The Immunomodulatory Function Of Bone Marrow-derived Mesenchymal Stem Cells In NK Cells During Liver Injury

Backgroud and Objective:The liver is the largest solid organ in the body and enriched in a variety of immune cells which interact with each other to modulate the innate and adaptive immunity.Emerging evidence has shown that the liver plays a key role in the innate immune defense against pathogens.More importantly,the lymphocytes are selectively enriched in the liver including natural killer(NK)cells which play a critical role in the defense against invading pathogens,modulation of liver inflammation and recruitment of circulating lymphocytes.Mesenchymal stem cells(MSCs)have been used in clinical studies due to their multiple differentiation potentials.During the last decade,there is a growing realization that MSCs also have immunomodulatory capacity which can suppress the proliferation,differentiation and function of T cells,B cells,NK cells,thus can be used in the prevention and cure immune diseases.However,it is still unknown how MSCs may affect immunity,especially NK cells during liver injury.This study was designed to explore the effect of bone marrow-derived MSCs(BM-MSCs)on hepatic NK cells in polyinosinic-polycytidylic acid(PolyI:C)-induced and hepatitis B virus(HBV)-induced liver injury.Methods:1.Mouse model of PolyI:C-induced liver injury:Male C57BI/6 mice,6-8 week-old.BM-MSCs(1×106)and an identical volume of PBS were injected into mice(n = 6/group)via the tail vein.After 24 hours,mice were intraperitoneally injected with PolyI:C at dose of 20μg/g body wt.Blood and liver tissue were harvested 24 hours after PolyI:C injection.2.Acute HBV infection mouse model:Male BALB/c mice,6-8 week-old BM-MSCs(1×106)were injected into mice(n = 6/group)through tail vein,24 h before hydrodynamic injection with pHBV1.3(a pUC18 vector containing 1.3-fold overlength HBV genome)or pUC18(10μg)within 6 s.Subsequently,mice were sacrificed after 4 or 7 days.The serum samples and liver tissues were collected for further analysis.3.Isolation and culture of primary cells;Identification of human BM-MSCs and mouse BM-MSCs according to their characteristic of phenotype.The liver MNCs were incubated with anti-NK1.1 and anti-CD69 to determine the number and activation of NK cells by flow cytometric analysis.4.The mRNA and protein levels of sphingosine-1-phosphate receptor type 5(S1PR5)and natural-killer group 2(NKG2D)on NK cell were measured by real-time PCR and western blot to analysis s;Hepatitis B surface antigen(HbsAg)and hepatitis B e antigen(HbeAg)were measured with enzyme-linked immunosorbent assay(ELISA)kit;HBV DNA was quantitated using PCR kit.The liver tissues were stained with hematoxylin and eosin or incubated with anti-HBc antibody to determine the morphologic changes and HBV core protein.Cell cytotoxicity was determined by the cell counting kit-8(CCK8).NK cell migration was measured using transwell assay.Results:1.BM-MSC treatment ameliorated PolyI:C-induced liver injury.The mechanism maybe,at least in part,the suppression of NK cell recruitment and activation caused by BM-MSC administration.2.BM-MSC treatment suppressed NK cell migration by reducing the elevation of sphingosine-1-phosphate receptor type 5(S1PR5)in the liver.3.In vivo imaging system showed that BM-MSCs were accumulated in the injured liver caused by pHBV1.3 injection.Further analysis revealed that they attenuated immune-mediated liver injury.4.BM-MSC-derived transforming growth factor-β1(TGF-β1)suppressed natural killer group 2,member D(NKG2D)expression on NK cells.As a consequence,the cytotoxicity of NK cells were also suppressed5.The results showed that BM-MSC treatment enhanced HBV gene expression and replication in vivo,suggesting the high risk in the clinical application.Conclusions:Our results demonstrated that adaptive transfer of BM-MSCs suppressed NK cell recruitment and activation in the liver,and thus ameliorating PolyI:C-induced liver injury.BM-MSCs also suppressed the cytotoxicity of NK cells by reducing NKG2D expression Consequently,BM-MSC administration attenuated immune-mediated liver injury and enhanced HBV gene expression and replication.These results further support the immunomodulatory function of MSCs and provide a theoretical basis for the therapeutic potential of BM-MSCs in immune-mediated liver injury.