Exploring The Relationship Between Airway Inflammatory Phenotype And Airway Microbiota In Bronchial Asthma And Chronic Obstructive Pulmonary Disease

BackgroundChronic inflammatory diseases of the airways,represented by bronchial asthma（asthma）and chronic obstructive pulmonary disease（COPD）,affect over 500 million people worldwide and consume enormous public health resources^[1],and have long been a focus of research in the field of respiratory medicine.Studies have confirmed that both asthma and chronic obstructive pulmonary disease can be classified into different airway inflammatory phenotypes based on the proportion of eosinophils and neutrophils in the patient’s induced sputum.Patients with the eosinophilic phenotype of both diseases showed better responsiveness to glucocorticoid therapy and improvement in symptoms and lung function,while patients with the neutrophilic phenotype of asthma and COPD showed more severe levels of airflow limitation and poorer responsiveness to glucocorticoid therapy^[2-6].However,the reasons for the different airway inflammatory phenotypes in the same disease are still unclear,and why different diseases can show similarities in inflammatory phenotypes needs to be further explored.With advances in high-throughput sequencing technology and the rapid development of research on human microbiota（mainly bacteria,fungi and viruses）,it has been recognized that the composition of the airway microbiota in asthma and COPD differs significantly from that of healthy individuals,and it has been suggested that the microbiota may have an impact on the airway inflammatory phenotypes.Patients with different phenotypes of asthma and COPD were found to differ significantly in the composition and diversity of their airway microbiota,and the abundance and load of specific bacterial genera（e.g.Haemophilus,Streptococcus,Moraxella,etc.）were clearly correlated with several key clinical indicators such as patient symptom scores,lung function,and frequency of acute exacerbations^[7-11].In addition,the role of fungal microbiota as an important factor influencing host immunity in chronic airway inflammatory diseases should not be overlooked.Most of the existing studies have focused on the relationship between eosinophilic asthma and fungal microbiota,the correlations between the relative abundance of several genera,such as Aspergillus,Cladosporium and Fusarium and indicators of eosinophilic inflammation,fractional exhaled nitric oxide（Fe NO）levels,lung function have been reported^[12,13].However,there is a lack of studies that have characterised the airway fungal microbiota in patients with neutrophilic asthma.In addition,the airway fungal microbiota of COPD patients has also not been characterised,and the question of whether there are differences in the fungal microbiota of patients with different phenotypes of COPD needs to be further investigated.To address these questions,in this study,the clinical data and induced sputum samples from patients with clinically stable asthma and COPD were included and collected,and the bacterial 16S r RNA gene and fungal ITS gene were sequenced by Pac Bio SMRT sequencing technology on induced sputum samples from enrolled subjects,to explore the airway microbiota characteristics of asthmatic and COPD patients with different phenotypes,respectively,and to further compare the similarities and differences in airway microbiota between subjects with the same phenotype.It enriches the data of airway microbiome studies in chronic airway inflammatory diseases and provides clues and directions for further research on the interaction between airway microbial colonization and host immune response,as well as the search for potentially treatable features in the future.Objective1.We aimed to explore whether similarities exist in the airway microbiota between eosinophilic asthma（EA）and eosinophilic COPD（EC）patients,between non-eosinophilic asthma（NEA）and non-eosinophilic COPD（NEC）patients.2.To explore the presence of biomarker species of airway microbiota in patients with different airway inflammatory phenotypes of COPD or asthma and to further focus on the characteristics of airway fungal and bacterial microbiota and their clinical significance in patients with stable neutrophilic asthma.Methods1.A total of 84 subjects were recruited from patients attending the Department of Respiratory Medicine at the Second Affiliated Hospital of the Army Medical University and participating in our previously registered clinical study（www.chictr.org.cn,NO.:Chi CTR1900024871）from August 2019 to February 2020,according to study inclusion and exclusion criteria.Subjects were defined as eosinophilic asthma（EA,n=26）/COPD（EC,n=22）group if their sputum eosinophil percentage was≥3%,otherwise,they were classified into non-eosinophilic asthma（NEA,n=19）/COPD（NEC,n=17）group.The sequencing of bacterial 16S r RNA genes and fungal ITS genes was performed on induced sputum samples from enrolled subjects to compare the differences in clinical data,αandβdiversity of airway microbiota between different phenotypes of the same disease（EA vs.NEA,EC vs.NEC）,and between subjects with the same phenotype of different diseases（EA vs.EC,NEA vs.NEC）,and to explore the relationship between the differential taxa and airway inflammatory phenotypes in each group.2.44 patients with stable neutrophilic asthma were enrolled from patients attending the Department of Respiratory Medicine at the Second Affiliated Hospital of the Army Medical University and participating in our previously registered clinical study（www.chictr.org.cn,NO.:Chi CTR1900024871）from August 2019 to February 2020,according to study inclusion and exclusion criteria.Relevant clinical data were collected from the subjects,and their induced sputum samples were sequenced for bacterial 16S r RNA genes and fungal ITS genes.Two subgroups（group A1,n=32;group A2,n=12）were identified by hierarchical clustering analysis of bacterial sequencing data,and the differences in their bacterial and fungal microbiota characteristics and their related clinical indices were further investigated.Results1.Subjects with eosinophilic airway inflammation phenotype,showed significantly differences in both fungal and bacterial microbiota than subjects with non-eosinophilic airway inflammation phenotype,that were observed in both asthma and COPD subjects.No significant differences in fungal microbiota diversity were observed in patients with the same airway inflammatory phenotype from different diseases.In terms of fungal microbiota,subjects with eosinophilic phenotypes in asthma and COPD（EA and EC groups）were significantly enriched in Aspergillus and Bjerkandera by LEf Se analysis,while the subjects with non-eosinophilic airway inflammation phenotype（NEA and NEC）were enriched in Rhodotorula,Papillotrema.In bacterial microbiota,the EA subjects enriched in Moraxella,Selenomonas,Prevotella pallens,Prevotella oris,Prevotella melaninogenica,Streptococcus thermophilus,Fusobacterium nucleatum,Campylobacter concisus,whereas Pseudomonas,Ochrobactrum and Stenotrophomonas were enriched in NEA subjects.Similar to asthmatics,Streptococcus thermophilus and Ochrobactrum were differential taxa between EC and NEC subjects.Further interaction network analysis also suggested similarities in the correlations between genera in asthma and COPD subjects.2.Hierarchical clustering analysis of bacterial sequencing data from 44 patients with stable neutrophilic asthma identified two clusters in patients with neutrophilic asthma.Compared to the larger cluster（A1 group,n=32）,the smaller cluster（A2 group,n=12）characterized by a predominance of Haemophilus in their bacterial microbiota,and patients in this group demonstrated a higher percentage of sputum neutrophils and poorer FEV1%pred.In bacterial microbiota,there were significant differences between the two groups.At the species level,LEf Se analysis revealed that the A1 group was significantly enriched in Streptococcus pneumoniae、Haemophilus parainfluenzae、Fusobacterium nucleatum、Lautropia mirabilis、Neisseria meningitidis、Porphyromonas gingivalis、Prevotella melaninogenica、Prevotella pallens、Ralstonia solanacearum,while A2 group was enriched in Haemophilus influenzae.In fungal microbiota,no significant differences were observed between the two groups,whereas significant differences were observed between theirβ-diversity.At the species level,LEf Se analysis revealed that the A1 group was enriched in Schizophyllum commune,A2 group was enriched in Trametes versicolor and Irpex lacteus.Interaction network analysis indicated that A2 group showed more correlations between genus than A1 group,and the strength of correlations between genus was more intense.Conclusion1.Airway inflammatory phenotypes in stable asthma and COPD are associated with specific bacterial and fungal microbiota characteristics,and heterogeneity exists between different phenotypes of the same disease.And there may exist some similarity in their fungal microbiota in patients with the same background of airway inflammation.2.Among patients with stable neutrophilic asthma,there is a subgroup characterized by the presence of Haemophilus influenzae as its airway predominant colonizing bacteria,which differs significantly from the non-Haemophilus influenzae dominant group in terms of bacterial and fungal microbiota,and presented clinically with a more severe level of airway inflammation and poorer lung function characteristics.