Toll-like receptor 2 has a prominent but nonessential role in innate immunity to Staphylococcus aureus pneumonia.

Staphylococcus aureus is an important cause of acute bacterial pneumonia. Toll-like receptor 2 (TLR2) recognizes multiple components of the bacterial cell wall and activates innate immune responses to gram-positive bacteria. We hypothesized that TLR2 would have an important role in pulmonary host defense against S. aureus TLR null (TLR2-/-) mice and wild type (WT) C57BL/6 controls were challenged with aerosolized S. aureus at a range of inocula for kinetic studies of cytokine and antimicrobial peptide expression, lung inflammation, bacterial killing by alveolar macrophages, and bacterial clearance. Survival was measured after intranasal infection. Pulmonary induction of most pro-inflammatory cytokines was significantly blunted in TLR2-/- mice 4 and 24 h after infection in comparison with WT controls. Bronchoalveolar concentrations of cathelicidin-related antimicrobial peptide also were reduced in TLR2-/- mice. Lung inflammation, measured by enumeration of bronchoalveolar neutrophils and scoring of histological sections, was significantly blunted in TLR2-/- mice. Phagocytosis of S. aureus by alveolar macrophages in vivo after low-dose infection was unimpaired, but viability of ingested bacteria was significantly greater in TLR2-/- mice. Bacterial clearance from the lungs was slightly impaired in TLR2-/- mice after low-dose infection only; bacterial elimination from the lungs was slightly accelerated in the TLR2-/- mice after high-dose infection. Survival after high-dose intranasal challenge was 50-60% in both groups. TLR2 has a significant role in early innate immune responses to S. aureus in the lungs but is not required for bacterial clearance and survival from S. aureus pneumonia.

Keratinocytes store the antimicrobial peptide cathelicidin in lamellar bodies.

Innate immune defense against microbial pathogens occurs by physical barriers, by recruitment of cells such as neutrophils, NK cells, and macrophages, and by secretion of molecules with antimicrobial activity. Such molecules are produced by various epithelia including skin. The importance of antimicrobial peptides has been shown in cathelicidin-deficient mice, which have increased susceptibility to skin infection by Streptococcus. Although keratinocytes increase cathelicidin expression upon injury, their role relative to neutrophil cathelicidin and their sites of peptide storage and activation have not been elucidated. Herein, it is reported that cathelicidin predominantly resides in granules of the superficial epidermis and partially localizes in lamellar bodies as determined by immunogold electron microscopy and immunoblot of lamellar bodies isolated from mice. In cultured keratinocytes, cathelicidin displays a granular distribution and partially localizes within the Golgi apparatus. Cathelicidin processing can be observed by western blot analysis in keratinocyte extracts but not in conditioned media. Further, fluorescent bacteria colocalize with cathelicidin in granules both intracellularly and at the cell surface. These observations illustrate the immune defense potential of keratinocytes acting directly through storage and processing of antimicrobial peptides.

Cutaneous defense mechanisms by antimicrobial peptides.

The skin actively contributes to host defense by mounting an innate immune response that includes the production of antimicrobial peptides. These peptides, which include but are not limited to the cathelicidin and defensin gene families, provide rapid, broad-spectrum defense against infection by acting as natural antibiotics and by participating in host cell processes involved in immune defense. This review discusses the biology and clinical relevance of antimicrobial peptides expressed in the skin. The importance of the epithelial contribution to host immunity is evident, as alterations in antimicrobial peptide expression have been associated with various pathologic processes.

Staphylococcus aureus exploits cathelicidin antimicrobial peptides produced during early pneumonia to promote staphylokinase-dependent fibrinolysis.

The increasing prevalence of Staphylococcus aureus strains isolated from hospital- and community-acquired respiratory tract infections is an important public health concern worldwide. The majority of S. aureus strains produce staphylokinase, a plasminogen activator capable of inactivating neutrophil alpha-defensins and of impairing phagocytosis via opsonin degradation. Cathelicidin antimicrobial peptides are present at sites of infection before the release of neutrophil alpha-defensins. Therefore, we hypothesized that staphylokinase interacts with cathelicidin during the early pathogenesis of S. aureus airway infection. In a mouse intranasal infection model, cathelicidin was strongly up-regulated in the airways during the development of staphylococcal pneumonia. In vitro, cathelicidin bound directly to staphylokinase and augmented staphylokinase-dependent plasminogen activation and fibrinolysis at concentrations consistent with those detected in the airways during infection. These data suggest that staphylokinase production may be a novel virulence mechanism by which S. aureus exploits cathelicidin to promote fibrinolysis, leading to enhanced bacterial dissemination and invasive infection.

Cathelicidin antimicrobial peptides block dendritic cell TLR4 activation and allergic contact sensitization.

Cathelicidins are antimicrobial peptides of the innate immune system that establish an antimicrobial barrier at epithelial interfaces and have been proposed to have a proinflammatory function. We studied the role of cathelicidin in allergic contact dermatitis, a model requiring dendritic cells of the innate immune response and T cells of the adaptive immune response. Deletion of the murine cathelicidin gene Cnlp enhanced an allergic contact response, whereas local administration of cathelicidin before sensitization inhibited the allergic response. Cathelicidins inhibited TLR4 but not TLR2 mediated induction of dendritic cell maturation and cytokine release, and this inhibition was associated with an alteration of cell membrane function and structure. Further analysis in vivo connected these observations because inhibition of sensitization by exogenous cathelicidin was dependent on the presence of functional TLR4. These observations provide evidence that cathelicidin antimicrobial peptides mediate an anti-inflammatory response in part by their activity at the membrane.

Keratinocyte production of cathelicidin provides direct activity against bacterial skin pathogens.

Immune defense at an interface with the external environment reflects the functions of physical and chemical barriers provided by epithelial and immune cells. Resident epithelial cells, such as keratinocytes, produce numerous peptides with direct antimicrobial activity but also provide a physical barrier against invading pathogens and signal the recruitment of circulating immune cells, such as neutrophils. Antimicrobial peptides such as cathelicidin are produced constitutively by neutrophils and are inducible in keratinocytes in response to infection. The multiplicity of antimicrobial peptides and their cellular sources has resulted in an incomplete understanding of the role of cathelicidin production by epithelial cells in cutaneous immune defense. Therefore, this study sought to evaluate keratinocyte antimicrobial activity and the potential contribution of keratinocyte cathelicidin to host protection against two leading human skin pathogens. Wild-type mice and those with a targeted deletion of the cathelicidin gene, Cnlp, were rendered neutropenic prior to cutaneous infection. Interestingly, Cnlp-deficient mice remained more susceptible to group A streptococcus infection than mice with Cnlp intact, suggesting the involvement of epithelial cell-derived cathelicidin in host immune defense. Keratinocytes were then isolated in culture and found to inhibit the growth of Staphylococcus aureus, an effect that was partially dependent on their ability to synthesize and activate cathelicidin. Further, lentivirus-mediated delivery of activated human cathelicidin enhanced keratinocyte antimicrobial activity. Combined, these data illustrate the potential contribution of keratinocyte cathelicidin to the innate immune defense of skin against bacterial pathogens and highlight the need to consider epithelial antimicrobial function in the diagnosis and therapy of skin infection.

Structure-function relationships among human cathelicidin peptides: dissociation of antimicrobial properties from host immunostimulatory activities.

Cathelicidins and other antimicrobial peptides are deployed at epithelial surfaces to defend against infection. These molecules have broad-spectrum killing activity against microbes and can have effects on specific mammalian cell types, potentially stimulating additional immune defense through direct chemotactic activity or induction of cytokine release. In humans, the cathelicidin hCAP18/LL-37 is processed to LL-37 in neutrophils, but on skin it can be further proteolytically processed to shorter forms. The influence of these cathelicidin peptides on keratinocyte function is not known. In the current study, DNA microarray analysis and confirmatory protein analysis showed that LL-37 affects the expression of several chemokines and cytokines by keratinocytes. Analysis of a synthetic peptide library derived from LL-37 showed that antimicrobial activity against bacterial, fungal, and viral skin pathogens resides within specific domains of the parent peptide, but antimicrobial activity does not directly correlate with the ability to stimulate IL-8 production in keratinocytes. IL-8 release was induced by d- and l-amino acid forms of cathelicidin and correlated with membrane permeability, suggesting that highly structure-specific binding to a cell surface receptor is not likely. However, this effect was inhibited by either pertussis toxin or AG1478, an epidermal growth factor receptor tyrosine kinase inhibitor, suggesting that cathelicidin may indirectly stimulate multiple signaling pathways associated with cell surface receptors. Taken together, these observations suggest that proteolytic processing may alter the balance between cathelicidin antimicrobial and host immunostimulatory functions.