Regulatory Effect Of Ouabain On HLA-DR Expression In Human Monocytes

Sepsis is characterized as a systemic inflammatory response syndrome that can be triggered by infections including trauma and stroke with a high morbidity and mortality.Sepsis syndromes include blood flow dysfunction,tissue hypoperfusion,and multiple organ failure.Unfortunately,few strategies have been proven to be effective for the treatment of sepsis in clinic.The pathogenesis of sepsis consists of two phases namely the excessive inflammation phase and the immunoparalysis phase.The excessive inflammation phase is characterized by release of numerous inflammatory cytokines such as TNF-α and IL-1β,which will cause vascular leakage and multiple organ failure.The immunoparalysis phase is characterized by loss of delayed type hypersensitivity,decreased ability of host immunity to clear pathogens and secondary infection.Increasing evidence demonstrates that immunoparalysis mainly accounts for the high mortality of sepsis.In clinic,the HLA-DR expression on monocytes has been assigned as an important maker to differentiate the immune status of sepsis patients.Patient is considered to be immunoparalysis once the HLA-DR expression is less than 30%(relative to normal people).Thus,increasing the expression of HLA-DR expression on the surface of monocytes has been a very useful approach to treat immunoparalysis.We reported for the first time that ouabain,as a cardiac glycoside,was able to reverse immunoparalysis both in vivo and in vitro by reprogramming the mRNA expression of several Thl-related cytokines at the post-transcriptional level Notably,we observed in previously study that ouabain can also increase HLA-DR expression on monocytes,but the underlying mechanism remains unknown.Ouabain can be produced either endogenously or exogenously.It was traditionally used in clinic for the treatment of cardiovascular disease.In 1968,ouabain was found to inhibit the proliferation of T lymphocytes induced by PMA.Now,ouabain is increasingly appreciated as a hormone-like immune regulatory factor,which plays important role in physiological and pathological settings.HLA-DR is a MHC class Ⅱ antigen encoded by the human leukocyte antigen complex,mainly expressed on the cell surface of antigen-presenting cells(APCs)such as monocytes,dendritic cells,and B lymphocytes.In the processes of immune responses,HLA-DR presents antigen peptides to CD4+T lymphocytes,resulting in release of pro-inflammatory cytokines such as TNF-α and IL-6,which helps host immunity to combat pathogen infection.In present study,we found that treatment with 50 nM ouabain alone up-regulated the mRNA expression of HLA-DRα/β in human monocytes THP-1.Moreover,compared with the cells treated with LPS(1μg/ml)alone,combined treatment with 50 nM and 1 μg/ml LPS increased the expression of HLA-DR in THP-1 cells,both at mRNA and protein levels,indicating the ability of ouabain to reverse the decreased expression of HLA-DR induced by LPS.MHC class Ⅱ transactivator(CIITA)is a co-activator for HLA-DR transcription.There are three distinct type of promoters for CIITA,namely pⅠ,pⅢ,and pⅣ In present study,we showed that ouabain activated CIITA4 mRNA expression,reversed the decreased expression of CIITA4 induced by LPS.Meanwhile,ouabain could activate IFNy expression in THP-1 cells,exerting a synergistic effect with IFNy to promote the mRNA expression of CIITA4,especially in the "two-hit" model of sepsis.Because of the importance of STAT1 and IRF1 in inducing CIITA4 expression,we found that CIITA4 and HLA-DR expression on the surface of THP-1 cells and monocytes isolated from normal donors were suppressed by IRF1 siRNA.Furthermore,it was demonstrated that the activation of STAT1 was suppressed by c-Src inhibitor PP2 and c-Src siRNA in THP-1 cells.Ouabain was also found to activate HLA-DR promoter.The full-length and the mutated(X-box)HLA-DRa promoter reporter plasmids were constructed,ouabain increased the promoter luciferase activity of the full-length HLA-DRa,but not the mutated one.Morover,ouabain-activated HLA-DR expression was suppressed in THP-1 cells after transfection with RFX5 siRNA.The novelties of this study can be concluded as following,1.Ouabain and IFNy have similar biological activities in regulating HLA-DR expression in human monocytes.2.CIITA4 plays a critical role in the upregulation of HLA-DR expression by ouabain.3.Ouabain/NA+,K+-ATPase/c-Src non-canonical signaling pathway involves as an important signaling pathway for the effect of ouabain on HLA-DR expression.