Study On Neonatal Respiratory Virus Infection Combined With Allergen Sensitization Promotes Childhood Allergic Asthma

Objective:Asthma is one of the most common chronic diseases in children.Investigation shows that infants with respiratory syncytial virus（RSV）or rhinovirus（RV）infection are the most important risk factors for childhood allergic asthma,and the mechanism is still unclear.RSV is the most important cause of lower respiratory tract infections in infants.More than 80%of children with bronchiolitis 3 to 6 months after birth are caused by RSV infection,therefore,the relationship between RSV infection and allergic asthma in children deserves special attention.Like human RSV,mouse pneumonia virus（PVM）belongs to the family Paramyxoviridae,Pneumovirus,although there is almost no direct amino acid sequence homology between the proteins encoded by the two viruses,PVM has a strong replication ability,and inoculation of a small amount of virus can cause symptomatic diseases,and PVM-infected mice can replicate most of human RSV infection symptom.In this study,PVM was used to infect newborn mice,and an allergen chicken ovalbumin（OVA）was used to stimulate the establishment of animal models in early childhood to study the mechanism of infection with respiratory viruses in childhood to promotes childhood allergic asthma.Methods:1. Culture and titration of PVM:PVM virus was cultured in 6-week-old female BALB/c mice and Suckling hamster kidney cells（BHK-21）were cultured for titer determination of PVM.2. Whether neonatal infection of PVM combined with OVA promotes childhood asthma.2.1 Experimental animal grouping,sensitization and challenge:BALB/c suckling mice born 3-5 days（recorded as day 0）were ran domly divided into 4 groups:PBS,PBS-OVA,PVM and PVM-OVA gr oups;sensitization by nasal inhalation on days 0,3,and 4,and OVA c hallenge by nasal inhalation on days 21-25 and then the correspondingd etection was carried out.2.2 Lung function tests:airway resistance and lung compliance.2.3 Mice lung tissues were taken for pathological section H&E staining,and the m RNA expression levels of IL-1,IL-5,IL-6,IL-13,IL-17,TNF-α,IFN-γwere detected by fluorescent quantitative PCR.3. Whether neonatal infection of PVM combined with OVA induces the formation of pulmonary Trained CD11b⁺cells.3.1 Experimental animal grouping,sensitization:BALB/c suckling mice born 3-5 days（recorded as day 0）were randomly divided into 2 groups:PBS、PVM-OVA group;sensitization by nasal inhalation on days 0,3,and 4,the single lung cells were detected by flow cytometry on the 21st day.3.2 Detection of lung cells surface markers CD11b,CD11c,Siglec F by flow cytometry.3.3 Mouse lung CD11b⁺cells were sorted by magnetic beads and stimulated by OVA/LPS for 5 h in vitro,then the surface markers CD69 and TLR4 were detected by flow cytometry,the relative expression of cytokines IL-1,TNF-α,MCP-1 and GRO-αwere detected by fluorescence quantitative PCR.4.Whether the adoptive transfer of Trained CD11b⁺cells affects the occurrence of allergic asthma.4.1 Experimental animal grouping,sensitization and challenge:After 21 days in PVM-OVA group,Trained CD11b⁺cells were sorted by magnetic beads and transferred to normal mice of the same age by tail vein injection and challenged for 5 consecutive days with nasal inhalation after 24hours,and then the corresponding detection was carried out.4.2 Lung function tests:airway resistance and lung compliance.4.3 Mice lung tissues were taken for pathological section H&E staining,and the relative expression levels of IL-1,IL-5,IL-13,IL-17,TNF-α,IFN-γwere detected by fluorescent quantitative PCR.5.Whether neonatal infection of PVM combined with OVA influences the formation of Trained CD11b⁺cells by affecting cell metabolic pathways.5.1 Experimental animal grouping,sensitization and challenge:BALB/c suckling mice born 3-5 days（recorded as day 0）were ran domly divided into 3 groups:PVM-OVA+Rapamycin、PVM-OVA+BPTE S and PVM-OVA group:sensitization by nasal inhalation on days 0,3and after 2 hours,Rapamycin or BPTES inhibitors were injected intrap eritoneally,only nasal inhalation immunization was performed on the 4t h day,and nasal inhalation challenge was performed on 21-25 days.Sin gle lung cells were detected by flow cytometry.5.2 Pulmonary CD11b⁺cells were detected by flow cytometry.6.Whether to inhibit allergic asthma by inhibiting the production of Trained CD11b⁺cells by inhibiting cell metabolic pathway6.1 Experimental animal grouping,sensitization and challenge:BALB/c suckling mice born 3-5 days（recorded as day 0）were ran domly divided into 4 groups:PBS、PVM-OVA+Rapamycin、PVM-OVA+BPTES、PVM-OVA group:sensitization by nasal inhalation on days 0,3,and after 2 hours,Rapamycin or BPTES inhibitors were injected intr aperitoneally,only nasal inhalation immunization was performed on the4th day,and nasal inhalation challenge was performed on 21-25 days.S ingle lung cells were detected by flow cytometry.6.2 Lung function tests:airway resistance and lung compliance.6.3 Cell surface markers F4/80,CD11b,and Gr-1 in alveolar lavage fluid of mouse were detected by flow cytometry.Mice lung tissues were taken for pathological section H&E staining,and the relative expression levels of IL-1,IL-5,IL-13,IL-17,TNF-α,IFN-γwere detected by fluorescent quantitative PCR.7.Inhibition of cell metabolic pathways during OVA challenge,whether it inhibits allergic asthma.7.1 Experimental animal grouping,sensitization and challenge:BALB/c suckling mice born 3-5 days（recorded as day 0）were ran domly divided into 3 groups:PVM-OVA、PVM-OVA+Rapamycin、PVM-OVA+BPTES group:sensitization by nasal inhalation on days 0,3,and4,and intraperitoneal injection of Rapamycin or BPTES on 21,23,and25 days,and nasal inhalation stimulation immediately after injection;22and 24 days only nasal inhalation stimulation,then the corresponding de tection was carried out.7.2 Lung function tests:airway resistance and lung compliance.7.3 Cell surface markers F4/80,CD11b,and Gr-1 in alveolar lavage fluid of mouse were detected by flow cytometry.Mice lung tissues were taken for pathological section H&E staining,and the relative expression levels of IL-1,IL-5,IL-13,IL-17,TNF-α,IFN-γwere detected by fluorescent quantitative PCR.Results:1.Successfully prepared PVM with TCID50=10^3.5/ml concentration.2.Neonatal infection of PVM combined with OVA promotes promotes childhood allergic asthma.2.1 Airway hyperresponsiveness:The airway resistance（RI）in PVM-OVA group was significantly higher than that in PBS,PBS-OVA and PVM groups;The pulmonary compliance（Cdyn）in PVM-OVA group was significantly lower than that in PBS and PBS-OVA groups.2.2 Pathological changes of lung tissue:The infiltration of inflammatory cells in alveolar cavity and around bronchi in PVM-OVA group was more than that in PBS,PBS-OVA and PVM groups,and the alveolar structure was incomplete.2.3 The expression levels of TNF-α,IFN-γand IL-17 in the PVM-OVA group were significantly higher than the PBS group,TNF-α,IL-5,IL-13,IFN-γand IL-17 were significantly higher than the PBS-OVA group,IL-1,IL-5,IFN-γand IL-17 were significantly higher than the PVM group,IL-5 was significantly lower than the PBS group;compared with the PVM group,the expression level of IL-1 in the PBS-OVA group was significantly increased,and TNF-α,IL-5,IL-13,and IFN-γwere significantly reduced;the expression level of IL-13 in the PBS-OVA group was significantly lower than the PBS group.3.Neonatal infection of PVM combined with OVA induces formation of pulmonary Trained CD11b⁺cells3.1 Compared with the PBS group,number of CD11b⁺cells in the PVM-OVA group was increased.3.2 CD11b⁺cells in the PVM-OVA group contained a small amount of3.3 Compared with the PBS group,the surface markers CD69 and TLR4in CD11b⁺cells of PVM-OVA group was increased.3.4 Trained CD11b⁺cells Compared with the PBS group,after OVA/LPS stimulation in vitro,the PVM-OVA group was increased.3.5 Compared with the PBS group,the relative expression of GRO-αin the PVM-OVA group after OVA stimulation increased significantly,and MCP-1 increased significantly after LPS stimulation in vitro.4. After adoptive transfer trained CD11b⁺cells in mouse model affects the occurrence of allergic asthma.4.1 Adoptive transfer trained CD11b⁺cells resulted in a significant increase in airway resistance and a significant decrease in lung compliance.4.2 Pathological changes of lung tissue:adoptive transfer of Trained CD11b⁺cells in lungs with incomplete alveolar structure and slight inflammatory cell infiltration.4.3 After adoptive transfer trained CD11b⁺cells,the relative expression of IL-5 in the PVM-OVA group was decreased,while MCP-1 was increased compared with PBS group.5. CD11b⁺cells was increased in lungs after inhibition of metabolic pathways with Rapamycin or BPTES.6. The production of trained CD11b⁺cells can be inhibited by inhibiting the cell metabolism pathways and allergic asthma can be inhibited.6.1 The airway hyperresponsiveness of mouse was inhibited after inhibition of metabolic pathways with Rapamycin or BPTES during sensitization.6.2 Pathological changes of lung tissue:After inhibition of metabolic pathways with Rapamycin or BPTES during sensitization could significantly reduce pulmonary inflammation and reduce the number of inflammatory infiltrating cells around the bronchus.6.3 After inhibition of metabolic pathways with Rapamycin during sensitization inhibited the proportion of neutrophils（F4/80^-CD11b⁺Gr-1⁺）in alveolar lavage fluid cells,while with BPTES during sensitization inhibited the proportion of eosinophils（F4/80^-CD11b⁺Gr-1^-）in alveolar lavage fluid.6.4 The relative expression levels of IL-5,IL-13,TNF-αand MCP-1 in the PVM-OVA+Rapamycin group were significantly higher than in the PVM-OVA group,while IFN-γwas significantly reduced.6.5 The relative expression level of IL-13 in the PVM-OVA+BPTES group was significantly higher than in the PVM-OVA group,while IL-1,TNF-α,IFN-γ,and MCP-1 were significantly reduced.7. Inhibiting the metabolic pathway of Trained CD11b⁺cells during OVA challenge can also inhibit allergic asthma.7.1 The airway hyperresponsiveness of mouse was inhibited after inhibition of metabolic pathways with Rapamycin or BPTES during challenge.7.2 Pathological changes of lung tissue:After inhibition of metabolic pathways with Rapamycin or BPTES during challenge could reduced lung inflammation in mice,and administration of Rapamycin could not reduced lung inflammation in mice.7.3 After inhibition of metabolic pathways with Rapamycin during challenge inhibited the proportion of eosinophil（F4/80^-CD11b⁺Gr-1^-）in al veolar lavage fluid cells,while with BPTES during challenge inhibited t he proportion of neutrophils（F4/80^-CD11b⁺Gr-1⁺）and eosinophil(F4/80^-CD7.4 The relative expression levels of IL-13 and MCP-1 in the PVM-OVA +Rapamycin group were significantly higher than in the PVM-OVA group,while IL-1 and TNF-αwere significantly reduced.7.5 The relative expression level of MCP-1 in the PVM-OVA+BPTES group were significantly higher than in the PVM-OVA group,while IL-1,TNF-αand IFN-γwere significantly reduced.Conlusions:1.Neonatal infection of PVM combined with OVA sensitization promotes Th1/Th17 severe asthma.2.Newborn infection of PVM induces production of lung trained CD11b⁺cells.3.Adoptive transfer of lung Trained CD11b⁺cells promotes allergic asthma.4.Neonatal respiratory virus infection combined with allergen sensitization promotes childhood allergic asthma by inducing trained CD11b⁺cells.