| BackgroundThe liver is a key and frontline immune organ,particularly enriched for innate immune cells including innate lymphoid cells(ILCs),natural killer T(NKT)cells,macrophages and γδT cells.These innate immune cells act coordinately to eliminate invading pathogens,as well as to maintain liver functional homeostasis.ILCs accounts for about 5%intrahepatic lymphocytes in mice and 25%in human at steady state and majorly consists of conventional natural killer(cNK)cells and liver-resident NK(LrNK)cells,and the latter is also known as liver type 1 ILCs(ILCls).Phenotypically,mouse LrNK cells can be identified as NK1.1+ NKp46+ CD49a+CD49b-,distinct from NK1.1+NKp46+CD49a-CD49b+cNK cells.LrNK cells reside in hepatic sinusoid and exhibit significant differences in terms of development,phenotype and effector functions compared to cNK cells.LrNK cells develop locally from Lin-Sca-1+ Mac-1+hematopoietic stem cells(LSM),which then differentiate into Lin-CD122+CD49a+cells,as precursor cells for LrNK.Both LrNK and cNK cells play important roles in maintaining local and systemic immune homeostasis and involve in anti-viral and anti-tumor immunity.Further unraveling the mechanisms by which the liver microenvironment supports the development and functional maturation of liver NK cells would provide insights for understanding the liver biology and developing therapeutic strategies for liver diseases.The liver and intestine are interconnected through the portal vein and biliary system.The liver is at the nexus of host-microbial interactions with respect to its unique anatomical location,allowing continuous blood flow from the gastrointestinal tract through the liver sinusoids.Importantly,accumulating evidence unraveled that gut microbiota plays crucial roles in the establishment and maintenance of liver immune homeostasis.Early life,namely perinatal period,newborn period and infant period,is a critical period for the development and colonization of gut microbiota,which determines the diversity of gut microbiota composition and functional integrity in adulthood.Simultaneously,early life is also a critical period for the interaction between gut microbiota and host immunity.A large number of data shows that the impaired development of gut microbiota in early life(the most common clinical cause is the use of preventive or therapeutic antibiotics)will cause irreversible gut microbiota disorder in adulthood and further increase the susceptibility of various immune diseases,such as IBD,psoriasis,etc.However,the importance of gut commensal in the regulation of hepatic NK cells remains unexplored.Methods and ResultsThis study aims to reveal the key role of early life gut microbiota in the development and functional maturation of liver NK cells.The main research methods and results are as follows:Ⅰ.Early-life gut microbiota is crucial for the maturation of LrNK cellsTo determine the impact of gut microbiota on liver NK cell development,we established a maternal antibiotic treatment mouse model.The number,phenotype and function of liver NK cells were detected by flow cytometry.Results showed that there were no significant differences in the percentage and number of LrNK cells and cNK cells and the precursor cells in early-Abx and control mice but significantly impaired the maturation and effector function of LrNK cells.We also found that early-life antibiotic exposure did not affect the number,phenotype and function of cNK cells.In addition,it was proved that early-life antibiotic exposure regulated the function of LrNK cells through an extrinsic manner by cell transfusion experiments.Ⅱ.Early-Abx induced abnormal maturation and dysfunction of LrNK cells accelerate HCC developmentThus,we wonder whether early-life antibiotics exposure also blunts LrNK-mediated antitumor effects.To address this,we introduced both c-myc/AKT-driven and STZ-HFD-induced hepatocellular carcinoma(HCC)mouse model.It was found that early-Abx mice developed more severe liver tumors than control mice in both HCC models.Importantly,depletion experiment confirmed that early-life antibiotic exposure promoted the progression of mouse liver cancer by inhibiting LrNK cells.Ⅲ.The persistent alteration of gut microbiota is responsible for LrNK cell maturation arrest in early-Abx treated miceTo determine the mechanism of early-life antibiotic exposure in inhibiting the maturation of LrNK cells,we analyzed gut microbiota using 16S rRNA sequencing with feces from control or early-Abx mice.The results showed that early-life antibiotic exposure significantly reduced the abundance and diversity of gut microbiota and changed the microbial community composition.To further demonstrate the role of gut microbiota in inhibiting the maturation of LrNK cell of early-life antibiotic exposure,we established a co-housing mouse model and the maturation and function of LrNK cells in the co-housing mice were detected by flow cytometry.As expected,co-housing treatment eliminated the differences of maturation and effector function between control and early-Abx mice.The above results suggest that early-life antibiotic exposure inhibits the maturation and function of LrNK cells by continuously affecting gut microbiota.Ⅳ.Microbiota-derived butyrate facilitates the maturation of LrNK cellsThe gut microbiota could impact the physiology of the host by generating abundant commensal metabolites.We detected the levels of microbial metabolites in feces of control and early-Abx mice by GC-MS.Results showed that butyrate was significantly reduced in feces of early-Abx mice.Notably,butyrate had close correlation with the abundance of the dominant bacterial in early-Abx mice.Moreover,compared to that of control mice,feces of early-Abx mice had significantly decreased abundance of butyrate producing microbiome.Next,we wonder whether microbiota-derived butyrate is responsible for the maturation of LrNK cells.Butyrate gavage could rescue the impaired maturation and effector function of LrNK cell in early-Abx mice.The above results suggest that butyrate plays an important role in inhibiting the maturation and function of LrNK cells by early-life antibiotic exposure.Ⅴ.Butyrate indirectly enhances LrNK cell maturation through acting on Kupffer cells and hepatocytes by GPR109A1.Early-life gut microbiota affects the function of LrNK cells in a cell-extrinsic mannerTo explore the mechanism that the reduced butyrate caused by early-life antibiotic exposure in inhibiting the function of LrNK cells,we transferred purified NK cells from control or early-Abx mice(CD45.2)into recipient CD45.1 mice and found that transferred LrNK cells from control and early-Abx mice had comparable IFN-y expression and CD 107a mobilization.In contrast,when we transferred NK cells(CD45.2)separately into control or early-Abx mice(CD45.1),donor LrNK cells in early-Abx mice had lower level of IFN-γ and CD 107a than those in control mice.The above results suggest Early-life gut microbiota affects the function of LrNK cells in a cell-extrinsic manner.2.Butyrate indirectly enhances LrNK cell maturationThe above results suggest that decreased butyrate play an important role in inhibiting the maturation and function of LrNK cell by early-life antibiotic exposure.To explore the role of butyrate on LrNK cells,the experiment of butyrate stimulated LrNK cells in vitro proving that butyrate indirectly regulates LrNK cells.In order to further clarify which cells butyrate indirectly regulates LrNK cells,hepatocytes and liver mononuclear cells(LMNCs)were isolated and co-cultured with LrNK cells,respectively,and stimulated with butyrate.Flow cytometry results showed that butyrate treatment significantly up-regulated the function of LrNK cells co-cultured with hepatocytes and LMNCs.The above results suggest that butyrate affects the maturation and function of LrNK cells by regulating the cellular interaction network in the liver microenvironment.3.Butyrate promotes LrNK cell function through acting on Kupffer cells and hepatocytes by GPR109AButyrate regulates cells by acting on GPCRs(GPR41,GPR43,GPR109A).To clarify the role of GPCRs dependent pathways in regulating the interaction network between hepatocytes/LMNCs and LrNK cells by butyrate,the expression of related GPCRs in hepatocytes and LMNCs was detected by RT-qPCR.The results showed that GPR109A is mainly expressed in hepatocytes,while GPR41,GPR43,and GPR109A were all expressed in LMNCs.Lentivirus interference with GPR109A expression in hepatocytes and Kupffer cells can eliminate the up-regulation effect of butyrate treatment on the function of LrNK cells in coculture system.These results suggest that butyrate indirectly promotes LrNK function by acting on GPR109A in hepatocytes and Kupffer cells.Ⅵ.Butyrate triggers IL-18 production in hepatocytes/Kupffer cells to improve the maturation and function of LrNK cellsTo gain insight into the mechanism underlying the indirect action of microbiota-butyrate on LrNK cell function,we first examined the important cytokines/chemokines in the liver.RTqPCR and ELISA found that early-life antibiotic exposure significantly reduced the level of IL18 in liver tissue.Intriguingly,intraperitoneal injection of recombinant IL-18 protein(rIL-18)markedly rescued the impaired maturation and effector function of LrNK cells in early-Abx mice.It is suggested that IL-18 plays an important role in the regulation of LrNK cell function maturation by gut microbiota.Then,we wonder whether microbiota-butyrate axis promotes LrNK maturation through upregulating IL-18 in liver microenvironment.RT-qPCR and ELISA found that addition of butyrate upregulated IL-18 level in both hepatocytes and Kupffer cells ex-vivo.More importantly,IL-18 blocking antibodies can eliminate the promoting effect of butyrate treatment on the function and maturation of LrNK cells co-cultured with hepatocytes/Kupffer cells.These data suggest that butyrate triggers IL-18 production in hepatocytes/Kupffer cells to improve the maturation and function of LrNK cells.Ⅶ.IL-18/IL18R promotes LrNK maturation through improving mitochondrial oxidative phosphorylationTo better understand how IL-18 facilitates the maturation and funcion of LrNK cells,flow cytometry was first used to analyze the phenotypic in IL-18Rα+ LrNK cells and IL-18Rα-LrNK cells and we found that IL-18Rα+ LrNK cells express higher levels of mature marker molecules CD11b and KLRG1,as well as stronger cytokine expression and degranulation abilities.IL18Ra KO LrNK cell transfer experiment showed that IL-18Rα Gene knockout LrNK cells did not exhibit significant functional differences between control and early antibiotic treated recipient mice,further proved that early-life antibiotic exposure effect LrNK cells depends on IL-18Rα.We performed RNA sequencing for IL-18Rα-positive and-negative liver CD3NK1.1+ cells.GSEA revealed that transcriptional signatures associated with mitochondrial oxidative phosphorylation pathway was positively enriched in IL-18Rα+CD3-NK1.1+ cells.Seahorse experiment and flow cytometry detected a relatively high level of basal and maximal oxygen consumption rate(OCR)as well as ATP production in IL-18Rα+LrNK cells.Conversely,LrNK cells from early-Abx mice showed decreased MMP and increased mitochondrial ROS,when compared to those cells from control mice.Intriguingly,inhibition of mitochondrial oxidative phosphorylation activity by Rotenone significantly decreased IFN-y production and abolished the difference between IL-18Rα+ and IL-18Rα-LrNK cells.These data suggest that IL-18/IL18R promotes LrNK maturation through improving mitochondrial oxidative phosphorylation.Ⅷ.Dietary Clostridium butyricum rescues the impaired maturation of LrNK cells in early-Abx treated miceTo explore intervention strategies for LrNK cell maturation disorders caused by early-life antibiotic exposure,Clostridium butyricum(C.butyricum)or a commercially Clostridium butyricum Powder(Boleyan)was administrated to early-Abx mice after weaning.The maturation and function of liver LrNK cells were detected by flow cytometry.The results showed that administration of Clostridium butyricum and commercial Clostridium butyricum Powder(Boleyan)could partially or completely recovered the function of liver LrNK cells in early-Abx mice and also restore the level of liver IL-18.These results suggest that dietary supplementation of butyrate-producing bacteria could recover the status of LrNK cells from early-Abx treated mice.Conclusion and Significance:Liver NK cells play an important role in the maintenance of local and systemic immune homeostasis in liver.We performed the early-life antibiotic exposure mice model to clarifing the role of gut microbiota in the regulation on the development and maturation of liver NK cell. The main research conclusions and significance are as follows:1.We found that early-life antibiotic exposure persistently affecting the composition and diversity of gut microbiota and the production of butyrate and then inhibiting the maturation,release of cytokine and anti-tumor efficiency of LrNK cells rather cNK cells.Our results provide new experimental evidence for revealing the role of gut microbiota in the regulation of liver immune microenvironment.2.We determined that butyrate derived from gut microbiota promotes the production of IL-18 by GPR109A on hepatocytes and Kupffer cells,and IL-18 promotes the maturation of LrNK cells by enhancing the mitochondrial oxidative phosphorylation levels.Our study uncovers an interplay network of gut-liver axis in regulating liver immunohomeostasis.3.Our study found that dietary supplementation of experimentally or clinically used Clostridium butyricum restored the impaired LrNK cell maturation and function induced by early-life antibiotic exposure which provided a potential intervention strategy for alleviating immune disorders caused by clinical antibiotic exposure. |
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