J. Wisler1, D. Dakhlallah2, T. Eubank2, C. Marsh2 1Ohio State University,Surgery,Columbus, OH, USA 2West Virginia University Robert C Byrd Health Sciences Center,Microbiology, Immunology And Cell Biology,Morgantown, WV, USA
Introduction: Systemic immunosuppression associated with severe sepsis is a poor prognostic marker and predisposes survivors to increased infection risk, morbidity and mortality. Immunosuppression can persist for years following the initial insult. We have previously presented this immunosuppression is related to epigentic regulators that can be transferred via circulating microvesicles (MV). Epigenetic events occur at the promoter sites of several key proinflmmatory genes including IL-1[beta] and TNF-[alpha]. We hypothesized that these circulating MVs can be targeted for therapy to prevent these epigenetic events from occuring and maintain a functional host immune response.
Methods: Naïve human monocytes were treated with MVs from patients with septic shock, sepsis, critical illness without sepsis, and non-septic normal controls. After incubation with MV, monocytes were stimulated with LPS and TNF-a expression measured and DNA methylation was assessed at the TNF-[alpha] promoter. Using a competitive inhibition model, dose response curves were generated to assess inhibition of uptake. Cells were also treated with a methylation inhibitor, 5'-Azacitidine, to prevent methylation events from occuring.
Results: Naïve monocytes treated with MVs from patients with sepsis demonstrated increased expression of DNMTs at 24 hours. Promoter methylation at TNF-[alpha] was increased significantly in recipient monocytes and these cells demonstrated a dramatically reduced TNF-[alpha] expression in response to LPS challenge (all p < 0.04, Fig 1). Treatment of naive cells with healthy donor derived MVs significantly reduced TNF-[alpha] promoter methylation. Additionally, treatment of naive cells with demethylating agents significantly reduced TNF-[alpha] promoter methylation events and these cells maintained their ability to generate TNF-[alpha] in response to LPS stimulation.
Conclusion: These data demonstrate that targeting of sepsis derived MV may prevent transfer of methylation events to naive recipient cells and prevent immunosuppressive consequences. The use of healthy donor derived MVs or demethylating agents significantly reduced the transfer of epigenetic elements and allowed naive monocytes to maintain their immunocompetence. This has potential as a novel therapeutic target during the septic process
