The Study On The Regulation Of Hepatic NK Cells By Tissue Microenvironment

Natural killer(NK)cells are innate immune cells,and unlike T cells and B cells,NK cells can directly kill cancer cells and virus-infected cells without antigen priming.NK cells are widely distributed in the body and are particularly abundant in the liver.For the first time,our group has identified a subpopulation of liver resident NK(LrNK)cells that are different from conventional NK(cNK)cells,with significant differences in phenotype,transcription factors,and effector functions.The developmental origin,heterogeneity,antitumor function,immune memory,and negative regulation of LrNK cells have been studied in our group in recent years,but the tissue microenvironmental factors affecting this cell population are still unclear.Kupffer cells are also liverresident cells with a high abundance in the liver sinusoids and a high overlap with the niche of LrNK cells.In addition,due to the specific physiology of the liver,a large number of enteric-derived substances,including commensal bacteria and food antigens,enter the liver with the blood.Whether Kupffer cells and commensal bacteria and their metabolites are involved in regulating the formation of immune features of LrNK cells remains unknown.Based on the above questions,we conducted a study on microenvironmental factors that might affect hepatic NK cells,with the following results.1.Contact between liver NK cells and Kupffer cells revealed by fluorescence imagingTo investigate the positional relationship between Kupffer cells and liver NK cells,we found that the spatial location of Kupffer cells and NK cells were close and in direct contact by immunofluorescence staining of liver tissues and in vivo imaging.2.Kupffer cells maintain hepatic NK cells at steady stateTo investigate whether there is regulation of Kupffer cells on hepatic NK cells,we used clodronate liposomes to deplete Kupffer cells and detected changes in hepatic NK cells using flow cytometry.We found that the proportion(of CD45+leukocytes)and number of hepatic cNK cells and LrNK cells were significantly decreased after depletion of Kupffer cells.In addition,the proliferation of LrNK cells was significantly decreased.These results confirmed the role of Kupffer cells in maintaining the liver NK cell subpopulation at steady state.3.Kupffer cells promote LrNK and cNK cell maturation in vivoAfter the depletion of Kupffer cells in vivo using clodronate liposomes,we further detected the maturation-related phenotypes and functional molecules of liver NK cell subpopulations.We found that the differentiation and maturation of LrNK cells and cNK were inhibited after Kupffer cell depletion,indicating that Kupffer cells can promote the maturation of liver NK cells in vivo.4.Kupffer cells promote LrNK and cNK cell maturation in vitroTo determine the role played by Kupffer cells in the maturation-promoting process of liver NK cells,we co-incubated Kupffer cell-containing and Kupffer cell-free liver non-parenchymal cells with Kupffer cell-free liver mononuclear cells.After 24 h incubation,the flow assay results showed that Kupffer cells could also promote maturation of hepatic NK cells in vitro.5.Microbiota affect the phenotype and function of LrNK cellsTo investigate the effect of commensal bacteria on liver NK cells,we used an antibiotic water-fed commensal clearance mouse model and a germ-free mouse model,and performed flow assays on liver mononuclear cells from mice.We found that commensal bacteria deficiency did not affect the ratio and number of liver NK cells.The phenotype of LrNK cells changed in the antibiotic-clearing commensal mouse model,and the decreased expression of the functional molecule granzyme C in LrNK cells was observed in germ-free mice.6.Regulation of liver NK cells by Kupffer cells is dependent on commensal bacteriaWe further investigated whether the regulation of liver NK cell maturation by Kupffer cells was dependent on commensal bacteria.Therefore,we depleted Kupffer cells from mice cleared of microbiota.The results of the flow assay showed no significant inhibition of maturation which was observed after depletion of Kupffer cells from microbiota-deficient mice,confirming that the regulation of liver NK cells by Kupffer cells NK cell regulation is partially dependent on commensal bacteria.7.Tlr4 deficiency does not affect hepatic NK cellsTLR4 receptors on Kupffer cells are classical symbiotic signaling(LPS)receptors.To investigate whether Kupffer cells receive symbiotic signals through TLR4 receptors and then regulate liver NK cells,we performed flow assays on Tlr4-deficient mice.We found that no significant changes in the number and maturation phenotype of liver NK cells,confirming that TLR4 signaling had not effect on the regulation of hepatic NK cells by Kupffer cells.In summary,Kupffer cells promote the maturation and differentiation of liver NK cells both in vitro and in vivo,and this regulatory effect is partially dependent on the microbiota but not on TLR4 signaling.Our study reveals an important role of hepatic microenvironmental factors in the formation of regional immune features of hepatic NK cells.