Toll like receptors are required for generation of protective lung mucosal

Toll like receptors are required for generation of protective lung mucosal immune responses against microbial pathogens. lung mucosal innate immunity a response that might be exploited therapeutically to prevent the development of Gram-negative bacterial infection of the respiratory tract. (PA) is an aerobic gram-negative bacterium that is the second most common cause of pneumonia in hospitalized patients with mortality rates as high as 60-90% in mechanically ventilated with Nrp2 pneumonia due to pneumonia 1(1 2 Lethality in PA pneumonia is usually caused by the propensity of these patients to develop bacteremia septic shock multiple organ failure and lung injury as compared to patients with pneumonia due to other bacterial pathogens (1-4). Toll-like receptors (TLRs) are a family of type I transmembrane receptors that respond to pathogen-associated molecular patterns (PAMPs) expressed by a diverse group of infectious microorganisms resulting in activation of the host’s immune system (5-7). Most PA strains express flagella which primarily consists of the protein flagellin (8). Flagellin is usually recognized by and activates several pathogen recognition receptors including Iressa TLR5 TLR2 and Ipaf a component of the NOD/inflammasome pathway (9-15). In the lung flagellin can induce neutrophil accumulation an effect that is dependent on TLR5 expression by lung structural cells rather than bone marrow-derived cells (16). In addition to mediating neutrophil influx flagellin can activate a broad array of protective innate responses. For instance the i.p. administration of Iressa purified flagellin guarded mice from lethal intestinal contamination rotavirus induced colitis and Iressa bacterial corneal contamination (17-19). Recently the repeated intranasal administration of flagellin has been shown to rescue TLR2/4 double deficient mice challenged with non-flagellated PA (20). Mechanism of protection in these models has not been defined but is usually felt to be partially due Iressa to stimulation of chemokines that facilitated the recruitment of inflammatory cells (17). Flagellin has also been shown to be protective in several non-infectious models including chemical-induced colitis and radiation pneumonitis (17 21 An important component of innate immunity of the respiratory tract is the release of molecules with antimicrobial activity at the mucosal surface. The two best characterized families of cationic antimicrobial peptides are defensins and cathelicidins (22 23 Cathelicidins are proteins that contain a highly conserved pre-pro region at the N terminus referred to as the cathelin domain name and substantial heterogeneity at the C terminal domain name (23-25). These peptides are stored intracellularly as inactive propeptide precursors that are proteolytically cleaved to active peptides upon stimulation (26). The single known human cathelicidin hCAP-18 is usually cleaved by proteinase 3 to form the active peptide LL-37. The murine homologue CRAMP is usually encoded by the gene (27). Cathelicidins are constitutively expressed in high levels by neutrophils (28). They are also inducibly expressed in response to contamination and injury by epithelial cells at mucosal surfaces (29-31). Cathelicidin peptides exert bactericidal activity against a broad range of both Gram-negative and Gram-positive organisms including PA. As compared to wildtype (WT) controls by bronchial epithelial cells isolated from patients with cystic fibrosis and the in-vivo pulmonary transgenic expression of LL-37 in mice challenged with PA simultaneously reduced lung bacterial burden and reduced inflammation (31 35 In addition to direct bactericidal properties cathelicidins exert unique immunomodulatory effects including binding to anionic molecules such as LPS resulting in reduced endotoxin immunotoxicity (36-38). In this study we evaluated the effect of flagellin on protective lung mucosal immune responses in a lethal murine PA pneumonia model. The intranasal delivery of purified PA flagellin induced strong protective immunity against PA which required in part the antimicrobial peptide CRAMP. Flagellin also prevented lung injury which was associated with reduced expression of caspase-3 in lung during pneumonia. Methods Reagents Anti-CRAMP antibodies used in Western immunoblotting were purchased from Genzyme. Purified recombinant murine CRAMP was obtained from Mary O’Riordan at the University of Michigan. For neutrophil depletion we treated mice with RB6-8C5 mAb. RB6-8C5 us a rat anti-mouse monoclonal.