Antimicrobial peptides CATH-2 and LL-37 produce no protective effect after intratracheal co-administration with LPS in vivo

BackgroundThe host inflammatory response produced in bacterial pneumonia is a major contributing factor of the pathophysiologic processes observed in a respiratory infection. Antimicrobial peptides (AMPs) have been shown to possess potent broad‐spectrum antimicrobial activity as well as immunomodulatory properties. As a potential therapy for bacterial pneumonia, we have previously investigated the anti‐bacterial properties of AMPs in the presence of a pulmonary delivery agent, exogenous surfactant. To further explore the properties of these compounds, the current study evaluates the effects of AMPs with and without exogenous surfactant on pulmonary inflammation in vivo.ObjectiveThe objective of this study was to investigate the potential of two cathelicidin peptides, CATH‐2 and LL‐37 and the exogenous surfactant/cathelicidin mixture BLES+CATH‐2, to reduce the inflammatory response produced upon LPS stimulation in vivo.HypothesisIntrapulmonary administration of AMPs, with and without exogenous surfactant, will reduce lipopolysaccharide (LPS) induced inflammation.MethodsTwo cathelicidin peptides, CATH‐2 and LL‐37, were used in this study. CATH‐2 was also administered in combination with bovine lipid‐extract surfactant (BLES), as previous studies have shown bactericidal activity and enhanced biophysical function of this combination in vitro. C57Bl/6 mice were anesthetized, intubated, and intratracheally instilled 2 mg/kg of LPS. Five minutes later, mice were intratracheally instilled with 50μL one of the following treatments: Sham (no LPS), Air, Saline, BLES (20 mg phospholipid/kg BW), CATH‐2 (100 μM), LL‐37 (100 μM), or BLES+CATH‐2. The mice were monitored for six hours, and were then euthanized. A FlexiVent was used to measure lung compliance, and bronchoalveolar lavage (BAL) fluid was collected in order to determine cell counts, cell differentials, protein content and inflammatory cytokine concentrations.ResultsAnimals that were administered LPS showed a significant increase in neutrophil recruitment and lavage IL‐6 content compared to sham control. Animals treated with CATH‐2 and LL‐37 showed a significant decrease in lung compliance compared to all other treatment groups, as well as an increase in lavage protein and IL‐6 content obtained from the BAL fluid. There was no significant difference among treatment groups with respect to total cells found in the BAL fluid, or percentage of neutrophils recovered between treatment groups.DiscussionThe results from this experiment did not support our initial hypothesis, as CATH‐2 and LL‐37 did not reduce the inflammatory response elicited by LPS challenge, but instead produced an exacerbated inflammatory response as well as decreased lung function, as observed through a decrease in lung compliance. It is possible that at the doses utilized, the cytotoxic effects of cathelicidins contributed to the inflammatory responses observed. The addition of exogenous surfactant BLES mitigated the deleterious effects of CATH‐2 administration; however it did not affect the markers of inflammation compared to controls.Support or Funding InformationCanadian Institutes of Health ResearchProgram of Experimental MedicineCystic Fibrosis Foundation