The Role And Mechanism Of Membrane-Bound And Secreted TL1A In Asthmatic Airway Remodeling

BackgroundBronchial asthma is one of the most common chronic diseases in the world,affecting over 300 million people globally,accounting for approximately 1%of the global disease burden.Studying the occurrence and progression mechanism of asthma,and controlling the disease in the early stage or even reversing its progression,has important clinical significance.Airway remodeling is the main pathological change leading to irreversible airflow restriction in asthmatic patients,and it is the main cause of rapid decline in lung function and fixed airflow obstruction in patients.Meanwhile,it is also considered as a prominent feature of many severe and refractory asthma patients.In the context of individualized treatment,it is necessary to accurately classify patients based on biological markers or other methods,including airway remodeling and other features,and further elucidate their mechanisms.Only on this basis,individualized intervention measures can be selected for patients with different phenotypes to achieve the idea balance state.TL1A(TNF ligand related molecule 1A)was initially discovered in 2002.It belongs to the TNF superfamily(TNFSF)protein and is encoded by the Tnfsf15 gene located on human chromosome 9q32 and mouse chromosome 4.Similar to other members of the TNF family,TL1A either exist in the form of membrane-bound protein(mTL1A)or be cleaved by matrix metalloproteinases and secreted into a soluble 20-kDa protein(sTL1A).The expression profile of TL1A shows that it plays a crucial role in both innate and adaptive immune responses,and exhibits a strong clinical correlation with inflammatory bowel disease and rheumatoid arthritis.Recent studies have suggested that membrane-bound and secretory TL1A may play different or even opposing roles in different contexts.DR3,encoded by the TNFRSF25 gene,is the key functional receptor of TL1A,and TL1A is the only confirmed DR3 ligand.The TL1A/DR3 signaling axis has been demonstrated to participate in the pathogenesis of asthma,inducing the activation of type 2 innate lymphoid cells,eosinophilic airway inflammation,and mucus hypersecretion.As such,it may serve as a promising therapeutic target with significant prospects for the treatment of asthma.While studies have confirmed that TL1A plays a role in asthma,the main focus of research has been on the immune cells,specifically type Ⅱ innate lymphoid cells,associated with secretory TL1A and T2 asthma.The role of TL1A and its receptor DR3 in airway remodeling in asthma has yet to be fully elucidated,including the its mutual transformation.ObjectiveThe purpose of this study is to investigate and explore the regulatory role of secretory and membrane-bound TL1A in airway remodeling in asthma,and to clarify its molecular mechanism.The aim is also to clarify the transformation process of TL1A in different forms during the development of asthma,further revealing the mechanism of the occurrence and development of asthma airway remodeling.Methods1.Utilizing the Human Protein Atlas database and immunoblotting experiments to investigate the expression levels of TL1A in human and mouse lung tissues.2.Collecting serum and clinical characteristics from asthma patients and healthy populations to determine the correlation between serum TL1A levels and asthma clinical features.3.Utilizing the GEO database for bioinformatics screening of potential inducers of TL1 A.Using experimental methods such as immunoblotting,immunofluorescence,RT-PCR,and ELISA to measure intracellular and secreted TL1A levels in bronchial epithelial cells.4.Confirming the effect of membrane-bound TL1A on airway epithelial cell fibrosis using TL1A small interfering RNA and membrane-bound truncated plasmids.5.Using bioinformatics analysis and tissue staining experiments to determine TL1A expression levels and collagen deposition in the airway epithelium of asthma patients and OVA-induced mouse models.6.Measuring TL1A expression levels in sputum and bronchoalveolar lavage fluid from asthma patients and mouse models using ELISA experiments.7.Examining the effect of intratracheal administration of TL1A on inflammatory cell infiltration and collagen deposition in the airway of wild-type and asthma model mice using tissue staining,immunofluorescence,and immunoblotting experiments.8.Constructing and validating a lentiviral vector to knock down DR3 expression using immunoblotting experiments and examining the effect of DR3 knockdown on inflammatory cell infiltration and collagen deposition in the airway of mouse models using tissue staining,immunofluorescence,and immunoblotting experiments.9.Breeding Tnfsf15 gene knockout mice(Tnfsf15-/-)and investigating the effect of Tnfsfl5 gene knockout on airway inflammation,collagen fiber deposition,and collagen protein and smooth muscle production in the airway using tissue staining and immunofluorescence experiments.10.Using transcriptome profiling via mouse lung tissue RNA-sequencing and enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes to identify critical differential enrichment pathways.Results1.TL1A is highly expressed in human and mouse lung tissue.2.TL1A levels are significantly upregulated in asthma patients’ serum,and closely correlated with asthma clinical features such as blood eosinophil count and lung function changes,compared to healthy controls.3.TGF-β1 can induce intracellular upregulation of TL1A in airway epithelial cells,without affecting its secretion.4.Membrane-bound TL1A participates in and assists TGF-β1-induced airway epithelial cell fibrosis under in vitro culture conditions.5.Membrane-bound TL1A expression is upregulated in the airway epithelium of asthma patients and OVA-induced mouse models,accompanied by increased collagen deposition.6.TL1A expression trended higher in asthma patients’ sputum and model mouse bronchoalveolar lavage fluid compared to healthy individuals.7.Exogenous airway instillation of TL1A in wild-type and asthma model mice directly induced inflammatory cell infiltration and collagen deposition,further exacerbating allergen-induced airway inflammation and remodeling.8.Validated lentiviral vectors that knock down DR3 expression in vivo were screened,and lentiviral-mediated DR3 knockdown helped alleviate allergen-induced airway inflammation and remodeling.9.Asthma model mice with Tnfsf15 gene knockout exhibited reduced airway inflammation,collagen fiber deposition,and collagen protein and smooth muscle production.10.Transcriptome sequencing and differential expression analysis revealed over 370 differentially expressed genes in gene-knockout asthma model mice and normal asthma model mice,with significant enrichment of downregulated genes in the NF-κB signaling pathway.ConclusionThis study has elucidated the differential expression patterns of membrane-bound and secretory TL1A in asthmatic patients(serum,sputum,and airway epithelium)and in asthmatic mouse models(alveolar lavage fluid and lung tissue).Additionally,the key inducer(TGF-β)of TL1A expression and its impact on the process of airway epithelial fibrosis have been identified.Furthermore,the study has clarified the roles of the two forms of TL1A(membrane-bound type and secretory type)in regulating airway remodeling induced by allergic inflammation,and demonstrated that TL1A,revealed through gene knockout in vivo model via the NF-κB signaling pathway,affects the molecular mechanisms underlying airway inflammation and remodeling in asthma Based on these findings,new possibilities for the discovery of key targets and the development of precise treatments for asthma airway remodeling have been proposed.