Levels of cholesteryl esters, novel lipid effector molecules of the innate immune response, in the airways of pediatric cystic fibrosis patients

Host-derived lipids including cholesteryl esters (CE), in particular cholesteryl linoleate (CL), have emerged as novel effector molecules of innate immunity in the airways with antibacterial activity against <italic>Pseudomonas aeruginosa (PA)</italic> and other bacteria. Cystic fibrosis (CF) is a genetic disorder that leads to chronic lung infection with <italic>PA</italic> and other bacteria and premature death. Studies have shown that the lipid composition of several body fluids is altered in CF. We hypothesized that in CF antimicrobial cholesteryl esters are decreased leading to colonization and subsequent chronic lung infection with <italic>P. aeruginosa</italic> and compared cholesteryl ester levels in bronchoalveolar lavage fluid (BALF) from CF patients and non-CF disease controls. BALF with and without protease inhibitor treatment were obtained from six pediatric subjects with CF (4 females, 2 males; ages 7.6 &ndash; 16.6 years) and seven pediatric subjects with non-CF chronic lung disease (4 females, 3 males; ages 2.2 &ndash; 11.7 years). First, we established a total lipid quantification assay for lipids in BALF using the fluorescent probe Nile red, then we compared untreated and treated BALF samples with respect to total lipid concentration, total protein, enzymatic lysozyme activity, and inherent antibacterial activity. We determined that protease inhibitor treatment had significantly altered the biological properties of BALF, increasing Nile red fluorescence, abolishing enzymatic lysozyme activity, and drastically killing <italic>PA</italic> (<italic>p</italic> = 0.000, <italic> p</italic> =0.016, <italic> p</italic> =0.000 in paired t-test, respectively). Subsequently, untreated BALF was lipid extracted and cholesteryl esters were identified and quantified by means of mass spectral analysis. We found that cholesteryl linoleate and cholesteryl arachidonate as well as all cholesteryl esters combined were statistically significantly elevated in CF BALF compared to non-CF disease control BALF (320.731 &plusmn; 105.089 pmol/mL and 62.548 &plusmn; 25.221 pmol/mL, respectively, <italic>p</italic> =0.04 in ANOVA). Since the statistical significance was lost after adjustment of the CE levels to albumin as an indicator for plasma transudation, as well as after adjustment to total protein concentrations and removal of samples from the statistical analysis that contained elevated erythrocyte and leukocyte concentrations, we concluded that the elevated CE concentrations in CF BALF were possibly contributed by increased inflammation with hemorrhagic events. Inflammation as a contributing factor was confirmed by significantly increased levels of human neutrophil peptides in CF BALF. However, the statistical significance of CE elevation in CF BALF was heightened when adjusted to total lipid content. This is in agreement with an increased relative contribution of CEs to the overall lipid content and thus, up-regulation of CE production in CF. Finally, realizing that BALF fluid of symptomatic patients with already established lung disease is not suited to test our hypothesis aimed at the development of CF associated lung disease, we began exploring the feasibility of a recently developed pig cystic fibrosis model to test our hypothesis. Pilot data indicate the CEs are readily detected in BALF collected from wild type and CF newborn piglets and future studies will be conducted to test our original hypothesis in this animal model