Memory NK Cells And Influenza Virus Infection In Mice

The innate immune system is the first line of defense towards influenza infection,which plays an important role in clearing virus and in inducing adaptive immuneresponse.Natural killer(NK)cells are the important part of innate immune system.Influenza is a common respiratory disease that caused by influenza virus.Influenzaviruses grow in the respiratory epithelial cells and may initiate the innate immuneresponse.More and more evidences demonstrated that NK cells play a dual role ininfluenza virus infection:clearing and controlling virus or exacerbating lungimmune injury.Although NK cells are classified as innate immune cells,recently a growingnumber of studies demonstrate that NK cells have been shown to possess features ofadaptive immunity with immunological memory in a manner similar to that of T andB cells.NK cells can become long-lived memory cells and contribute to secondaryimmune responses.Three major viewpoints of NK cell memory initially arose fromthe studies of NK cell memory to recall to mouse cytomegalovirus(MCMV),cytokine and skin contact hypersensitive chemical antigens.Currently,NK cellmemory has been reported in acute infection of mouse HSV,HCMV and simianimmunodeficiency virus(SIV).Whether memory NK cells can provide protectionagainst subsequent influenza virus infection and the related immune mechanisms arepoorly understood.In this study,we built a mouse model of influenza virus intranasal infection andthen studied the phenotype and function of memory NK cells.The function andspecific of NK cells were determined by intracellular staining after stimulated byinactivated influenza.The viral titers in the lung were determined throughquantitative RT-PCR analysis and Madin-Darby canine kidney(MDCK)celldetermination assay.Adoptive transfer experiments were used to detect the function of memory NK cells.In Rag1-/-mice,we determined the generation and function ofmemory NK cells by intranasal infection of influenza virus and subcutaneousimmunized with inactivated influenza virus.The major results of our studied areshown as follows:1.Lung NK cells are activated rapidly after influenza virus infection.Firstly,the C57BL/6 mice were infected with influenza virus,and we found that lung NK cells were recruited to the lung and could be activated rapidly on day 3 after infection.The expressions of the activating receptors and molecules were increased and the inhibitory receptors were decreased.Accompanying with activation,the expression of functional molecule IFN-? of lung NK cells was increased.Furthermore,the level of cytokine IFN-a in BALF was increased on day 1 after infection.Thus NK cells were recruited to the lung and activated during influenza virus infection,raising a possibility that NK cells are involved in host antiviral immune response.2.Defects or depletion of NK cells results in delayed viral clearance.Next,to examine the function of NK cells in controlling influenza virus infection,we compared changes in lung histology,influenza virus titration and body weight in Nfil3-/-mice,NK depleted mice(AsGM1)and wild type mice during influenza virus infection.We found that defects or depletion of NK cells significantly delayed virus clearance and increased bodyweight loss.However,there was no significant difference in lung histopathology injury on day 3 and 7 after infection.Thus NK cells would eliminate virus after primary influenza virus infection.3.Memory lung NK cells mediate a modest protection against viral challenge.Further,we detected the expressions of memory makers on NK cells in lung on day 30 after infection,and found that NK cells expressed higher levels of memory markers Ly6c and KLRG1 and lower levels of CD62L.We stimulated lung lymphocytes with inactivated influenza virus,and observed that lung NK cells from PR8 infected-mice expressed higher levels of IFN-y compared with control mice.This suggested that lung NK cells from influenza virus-infected mice possess antigen-specific characteristics.Then adoptive transfer of lung NK cells from influenza virus-infected mice to naive C57BL/6 mice showed a slightly slow weight loss,and the survival rate of the recipient mice was not significantly increased after PR8 infection.Therefore,lung NK cells from influenza virus-infected mice possess a memory-like phenotype but mediate a modest protection against viral challenge.4.Liver NK cells are activated rapidly after influenza virus infection.Similarly to lung NK cells,we found that liver NK cells could be activated rapidly.The expression of functional molecule IFN-?of liver NK cells was increased on day 3 after infection.However,why influenza virus could activated NK cells in the liver where couldn't detect any influenza.Increased cytokines expression like IFN-a were induced during influenza virus infection and these cytokines were likely enter the liver through the bloodstream to activate NK cells.5.Memory liver NK cells mediate an effective protection against viral challenge.To determine whether PR8 activated liver NK cells could memory influenza virus,we analyzed the phenotype and function of liver NK cells using flow cytometry and stimulated with inactivated PR8.We found that liver NK cells displayed a memory-like phenotype on.day 30 after infection.Functional analysis showed that liver NK cells from PR8-infected mice possess antigen-specific characteristics stimulated with inactivated PR8.Importantly,the adoptive transfer of liver NK cells from PR8-infected mice protected recipient mice against PR8 infection-induced weight loss and death.These results suggested that respiratory influenza virus infection induces liver memory-like NK cells,which protects mice against influenza virus infection-induced death.6.Liver DX5-CD49a+NK cells memory of influenza virus The results showed that liver DX5-CD49a+ NK displayed a memory-like phenotype and increased expression of IFN-?.Further liver DX5-CD49a+ memory NK cells expressed higher genes involved in immune memory(Hopx).The adoptive transfer of liver DX5-CD49a+ NK cells from PR8-infected mice to Rag1-/-mice significantly decreased the viral titer in the lungs of recipient mice.Furthermore,the adoptive transfer of liver DX5-CD49a+NK cells from PR8-infected mice to naive C57BL/6 mice protected recipient mice against PR8 infection-induced death.So,these results demonstrated that liver DX5-CD49a+ NK cells were the primary source of memory NK cells against influenza virus.7.Inactivated PR8 induced memory NK cells in Rag1-/-mice.In this part,we found that primary inactivated PR8 protected Rag1-/-mice against secondary PR8 infection-induced death and decreased the viral titer in the lungs.Then,compared Rag1-/-Nfil3-/-and Rag1-/-tbx21-/-mice,we demonstrated that liver DX5-CD49a+ NK cells plays important role in the recall response to influenza virus after immunized with inactivated influenza.Taken together,these results suggest that generation of memory-like NK cell was T cell-and B cell-independent and influenza virus-specific memory-like NK cells of Rag1-/-mice resided in the livers.In conclusion,we used a well-established mouse model of respiratory influenza virus infection to characterize influenza virus-specific memory-like NK cells and observed that liver-resident DX5-CD49a+ NK cells but not lung NK cells possess a memory-like phenotype and protect mice against influenza virus reinfection.Thus,our study will provide an adequate platform to investigate the mechanisms underlying the generation,migration and function of memory NK cells.