Development Of Mouse Immune System Affects Susceptibility Of EV71

IntroductionEnterovirus 71(enterovirus 71,EV71)is a common causative agent of hand,foot,and mouth disease(HFMD)and complications of severe nervous system,which is mainly prevalent in the asia-pacific region.The HFMD caused by EV71 infection may lead to serious complications,including myocarditis,acute flaccid paralysis,aseptic meningitis,brain stem encephalitis,neurogenic pulmonary edema,and even death.EV71 mainly infects children under 5 years old and infants,usually adults are not susceptible.In the mouse model,EV71 was only susceptible to the death of normal mice under 14 days old and was not susceptible to mice aged 14 days or above.With the development of different mice adapted strains and the improvement of different infection methods,the age of EV71 infected mice increased.The age of mouse model is increasing.However,the age of mice was still the key factor limiting the EV71 susceptibility in mice.It is speculated that the development of mice has a great influence on EV71 susceptibility.In the process of the development of mice after birth,there are changes in each system,including metabolism,amount and type of symbiotic microbes,the structure and function of tissues and organs.Among these changes,there are factors affecting EV71 susceptibility in mice.ObjectiveIt is generally believed that innate immunity plays an extremely important antiviral role in the process of EV71 infection.Therefore,it is speculated that the maturity of the immune system plays an important role in various factors affecting EV71 susceptibility in the development process.In immune organs,intestines as the second largest immune organ,exercise important immune function.At the same time,intestines are in the process of natural infection EV71 virus first infected organs,are important target organs at early stage of infection EV71 virus replication.Therefore,this study focused on analyzing the intestinal immune development in mice of different age,with a view to finding the key factors affecting EV71 susceptibility and disease progression in the course of intestinal development.By organizing RNA group analysis,we speculated that one of the immune function pathways associated with ILC3s played an important role in EV71 infection.At the same time,this study used EV71 mouse infection model of different age to verify this hypothesis.MethodsModel:To establish a better EV71 infection mouse model.First,the EV71 virus isolated from the clinical patients was infected by intraperitoneal injection to infecte mice,and the clinical symptoms of EV71 infected mice were observed.The distribution and replication dynamics of EV71 virus in mouse tissues were detected.The death model of BALB/c mice aged 15-18 days was established and the model of eye disease infection of 19-21 days was established.The death rate was 100%within 7 days after the infection of 15-18 days of mice,all of which showed weight loss,paralysis of hind limbs and inflammation of the eyes.No death was found in the aged mice aged 19-21,and the infection was shown to be vertical hair and inflammation of the eyes,which can be detected in the eye mucosa for 5 days after infection.Immune development analysis:The immune development of spleen,lymph nodes,thymus,intestine and bone marrow in 1,7,14,18 and 21-day-old mice were analyzed by tissue pathology,flow cytometry and tissue mRNA and IncRNA high-throughput sequencing analyze.The basic data collection of immunological development after birth of mice was established.The results of histopathological examination in mice showed that the proportion and number of bone marrow cells in 18 days were close to adult mice.There were obvious cortical areas and medullary demarcation in the thymus of 7 days old mice,and the structure was close to maturity.The spleen of 7-day-old mouse began to appear in the peripheral lymphatic sheath and spleen,the boundary was not obvious.The lymphatic sheath and splenic junction in the spleen arteries of the 14-days-old mice showed obvious boundary.7-day-old mice started to submucosal lymphoid nodules of small intestine in mice,small intestine of 14-day-old mice started to set lymph nodules,21-day-old mice set lymph nodules appear kind of germinal center structure,but not mature.The lymph nodes in the 14-day-old mouse began to appear in the lymph nodes,and the lymph nodes in the 18 to 21-days were close to maturity,but there was no growth center.Flow cytometry analysis results showed that in the spleen,lymph nodes,bone marrow and thymus,comparison between different day age,B cells,alpha,beta,gamma delta T cells,T cells DC cells,NK cells,the proportion is similar.Among them,the proportion of B cells in the spleen was the largest,with the largest proportion of CD4+T cells in the thymus and lymph nodes.The proportion of immune cells detected in the small intestine of 1 to 7 days old mice was similar.The percentage difference of various immune cells in the small intestine of 14 to 21 days old mice increased gradually with the increase of day age.The high-throughput sequencing analysis of small intestinal tissue RNA in mice showed that the expression of small intestine RNA in 1 day,7 and 14 days old mice and 18 days old and 21 days old mice showed different patterns.It was proved that the gene expression of small intestine in mice was different in 14 days,and the development of small intestine in mice may enter a new stage.Key immune factors analysis:Through the analys on mice immune organ and cell development after birth,found in the age of 7,14,18 and 21 in mice,the organizational structure of the small intestine and the proportion of immune cells have a more significant change.Furthermore,high-throughput sequencing analysis of small intestine RNA before and after infection of mice with different day age mice was found to have significant differences in the expression of cytokine gene expression associated with ILC3s.Therefore,it is speculated that there may be a functional pathway or functional network centered on ILC3s to play an important role in the process of EV71 infection.Through the analysis of the relative expression levels of flow cytometry and cytokine RNA,it was found that ILC3s and its related cytokines showed a significant change in correlation with the age of mice before and after infection.After EV71 infection of mice,the ILC3s loss rate and IL-22 expression increased multiple were positively correlated with the rate of death rate of mice,the rate of increase of clinical scores and the degree of histopathological damage.It is proved that ILC3s and its related cytokines IL-22 play an important role in EV71 infection.Prove the existence of the ILC3s centered at the same time,the beta,IL-1,IL-23,ILC3s,IL-22,TNF α,small intestinal mucosa epithelial cells and IL-10 of network function,and the function of network in EV71 infection disease pathology play an important role in the process.ResultsModel:The main clinical manifestations of EV71 infection were weight loss,paralysis of hind limbs,eye lesions and death.The lethal model of BALB/c mice of 15-18 days and the infection model of 19-21 days old were established for the first time by constructing the mouse adaption strain of EV71 clinical isolates.The death rate was 100%in mice aged 15-18 days after infection,all of which showed weight loss,paralysis of hind limbs and inflammation of the eyes.The mouse model of 19-21 days was not dead,and the infection was shown to be vertical hair and inflammation of the eyes,and the eye mucosa was detected within 5 days after infection.Immune development analysis:In this study,the immune development process of BALB/c mice from 1 to 21 days old was observed,and the basic data collection of post-natal immune development was established.The results of histopathological examination in mice showed that the proportion and number of bone marrow cells in 18 days were close to adult mice.There were obvious cortical areas and medullary demarcation in the thymus of 7 days old mice,and the structure was close to maturity.The spleen of 7 days old mouse beganed to have the peripheral lymphatic sheath and splenic corpuscle,the boundary was not obvious.The lymphatic sheath and splenic junction in the spleen of the 14 days old mice showed obvious boundary.The lymphatic sheath and splenic corpuscle of the 18 days old mice were close to maturity.The small intestine of 7 days old mouse beganed to have submucosal lymph nodes.The small intestine of the 14 days old mouse beganed to have aggregated lymphoid nodule.In the 21 days old mouse,the the germinal cortex appeared in the aggregated lymphoid nodule,but not mature.The lymph nodes of the 14 days old mouse beganed to show obvious lymphatic nodules.The lymph nodes of 18-21 days old mouse were close to ripeness,but there was no germinal cortex.Flow cytometry analysis results showed that in the spleen,lymph nodes,bone marrow and thymus,comparison between different day age,B cells,α β T celld,γ δ T cells,DC cells,NK cells,the proportion is similar.Among them,the proportion of B cells in the spleen was the largest,with the largest proportion of CD4+T cells in the thymus and lymph nodes.The proportion of immune cells detected in the small intestine of 1 to 7 days old mice was similar.The percentage difference of various immune cells in the small intestine of 14 to 21 days old mice increased gradually with the increase of day age.The high-throughput sequencing analysis of small intestinal tissue RNA in mice showed that the expression of small intestine RNA in 1,7 and 14 days old mice and 18 and 21 days old mice showed different patterns.This shows that the gene expression of the small intestine of mice is different after the age of 14th day,and the development of the small intestine may enter a new stage.Key immune factors analysis:Through analysis on mice immune organ and cell development after birth,we found that in the age of 7,14,18 and 21 days in mice,the organizational structure of the small intestine and the proportion of immune cells have a more significant change.Furthermore,high-throughput sequencing analysis was performed on the small intestine RNA group before and after infection in mice with different age mice.We found significant differences in the expression of cytokine gene expression associated with ILC3s.Therefore,it is speculated that there may be a functional pathway or functional network centered on ILC3s which played an important role in the process of EV71 infection.Through the analysis of the flow and expression levels of cytokine relative RNA,it was found that ILC3s and its related cytokines showed a significant change in correlation with the age of mice before and after infection.After EV71 infection of mice,the ILC3s loss rate and IL-22 expression increased multiple were positively correlated with the death rate of mice,the rate of increase of clinical scores and the degree of histopathological damage.This proved that ILC3s and its related cytokines IL-22 play an important role in the process of EV71 infection.At the same time,this proved the existence of the ILC3s centered network,which composed of IL-1 β,IL-23,ILC3s,IL-22,TNF a,small intestinal mucosal epithelial cells and IL-10,and its important role in the process of EV71 infection.Conclusion1.This study constructed an adaptive strain of enterovirus 71(EV71)mice,demonstrating that viral genome variation has a great impact on its pathogenicity.It is very important to monitor the pathogen of enterovirus,especially EV71,because it is easy to mutate and reorganize.2.This study used mice adapted strains to construct multiple EV71 mouse infection models at different stages of development,which provided a rich research tool for EV71 related research.The lethal model of EV71 mice aged 15 to 18 days and the EV71 infection model of 19 to 21 days were constructed for the first time,which provided the possibility for the study of the interaction between postnatal and EV71.3.This study based on different day age mice immune organ tissue pathology and cytology analysis,found in EV71 infection in mice model in the process of building a few key points in time(7,14,18 and 21 days of age),the small intestine mucosa immune cells in the lymph node tissue structure and the small intestine under the proportion have a more significant change.4.Through the analysis of the flow and expression levels of cytokine relative RNA,it was found that ILC3s and its related cytokines IL-22 play an important role in the process of EV71 infection.At the same time,this proved the existence of the ILC3s centered network,which composed of IL-1 β,IL-23,ILC3s,IL-22,TNF a,small intestinal mucosal epithelial cells and IL-10,and its important role in the process of EV71 infection.IL-1 β,IL-23,IL-22,TNF a and IL-10 may be potential markers for the identification of severe cases.5.After the mice were born,the immune development affected the EV71 susceptibility and post-infection disease process.Innate immunity,especially the innate lymphoid cell group 3(ILC3),has important influence on the EV71 susceptibility and post-infection disease process in mice.