Critical to understanding these peptides and utilising their properties therapeutically is the need to clarify their modes of action in vivo in infectious contexts. In this study, an acute murine pulmonary infection model with P. aeruginosa was utilised in order to evaluate the capacity of cathelicidins to enhance host defence against infection with a microbe which is largely resistant to these peptides under physiological conditions in vitro. Under favourable in vitro conditions in which microbicidal properties are evident for LL-37, this peptide has been shown to permeabilise bacterial membranes within minutes. However, we found no evidence for direct microbicidal activity against P. aeruginosa after coincubation with LL-37 in vivo, yet exogenously delivered LL-37 was found to significantly enhance pathogen clearance over 24 hours. Although we cannot exclude some alternative form of late direct microbicidal activity of LL-37, even by 6 hours after infection no significant impact on bacterial load of the whole lung could be demonstrated in response to LL-37 treatment, although interestingly a therapeutic bolus of peptide was found to diminish the number of live bacteria accessible to bronchoalveolar lavage at this time point. The reason for this is unclear, but may relate to early removal of the most accessible bacteria by the enhanced neutrophil influx observed. A previous study using adenoviral vectors carrying the cDNA for hCAP18/LL-37, to overexpress the human cathelicidin in the murine lung over the 5 days prior to infection, resulted in the promotion of a small, but significant enhancement of P. aeruginosa clearance from the murine lung over a 24 hour period. This was observed to be accompanied by decreased pulmonary TNF levels, but the mechanism underpinning this therapeutic effect was not evaluated and was assumed to be microbicidal. In contrast, we found no evidence to support a microbicidal effect, but demonstrate a peptide-mediated enhanced early neutrophil influx in vivo. Prior research has demonstrated the capacity for cathelicidins to have direct chemotactic activity for human neutrophils and monocytes and murine leukocytes in vitro and for murine leukocytes in an experimentally-formed murine air pouch model. In that in vivo model, injection of 2 mM LL-37 or mCRAMP into the air pouch significantly enhanced the influx of neutrophils and monocytes within a 4 hour period. This is in contrast to the complete absence of neutrophils observed in our studies in the murine lung 6 hours after instillation of LL-37 alone. In addition, LL-37 was not found to mediate any significant effects on the number of monocytes in the BALF, in contrast to the previously published findings in other systems. A small, but significant neutrophil response was observed in the lungs of LL-37treated uninfected mice at 24 hours after instillation, demonstrating some LL-37-mediated neutrophil influx.