mRNA-based approach to monitor recombinant gamma-interferon restoration of LPS-induced endotoxin tolerance.

Abstract

It is now well accepted that sepsis is associated with the development of a pronounced immunosuppressive state, characterized by severe immune alterations (e.g. reduced proliferative capacity, endotoxin tolerance, apoptosis) participating in increased mortality and susceptibility to nosocomial infections. Efforts are currently aimed at restoring a functional immune response in septic patients. Successful therapy depends on the identification of appropriate immunostimulatory drugs and on the development of suitable biomarkers that could be used to stratify patients and to follow response to treatment. In this study, we evaluated the ex vivo effect of recombinant interferon gamma (rIFN-γ) in restoring monocyte functionality (endotoxin-induced Tumor Necrosis Factor-α production) in a two-hit model of endotoxin tolerance (ET) with peripheral blood mononuclear cells from healthy volunteers and in whole blood of septic shock patients. Importantly, we used quantitative-reverse transcription polymerase-chain reaction to monitor the effect of rIFN-γ on the expression of seven genes known to participate in ET (TNF-α, IL-10, HLA-DRA, CIITA, IRAK-M, ABIN-3 and LY64). Expression analysis of those genes confirmed the presence of an immunosuppression state and the ex vivo restoration of immune functions by rIFN-γ. We show for the first time that rIFN-γ is able to bypass, at the mRNA level, the effect of negative regulators of the LPS signalling pathway such as IRAK-M, ABIN-3 and LY64. Overall, mRNA expressions of a panel of genes could represent promising candidates for the ex vivo evaluation of rIFN-γ effect on monocyte functionality. This ex vivo translational research study demonstrates the potential of a mRNA-based approach to successfully monitor drug efficacy.