The Effect And Mechanism Of The Pattern Recognition Receptor Signaling Pathway Of Liver Sinusoidal Endothelial Cells In Regulating Intrahepatic NK Cells

Objective:To explore the immune regulation effect of LSECs on intrahepatic NK cells under physiological state and pattern recognition receptor signaling pathway activation state,and to clarify the effect of LSECs on the phenotype and function of NK cells and its specific mechanism.Methods:1、Conventional methods were used to separate liver infiltration lymphocytes(LILs),NK cells and LSECs from mice.The mouse LSECs were co-incubated with NK cells or LILs in an appropriate ratio in vitro,and flow cytometry was used to analyse the phenotypic and functional changes of NK cells or the NK cells in liver infiltrating lymphocytes after stimulation by different pattern recognition receptor ligands(TLR-3-poly I:C /TLR-4-LPS/NOD1-DAP).2 、 The chronic HBV replication mouse model was constructed by injection of p AAV/HBV1.2 plasmid with high-pressure water from the tail vein.The chronic HBV replication mouse LSECs and liver infiltrating lymphocytes were co-incubated under the stimulation of different pattern recognition receptor(TLR-3/TLR-4/NOD1)ligands(poly I:C/LPS/DAP),and flow cytometry was used to analyse the phenotypic and functional changes of NK cells in liver infiltrating lymphocytes.Results:1、LSECs and NK cells were co-incubated without any pattern recognition receptor ligand in vitro in the way of direct contact and transwell respectively.The results showed that the expression of CD69 and IFN-γ on NK cells was down-regulated regardless of direct contact or transwell.2、Direct stimulation of pattern recognition receptor ligands regulated the function of intrahepatic NK cells.In naive mice,after poly I:C,DAP,LPS and P3 C stimulation,intrahepatic NK cells had a certain degree of activation.The co-incubation of LSECs with NK cells after poly I:C,DAP,LPS and P3 C activated different pattern recognition receptors,respectively,reversed the negative regulatory effect of LSECs on NK cells.3、Compared with purified NK cells,direct stimulation of pattern recognition receptor ligands weakened the regulation of NK cells in LILs.In naive mice,LSECs had a negative regulatory effect on NK cells in LILs.Compared with purified NK cells,activation of LSECs pattern recognition receptor pathway has weakened the reversal effect of LSECs negative regulation of NK cells in LILs.Poly(I:C)stimulation of LILs did not activate NK cells in LILs;only DAP and LPS stimulation activated NK cells in LILs.4、Compared with naive mice,direct stimulation of pattern recognition receptor ligands weakened the regulation of NK cells in LILs of HBV mice.In HBV mice,as a whole,LSECs negatively regulated NK cells in LILs,but up-regulated the expression levels of TRAIL and Fas L on NK cells.After activating the LSECs pattern recognition receptor pathway,it failed to reverse the negative regulatory effect of LSECs on NK cells in LILs of HBV mice.Conclusion:1、Liver sinusoidal endothelial cells were involved in regulating the function of intrahepatic NK cells.The regulation of NK cells by sinusoidal endothelial cells depended on both the direct contact of cells and the effect of indirect cytokines.2、In vitro,mouse LSECs regulated NK cells in a negative way,but in the presence of pattern recognition receptor ligands,the negative regulatory effect of liver sinusoidal endothelial cells on NK cells could be reversed to a certain extent,especially in the action of TLR-4 and NLR-1 ligands.In the complex immune system such as LILs and HBV replication mouse model,pattern recognition receptors had limited regulation of NK cells,and in the same time,their influence on LSECs in regulating NK cell functions was further weakened.We speculated that other immune cells in the liver and HBV virus might also participate in the regulation of NK cells by LSECs.