Group 2 Innate Lymphoid Cells Mediate The Activation Of CD4~+T Cells And Aggravate Th1/Th2 Imbalance Via MHCⅡ Molecules During Respiratory Syncytial Virus Infection

Objective:Respiratory syncytial virus(RSV)belongs to Paramyxovirus family of respiratory viruses.In autumn and winter,RSV is the main pathogen causing severe respiratory diseases in infants,the elderly and immunocompromised people.Epidemiological investigations have found that almost 100%of infants who under 2 years of old have infected RSV,and RSV infection is the most important risk factor for wheezing in infants.Viral infection can affect airway function,leading to asthma like symptoms such as hyperresponsiveness,hypermucus production,and increased eosinophil granulocyte infiltration and so on.Many studies have shown that T lymphocyte plays an integral role in respiratory tract infections caused by RSV,and the imbalance between Th1 and Th2 cells plays an important role in the progression and severity of the disease.During RSV infection,Th1cells are thought to play a protective role by secreting the cytokine IFN-γ,which inhibits eosinophil granulocyte accumulation in the lungs by reducing the production of chemokines CCL11,CCL17 and CCL22,and IFN-γcan directly inhibit the development of Th2 cells.While Th2 cells act through the production of Th2-type cytokines,such as IL-4,IL-5,IL-13,to increase the production of Ig E,recruitment and activation of eosinophils and mucus secretion,increase airway inflammation and AHR.Therefore,it is of great significance to elucidate the mechanism of Th2 cell activation and regulate it in the treatment of RSV infection.Group 2 innate lymphoid cell type is a non T,non B lymphoid cells that were first detected in 2010 in intestinal fat related tissues and subsequently in liver,spleen,and lungs.CD3,CD4,CD5,CD8,CD11b,Gr-1,CD19,B220,NK1.1,TCRδwere not expressed but c-Kit,Sca-1,Thy1.2,and IL-33 receptor ST2 were expressed in ILC2s.At the same time,IL-25,IL-33 or TSLP can stimulate the proliferation of ILC2s and secretion of IL-5,IL-9,IL-13 and other Th2-type cytokines.In the early stage of respiratory virus infection,ILC2s can mediate inflammation by secreting type 2 cytokines.The activation of ILC2s was earlier than that of Th cells.Our previous work showed that ILC2s can modulate the proliferation and activation of CD4~+T cells through OX40/OX40L,while transwell can modulate the biological activity of CD4~+T cells.However,it is not clear whether ILC2s can affect the biological activity of T cells in other ways besides OX40/OX40L axis.It is well known that for initial CD4~+T cell activation,Major Histocompatibility Complex II(MHCⅡ)and B7 molecules from antigen presenting cells are dual signals necessary.Usually,as dedicated antigen presenting cells,dendritic cells play a key role in activating the initial CD4~+T cells.Recently,however,it has been shown that ILC2s can also act as an antigen-presenting cell in intestinal helminth infection,regulating the differentiation and proliferation of CD4~+T cells in intestinal mucosa through the expression of MHCⅡ molecules on its surface.However,it is not clear whether ILC2s can also act as antigen presenting cells to influence T cell proliferation and differentiation in RSV-mediated acute respiratory infection.In this study,we designed a mouse model of RSV acute respiratory infection to investigate the role of ILC2s in the regulation of Th cell differentiation and Th1/Th2homeostasis.On the one hand,we show that CD4~+T cells are important effector cells in the pathogenesis of acute RSV infection through clinical RSV infection in children and acute RSV infection in mice.On the other hand,it was confirmed that ILC2s caused the imbalance of Th1/Th2 in the lungs of the model mice by adoptive transfusion.Then we detected the expression of MHCⅡ and B7 on the surface of ILC2s in the lungs of model mice,and confirmed that ILC2s mediates the proliferation and activation of CD4~+T cells and the regulation of Th1/Th2 balance in RSV infection via MHCⅡ in vitro,to provide experimental evidence for elucidating the immunological pathogenesis of respiratory diseases caused by RSV infection.Methods:1.Inclusion of clinical cases.Children with respiratory infections and wheezing from 2021.10 to 2021.12 were admitted to the pediatric of the China Medical University,the respiratory syncytial virus(RSV)/Adenovirus Test Kit(immunochromatography,V740001AC)was used to test the nasal test paper.RSV positive children were in the experimental group,and RSV negative children were in the control group,children between 2 and 6 years of age who were admitted to hospital at the same time as the appeal and who were scheduled for strabismus surgery served as a healthy control group and required blood tests before minor surgery.All specimens were taken with the informed consent of the child’s guardian.Exclusion criteria:children with systemic hormone and antiviral drug use in the past 1 month and children with autoimmune diseases.2.Data collection and sample processing of clinical children.Unified information questionnaire was used to collect data for children,including general information,history of present disease,past history,birth situation,feeding situation,family history,clinical symptoms,signs,auxiliary examination and treatment,etc.Peripheral blood mononuclear cells(PBMC)were prepared by collecting 1ml of venous blood using heparin sodium anticoagulant vessels.Flow cytometry was used to analyze the expression of CD4~+T cells,T-bet~+CD4~+T cells and GATA3~+CD4~+T cells in PBMC of children.3.Animal modelsSPF 6-8 week old female BALB/c mice were purchased from Benxi Changsheng Biotechnology Co.Ltd.20μl RSV suspension containing 2×10~6 TCID50(half of the infected cells)was dripped into the left nostril of mice.In the depletion of CD4~+T cell experiment,BALB/c mice in the experimental group were intraperitoneally injected with250μg anti-CD4 monoclonal antibody 24 hours before RSV infection.In the reinfusion model,2×10~5 ILC2s(isolated from the lung tissue of BALB/c mice on Day 3 of infection)were reinfused into normal BALB/c mice via caudal vein two hours before infection.4.Detection of pneumonia in miceThe left lower lobe of the model lung was fixed with 4%paraformaldehyde,dehydrated with gradient alcohol,paraffin-embedded sections were stained with HE,and the morphology and inflammatory infiltration of the lung were observed.BALF were collected and centrifuged into 1.5 ml centrifuge tube and stored in-80°c refrigerator to detect cytokines.Cell precipitation was resuspended with 100μl PBS and 10μl solution was counted.The remaining 90μl cell smear was prepared.A total of 200 cells were counted at random in the field of vision,and the number and percentage of neutrophil,eosinophilic cells,lymphocyte and macrophages were counted according to morphological characteristics.5.Detection of CD4~+T cells and ILC2s in lung tissues of mice by using Flow cytometry.The number of CD4~+T cells and the number of secretinf IFN-γ,IL-4,IL-5,IL-13CD4~+T cells,and the expression of MHCⅡ and B7 on ILC2s in lung tissues of mice infected with RSV infection were analyzed by flow cytometry.6.The levels of Th1 and Th2 cytokines in lung tissue and BALF were detected by El ISA.The levels of IFN-γ,IL-4,IL-5 and IL-13 cytokines in lung tissue and BALF were detected by Elisa Kit according to the instructions.7.Real-time PCR was used to detect the expression of Th1,Th2 cytokines in the lung tissue and co-cultured CD4~+T cells of the model mice,and the m RNA of MHCⅡ and B7in the lung ILC2s of the model mice.Total RNA was extracted from the lung of the model mice,total RNA was extracted from the lung ILC2s of the selected model mice,turn into c DNA.The expression of m RNA of IFN-γ,IL-4,IL-5,IL-13 in CD4~+T cells and lung tissue were detected by Real-time PCR.And the expression of m RNA of CD80,CD86 and MHCⅡ in ILC2s were also detected by Real-time PCR.8.Western Blot was used to detect the expression levels of MHCⅡ,CD80 and CD86 of ILC2s in lung tissue.ILC2s was isolated by Magnetic activated cell sorting(MACS)after RSV infection,and the protein expression of MHCⅡ,CD80 and CD86 were detected by Western Blot.Results:1.Proliferation of CD4~+T cells and Th2 polarization in PBMC of children infected with RSV:The percentage of CD4~+T cells in PBMC of who infected with respiratory tract infection was significantly higher than that of children who were normal,the increase of Th2 in respiratory tract infection of RSV(+)leads to imbalance of Th1/Th2 in peripheral blood,and the balance of Th1/Th2 tends to Th2.2.Non-specific symptoms and signs in children with RSV infection:compared with children with respiratory disease caused by non-RSV infection,children with RSV infection had no specific symptoms and signs,both had cough,fever and wheezing,lung X-ray or CT imaging changes were found in some children.While the result of blood cell analysis showed that the lymphocyte of RSV(+)children was elevated obviously.3.RSV infection induced CD4~+T cells activate to Th2 type cells:RSV infection increased the number of CD4~+T cells and IFN-γ~+CD4~+T、IL-4~+CD4~+T、IL-5~+CD4~+T、IL-13~+CD4~+T in the lung tissue of model mice,while Th2CD4~+T cells were mainly increased,and the balance was partial to Th2.4.CD4~+T cells are the key effector cells mediating airway inflammation of RSV.Blocking CD4~+T cells in vivo resulted in reduced infiltration of inflammatory cells in the lung tissues of model mice,decreased the expression of Th2 cytokine m RNA,decreased number of inflammatory cells in BALF and decreased Th2 cytokine in its tissue,confirming that CD4~+T cells are important effector cells mediating acute pneumonia of RSV.5.ILC2s aggravates the airway inflammation after RSV infection:The local inflammation of the lungs of the mice infected with RSV increased after adoptive transfer of ILC2s,the inflammatory cell infiltration in BALF increased and the airway wall became deformed,the level of Type 2 cytokines in supernatant and Th2 cytokines in lung tissue were significantly increased.It is suggested that ILC2s is an important factor to aggravate RSV airway inflammation.6.ILC2s promotes the proliferation of CD4~+T cells in the lung tissue of mice infected with RSV.Compared with the mice not infected with RSV,the proliferation of CD4~+T cells in the lung tissue of mice which adoptive transfer of ILC2s before RSV infection were significantly increased,the percentage and absolute number of CD4~+T cell subsets secreting Th2 cytokines IL-4,IL-5 and IL-13 were significantly increased,and the Th1/Th2 ratio in lung tissue was decreased,it is suggested that ILC2s plays an important role in the proliferation,activation and Th2 differentiation of lung CD4~+T cells induced by RSV.7.RSV infection can promote the proliferation of ILC2s and the expression of MHCⅡ and B7.The proliferation of ILC2s and the expression of MHCⅡ,CD80 and CD86 of ILC2s were increased in the model mice infected with RSV,the m RNA and protein levels of MHCⅡ,CD80 and CD86 were also increased.8.MHCⅡ molecules are important membrane molecules that ILC2s regulate the proliferation and activation of CD4~+T cells:The lung ILC2s of BALB/c mice infected with RSV on day 3 and the CD4~+T cells of the spleen of normal mice were sorted,and the number of CD4~+T cells and Th2CD4~+T cells were significantly reduced in the MHCⅡ blocking group.The Th1/Th2 ratio of CD4~+T cells increased significantly,and the expression levels of IL-4,IL-5 and IL-13 m RNA of CD4~+T cells decreased significantly.That is suggested that ILC2s may mediate the proliferation of CD4~+T cells in RSV infection through MHCⅡ.Conclusion:1.RSV infection can induce the differentiation of CD4~+T cells into Th2 cells in PBMC of patients and lung tissues of BALB/c mice,and CD4~+T cells are important effector cells mediating airway inflammation when RSV infection.2.ILC2s promotes the proliferation and activation of CD4~+T cells and exacerbates airway inflammation.The expression of MHCⅡ and B7(CD80 and CD86)on ILC2s was significantly increased by RSV infection.3.MHCⅡ play an important role for the proliferation of CD4~+T cells and aggravate Th1/Th2 imbalance induced by ILC2s.