The Role Of Human Hepatocytes In The Development Of Human Immune Cells In Humanized Mice With Human Chimeric Liver And Immune System Reconstitution

BackgroundHumanized mouse models engrafted with functional human cells and/or tissues,or transgenically expressing human genes have become more and more valuable platforms in the studies of human specific diseases and biology.In the field of immunology,immunodeficient mice reconstituted with functional human immune systems through transplantation of human hematopoietic stem/progenitor cells（HSPCs）and/or hemato-lymphoid tissues are termed as immune system humanized mice（hu-mice）.With 30 year’s development,the compositions and functions of human immune cells in hu-mice have been more and more of fulfillment,and they have been widely used to the studies of human diseases including infections,autoimmune diseases,cancer and so on.Among the current models,the hu-mice constructed by combined transplantation of human fetal thymus tissues（FTHY）and autologous fetal liver derived CD34⁺HSPCs（i.e.FTHY/HSPCs hu-mice or TH hu-mice in this study）attract a lot of focuses,as they can develop multi-lineage profiles of human adaptive and innate immune cells,such as T cells,B cells,monocyte/macrophages,and DCs.Importantly,the human T cells in TH hu-mice develop in human thymus and are HLA-restricted,which are able to optimize the human T cell functions.Besides,the spleens and lymphoid nodes of TH hu-mice form secondary lymphoid architectures,such as splenic white pulp.However,the levels of human immune cells in the livers of TH hu-mice remain relatively low.Hepatocytes account for 70-80%of all cells in liver.Liver’s functions include metabolism,detoxification,biliary secretion,protein synthesis and storage of glycogen,all of which are achieved mainly by hepatocytes.Liver has been considered as an immunological organ with immune tolerance property,it can mount robust immune responses in some settings as well,such as HBV-induced fulminant hepatitis,both of which are involved in the liver immune cell profiles including Kupffer cells,NK cells,NK-like T（NKT）cells,DCs,and so on.Hepatocytes,according to reported studies,play important roles in the induction of liver immune tolerance.For examples,hepatocyte transplantation can induce immune tolerance to allogeneic graft;hepatocytes can promote the generation of tolerant CD4⁺T cells through up-regulated expression of Jagged1,the ligand of Notch on T cells.However,what roles hepatocytes play in the development of immune cells have been poorly elucidated.Humanized mice with human chimeric liver and immune system reconstitution have been widely used for the studies of HBV and HCV specific immune responses and associated liver immunopathogenesis.However,how human hepatocytes influence the development of human immune system in these humanized mcie remain to be investigated deeply.The liver injury characteristics of the host immunodeficint mice are critical to optimize the generation of such models.uPA/SCID mouse,the SCID mouse with liver specific overexpression of uPA,is liver injured and immunocompromised.These mice can be effectively repopulated by human hepatocytes.However,they also have some disadvantages,including narrow experimental framewindow（these mice have to be transplated with health hepatocytes within 2 weeks old）,low capability of breeding,and transgenic uPA lost caused by homologous recombination.These disadvantages dramatically limit the wide use of these mice.Therefore,development of a strategy to regulate uPA function that can overcome these limitations,at least partially,may be of high improtance to generate a nicer host mouse for human hepatocyte repopulation.AimsIn the present study,the aim 1 are to optimize the protocol of anti-mouse Fas（Jo2）antibody-induced liver injury in immunodeficient mice(NOD-Prkdc^em26Il2rg^em26/Nju,NCG),and to detect human fetal hepatocyte repoppulation in Jo2 treated NCG mice;the aim 2 are to reconstitute functional human immune systems in human fetal hepatocyte engrafted NCG mice and to compare their immune cell levels to those mice with human immune cell reconstitution only;the aim 3 are to in vitro test a feasible strategy（i.e.whether ERT2 can regulate uPA function in uPA-ERT2 fused construct）,and to generate NOD/SCID mice transgenic expressing uPA-ERT2 under control of mouse albumin promotor（NOD/SCID-uPA-ERT2 mice）and in vivo test the regulation of uPA activity and function by ERT2.MethodsCharacterizations of the Jo2 induced NCG mouse liver injury by ALT level and H&E histology analysis,of human fetal hepatocyte repopulation in Jo2 treated NCG mice by IHC staining and qPCR analysis,of human immune cell levels in humanized mice repopulated with human immune system without or with human hepatocyte engraftment by Flow cytometry,IHC and muti-color IHC staining,of the expression of human cytokines and chemokines in human hepatocytes repopulated in NCG mice by qPCR,of human immune cell survival and expansion cultured with or without human hepatocytes by cell counting and Flow cytometry.Characterizations of uPA functions regulated by ERT2 trough 4OH-T by cell migration assay and Urokinase activity assay.Mouse albumin promoter/enhancer-uPA-ERT2-pA construct was inserted into Tol2transposon vector.This qualified and extracted fragment,along with Tol2 transposase mRNA were injected into NOD/SCID zygotes.The uPA-ERT2 expression in NOD/SCID-uPA-ERT2 mouse was assessed by RT-PCR and qPCR,liver injury by ALT level and H&E histology,in vivo induction of uPA activity in these mice by oral gavage of Tamoxifen.ResultsTreatment with Jo2 at different doses 0,0.2,0.4,0.9mg/kg led to respectively 20±3,88±27,521±83,3044±1788 U/L（mean±SEMs）of ALT in the serum of NCG mice within 3 hours,HE analysis indicated the mice underwent different degree of liver damage.Jo2 induced hepatocyte apoptosis mainly concentrated around the veins.Treatment with 0,0.3,0.4,0.5mg/kg Jo2 led to 100%,100%,67%and 33%respectively survival rates of mice within 48 hours.2 time（interval 24 hour）injections of Jo2 at0.3mg/kg dose led to durable mouse liver injury.Long term injections of Jo2 at0.3mg/kg/3d for 10weeks did not affect the mouse body weight changes.Human fetal hepatocytes were detected migration from spleens to livers of NCG mice at early 24hour post-transplantation.Human albumin was detected durable secretion in the human fetal hepatocyte engrafted NCG mice underwent Jo2 treatment,demonstrating efficient repopulation of human hepatocytes in the mouse livers,which was further evidenced by IHC staining.qPCR analysis for the expressions of human hepatocyte specific metabolic genes including CYP1A2（Cytochrome P450 1A2）,CYP2D6（Cytochrome P450 2D6）,UGT1A1（Bilirubin UDP-glucuronosyltransferase 1-1）,Transpotors MRP2（Multidrug resistance-associated protein 2）and NTCP（Na+/taurocholate Cotransporting）in human fetal hepatocyte-repopulated NCG mouse liver tissues,human fetal liver tissues and adult liver tissues indicated that human fetal hepatocytes in NCG mouse livers developed toward,but still did not mature as,human adult hepatocytes.We next constructed LTH humanized mice by transplantation of human fetal thymus tissues and fetal liver derived CD34⁺HSPCs into human fetal hepatocyte engrafted NCG mice（termed LTH hu-mice,liver chimeric FTHY/HSPC humanized mice）,of which human hepatocyte repopulation was evidenced by durable human albumin secretion and IHC staining.The levels of CD45⁺human immune cells in periphery blood and spleens,and the percentages of CD3⁺CD4⁺T,CD3⁺CD8⁺T,CD19⁺B,and CD14⁺monecyes/macrophages in the spleens of LTH mice,were comparable to those in TH hu-mice（Thymus/HSPC humanized mice）,whereas the human immune cells including CD68⁺macrophages,CD11c⁺cells（likely DCs）and CD94⁺NK cells in the livers of LTH hu-mice were significantly improved.Notably,human immune cells,including Kupffer cells,dendritic cells and natural killer cells,were shown to be closely colocalized with human hepatocytes in the livers of LTH hu-mice.In addition,human hepatocytes engrafted in the mouse livers were found to produce IL-3,IL-15,GM-CSF,M-CSF,MCP-1,CXCL-1,and CXCL-10,which are known to be important for immune cell development,differentiation,tissue migration and retention,and have no or poor cross-reaction between humans and mice.Furthermore,human hepatocytes were able to support human immune cell survival and expansion in an in vitro co-culture assay.The 4T1-uPA cells migrated faster than both 4T1-GFP and 4T1-uPA-ERT2 cells,whereas the migration widths of 4T1-uPA-ERT2 and 4T1-GFP cells were comparable,suggesting that uPA-ERT2 did not show significant uPA activity to promote cell migration（maybe uPA activity was repressed by ERT2）.When treated with 4OH-T,the migration widths of 4T1-uPA-ERT2 cells become similar to 4T1-uPA cells,both of which were significantly higher than 4T1-GFP cells,indicating that the uPA-ERT2exhibited similar physiological effects to uPA under 4OH-T treatment.Although 4OH-T inhibited ER-negative 4T1 cell migration（including 4T1-GFP,4T1-uPA,and 4T1-uPA-ERT2 cells）,the 4T1-uPA-ERT2 cell migration was comparable between with MOCK and with 4OH-T treatment,demonstrating that the 4OH-T treatment counteracted the migration inhibition by 4OH-T in 4T1-uPA-ERT2 cells,and promoted its migration.Furthermore,the uPA activity of 293FT-uPA-ERT2 cells treated with4OH-T,both in supernatant and cytoplasmic crude proteins,was dramatically higher than those treated with MOCK control,and the 4OH-T mediated increasing of uPA activity of uPA-ERT2 was dose dependent.Collectively,these data demonstrated that ERT2 can regulate uPA activity and function through 4OH-T.Futher,NOD/SCID-uPA-ERT2 mice were generated（20 F0 NOD/SCID mice were positive for uPA-ERT2 by PCR,random 10 of them were further verified by DNA sequence）.RT-PCR showed uPA-ERT2 expression was specific in the livers of the NOD/SCID-uPA-ERT2 mice.These mice had mild and chronic liver injury,which may due to the leak of uPA activity in uPA-ERT2 fusion.The uPA activity of hepatocyte culture isolated from NOD/SCID-uPA-ERT2 mice was improved upon treatment with 4OH-T.Furthermore,treatment with Tamoxifen improved the uPA activity and ALT level in the serum of NOD/SCID-uPA-ERT2 mice.ConclusionHuman fetal hepatocytes can effectively repopulate in the livers of Jo2 treated NCG mice,they develop toward,but still do not mature as,adult human hepatocytes.Human hepatocyte repopulation improves the immune cell reconstitution in the liver,but not in the peripheral blood and spleens,of humanized mice.Human hepatocyte repopulated in mice can express a series of cytokines and chomkines.Human hepatocytes support survival and expansion of human immune cells in vitro.Through in vitro,ex vivo and in vivo experiments,we demonstrated that ERT2 can regulate uPA activity and function via 4OH-T/Tamoxifen.The liver injury of NOD/SCID-uPA-ERT2mice can be further induced by Tamoxifen treatment.NOD/SCID-uPA-ERT2 mice may be nicer hosts for human hepatocyte repopulation.