Therapeutic Inhaled Sphingosine for Treating Lung Infection in a Mouse Model of Critical Illness.

Abstract

Sphingosine, a sphingoid long chain base, is a natural lipid with antimicrobial properties. Recent animal studies have shown that preventive sphingosine inhalation can rescue susceptible mice, such as cystic fibrosis-, burn injured- or aged mice from bacterial pulmonary infection. While preventing lung infections in susceptible patients has obvious clinical merit, treatment strategies for an established infection are also direly needed, particularly in the times of rising antibiotic resistance. Here, we tested the potential of sphingosine in treating an established pulmonary infection. We used a cecal ligation and puncture (CLP) model in male CF-1 mice and a Pseudomonas aeruginosa strain that was isolated from a septic patient (P. aeruginosa 762). We determined susceptibility to intranasal infection and ascertained when the pulmonary infection was established by continuous core body temperature monitoring. We quantified sphingosine levels in the tracheal epithelium by immunohistochemistry and studied the effects on sphingosine on bacterial membrane permeabilization and intracellular acidification using fluorescent probes. We first determined that septic mice are highly susceptible to P. aeruginosa infection 2 days after indu-cing sepsis. Additionally, at this time, sphingosine levels in the tracheal epithelium are significantly reduced as compared to levels in healthy mice. Secondly, upon intranasal Pseudomonas inoculation, we ascertained that pulmonary infection was established as early as 2.5 h after inoculation as evidenced by a significant drop in core body temperature. Using these times of infection susceptibility and detection (2 days post CLP, 2.5h after inoculation) we treated with inhaled sphingosine and observed pulmonary bacterial loads reduced to levels found in infected healthy mice after inoculation and decreased infection-associated mortality. Further, our data demonstrate that sphingosine induces outer membrane permeabilization, disrupting the membrane potential and leading to intracellular acidification of the bacteria. Sphingosine shows efficacy in treating P. aeruginosa lung infections not only prophylactically, but also therapeutically.