Study On The Mechanism Of Action Of IDO And TLR7-NF-κB Axis In The Pathogenesis Of Bronchial Asthma In Mice

Research background and purpose:Bronchial asthma is an allergic disease characterized by airway hyperresponsiveness and chronic airway inflammation caused by the body’s response to allergen stimulation.Immune tolerance mechanism deficiency may be the primary cause of asthma,and Th1/Th2 cell imbalance is the key link.Immunologic tolerance is a state of specific immune non-response to antigens produced by the body’s immune system after exposure to antigenics.Studies have found that by inducing immune tolerance in the body,diseases that respond to foreign antigens and over-activate their own antigens,such as transplant rejection,tumors and allergic diseases,can be prevented.Indoleamine 2,3-dioxygense(IDO)mediated tryptophan metabolism has been confirmed to play an important role in peripheral immune tolerance.The expression of IDO gene at the transcriptional level was stimulated by various specific inflammatory factors,such as interferon-α,β,γ,(IFN-α,β,γ),Lipopolysaccharide(LPS),IFN-γ was the typical representative of such inflammatory factors.IFN-γ is a typical inducer of IDO,and the binding site of IFN-γ exists in the promoter region of IDO gene.Once the two bind,the expression of IDO will be stimulated in key cells of the immune system,such as monocytes-macrophages and dendritic cells(DC),so as to induce various immune regulatory functions.The expression pathway of IDO induced by IFN-γ is regulated by a variety of pro-inflammatory cytokines,such as Tumor necrosis factor-α(TNF-α)and Nuclear factor kappa-B(NF-κB).These pro-inflammatory cytokines are regulated by downstream products of TLRs signal transduction pathway.Studies have shown that the inhibitor 1-methyl-tryptophan(1-MT)of IDO can interfere with TLR signal transduction in dendritic cells independently of the activity of IDO.A number of studies on immunomodulation mechanism have suggested that there is a phase between IDO and TLRs family in immune-related diseases.However,the role of IDO and members of the TLRs family in the pathogenesis of bronchial asthma and their correlation have not been reported.The role of TLRs as pattern recognition receptors in the pathogenesis of allergic diseases has been widely concerned.Recent studies have shown that activation of TLR7 plays a significant role in the occurrence and development of allergic diseases and autoimmune diseases.Recent studies on TLR7 in allergic diseases mainly focus on the pathogenesis and treatment of allergic rhinitis,and the main allergen of allergic rhinitis is dust mite.At present,WHO has put forward the concept of "one airway,one disease" for asthma,which not only reminds clinicians to pay attention to the prevention and treatment of allergic rhinitis,but also shows the close correlation between allergic rhinitis and asthma.The cytokines IFN-α,IL-12 and TNF-α produced after activation of TLR7 will mediate the immune response of the body to the direction dominated by Th1 cells,so that the Th2 cell dominance phenomenon can be corrected in asthmatic patients,the expression of IL-4 and IL-13 can be reduced,and the purpose of alleviating asthma symptoms is achieved.In a mouse model of asthma,a topical application of the TLR7 agonist Imiquimod showed a decrease in alveolar macrophages,B cells and TNF-α,as well as an increase in the secretion of natural killer cells and IL-10,suggesting a therapeutic effect on asthmatic mice.This also indicates that TLR7 as a target in the treatment of asthma has a great potential.Nuclear factor κB(NF-κB)is the main signal transduction pathway of TLRs.NF-κ B is associated with factors such as IKK α(Inhibitor of nuclear factor kappa-B kinase)and NF-κBp65(Nuclear factor kappa-Bp65),which plays a pivotal role in inflammation and immune responses by regulating the cascade between immune and inflammation-related factors and inflammatory mediators to amplify the cascade effect.It has been reported that NF-κB is activated during airway tissue remodeling in asthma,including hyperplasia of airway epithelial cells,fibrosis of surrounding airway tissue,and hyperplasia of smooth muscle cells.To investigate the association between TLRs and TLRs,TLRs can self-dimerize through binding with their ligands,and bind to the connector myeloid differentiation protein 88,thus activating NF-κB,promoting the synthesis and release of inflammatory factors and interleukin,and thus inducing the cascade inflammatory response in the body.In summary,there are few reports on the mechanism of action between IDO and TLR7-NF κ B pathway in the pathogenesis of bronchial asthma and their relationship in the pathogenesis of bronchial asthma.In view of this,this study selected clean C57BL/6J mice for the experiment to establish a bronchial asthma mouse model and isolate and culture mouse airway smooth muscle cells(ASMCs),to clarify the expression of IDO and TLR7 in the airway epithelium and smooth muscle cells of asthmatic mice,as well as the role of intervention of IDO and TLR7 in asthmatic mice.To further explore the mechanism of IDO and TLR7-NFκB pathway in the pathogenesis of bronchial asthma in mice.The major purposes of this experiment are described as follows:1.To detect TLR7 expression in the airway of asthmatic mice.To predict the role of TLR7 expression and NF-κB signaling pathway in the pathogenesis of asthma;2.To detect the expression level of factors related to TLR7 mediated NF-κB signaling pathway by IDO and TLR7 expression intervention,and to verify the role of IDO and TLR7 in the pathogenesis of asthma and their correlation.3.To further analyze the mechanism of TLR7 mediated NF-κB signaling pathway in the pathogenesis of bronchial asthma on the molecular level.Experimental Methods:(3 parts)Part Ⅰ:Establishment and observation of bronchial asthma mouse model and detection of TLR7,IKKα and NF-κBp65 expression.Aim:TLR7 expression was detected in the airway of asthmatic mice.To predict the role of TLR7 expression and NF-κB signaling pathway in the pathogenesis of asthma.Methods:Clean C57BL/6J mice were selected for the experiment and divided into Normal group and Asthma group,with 10 mice in each group.Normal group did not do any modeling.Asthma group used dust mite antigen extract/aluminum hydroxide to construct dust mite induced Asthma mouse model.All mice were sacrificed within 24 h after the last stimulation.The tracheoalveolar lavage fluid(BALF)was extracted and frozen,and cells were classified and counted.After alveolar lavage,the left bronchial tissues of mice were collected and used for hematoxylin-eosin(HE)and immunohistochemical detection.Statistical software(SPSS 21.0)was used for data analysis in this chapter.Results:1.Behavioral changes of mice in each group after asthma modeling:There was no abnormality in body weight,respiration,hair color,activity,urine and feces of mice in the Normal group.While mice in the Asthma group showed different degrees of shortness of breath,vertical hair,bow back,scratching ears and gills,irritability,increased frequency of urination and defecation and other symptoms.In severe cases,the manifestations included slowed rate of breathing,slow action,incontinence,etc.2.HE staining for observing the pathological changes of lung tissue:In Normal group,there were smooth airway epithelium,no inflammatory cell infiltration,no airway smooth muscle thickening and no inflammatory exudate infiltration.While in Asthma group,there were a large number of inflammatory cells infiltrated in the bronchi and around the blood vessels,increased inflammatory cells and exudates increased,and formation of mucus thrombus.3.The positive expression rates of TLR7,IKKα and NF-κBp65 detected by immunohistochemistry:the staining intensity of TLR7 was strongly positive in the Normal group and weakly positive in the Asthma group.Furthermore,IKKα and NF-κBp65 were weakly positively stained in the Normal group,but strongly positively stained in the Asthma group.Besides,compared with Normal group,Asthma group had significantly decreased positive expression rate of TLR7,while obviously increased positive expression rates of IKKα and NF-κBp65(All P<0.05).4.Detection of cell count and inflammatory factors in BALF of asthmatic mouse model:Compared with the Normal group,the total number of cells,percentage of eosinophils,percentage of neutrophils,percentage of lymphocytes and percentage of macrophages in BALF were significantly increased in the Asthma group(All P<0.05);besides,there were evident decreased contents of IL-4 and IL-10,as well as increased contents of IL-12 and IFN-γ in the supernatant of BALF(P<0.05).Conclusions:1.The clinical and pathological manifestations of the mice indicated that the asthma mouse model made by dust mite/aluminum hydroxide was successfully established;2.The levels of inflammatory cells such as eosinophils and macrophages and inflammatory factors such as IL-12 and IFN-γ in the airway tissues of asthmatic mice were significantly increased,suggesting that there is an obvious inflammatory response in the pathogenesis of asthma;3.In asthmatic mice,the expression of TLR7 was low,while the expression of IKKα and NF-κBp65,which are two key factors in NF-κB signaling pathway,were high,indicating the activation of NF-κB signaling pathway.These results suggest that TLR7 and NF-κB signaling pathways are involved in the inflammatory pathogenesis of asthma through the negative regulation mechanism.Part Ⅱ:The relationship of IDO and TLR7 as well as the effect and potential mechanism of intervening IDO and TLR7 on the pathogenesis of bronchial asthma in mice.Aim:To detect the expression level of factors related to TLR7 mediated NF-κB signaling pathway by IDO and TLR7 expression intervention,and to verify the role of IDO and TLR7 in the pathogenesis of asthma and their correlation.Methods:Clean grade C57BL/6J mice were divided into Normal group,Asthma group,Model+1-MT group,Model+IFN-γ group,Model+TLR7 agonist group,TLR7 knockout group,Model+TLR7 knockout group.All mice were sacrificed within 24 h after the last excitation for BALF extraction and cryopreserved.After the alveolar lavage,the bronchial tissues of the left lung of mice were collected for HE staining and immunohistochemical detection.HE staining was used to observe the pathological changes of lung tissue of mice in different treatment groups.After asthma modeling,the contents of IL-4,IL-10,IL-12 and IFN-γ in BALF supernatant of mice in different treatment groups were determined by ELISA.mRNA expression levels of TLR7,IDO,IKKα and NF-κBp65 in lung tissues were detected by RT-PCR.The protein expression levels of TLR7,IDO,p-IKKα and NF-κBp65 in lung tissues were detected by Western Blot.The data in this chapter were analyzed by SPSS 21.0.Results:1.Effect of IDO on pathological characteristics,inflammatory response and NF-κB pathway in asthmatic mice:According to the experimental results,Model+1-MT group showed increased infiltration of inflammatory cells and inflammatory levels(All P<0.05);besides,there was significant decrease in the mRNA and protein expression levels of TLR7 and IDO(All P<0.05)and obvious increase in the protein expression levels of p-IKKα and NF-κBp65(All P<0.05).In addition,in Model+IFN-γ group,there was a decreased trend in the number of inflammatory cell count and inflammatory levels(All P<0.05);meanwhile,the mRNA and protein expression levels of TLR7 and IDO were significantly increased(All P<0.05),while there was obvious decrease in the protein expression levels of p-IKKα and NF-κBp65(All P<0.05).2.Effect of TLR7 on pathological characteristics,inflammatory response,IDO expression and NF-κB pathway in asthmatic mice:According to the experimental results,the airway epithelium of TLR7 knockout group was smooth,there was no inflammatory cell infiltration and cilia abscission under the mucosa,no airway smooth muscle thickening,no inflammatory exudate infiltrating into the alveolar cavity;no significant change in the contents of IL-4,IL-10,IL-12 and IFN-γ in BALF supernatant(All P>0.05);besides,no obvious change was observed in the mRNA and protein expressions of TLR7 and IDO,as well as the protein expressions of p-IKKα and NF-κBp65(All P>0.05).In Model+TLR7 agonist group,there were reduced inflammatory cell infiltration and decreased inflammatory levels(All P<0.05);besides,there were significantly increased mRNA and protein expression levels of TLR7 and IDO,as well as evidently increased protein expressions of p-IKKα and NF-κBp65(All P<0.05).Furthermore,in Model+TLR7 knockout group,there were increased inflammatory cell infiltration and increased inflammatory levels(All P<0.05);meanwhile,there were significantly decreased mRNA and protein expression levels of TLR7 and IDO,as well as evidently decreased protein expressions of p-IKKα and NF-κBp65(All P<0.05).Conclusions:1.IDO inhibitor 1-MT and TLR7 knockout induced inflammatory response and activated NF-κB signaling pathway.IDO agonist IFN-γ treatment and TLR7 agonist treatment inhibited inflammatory response and inhibited NF-κB signaling pathway activation;2.During the inflammatory response of asthma,the expression of IDO and TLR7 was correlated in the same direction.It was negatively correlated with the activation level of NF-κB signaling pathway;3.IDO and TLR7 inhibit the inflammatory response of asthma through the reverse regulation of NF-κB signaling pathway.Part Ⅲ:The mechanism of TLR7 mediating NF-κB signaling pathway in ASMCS of asthmatic mice.Aim:To further investigate the mechanism of TLR7 mediated NF-κB signaling pathway in the pathogenesis of bronchial asthma at the molecular level.Methods:Clean C57BL/6J mice were selected for the experiment and divided into Normal group,Asthma group,Model+1-MT group,Model+IFN-γ group,Model+TLR7 agonist group,TLR7 knockout group,In the Model+TLR7 knockout group,mice are killed after successful treatment,and all their main trachea is taken aseptically.Tracheal smooth muscle cells(ASMCs)were cultured and transfected as required.TLR7 mRNA expression level after ASMCS transfection was detected by RT-PCR.The protein expression levels of TLR7,p-IKKα and NF-κBp65 after ASMCS transfection were detected by Western Blot.MTT assay was used to detect cell proliferation after ASMCS transfection.Cell invasion after ASMCS transfection was detected by Transwell assay.Flow cytometry was used to detect the cell cycle and apoptosis of transfected ASMCs in each group.The data in this chapter were analyzed by SPSS 21.0.Results:1.RT-PCR to detect the expression of TLR7 mRNA in ASMCS cells after transfection:Compared with the Control group,there was significantly decreased mRNA expression level of TLR7 in other groups(All P<0.05).No obvious difference was found among pcDNA-TLR7 NC group,siRNA-TLR7 NC group and pcDNA-TLR7+Asatone group(P>0.05).Furthermore,compared with corresponding NC group,pcDNA-TLR7 group had significantly increased mRNA expression of TLR7,and siRNA-TLR7 group showed the opposite trend(All P<0.05);Asatone group and Triptolide group showed no obvious change in the mRNA expression of TLR7(All P>0.05).Besides,there was evidently increased mRNA expression of TLR7 in the pcDNA-TLR7+Asatone group(All P<0.05).2.Western blot to detect the expression of TLR7,p-IKKα and NF-κBp65 protein in ASMCS cells after transfection:Compared with the Control group,there was significantly decreased protein expression level of TLR7 while obviously increased protein expressions of p-IKKα and NF-κBp65 in other groups(All P<0.05).No obvious difference was found among pcDNA-TLR7 NC group,siRNA-TLR7 NC group and pcDNA-TLR7+Asatone group(P>0.05).Furthermore,compared with corresponding NC group,pcDNA-TLR7 group had significantly increased expression of TLR7 while obviously decreased expressions of p-IKKα and NF-κBp65,and siRNA-TLR7 group showed the opposite trends(All P<0.05);no obvious change was found in TLR7 expression in Asatone group and Triptolide group(All P>0.05),while the expressions of p-IKKα and NF-κBp65 were significantly increased in the former group and decreased in the latter group(All P<0.05).Besides,there was remarkable increase in the expression of TLR7 in pcDNA-TLR7+Asatone group(P<0.05),while no obvious change was observed in the expressions of p-IKKα and NF-κBp65(P>0.05).3.MTT assay to detect cell proliferation condition in ASMCS cells after transfection:Compared with the Control group,there was significantly increased level of proliferation in other groups(All P<0.05).No obvious difference was found among pcDNA-TLR7 NC group,siRNA-TLR7 NC group and pcDNA-TLR7+Asatone group(P>0.05).Furthermore,compared with corresponding NC group,pcDNA-TLR7 group and Triptolide group showed significantly reduced ability of proliferation;while siRNA-TLR7 group and Asatone group showed promoted ability of cell proliferation(All P<0.05).In addition,compared with pcDNA-TLR7 group,pcDNA-TLR7+Asatone group indicated significantly upregulated ability of cell proliferation(P<0.05).(4.Transwell assay to detect cell invasion condition in ASMCS cells after transfection:Compared with the Control group,there was significantly increased invasion ability in other groups(All P<0.05).No obvious difference was found among pcDNA-TLR7 NC group,siRNA-TLR7 NC group and pcDNA-TLR7+Asatone group(P>0.05).Furthermore,compared with corresponding NC group,pcDNA-TLR7 group and Triptolide group showed obviously suppressed cell invasive ability;siRNA-TLR7 group and Asatone group had significantly stimulated cell invasive ability(All P<0.05).Besides,compared with pcDNA-TLR7 group,pcDNA-TLR7+Asatone group showed obviously promoted cell invasive ability(P<0.05).5.Flow cytometry to detect cell apoptosis condition and cell cycle distribution in ASMCS cells after transfection:Compared with the Control group,there was significantly shortened G0/G1 phase(decreased cell proportion),prolonged G2+S phase(increased cell proportion)and decreased cell apoptosis in other groups(All P<0.05).No obvious difference was found among pcDNA-TLR7 NC group,siRNA-TLR7 NC group and pcDNA-TLR7+Asatone group(P>0.05).Furthermore,compared with corresponding NC group,pcDNA-TLR7 group and Triptolide group had prolonged G0/G1 phase,shortened G2+S phase,and significantly increased rate of cell apoptosis(All P<0.05).While siRNA-TLR7 group and Asatone group had shortened G0/G1 phase,prolonged G2+S phase,and significantly decreased rate of cell apoptosis(All P<0.05).At the same time,compared with pcDNA-TLR7 group,pcDNA-TLR7+Asatone group displayed shortened G0/G1 phase,prolonged G2+S phase,and significantly decreased rate of cell apoptosis(All P<0.05).Conclusions:1.The transfection and stimulation strategies of ASMCS in different model mice were successful;2.Up-regulation of TLR7 expression can potentially inhibit the activation of NF-κB signaling pathway;3.The up-regulation of TLR7 expression and NF-κB inhibitor treatment can reduce the proliferation level of ASMCS;4.At the molecular level,the up-regulation of TLR7 expression and the inhibition of NF-κB signaling pathway can effectively alleviate the proliferation and remodeling of smooth muscle cells in asthma.