Telomerase Reverse Transcriptase Acts In A Feedback Loop With NF-?B Pathway To Regulate Macrophage Polarization In Alcoholic Liver Disease

Alcoholic liver disease(ALD)is one of the common chronic liver disease,which is due to the long-term heavy drinking.Fatty liver usually presents at the early stage,then it can develop into alcoholic hepatitis,liver fibrosis and cirrhosis.Since the molecular mechanisms are complex,there is no effective medicines.Acute and chronic exposure to alcohol can activate Kupffer cells(KCs).As liver resident macrophages,KCs play an important role in the pathogenesis of ALD.It has lots of functions including cytokines secretion,phagocytosis and immune regulation.With its unique plasticity and heterogeneity,macrophages may change the function and phenotype according to the external environment.Firstly,a mouse model of ALD was established by using NIAAA recommended Lieber-DeCarli liquid diet plus alcohol gavage.Histopathological analysis showed that all of male C57BL/6J mice with chronic alcohol feeding were characterized by immune cell activation,inflammation,injury and steatosis in the liver.Immunohistochemical(IHC)staining analysis showed that more CD68+ cells in liver tissue were detected in the EtOH group compared to the control group.In FACS analysis of KCs isolated from the liver,there was an approximate 1-fold increase in the frequency of CD45+F4/80+CD11b+ cells in the EtOH group compared to the control group.caM? macrophage markers TNF-?,IL-1?,NOS2 and CCL2 were remarkably up-regulated in the EtOH group.Similarly,there was substantial difference in the level of the aaM? macrophage markers Arg-1,IL-10,Mrc2 and CD163 between the two groups.Therefore,all above data suggests that chronic and binge ethanol feeding induces a mixed caM?/aaM? macrophage phenotype in vivo.Since TA occurs at critical stages of myeloid and lymphoid cell activation,we herein investigated the role of telomerase reverse transcriptase(TERT),the determining factor of telomerase,in macrophage activation during ALD.Results of the double immunofluorescent(IF)analysis showed the representative colocalization of TERT with macrophage CD68 immunoreactivity in liver tissue.TERT expression were remarkably increased in liver tissue and isolated KCs of EtOH-fed mice.Quantification of TA revealed that the development of ALD might be associated with a more than 6-fold increase in TA.Furthermore,ethanol stimulation can induce TERT expression in vitro and TERT was up-regulated in murine proinflammatory caM? macrophages.Thus,it indicates that up-regulation of TERT may have key roles in the development of ALD,a process driven primarily by macrophages.To explore the potential role of TERT in caM? macrophage polarization,TERT knockdown and over-expression in LPS-stimulated RAW 264.7 cells were realized by introducing siRNA and plasmids,respectively.The mRNA levels of caM? macrophages biomarkers including TNF-?,IL-1?,NOS2 and CCL2 were substantially decreased when TERT was blocked.In line with the above data,the secretion of proinflammatory cytokines including TNF-?,IL-1?,IL-6 and IL-12 was significantly decreased in TERT-knockdown caM? macrophages compared with that in caM? macrophages transfected with the control-siRNA.Conversely,over-expression of TERT significantly up-regulated these markers except CCL2 in LPS-treated macrophages transfected with TERT plasmid compared with vector control.Further study showed that NF-?B signaling pathway was activated in the progression of ALD as well as caM? macrophage differentiation.More importantly,TERT regulated murine caM? macrophage polarization through NF-?B pathway.In addition,PDTC,a chemical inhibitor for NF-?B,obviously inhibited the protein level of TERT in caM? macrophages and suppressed the expression of caM? macrophage markers.Therefore,our study unveils the role of TERT in macrophage polarization and the cross-talk between TERT and p65,which may provide a possible explanation for the ethanol-mediated hepatic proinflammatory response and caM? macrophage polarization.