The Distribution Characteristics Of Upper And Lower Respiratory Tract Microbiota In Acute Asthmatics With Type 2 Inflammation

Objective:The distribution characteristics of airway microbiota are closely related to the incidence frequency and clinical prognosis of asthma,while the characteristics of upper and lower respiratory tract microbiota in different asthma phenotypes are not clear.This study investigated the distribution characteristics of upper and lower respiratory tract microbiota in acute asthmatics with type 2inflammation and its relationship with asthma severity.Meanwhile,we verified the correlation between the characteristic bacteria of upper and lower respiratory tract and Th2 inflammatory factors,so that we can provide reliable clinical evidences for airway microecological researches,individualized diagnosis and therapy in patients with asthma.Methods:A total of 45 acute asthmatics with type 2 inflammation were enrolled in the study from Second Clinical Medical College of Jinan University(Shenzhen People’s Hospital)from June 2020 to September 2021.Sixteen healthy controls were matched by gender and age during the same period.All asthma patients were divided into two groups according to the asthma severity:(1)23 mildto-moderate type 2 asthmatic patients;(2)22 severe type 2 asthmatic patients.We collected the demographic datas,samples of nasopharyngeal swab,and induced sputum for 16 S r RNA sequencing from all subjects.For all asthmatics,we recorded their blood cell count,induced sputum differential cells counts,pulmonary function indices,and collected their peripheral blood samples for detecting interleukin(IL)-4,IL-5,and IL-13.Statistical analyses were performed in SPSS 26.0 software.The t test,ANOVA and non-parametric test were used for analyzing measurement data between groups,and chi-square test was used for enumeration data.The α-diversity indices of airway microbiota were calculated with Mothur software,and the differences between groups were analyzed by wilcoxon rank sum test.The β-diversity were assessed by BrayCurtis and unweighted Uni Frac distance measurement,and the difference between groups were analyzed by ANOSIM analysis.Wilcox rank sum test was employed to analyze the difference in species abundance,and Linear discriminant analysis was conducted to estimate the effects of different species on difference between groups.Finally,Spearman Rank Correlation Test and multivariate association with linear models(Ma As Lin)were used to identify specific bacteria that were significantly associated with th2 inflammatory factors.Results:(1)The distribution characteristics of upper respiratory tract microbiota in type 2 asthma were as follows:1)Compared with healthy control group,the community richness of upper respiratory tract was significantly decreased in type 2 asthma(all P≤0.01),while the community diversity and evenness were prominently increased(all P<0.05).There was significantly difference in β-diversity between healthy controls and type 2 asthmatics(P=0.006).In community composition,upper respiratory tract of asthmatics were enriched for Proteobacteria at the phylum level(P=0.049),while Lactobacillus was significantly reduced in type 2 asthma group than control group at the genus level(P=0.001).2)There were no statistical difference in microbiota community richness,diversity,eveness,β-diversity and community composition at both phylum and genus levels between mild-tomoderate asthma group and severe asthma group(all P>0.05).3)For all type 2 asthmatics,the relative abundance of Proteobacteria was negatively correlated with IL-13(Coeffencient=-0.009,P=0.033),while the relative abundance of Corynebacterium was positively correlated with IL-13(Coeffencient=0.014,P=0.031).The abundance of Actinobacteriota was positively correlated with the level of IL-5(Coeffencient=0.065,P=0.030).In addition,there was a negative correlation between the abundance of Bacteroidota,Prevotella,Acinetobacter and peripheral blood eosinophil counts(Coeffencient=-0.103,-0.076,-0.175;P =0.046,0.015,0.017;respectively).(2)The distribution characteristics of lower respiratory tract microbiota in type 2 asthma were as follows:1)As to the lower respiratory tract microbiota,there were no statistically significant differences in the community richness,diversity,evenness and the relative abundance of each dominant bacteria at the phylum level between asthma patients and healthy controls(all P>0.05).However,the β-diversity in severe asthmatics was significantly different with controls(P=0.004).At the genus level,the relative abundance of Neisseria was significantly decreased(P=0.010),and the abundance of Veillonella was significantly increased in asthmatics(P=0.002).2)Compared with the mild-to-moderate asthma group,no statistically differences were found in the community richness,diversity,evenness and the abundance of each dominant bacteria at the phylum level in the severe asthma group(all P>0.05).As to the community composition,severe asthma group were prominently enriched for Veillonella(P=0.002),while Neisseria,Parvimonas and Rhodococcus were statistically enriched in the mild-tomoderate asthma group(P=0.010,0.043,0.001;respectively).3)In the lower respiratory tract microbiota,the relative abundance of Peptostreptococcus was positively correlated with the level of IL-5 in all type 2 asthmatics(Coeffencient=0.024,P=0.002),while the relative abundance of Capnocytophaga was negatively correlated with IL-5(Coeffencient=-0.011,P=0.001).The abundance of Peptostreptococcus,Parvimonas and Actinomyces was positively correlated with levels of IL-13(Coeffencient=0.004,0.001,0.004;P=0.007,0.026,0.049;respectively).Moreover,there was a negative correlation between the relative abundance of Actinobacteriota and induced sputum eosinophil counts(Coeffencient=-0.009,P=0.019).(3)The comparison of upper and lower respiratory tract microbiota in severe type 2 asthma was as follows:1)The community diversity and evenness of microbiota in the lower respiratory tract were significantly higher than those in the upper respiratory tract in patients with type 2 severe asthma(all P<0.01).2)Compared with healthy controls and mild-to-moderate asthmatics,the community structure difference of microbiota between the upper and lower respiratory tracts was reduced in severe asthma(R=0.441,P=0.001).3)At the phylum level,Proteobacteria and Actinobacteria were significantly enriched in the upper respiratory tract of severe asthma(P=0.020,0.004,respectively),and Bacteroidota,Fusobacteriota,Patescibacteria and Spirochaetota were significantly enriched in the lower respiratory tract(all P<0.001).At the genus level,Staphylococcus,Corynebacterium,Rhodococcus,Ralstonia,Acinetobacter,Cutibacterium and Pseudomonas were significantly enriched in the upper respiratory tract of severe asthmatics(all P<0.05),wihle Streptococcus,Prevotella,Veillonella,Neisseria,Actinomyces,Porphyromonas,Haemophilus,Fusobacterium and Capnocytophaga were significantly enriched in the lower respiratory tract(all P<0.01).Conclusion:(1)There were prominent differences in the composition and structure of upper or lower respiratory tract microbiota in healthy,type 2 mild-to-moderate and severe asthmatics.(2)The relative abundance of specific bacteria including Actinobacteriota,Peptostreptococcus and Actinomyces in the upper and lower respiratory tract were closely related to the levels of IL-5 and IL-13 in type 2 asthmatics.(3)The structure of microbiota in the upper and lower respiratory tract of severe type 2 asthmatics have certain degrees of similarity,but the relative abundance of Corynebacterium,Streptococcus,Veillonella,Neisseria,and other specific bacteria were significantly different.