The Relationship Between The Difference Of Immune Reactions Against MCMV And The Infectious Outcomes In BALB/c And C57BL/6 Mice

Background Human cytomegalovirus(HCMV)is widespread in the world.Although HCMV infection is usually asymptomatic in immunocompetent individuals,it causes serious consequences in immunocompromised individuals including transplant recipients and infants suffering intrauterine infection.Mice infected with MCMV are widely used to study the mechanism of HCMV infection.Similarly,the susceptibility to MCMV also varies among different mouse strains.MCMV infection in BALB/c mice is serious and usually ends up with chronicity,while C57BL/6 mice are resistant to MCMV and capable of removing virus in acute infection period.The different infection outcomes are usually attributed to specific Ly49H-m157 pathway of NK cells in C57BL/6 mice.Nevertheless,considering the huge genome of MCMV and complicated antiviral responses in vivo,other factors are probably involved as well.As an important component of innate immunity,NK cells can recognize pathogens and defend against them immediately.Cytotoxicity of NK cells and IFNγ+NK cells play an important role in this process.NK cells of C57BL/6 mice make a significant contribution to resisting infection;relatively,although innate immunity of BALB/c mice plays a role in eliminating infection to some degree,it is far from enough.The spleen is a downstream central lymphoid organ and a common site of MCMV infection,which adopts various immune strategies to resist infection.The spleen contains abundant immune cells such as NK cells,dendritic cells(DCs)and T cells.NK cells can influence the function of CD8 T cells.The roles of IFNγ,IL10 and IL18 in antiviral responses of MCMV infection have been reported as well.So we detect the function of NK cells and m RNA levels of related inflammatory factors in BALB/c and C57BL/6 mice after MCMV infection,trying to find the difference between them and analyzing the relationship with the different outcomes of MCMV infection.Absent in melanoma 2(AIM2)inflammasome is a crucial link bridging the innate host defense and the subsequent adaptive immunity when activated by double stranded DNA(ds DNA).Once activated,AIM2 recruits apoptosis-associated speck-like protein(ASC)containing a caspase recruitment domain to cleave pro-caspase-1 and finally converts pro-IL1β and pro-IL18 into their mature molecule,IL1β and IL18.Through the production of the two important proinflammatory cytokines,AIM2 inflammasome is crucial for innate and subsequent adaptive immune response during various virus infections including MCMV.AIM2 inflammasome may have different activation status between BALB/c and C57BL/6 mice,which relates to the antiviral responses and infection outcomes and finally contributes to the chronic MCMV infection.Objectives: 1.Through building models of MCMV infected mice,we focuse on the antiviral mechanism of NK cells in spleens of BALB/c and C57BL/6 mice,analyzing the difference between them.We further evaluate the m RNA levels of inflammatory factors and pathological changes in spleens of the two mouse strains.Through analyzing the difference in antiviral responses,we try to find the relationship between antiviral functions and different outcomes of MCMV infection in BALB/c and C57BL/6 mice.2.Through building models of disseminated MCMV infection in BALB/c and C57BL/6 mice,we evaluate their dynamic expressions of AIM2 inflammasome components and the relationship with pathological damage and viral replication,trying to figure out whether AIM2 inflammasome relates to the chronic mechanism of MCMV.Methods: Part 1: The effects of IL10 and NK cells on the susceptibility to MCMV in BALB/c mice despite compensation of IFNγ 1.Building models of MCMV infection in BALB/c and C57BL/6 mice: female BALB/c and C57BL/6 mice(five weeks old)received intraperitoneal injection of 5×104 PFU MCMV Smith.Mice of the control group received the same volume of DMEM medium.2.Mice were sacrificed on day 0,1,3,7,14 and 28 post infection and spleens were separated from mice sterilely.NK cells were enriched from splenocytes using a negative-selection NK Cell Isolation Kit.Cytotoxicity of NK cells was determined by detecting lactate dehydrogenase(LDH)contents.Flow cytometry was used to detect activated NK cells,IFNγ+ NK cells and total NK cells in spleens of each group.3.Total RNA of spleens was extracted and c DNA was synthesized as well.q RT-PCR was performed to detect m RNA levels of Thpok,IFNβ,IL10,IL18 and IFNγ in spleens.4.The pathological damage of spleens in each group was evaluated after HE staining,and livers was also evaluated according to the instruction of Knodell histological activity index(HAI).5.q RT-PCR was used to detect m RNA levels of MCMV g B in spleens,livers and salivary glands.The viral titer of MCMV in salivary glands was also detected by standard plaque assay.Part 2: The short-lived AIM2 inflammasome activation relates to the chronic MCMV infection in BALB/c mice 1.Building models of disseminated MCMV infection in BALB/c and C57BL/6 mice,methods were the same with the former.2.BALB/c and C57BL/6 mice were sacrificed on day 0,1,3,7,14 and 28 post infection.Expressions of AIM2,pro-caspase-1,caspase-1 p20,pro-IL1β and mature IL1β in primary peritoneal macrophages(PMs)and spleens were detected by Western blot.3.Levels of IL18 in serum of BALB/c and C57BL/6 mice were assessed by ELISA.4.Expression levels of p202: Livers of BALB/c and C57BL/6 mice were sectioned.Then we assessed the m RNA levels of p202,which was negative related to AIM2 inflammasome.5.The pathological damage and viral loads of organs were the same with the former.Results: Part 1: The effects of IL10 and NK cells on the susceptibility to MCMV in BALB/c mice despite compensation of IFNγ 1.Spleen NK cells of BALB/c mice possessed more potent cytotoxicity on day 1,3 and 7 post infection.Cytotoxicity of NK cells in C57BL/6 mice was enhanced on day 1,3,7,14 and 28 post infection.Except on day 3,NK cells of C57BL/6 mice had much more powerful cytotoxicity than BALB/c mice in MCMV infection.2.In BALB/c mice,activated NK(CD69+NK)cells increased on day 1,and declined afterwards.In contrast,activated NK cells of C57BL/6 mice were more than the control group on day 1,3 and 28 post infection.Compared with BALB/c mice,activated NK cells of C57BL/6 mice were higher on day 3,7,14 and 28 post infection.3.When infected with MCMV,IFNγ+NK cells in BALB/c mice did not increase and were even less than the control group at the late stage of infection.When it came to C57BL/6 mice,IFNγ+NK cells increased on day 1 and day 3.Compared with BALB/c mice,C57BL/6 mice had more IFNγ+NK cells during MCMV infection.4.C57BL/6 mice had more spleen NK cells than BALB/c mice during the whole infection.5.BALB/c mice expressed more Thpok than the control group on day 1,3,7 and 28 post infection,while Thpok in C57BL/6 mice maintained a lower level.BALB/c mice had increased IFNβ only on day 1,lower than the control group subsequently.In contrast,C57BL/6 mice expressed more IFNβ on day 3,7 and 28.Compared with C57BL/6 mice,BALB/c mice contained more Thpok after infection.For IFNβ,BALB/c mice had more advantage on day 1 and day 3,and then declined,less than C57BL/6 mice on day 7,14 and 28.6.BALB/c mice had increased IFNγ on day 1,3 and 7 post infection,while IL18 only increased on day 1.As for C57BL/6 mice,IFNγ merely increased on day 3,and then decreased.IL18 did not increase and was even less than the control group later.When compared with C57BL/6 mice,it was obvious that BALB/c mice expressed more IFNγ and IL18 during the infection.The expression tendency of IL10 in each group of BALB/c mice was similar to C57BL/6 mice.Both of them expressed more IL10 on day 3,and declined later.Comparison of the two mouse strains indicated a dynamic process of IL10 production,which was similar to the expression of IFNβ.7.The pathological damage of spleens and livers in BALB/c mice was more serious than C57BL/6 mice.8.The more viral loads in spleens,livers and salivary glands of BALB/c mice also indicated the severer virus dissemination than C57BL/6 mice.Part 2: The short-lived AIM2 inflammasome activation relates to the chronic MCMV infection in BALB/c mice 1.Primary peritoneal macrophages: BALB/c mice expressed more AIM2 on day 1 and day 7 post infection.Pro-caspase-1 and pro-IL1β were constitutively expressed in BALB/c mice,while caspase-1 p20 and mature IL1β increased on day 7.As for C57BL/6 mice,contents of AIM2 in PMs increased on day 3,7 and 14 post infection.C57BL/6 mice showed a similar expression of pro-caspase-1 and pro-IL1β to BALB/c mice.Notably,the increased caspase-1 p20 and mature IL1β of C57BL/6 mice appeared on day 7 and continued into the day 28 post infection,which was obviously different from BALB/c mice.2.Spleens: Contents of AIM2 in BALB/c mice increased on day 1 and day 3 post infection.The expression of AIM2 reduced later,showing no difference to the control group.Unlike peritoneal macrophages,expressions of pro-caspase-1 and pro-IL1β varied during MCMV infection.Levels of caspase-1 p20 increased on day 1 and day 3 post infection.The mature form of IL1β(IL1β p17)increased on day 7.As for C57BL/6 mice,contents of AIM2 increased on day 3,7,14 and 28 post infection.C57BL/6 mice showed a similar expression of pro-caspase-1 and pro-IL1β to BALB/c mice.The increased caspase-1 p20 and mature IL1β in C57BL/6 mice appeared on day 7,14 and 28 post infection.3.In serum of BALB/c mice,IL18 only increased on day 3 post infection and held a relatively stable content in all other groups.In sharp contrast,expressions of IL18 in C57BL/6 mice were enhanced during the whole process of infection,which started on day 1 and showed a double peak curve.4.Livers of BALB/c mice contained a certain level of p202 m RNA during MCMV infection,while expressions of p202 in C57BL/6 mice were too low to detect.5.The pathological damage and viral loads of organs were the same with the formerConclusions: 1.Spleen NK cells of C57BL/6 mice worked effectively to eliminate infection.At the same time,the appropriate expression of IL10 made the resistance more efficient.2.As for BALB/c mice,cytotoxicity of NK cells in BALB/c mice only took some effect during the early period of infection,which was far from enough.Although the increased IFNγ of BALB/c mice compensated to some degree,the combined effect was still insufficient.3.The percentages of Ly49H+CD69+/IFNγ+NK cells in CD69+/IFNγ+NK cells of C57BL/6 mice only increased at the early stage of infection.And even at this moment,the percentages were still no more than 50%.We did not see much increase at the later stage of infection.In our study,C57BL/6 mice had a persistent advantage over BALB/c mice in resisting MCMV,which implied the noteworthy role of NK cells without the Ly49H-m157 pathway.4.Comparing with the two mouse strains,the activation of AIM2 inflammasome in BALB/c mice lasted no longer than 7 days post infection.C57BL/6 mice held a longer duration of AIM2 inflammasome activation,which could continue to the day 28 post infection.5.Levels of BALB/c mice contained much more p202 than C57BL/6 mice,which could suppress the activation of AIM2 inflammasome.6.The activation of AIM2 inflammasome in BALB/c mice was short-lived,which quite possibly related to the chronicity of MCMV infection.