L. K. Winer1, A. M. Pugh1, E. Gulbins3, C. C. Caldwell1, V. Nomellini2 1University Of Cincinnati,Division Of Research, Department Of Surgery,Cincinnati, OH, USA 2University Of Cincinnati,Division Of Trauma, Critical Care, And Acute Care Surgery, Department Of Surgery,Cincinnati, OH, USA 3University Of Duisburg-Essen,Department Of Molecular Biology,Essen, GERMANY, Germany
Introduction: Critically ill patients who survive the acute phase of sepsis can progress to persistent inflammation, immunosuppression and catabolism syndrome (PICS), and are at risk for secondary infection, organ failure, and death. Characteristics of PICS include increased circulating myeloid cells, decreased lymphocytes, and muscle loss. We recently reported that 8 days after cecal ligation and puncture (CLP), mice concurrently display the aforementioned features of PICS and are susceptible to lung infection. During lung infection, bronchial alveolar lavage analysis reveals impaired neutrophil accumulation and increased bacterial loads in PICS mice. However, the mechanisms by which PICS weakens the host response to infection remain unclear. Previous studies suggest that elevated levels of the membrane lipid ceramide induce cellular dysfunction. Here, we test the hypothesis that ceramide modulates neutrophil reactive oxygen species (ROS) production and chemotaxis during PICS.
Methods: Male CF-1 mice underwent 33% cecal ligation with a single 25-gauge needle puncture. Neutrophil dihydrorhodamine (DHR), a surrogate for ROS production, was measured by flow cytometry. Neutrophil chemotaxis was conducted on transwell plates with KC as the chemoattractant. Separate cohorts of healthy cells were treated with 10 μ M of C16-ceramide for 30 minutes and washed prior to DHR and chemotaxis assays. A student t-test was used for statistical comparison, and p-values ≤ 0.05 were considered statistically significant.
Results: We performed DHR and chemotaxis assays to (1) characterize neutrophils during PICS and (2) measure the effect of ceramide on neutrophil activity. We found that neutrophils isolated from PICS mice demonstrate a nearly 50% DHR increase (p<0.05, n=17) and a greater than 50% reduction in chemotaxis (p<0.05, n=17) compared with healthy controls. These effects were mimicked by ex vivo addition of C16-ceramide to healthy neutrophils, which increased DHR by nearly 10% (p<0.05, n = 6). Ceramide also decreased neutrophil chemotaxis by more than 30% (p<0.05, n = 7). Altogether, both PICS and ceramide increase neutrophil ROS and suppress chemotaxis.
Conclusion: This study demonstrates that during PICS, there is a marked increase in neutrophil ROS production and impaired neutrophil chemotaxis. This combination of factors may underlie the decreased bacterial clearance and increased mortality previously observed in PICS mice. When applied to healthy mouse neutrophils, exogenous C16-ceramide produces a similar pattern of DHR expression and chemotaxis as that seen in vitro. We therefore speculate that C16-ceramide is associated with PICS neutrophil dysfunction, and may represent a therapeutic target in critical illness.