T. C. Rice1, E. F. Midura1, E. Gulbins1,2, M. J. Edwards1, C. C. Caldwell1 1University Of Cincinnati,Division Of Research,Cincinnati, OH, USA 2University Hospital Essen,Department Of Molecular Biology,Essen, NORTH RHINE – WESTPHALIA, Germany
Introduction: Burn-injured patients are susceptible to severe pulmonary infection by Pseudomonas aeruginosa (PA). Here, we investigate a potential role of the ceramide/sphingosine axis in increased susceptibility. Ceramide levels are regulated by acid sphingomyelinase (Asm) activity that converts the membrane phospholipid sphingomyelin to ceramide. Subsequently, ceramide is hydrolyzed via acid ceramidase to the long chain base, sphingosine. Beyond its cellular localization, sphingosine can be observed extracellularly in microparticles (MPs). Although it is known that sphingosine treatment can be used to combat infection, the role of cellular sphingosine or sphingosine-containing MPs in the defense against pulmonary infection in burn patients has not been elucidated. Altogether, we hypothesize that 1) injury alters Asm activity and subsequently sphingosine levels such that susceptibility to infection is increased and 2) sphingosine treatment will prevent lung PA infection in burn injured mice.
Methods: Outbred mice were subjected to a dorsal 28% total body surface area, full thickness scald injury. First, we characterized sphingosine expression in the lungs (by immunohistochemistry) and in bronchoalveolar lavage (BAL) derived MPs (by Nanoparticle Tracking Analysis) on post burn day one (PBD1). Second, Asm activity and MP numbers were determined in BAL fluid in mice 24 hours following inoculation with PA. Finally, to determine the effects of sphingosine on infection, some PBD1 mice were pre-treated with sphingosine. All mice were then inoculated with PA and four hours later, colony forming units (CFUs) were enumerated.
Results: Similar to what is seen clinically, infected burn-injured mice had increased mortality as compared to infected uninjured mice (50.0% vs 18.2%, p=0.048). To determine underlying mechanisms of this mortality, we first observed on PBD1, there is a four-fold reduction in bronchial epithelial sphingosine expression in burn injured mice compared to sham. Further, these mice have a significant reduction in total MPs in BAL fluid. Secondly, there was a significant reduction in Asm activity and total MPs in BAL fluid in infected burn mice compared to infected uninjured mice. The BAL MPs from burn infected mice have significantly reduced sphingosine expression. Finally, following PA inoculation, burn mice have a three-log increase in pulmonary infection compared to sham. However, when burn mice are treated with inhaled sphingosine prior to inoculation, there is a significant reduction in pulmonary infection to a level similar to control mice.
Conclusion: The data demonstrate that 1) burn decreases bronchial sphingosine levels and MP numbers, 2) upon infection, injured mice have decreased Asm activity and MP sphingosine levels, and 3) sphingosine treatment prevents lung infection of PA in burn injured mice. These data suggest that in burn patients, treatment with sphingosine will reduce subsequent pulmonary infection.