Studies On Mechanism Of Allergic Airway Inflammation Based On Microbiome And Metabolomics

The airway mucosa is continuous from the nostril to the end of the alveoli,and the upper and lower airways are anatomically and histologically similar.Therefore,upper and lower airway diseases often coexist.Allergic rhinitis(AR)and allergic asthma(AA),described as "one airway,one disease",are collectively referred to as allergic airway inflammation(AAI).AAI is a type I allergic disease mediated by immunoglobulins(Ig E)in atopic individuals exposed to allergens,and it is a major global health problem that imposes a significant burden on individuals and society by seriously affecting patients’ quality of life and social life and incurring substantial economic costs.Therefore,studying the pathogenesis of AAI is essential to control the onset and progression of the disease.Allergic diseases are inextricably linked to the human living environment.The airways are protected to varying degrees by normally colonized microorganisms,but dysbiosis induces inflammatory responses and increases susceptibility to allergic diseases.It has been found that a large number of microbial communities are present throughout the airway,but there are differences in the abundance and diversity of microbial community structure in different locations of the airway,which may play a role in the development of AAI.The upper airway has different epithelial cell types and is exposed to a variety of environmental factors,and abnormal changes in the upper airway microbiota may occur in response to allergen stimulation.At the same time,upper airway inflammation can spread to the lower airways.Therefore,exploring the upper airway microbiota in allergic airway inflammation is important to elucidate the pathogenesis of AAI.Based on the above background,this study compares the differences in upper airway microorganisms between patients with allergic rhinitis and normal individuals based on microbiome and metabolome approaches,and then investigates the role and mechanism of S.aureus in allergic airway inflammation through in vivo and in vitro experiments.To provide new experimental data for understanding the role of flora in the development of allergic airway inflammation and to provide new strategies for clinical treatment of allergic airway inflammation.1.Airway microbiomics and serum metabolomics in patients with allergic rhinitisMETHODS: Using patients with allergic rhinitis(AR)as well as healthy volunteers(HC)as study subjects.(1)Analysis of the abundance and diversity of respiratory microorganisms in nasal swab samples using 16 S r RNA high-throughput sequencing technology to study the structure and composition of the microbiota.(2)Metabolomic analysis of serum samples by ultra-high performance liquid chromatography-high resolution mass spectrometry.RESULTS:(1)Significant differences in species Beta diversity were found between the AR and HC groups(p<0.01).At the phylum level,Proteobacteria,Firmicutes and Bacteroidete were the most abundant among upper respiratory tract microorganisms.At the genus level,Stenotrophomona,Sphingomonas and Faecalibacterium were the most abundant among upper respiratory tract microorganisms.Compared with the HC group,the abundance of eight microorganisms,including Staphylococcus,was higher in the AR group,while the abundance of Sphingomonas was lower.(2)Based on the constructed OPLS-DA model,26 differential metabolites and 16 related metabolic pathways were identified,of which linoleic acid metabolism,arachidonic acid metabolism and caffeine metabolism were the top three metabolic pathways,with arachidonic acid(AA)metabolic process producing important inflammatory mediators that are thought to be markers of several inflammation-related diseases,including allergic asthma.2.Role of S.aureus in OVA-induced allergic airway inflammation in mice.METHODS: S.aureus infected AAI mouse model with OVA was constructed.(1)Observe the behavioral and body weight changes of mice in Control,SA,OVA and OVA+SA groups.(2)Detection of Ig E and OVA-s Ig E in mice by ELISA.(3)Detection of airway hyperresponsiveness in each group of mice using the airway resistance and lung compliance detection system(RC system)of the small animal respiratory lung function instrument.(4)HE staining of nasal mucosa in mice.(5)HE staining and PAS staining of mouse lung tissues.(6)Metabolomic analysis of serum,lung tissues and nasal mucosa.RESULTS:(1)Mice in the Control group showed normal activity and increased body weight,while mice in the SA group showed scratching behavior,reduced activity and decreased body weight.mice in the OVA and OVA+SA groups often showed eye scratching,nose scratching and sneezing behaviors,reduced daily activities and decreased body weight,and mice in the OVA+SA group showed a more obvious decrease in body weight.(2)Compared with the Control group and SA group,the Ig E level of mice in the OVA group was significantly increased(p<0.05).The Ig E levels of mice in the OVA+SA group were significantly increased compared to the OVA group(p<0.05).(3)Compared with the Control group,the airway resistance of mice in the SA,OVA and OVA+SA groups was increased.Airway resistance was significantly increased(p<0.05)and pulmonary compliance was highly significantly reduced(p<0.01)in the OVA+SA group compared to the OVA group,demonstrating airway hyperresponsiveness.(4)Compared with the OVA group,the mice in the OVA+SA group showed epithelial cell damage in the nasal mucosa,significant epidermal cell detachment,increased edema,disorganization of the lamina propria,significant vasodilatation and congestion,glandular hyperplasia and massive infiltration of inflammatory cells.(5)Compared with the OVA group,the lung tissue of mice in the OVA+SA group showed increased bronchial thickness,marked focal atrophy,more incomplete mucosal epithelium,marked thickening of the alveolar septum,increased mucus secretion and massive infiltration of inflammatory cells in all layers of the duct wall.(6)Serum and lung levels of linoleic acid,9,10-epoxyoctadecenoic acid,and12,13-Ep OME were significantly changed in both groups of mice in the linoleic acid metabolic pathway.9,10-epoxyoctadecenoic acid and 12,13-Ep OME were reduced in both serum and lung tissue of mice in the OVA+SA group,but linoleic acid was different in that it was The levels of linoleic acid in serum and lung tissues were reduced in the OVA+SA group mice,but linoleic acid levels were increased in lung tissues.In addition,increased levels of metabolites related to the pentose phosphate pathway were found in the nasal mucosa of mice in the OVA+SA group.3.Study on the mechanism of action of Staphylococcus aureus in aggravating allergic airway inflammationMETHODS: S.aureus infected AAI mouse model with OVA and DNP-Ig E/HSA induced RBL-2H3 cell degranulation model was constructed.(1)Flow cytometry to detect the number of immune cells in BALF of mice.(2)ELISA assay to detect the amount of cytokines in mice.(3)Western blot assay to detect the expression of NF-κB and MAPK signaling pathway-related proteins.(4)Microscopic observation of the morphology of each group of toluidine blue-stained cells.(5)Fluorescence microscopy observation of each group of ghost pen cyclic peptide stained cytoskeleton F-actin.(6)Detection of cytokine and TLR2 expression levels in each group of cells by real-time fluorescence quantitative PCR.(7)To detect the release rate of β-HEX in the degranulation model of S.aureus-infected RBL-2H3 cells treated with C29,an inhibitor of TLR2.RESULTS:(1)The number of total cells,macrophages and eosinophils in the BALF of mice in the OVA+SA group was significantly higher compared to the OVA group(p<0.05).(2)The levels of IL-4,IL-5,IL-9 and IL-13 in the serum of mice in the OVA+SA group were significantly increased compared with the OVA group(p<0.05).(3)The ratios of p-p65/p65 and p-p38/p38 in the lung tissues of mice in the OVA+SA group were significantly higher compared with the OVA group(p<0.05).(4)S.aureus caused a large number of purple particles to be released outside the cells and the cell membrane to become round and irregular.(5)S.aureus led to remodeling and significant loosening of the mast cell cytoskeleton.(6)C29,an inhibitor of TLR2,significantly reduced(p<0.01)the β-HEX release rate(OD405 nm)in a degranulation model of S.aureus-infected RBL-2H3 cells.CONCLUSION:(1)Microorganisms in the upper respiratory tract of patients with allergic rhinitis were disturbed,including a significant increase in the abundance of eight differential genera,including Staphylococcus spp.Systemic metabolic profiles were altered,and26 differential metabolites,including prostaglandin H2 and prostaglandin D2,and 16 related metabolic pathways,including linoleic acid and arachidonic acid metabolism,were identified.(2)S.aureus promoted the development of allergic airway inflammation by increasing Ig E levels in vivo,promoting airway hyperresponsiveness,inflammatory infiltration of nasal and lung tissues,and altering systemic and local metabolic patterns in mice with allergic airway inflammation by modulating 10 metabolic pathways including linoleic acid metabolism and the pentose phosphate pathway.(3)Allergic airway inflammation occurs when S.aureus affects mast cell degranulation via TLR2 by a mechanism that may be related to NF-κB and MAPK signaling pathways.