Investigation Of Immune Responses To Influenza A Virus Infection In Human Lung Using Humanized Mouse Models

Aims:Influenza viruses belong to the Orthomyxoviridae family and are divided into four types:A,B,C,and D.Influenza A viruses(IAVs)are the main cause of human influenza pandemic and pose a potential threat to public health.At present,the main preventive measure for IAVs is the application of influenza virus vaccines.However,due to antigen drift and antigen shift,this method is difficult to play a better protective role,and one kind of vaccine is difficult to neutralize different viral strains.Many studies have found that Tissue resident memory T cells(Trm cells)may have a certain role in resisting influenza virus infection.Therefore,research on T cell vaccines has gradually been carried out in recent years.The study of influenza A virus mainly depends on the mouse model,but there are some differences between the lung tissue structure and cell ratio of mouse and human lung,so the mouse lung may not fully reflect the situation in human lung.However,lung tissue samples of patients with mild to moderate symptoms of influenza virus are difficult to obtain.Most of the samples come from tissues adjacent to cancer and organ donation.These samples are difficult to carry out dynamic observation research on immune cells during influenza virus infection.Therefore,based on reconstructing human immune system(HIS)in NCG immunodeficient mice,we established HIS with autologous lung xenograft(HISL mice)model by subcutaneous transplantation of homologous HL tissue on the back of mice.Influenza A virus was inoculated into human lung implants of HISL mice to study the human immune response in human lung and various organs after infection,and to explore the important clinical significance of this model.Methods:(1)The HISL mouse model was established by transplanting human fetal thymus tissue under the renal capsule of NCG mice,subcutaneously transplanting homologous lung tissue(HL)on the back of NCG mice and injecting CD34~+hematopoietic stem cells derived from homologous liver via tail vein;(2)Inoculate transplanted human lung tissues of HISL mice with H1N1influenza virus and the control group was given PBS with the same volume to study the changes of immune cells,the production of specific antibodies,the characteristics of transcriptome between the two groups;(3)HISL mice were pre-vaccinated with H1N1 virus in subcutaneous HL,and then nasally inoculated with lethal dose of H1N1 virus to HISL mice to explore the protective effect of pre-vaccination on HISL after receiving lethal dose of H1N1nasal infection.Results:(1)After whole-body irradiated NCG mice underwent embryonic thymus tissue transplantation under the renal capsule,subcutaneous homologous embryo lung tissue transplantation,and tail vein injection of CD34~+hematopoietic stem cells derived from homologous embryonic liver,the HISL mouse model was successfully established.The chimeric ratio of human CD45~+cells of this model can reach more than 90%,with T cells and B cells as the main cells,while the proportion of myeloid cells and natural killer cells was relatively low.Human lung implants in mice can show complete alveoli and other structures like those in normal adult lung after the model was established successfully.(2)After H1N1 virus infects HL in HISL mice,the proportion of HLA-DR~+CD11c~+cells and CD20~+cells in human lungs of HISL mice in H1N1group was significantly higher than that in control group,and the levels of CD4~+Trm and CD8~+Trm in HL of H1N1 mice were significantly higher than that in PBS group,and their levels were positively correlated with the ratio of HLA-DR~+CD11c~+cells and CD20~+cells;The proportion of HLA-A*0201 restricted H1N1 antigen-specific CD8~+T cells in HL of HISL mice in H1N1 group was significantly higher than that in control group;H1N1-specific Ig and IgG antibodies were produced in the serum of H1N1 group;There are significant differences in the expression genes of the two groups of HL after transcriptome sequencing.The expression of H1N1influenza virus infection related and immune related genes in HL of H1N1 group was significantly up-regulated.(3)After a single inoculation of 5000TCID50 H1N1 virus in the lung implants of HISL mice,the mice died of nasal inoculation of lethal dose(5000TCID50)virus.After repeated inoculation of 500TCID50 H1N1 virus,HISL mice can still survive after lethal dose of virus inoculated through nose.Conclusions:(1)HISL mice model can be used to study human lung tissue and immune cells.(2)HISL mice can study the changes of immune cells in human lung tissue after influenza virus infection,and can further study the characteristics of T cell subtypes,genomic difference and the production of specific antibodies in serum.(3)Repeated low-dose H1N1 virus inoculation into the human lung implants of HISL mice can prolong the survival time when receiving lethal dose of virus infection,and improve the survival rate.In summary,for the first time this project established a mouse model of human lung tissue and immune system that can be used for influenza virus research,for the further study of changes in the immune system after infection,the characteristics of T cell phenotypes,and the production of specific antibodies and the differences in transcription levels.This model provides powerful tools and are of great significance for the development of vaccines and the research of other pulmonary infectious diseases.