The Role And Mechanism Of CARDS Toxin-induced Typel Immune Positive Feedback Loop In Mycoplasma Pneumoniae Pneumonia

Background:In recent years,Mycoplasma pneumoniae(MP)has become one of the major pathogens of community-acquired pneumonia in children.Mycoplasma pneumoniae pneumonia(MPP)is generally relatively mild,and macrolide antibiotics are the first choice for treatment;the incidence of severe mycoplasma pneumoniae pneumonia(SMPP)is increasing year by year and can leave bronchiectasis,bronchiolitis obliterans and other sequelae,and even life-threatening in severe cases.The pathogenic mechanism of MP infection has not been fully clarified.Currently,it is believed that MP directly invades the bronchial and lung tissues and MP stimulates the body’s excessive immune inflammatory response.Studies have confirmed that the interactive inflammatory network formed by innate and adaptive immunity plays an important role in MPP and is closely related to the severity of diseases.The type 1 immune response,which involves Thl cells,IFN-γ and M1 macrophages together and exerts immune effector function,plays an important role in the pulmonary immune inflammatory damage among MPP.The Thl-type cytokine IFN-γinduces Ml-type macrophages to produce the chemokine CXCL9.CXCL9 recruits Th1 cells to the site of inflammation and forms a cascade of amplified inflammatory circuits locally.Therefore,IFN-γ/CXCL9 is the core of the type I immune response link.Recent studies have found that community-acquired respiratory distress syndrome toxin(CARDS Toxin)is a new 68KDa protein encoded and expressed by MP gene MPN372,which can cause loss of ciliary function and other airway mucosal damage and has the biological activity of vacuolizing airway epithelial cells.CARDS Toxin is closely related to the Thl-type immune inflammatory response in the lungs after MP infection.At present,the specific regulatory mechanism of excessive immune inflammation after MP infection is unknown,and the existing treatment methods are limited.Studies have shown that IFN-γregulates the polarization of Ml macrophages by activating the JAK/STAT1 pathway,producing a large amount of CXCL9,while RNA Interference is a post-transcriptional gene silencing mechanism,which is often achieved by small interfering RNA(siRNA),transfection of STAT1-siRNA can down-regulate the expression of STAT1.In view of this,starting from the inflammatory factor IFN-γ/CXCL9,this paper intends to explore the positive feedback effect and related regulatory mechanism of type 1 immune response in Mycoplasma pneumoniae pneumonia under the background of CARDS Toxin induction.Part Ⅰ.Expression and clinical significance of type 1 immune response inflammatory factor IFN-γ/CXCL9 in children with MPPObjective:To analyze the expression and clinical significance of type 1 immune response inflammatory factor IFN-γ/CXCL9 in MPP,and to provide a new biological marker for early identification,comprehensive assessment of the disease,guidance of treatment and prognosis.Methods:1.The high-throughput protein screening of the previous protein microarray found that CXCL9 was differentially expressed after MP infection,and then analyzed its biological function and screened possible signaling pathways.2.From July 2019 to October 2019,117 children who were hospitalized in the Department of Respiratory Medicine,Children’s Hospital of Soochow University and met the diagnostic criteria of MPP were selected as the research subjects,namely the experimental group A,and the correlation between the expression levels of CXCL9 in peripheral blood was carried out.3.In order to further study the correlation between IFN-γ/CXCL9 and pulmonary immune inflammatory injury,children(23 cases)who were hospitalized in our department from May 2021 to October 2021 and met the diagnostic criteria of MPP were selected as the research subjects,namely the experimental group B.Peripheral blood of experiment group B admitted to the hospital for within 24 hours was collected,and electronic bronchoscopy and adjuvant therapy were performed for bronchoalveolar lavage fluid(BALF),and completed the determination of IFN-γ and CXCL9 in blood and BALF.The peripheral blood of 30 children who underwent physical examination in the outpatient department of the Children’s Health Department of our hospital during the same period was selected as the control group 1,and the determination of blood IFN-γ and CXCL9 was completed;BALF of 9 children who underwent emergency surgery for foreign bodies in the ENT department of our hospital during the same period were selected as the control group 2,and the determination of IFN-γ and CXCL9 in BALF was completed.Results:1.High-throughput protein screening results of peripheral blood protein microarrayProtein microarray high-throughput protein screening and validation revealed differential expression of protein CXCL9 after MP infection(P<0.05);GO analysis indicated that the differentially expressed proteins were related to CXCR3 chemokine receptor binding,cytokine response,cell chemotaxis and migration.KEGG analysis showed that the differentially expressed proteins might be related to JAK/STAT signaling pathway,cytokine receptor signaling pathway,chemokine signaling pathway and viral protein cytokine and cytokine receptor signaling pathway.2.Expression level and clinical significance of CXCL9 in peripheral blood of children with MPP in experimental group A(1)Comparison of clinical characteristics of children with mild MPP and severe MPP:the children presented with different degrees of cough and fever and other respiratory symptoms;the fever duration and fever peak in the severe MPP group were higher than those in the mild MPP group(P<0.05);CRP,CD3+CD4+and CD4/CD8 in the severe MPP group were higher than those in the mild MPP group(P<0.05);CD3+ CD8+in the severe MPP group was significantly lower than that in the mild MPP group,and the difference was statistically significant(P<0.05).(2)Correlation analysis of CXCL9 and clinical indexes of children with MPP:the expression level of CXCL9 in the blood was higher in the severe MPP group than in the mild MPP group,but the difference was not significant(P>0.05),and the higher the fever peak,the higher the CXCL9 expression level(P<0.05);the expression level of CXCL9 in the blood in the fever>7 days group was significantly higher than that in the fever≤7days group,and the higher CXCL9 expression level in the group with clinically pleural effusion,lobar or segmental imaging manifestations,or decreased breath sounds on lung auscultation(P<0.05);the blood CXCL9 expression level was lower in the group with positive allergic family history/allergic history than in the group with negative allergic family history/allergic history(P<0.05).(3)Correlation analysis of CXCL9 and laboratory indexes in children with MPP:the blood CXCL9 level was positively correlated with N%and CRP inflammatory indicators(r=0.290,0.271,P<0.05).3.Expression level and clinical significance of IFN-γ/CXCL9 in peripheral blood and bronchoalveolar lavage fluid of children with MPP in experimental group B(1)Expression levels of IFN-γ/CXCL9 in children with MPP:the expression levels of IFN-γ and CXCL9 in peripheral blood were higher than those in the control group 1,and the expression level of CXCL9 in BALF were higher than those in the control group2(P<0.05).(2)Correlation analysis between IFN-γ and CXCL9:the expression level of blood CXCL9 and blood IFN-γ was positively correlated(r=0.502,P<0.05),and the expression level of CXCL9 in BALF was higher than that of CXCL9 in peripheral blood(P<0.05).Conclusion:The inflammatory response in vivo after MP infection is mainly type 1 immune response,and the expression level of inflammatory factor IFN-γ/CXCL9 is closely related to the degree of immune inflammation injury of lung.Part Ⅱ.CARDS Toxin induces DCs to promote Th1 cell differentiationObjective:To further explore the role of CARDS Toxin in inducing DCs activation of co-stimulatory molecules and promoting Th1 cell differentiation through in vitro assays,and to provide new ideas for future exploration of immune interventions for MPP.Methods:1.Preliminary construction of CARDS Toxin and its biological function verification.2.Isolate mouse bone marrow mononuclear cells,induce F-DC with Flt3L,co-incubate with naive CD4+T cells under the stimulation of CARDS Toxin,and detect expression of CD4+IFN-γ+Th、CD4+IL-4+Th and CD4+IL-17+Th by flow cytometry.3.Isolate mouse bone marrow mononuclear cells,induce F-DC with Flt3L,stimulate with CARDS Toxin,and detect the mRNA expression levels of costimulatory molecules B7-H1,B7-H3 and B7-DC on F-DC by qPCR.4.Use transcriptome sequencing technology(RNA-seq)to preliminarily explore the upstream regulatory mechanism of CARDS Toxin-stimulated F-DC expression of costimulatory molecules,and in vitro experiments to verify the differential gene expression level after CARDS Toxin(10ng/ml)stimulated F-DC.Results:1.The constructed CARDS Toxin had biological functions,and F-DC stimulated with CARDS Toxin promoted the differentiation of CD4+IFN-γ+Th(Th1)cells,while it did not affect the differentiation of Th2 and Th17 cells(P<0.05).2.CARDS Toxin did not induce high expression of co-stimulatory molecules B7-H1 and B7-H3 in F-DC,but could induce high expression of B7-DC in F-DC(P<0.05).3.A total of 144 genes were differentially analyzed by transcriptome sequencing technology(RNA-seq),among which 59 were up-regulated and 85 were down-regulated,in which the transcription factor NF-κB was highly expressed,therefore,CARDS Toxin induced F-DC to highly express B7-DC most likely closely related to NF-κB signaling pathway.4.Increased NF-κB expression was found after stimulation of F-DC with CARDS Toxin(10ng/ml)(P<0.05).Conclusion:CARDS Toxin is closely related to the Thl-type immune inflammatory response in the lungs after MP infection.The mechanism may be that CARDS Toxin activates the NF-κB signaling pathway and induces the high expression of costimulatory molecule B7-DC in F-DC,thereby promoting Th1 cell differentiation.Part Ⅲ.Role and mechanism of IFN-γ-induced CXCL9 secretion by Mφ and promotion of Th1 cell migration by CXCL9Objective:In order to further explore the related mechanism of CARDS Toxin-induced type 1 immune response circuit after MP infection,to study the pathway of IFN-γ-induced Mcp secretion of CXCL9 and the effect of CXCL9 on Th1 cell migration through vitro experiments.Methods:1.Flow cytometry was used to determine the count of BALF neutrophils,Mφ,and lymphocytes in the experimental group B and control group2 in part I,and to analyze the Mcp phenotype.2.The human monocyte cell line THP-1 was cultured in vitro,and different concentrations of IFN-γ(0ng/ml,lng/ml,10ng/ml)stimulated Mφ(THP-1+PMA indued),using different methods(qPCR,ELISA)to determine the expression of CXCL9 in Mφ.3.IFN-γ(1ng/ml)stimulated Mφ and Mφ which was transfected with STAT1-siRNA-1,then,different methods(qPCR,Western blot,ELISA)were used to determine the expression of CXCL9,p-STAT1 and STAT1 in Mφ.4.Validation of CXCL9-induced Th1 cell migration assay by using Transwell chambers.Results:1.Cell count and Mφ expression in alveolar lavage fluid of children with MPPThe number of neutrophils and Mφ cells in the BALF of children with MPP in the experimental group B was higher than that in control group2(P<0.05),and flow cytometry assay revealed that Mφ dominantly showed M1 phenotype(CD16+CD64+CD163-).2.IFN-γ induces Ml-type Mφ to express CXCL9IFN-γ could stimulate M1-type Mφ to promote high expression of CXCL9,and CXCL9 expression level was dose-dependent(P<0.05)at the same cut-off point;CXCL9 expression level was time-dependent(P<0.05)at the same concentration stimulation(IFN-γ 1ng/ml group and IFN-γ 10ng/ml group);the expression level of CXCL9 in IFN-γOng/ml group did not show significant time dependence(P>0.05).3.The possible mechanism of IFN-γ-induced high expression of CXCL9 in Ml-type Mφ(1)STATl-siRNA could down-regulate the expression level of STAT1 gene.(2)After IFN-γ(lng/ml)stimulated Mφ(THP-1+PMA induced)for 24h,the expressions of CXCL9,p-STAT1 and STAT1 were significantly increased(P<0.05).(3)Mφ transfection of STAT1-siRNA-1 down-regulated the expression of CXCL9,p-STAT1 and STAT1(P<0.05).4.CXCL9 induced Th1 cell migration(1)After IFN-γ(lng/ml)stimulated Mφ(THP-1+PMA induced)for 24h,the expression of CXCL9 in the lower chamber of Transwell increased,and the number of Thl cells migrated increased(P<0.001).(2)After Mφ was transfected with STAT1-siRNA-1,the expression of CXCL9 in the lower chamber of Transwell decreased,and the number of Th1 cell migration was significantly decreased(P<0.001).Conclusion:IFN-γ can induce high expression of CXCL9 in M1-type Mφ in a doseand time-dependent manner,and its mechanism may be related to the activation of JAK/STAT1 signaling pathway;CXCL9 can promote Thl cell migration.Full text conclusion:1.The inflammatory response in vivo after MP infection is dominated by type 1 immune response.The expression level of inflammatory factor IFN-γ/CXCL9 is closely related to the degree of pulmonary immune inflammatory damage,which provides new biological markers for early identification,comprehensive assessment of the disease,guidance of treatment and prognosis.2.CARDS Toxin is closely related to the Thl-type immune inflammatory response in the lungs after MP infection.The mechanism may be that CARDS Toxin activates the NF-κB signaling pathway and induces the high expression of costimulatory molecule B7-DC in F-DC,thereby promoting Thl cell differentiation;IFN-γ activates the JAK/STAT1 signaling pathway and promotes the secretion of CXCL9 by Ml type Mφ;CXCL9 promotes the migration of more Thl cells to the inflammatory response center,thereby forming a type 1 immune positive feedback loop,amplifying the inflammatory cascade,and aggravating the immune damage of lung tissue.3.By studying the role of CARDS Toxin-induced type 1 immune positive feedback loop in MPP,the pathogenesis of MP infection can be better explained,which provides a new idea for exploring the immune intervention methods of MPP in the future.