of S. aureus from the airways and lung tissue. Ifnar−/− mice also had improved clearance. This enhanced clearance in Tlr9−/− mice was not due to differences in the numbers of recruited neutrophils into the airways, but instead correlated with decreased induction of TNF. Thus, we identified TLR9 as the critical receptor mediating the induction of type I IFN signaling in DCs in response to S. aureus, illustrating an additional mechanism through which S. aureus exploits innate immune signaling to facilitate infection.

The Type III Toxins of Pseudomonas aeruginosa Disrupt Epithelial Barrier Function

The type III secreted toxins of Pseudomonas aeruginosa are important virulence factors associated with clinically important infection. However, their effects on bacterial invasion across mucosal surfaces have not been well characterized. One of the most commonly expressed toxins, ExoS, has two domains that are predicted to affect cytoskeletal integrity, including a GTPase-activating protein (GAP) domain, which targets Rho, a major regulator of actin polymerization; and an ADP-ribosylating domain that affects the ERM proteins, which link the plasma membrane to the actin cytoskeleton. The activities of these toxins, and ExoS specifically, on the permeability properties of polarized airway epithelial cells with intact tight junctions were examined. Strains expressing type III toxins altered the distribution of the tight junction proteins ZO-1 and occludin and were able to transmigrate across polarized airway epithelial monolayers, in contrast to DeltaSTY mutants. These effects on epithelial permeability were associated with the ADP-ribosylating domain of ExoS, as bacteria expressing plasmids lacking expression of the ExoS GAP activity nonetheless increased the permeation of fluorescent dextrans, as well as bacteria, across polarized airway epithelial cells. Treatment of epithelial cells with cytochalasin D depolymerized actin filaments and increased permeation across the monolayers but did not eliminate the differential effects of wild-type and toxin-negative mutants on the epithelial cells, suggesting that additional epithelial targets are involved. Confocal imaging studies demonstrated that ZO-1, occludin, and ezrin undergo substantial redistribution in human airway cells intoxicated by ExoS, -T, and -Y. These studies support the hypothesis that type III toxins enhance P. aeruginosas invasive capabilities by interacting with multiple eukaryotic cytoskeletal components.

Immunopathogenesis of Staphylococcus aureus pulmonary infection

Staphylococcus aureus is a common human pathogen highly evolved as both a component of the commensal flora and as a major cause of invasive infection. Severe respiratory infection due to staphylococci has been increasing due to the prevalence of more virulent USA300 CA-MRSA strains in the general population. The ability of S. aureus to adapt to the milieu of the respiratory tract has facilitated its emergence as a respiratory pathogen. Its metabolic versatility, the ability to scavenge iron, coordinate gene expression, and the horizontal acquisition of useful genetic elements have all contributed to its success as a component of the respiratory flora, in hospitalized patients, as a complication of influenza and in normal hosts. The expression of surface adhesins facilitates its persistence in the airways. In addition, the highly sophisticated interactions of the multiple S. aureus virulence factors, particularly the α-hemolysin and protein A, with diverse immune effectors in the lung such as ADAM10, TNFR1, EGFR, immunoglobulin, and complement all contribute to the pathogenesis of staphylococcal pneumonia.

Induction of Type I Interferon Signaling Determines the Relative Pathogenicity of Staphylococcus aureus Strains

The tremendous success of S. aureus as a human pathogen has been explained primarily by its array of virulence factors that enable the organism to evade host immunity. Perhaps equally important, but less well understood, is the importance of the intensity of the host response in determining the extent of pathology induced by S. aureus infection, particularly in the pathogenesis of pneumonia. We compared the pathogenesis of infection caused by two phylogenetically and epidemiologically distinct strains of S. aureus whose behavior in humans has been well characterized. Induction of the type I IFN cascade by strain 502A, due to a NOD2-IRF5 pathway, was the major factor in causing severe pneumonia and death in a murine model of pneumonia and was associated with autolysis and release of peptidogylcan. In contrast to USA300, 502A was readily eliminated from epithelial surfaces in vitro. Nonetheless, 502A caused significantly increased tissue damage due to the organisms that were able to invade systemically and trigger type I IFN responses, and this was ameliorated in Ifnar -/- mice. The success of USA300 to cause invasive infection appears to depend upon its resistance to eradication from epithelial surfaces, but not production of specific toxins. Our studies illustrate the important and highly variable role of type I IFN signaling within a species and suggest that targeted immunomodulation of specific innate immune signaling cascades may be useful to prevent the excessive morbidity associated with S. aureus pneumonia.

Staphylococcus aureus Activation of Caspase 1/Calpain Signaling Mediates Invasion Through Human Keratinocytes

The USA300 strains of Staphylococcus aureus are the major cause of skin and soft tissue infection in the United States. Invasive USA300 infection has been attributed to several virulence factors, including protein A and the α-hemolysin (Hla), which cause pathology by activating host signaling cascades. Here we show that S. aureus exploits the proinflammatory bias of human keratinocytes to activate pyroptosis, a caspase 1-dependent form of inflammatory cell death, which was required for staphylococci to penetrate across a keratinocyte barrier. Keratinocyte necrosis was mediated by calpains, Ca(2+)-dependent intracellular proteases whose endogenous inhibitor, calpastatin, is targeted by Hla-induced caspase 1. Neither Panton-Valentine leukocidin nor protein A expression was essential, but inhibition of either calpain or caspase 1 activity was sufficient to prevent staphylococcal invasion across the keratinocytes. These studies suggest that pharmacological interruption of specific keratinocyte signaling cascades as well as targeting the Hla might prevent invasive skin infection by staphylococci.

Participation of CD11c+ Leukocytes in Methicillin-Resistant Staphylococcus aureus Clearance from the Lung

ABSTRACT Staphylococcus aureus causes especially severe pulmonary infection, associated with high morbidity and mortality. In addition to the effects of specific virulence factors, it appears that the intensity of the host proinflammatory response, particularly in the initial stages of infection, contributes substantially to pulmonary damage. We tested the hypothesis that the CD11c+ leukocytes are important in the host response to pulmonary infection with methicillin-resistant S. aureus (MRSA) USA300. Clodronate-induced depletion of the alveolar macrophage population resulted in increased numbers of dendritic cells (DCs) and CD4+ cells in bronchoalveolar lavage (BAL) fluid and was associated with significantly increased mortality by 18 h following S. aureus inoculation but had no effect on bacterial load or polymorphonuclear leukocyte (PMN) numbers in the lung. These clodronate-treated mice also had increased expression of interleukin-17A/F (IL-17A/F) and CXCL10 but not of gamma interferon (IFN-γ) or tumor necrosis factor (TNF). Depletion of the dendritic cell population in mice expressing a CD11c-enhanced green fluorescent protein (EGFP)-diphtheria toxin receptor (DTR) transgene was associated with an increased bacterial load in the lung but not increased mortality. Both DCs and airway epithelial cells produced CXCL9, -10, and -11 in response to S. aureus. Pretreatment of mice with an anti-CXCR3 antibody prior to inoculation with MRSA substantially reduced CD4+ cells and decreased pulmonary inflammation at 18 h postinfection compared to pretreatment with an IgG control. The results of these experiments suggest that CD11c+ cells, the induction of CXCR3 ligand expression, and subsequent CD4+ cell recruitment have an important role in the pathogenesis of severe MRSA pulmonary infection.