View Abstract

A7569 - Defective Pulmonary Host Defense Against Klebsiella Pneumonia in 25-Hydroxycholesterol Knockout Mice
Author Block: P. Mancuso1, L. Russo2, C. Lumeng3; 1Dept of Nutritional Sciences, Univ of Michigan, Ann Arbor, MI, United States, 2Dept of Pediatrics and Communicable Diseases, Univ of Michigan, Ann Arbor, MI, United States, 3Pediatrics and Communicable Diseases and Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, United States.
RANTIONALE: Oxysterols are metabolites of cholesterol whose synthesis increases in response to Toll-like receptor activation. The oxysterol, 25-hydroxycholesterol (25-HC), is a product of cholesterol hydroxylation by the action of cholesterol 25-hydroxylase (Ch25h). The expression of Ch25h increases several-fold in macrophages and dendritic cells in response to bacterial or viral antigens resulting in marked increases in 25-HC at the site of infection. While Ch25h KO mice are protected from Influenza A infection, a role of 25-HC in response to Klebsiella pneumoniae, a common cause of nosocomial pneumonia, has not been reported.
OBJECTIVE: Determine if Ch25h plays a role in pulmonary host defense against K. pneumoniae in vivo and in alveolar macrophage (AM) and glycogen-elicited peritoneal neutrophil (PMN) phagocytosis and killing of bacteria in vitro.
METHODS: Female wild type and Ch25h KO C57BL/6 mice were infected with 5000 CFUs of K. pneumoniae via the intratracheal route. Bacterial burdens in lungs and spleens and concentrations of cytokines (IFN-β, IL-1α, IL-1β, IL-6, IL-10, M-CSF, MIP-2, and TNF-α) in the lung homogenates were assessed 24 and 48 h following infection. In a separate group of mice, AMs and PMNs were recovered from uninfected animals and assessed for phagocytosis and killing (AMs only) of K. pneumoniae opsonized with immune serum in vitro.
RESULTS: Bacterial burdens were approximately 2-log fold higher in the lungs and 1-log fold higher in the spleens of Ch25h KO mice 24 and 48 h after infection. IFN-β, IL-1β, IL-6, and MIP-2 were higher in lung homogenates of Ch25h KO mice 24 h after infection. Lung homogenate levels of IL-10 and IFN-β were higher in Ch25h KO mice 48 h after infection. Phagocytosis of K. pneumoniae opsonized with immune serum was lower in PMNs obtained from Ch25h KO mice but not different in AMs. In contrast, we observed reduced killing of K. pneumoniae in AMs recovered from Ch25h KO mice.
CONCLUSIONS: Ch25h plays a protective role in the pulmonary host responses against K. pneumoniae. The elevated levels of pulmonary IFN-β, IL-1β, IL-6, and MIP-2, but not IL-1α, M-CSF, or TNF-α, in Ch25h KO mice suggest that 25-HC plays an important regulatory role in proinflammatory cytokine synthesis following K. pneumoniae infection. The reduced phagocytosis in PMNs and defective killing of K. pneumoniae in AMs from Ch25h KO mice suggests 25-HC plays an essential role in leukocyte bactericidal functions and that products of Ch25h may be novel therapeutic agents for the treatment of gram-negative pneumonia.