Correlation Analysis Of Lung Microbiota And Lung Function In Lung Transplant Recipients

BackgroundLung transplantation is the sole effective treatment for end-stage lung diseases.However,the long-term outcomes of lung transplantation remain suboptimal.The major obstacle to improving long-term prognosis after lung transplantation is chronic lung allograft dysfunction（CLAD）,which can be categorized into two main types:bronchiolitis obliterans syndrome（BOS）and restrictive allograft syndrome（RAS）.BOS is the primary phenotype of CLAD and is characterized by obstructive ventilatory dysfunction.In lung transplantation,lung ventilatory function serves as a crucial indicator for predicting recipient prognosis and as a diagnostic criterion for the most significant long-term complication after transplantation,CLAD.Therefore,lung function testing plays a vital role in evaluating the recovery of lung transplant recipients,assessing graft function,and predicting prognosis.Continuous monitoring of lung function is routinely performed after lung transplantation,typically utilizing forced expiratory volume at one second（FEV₁）as an index to evaluate graft status and function.However,there has been insufficient research on the temporal changes in forced vital capacity（FVC）,FEV₁/FVC ratio,and pulmonary diffusion function.Postoperative lung function in lung transplant recipients gradually recovers during the early period and reaches a plateau or stable phase after achieving optimal lung function.Studies conducted abroad have shown that post-transplant lung ventilatory function typically peaks between 3 and 17 months postoperatively,although there are significant variations among different studies.However,there is currently a lack of investigation into the dynamic changes in lung function after lung transplantation among domestic populations.With the development of bacterial 16S ribosomal RNA（r RNA）gene sequencing and high-throughput sequencing technologies,it has become possible to quantify the relative abundance of microorganisms in respiratory samples.Research has indicated that dysbiosis of the lung microbiota plays a significant role in the progression of various chronic respiratory diseases such as asthma,chronic obstructive pulmonary disease,and pulmonary fibrosis.The lung microbiota of lung transplant recipients differs from that of healthy individuals,and there exist complex interactions between the lung microbiota and the host.There is an increasing amount of research internationally on the lung microbiota and CLAD.Recent studies have discovered that the presence of different pathogenic bacterial communities in the lungs can directly impact the activation of fibroblasts,potentially being a significant factor contributing to the loss of chronic graft lung function.Phenotypes dominated by actinomycetes are associated with a reduced risk of bronchiolitis obliterans compared to those dominated by Pseudomonas.Both domestic and international studies have indicated that the lung microbiota has a substantial influence on post-lung transplant graft function and prognosis.Therefore,we conducted an investigation into the postoperative lung function characteristics and influencing factors of lung transplant recipients at our transplantation center.Additionally,we analyzed the correlation between changes in lung function and the lung microbiota.Furthermore,we hypothesized that the lung microbiota has an impact on postoperative lung function.Objective:Investigating the characteristics of postoperative lung function and its influencing factors in lung transplant recipients at our center.Examining the characteristics of lung microbiota in stable lung transplant recipients with different lung functions after surgery.Exploring the correlation between lung microbiota and lung function.Part 1:Analysis of postoperative lung function characteristics and influencing factors in lung transplant recipients at our centerObjective:To retrospectively analyze the changes in pulmonary ventilation and diffusion function of lung transplant and heart-lung transplant recipients at our center three years after surgery,and to analyze their influencing factors,in order to provide data support for the characteristics of postoperative lung function changes in lung transplant recipients in China.Method:This study was a retrospective study.The study subjects were recipients who underwent allogeneic lung transplantation and heart-lung transplantation at The First Affiliated Hospital of Guangzhou Medical University from June 2016 to January2022.The pulmonary ventilation and diffusion function of the enrolled patients were measured at 1 month,3 months,every 3 months（3 to 18 months after surgery）,and every 6 months（18 to 36 months after surgery）.The measured values included forced vital capacity（FVC）,forced expiratory volume in one second（FEV₁）,FEV₁/FVC ratio,carbon monoxide diffusing capacity（D_LCOc）,and the ratio of D_LCOc to alveolar volume（D_LCOc/VA）.The effects of the primary disease type,age,gender,body mass index（BMI）,postoperative mechanical ventilation time,postoperative ICU stay time,and hospitalization due to infection within one year on the measured values of pulmonary function as a percentage of predicted were analyzed.Results:1.Basic informationA total of 81 subjects were included in this study.2.Postoperative lung function characteristics of lung transplant recipients at different time pointsThe pulmonary ventilation function of lung transplant recipients showed a gradual upward trend after surgery,and FVC%pred and FEV₁%pred gradually increased within 9 months after surgery,but there was no statistically significant change from 9to 36 months after surgery.FEV₁/FVC%pred showed a downward trend in the early postoperative period,and gradually decreased within 6 months after surgery,but there was no statistically significant change from 6 to 36 months after surgery.The diffusion function of lung transplant recipients increased in the early postoperative period,and DLCOc%pred gradually increased within 3 months after surgery,but there was no statistically significant change from 3 to 36 months after surgery.3.Best lung function characteristics of lung transplant recipients after surgeryPost-lung transplant recipients have a postoperative FVC%pred of（75.84±1.99）%,FEV₁%pred of（79.95±2.37）%,and FEV₁/FVC%pred of 106.76（98.96-113.46）%.Of the 81 recipients,36（44.44%）had normal FVC%pred,41（50.62%）had normal FEV₁%pred,and 76（93.83%）had normal FEV₁/FVC%pred as their best post-transplant lung function.Postoperative D_LCOc%pred was（66.12±1.95）%,and D_LCOc/V_A%pred was（93.10±2.02）%.Of the 81 recipients,16（19.75%）had normal D_LCOc%pred and 63（77.78%）had normal D_LCOc/V_A%pred as their best post-transplant diffusion function.Recipients with obstructive lung function as the underlying disease had a postoperative FEV₁%pred of（78.40±15.87）%,FEV₁/FVC%pred of 110.12（102.65-116.05）%,and D_LCOc%pred of（72.18±18.28）%.Recipients with restrictive lung function as the underlying disease had a postoperative FEV₁%pred of（76.13±21.43）%,FEV₁/FVC%pred of（104.15±14.08）%,and D_LCOc%pred of（61.96±15.98）%.There was a significant difference in lung function between the two groups with different underlying diseases.Recipients who developed PGD had a postoperative D_LCOc%pred of（61.21±13.69）%,while those who did not develop PGD had a postoperative D_LCOc%pred of（68.58±18.88）%.There was a significant difference in the best lung function between the two groups with and without PGD.4.Factors influencing post-lung transplant lung functionPostoperative FEV₁,FVC,and FEV₁/FVC were positively correlated with age（p<0.05）.Postoperative FEV₁/FVC was positively correlated with BMI（p<0.05）,and recipients with restrictive lung disease had a lower FEV₁/FVC（p<0.05）.Postoperative mechanical ventilation time and ICU stay were negatively correlated with FVC（p<0.05）.Age was positively correlated with D_LCOc（p<0.05）,and recipients with restrictive lung disease had a lower D_LCOc（p<0.05）.Conclusions:Most post-lung transplant recipients experience moderate restrictive lung function and diffusion impairment.Post-lung transplant lung function gradually improves within the first 9 months and reaches a plateau.Age,BMI,mechanical ventilation time,ICU stay,and underlying lung disease all have an impact on post-lung transplant lung function.Part 2:Characteristics of Lung Microbiota in Lung Transplant Recipients with Different Lung Function Grades in the Stable Period of our CenterObjective:To investigate the differences in lung microbiota between the normal lung function group and the impairment group after lung transplantation in our center.Methods:This study was a retrospective study.Lung transplant recipients who underwent allogeneic double lung transplantation or heart-lung combined transplantation at the The First Affiliated Hospital of Guangzhou Medical University from June 2016 to January 2022,with a survival time of more than 1 year and stable clinical status（defined as no clinical manifestations of respiratory tract infection and no new changes in lung imaging）at the time of enrollment were included in this study.The lung transplant recipients were instructed to regularly follow up in the hospital after surgery,and their lung ventilation and diffusion function were measured within 1 month,including forced vital capacity（FVC）,forced expiratory volume in one second（FEV₁）,and FEV₁/FVC ratio.Lung ventilation function was considered normal if FVC%pred and FEV₁%pred were both≥80%,and FEV₁/FVC%pred ratio was≤92%.BALF samples were obtained by experienced bronchoscopists using a standardized procedure from the transplanted lungs of the recipients,and BALF samples were transported and tested within 2 hours at room temperature（about 25°C）.Nucleic acid extraction,library preparation,and sequencing were performed,and low-quality,short,and adapter sequences were filtered out to generate high-quality data.Human sequences were removed,and samples with less than 2000 unique microbial sequences were filtered out.The Shannon index was used to evaluateαdiversity（within-sample diversity）.PCo A based on the Bray-Curtis algorithm was used to calculateβdiversity（between-sample differences）.LEf Se analysis with an LDA score threshold of 2.0 was used to identify differentially abundant bacterial taxa between different groups.Result:1.Basic informationThis study included 67 participants,with 29 participants in the FEV₁ normal group and 38 in the FEV₁ impairment group.There were 24 participants in the FVC normal group and 43 in the FVC impairment group.The FEV₁/FVC%pred normal group had 60 participants,while the FEV₁/FVC%pred impairment group had 7participants.2.Differences in microbial communities between different lung functionsThere were no significant differences inαdiversity andβdiversity between the groups.3.Changes in microbial communities between different lung functionsIn the analysis of the FEV₁ normal and impairment groups,two different taxa were identified.Lactobacillus was more abundant in the FEV₁ impairment group,while Rothia was more abundant in the FEV₁ normal group.In the analysis of the FVC normal and impairment groups,eight different taxa were identified.Prevotella,Streptococcus,Pseudomonas,and several other taxa were more abundant in the FVC impairment group,while Burkholderia was more abundant in the FVC normal group.In the analysis of the FEV₁/FVC%pred normal and impairment groups,nine different taxa were identified.Circoviridae,Circovirus,Hemophilus,Shewanella,and several other taxa were more abundant in the FEV₁/FVC%pred impairment group,while Staphylococcus,Paracoccus,and Pseudomonas were more abundant in the FEV₁/FVC%pred normal group.Conclusion:Lactobacillus,Rothia,Prevotella,Streptococcus,Pseudomonas,Burkholderia,Circoviridae,Circovirus,and Hemophilus may be potential indicators for assessing the recovery of lung function after lung transplantation.Part Ⅲ:Correlation between lung microbiota and lung function in lung transplant recipientsObjective:To explore the correlation between lung microbiota and lung function.Methods:This study is a retrospective study.Recipients who underwent allogeneic lung transplantation surgery at The First Affiliated Hospital of Guangzhou Medical University from June 2016 to January 2022 and survived for more than 1 year and were clinically stable（stable period defined as no clinical manifestations of respiratory tract infection and no new changes in pulmonary imaging）were included.The enrolled recipients were regularly followed up after surgery and underwent pulmonary ventilation function tests at two time points,at the time of enrollment and one year after enrollment.Forced vital capacity（FVC）,forced expiratory volume in one second（FEV₁）,and FEV₁/FVC were measured.BALF samples were lavaged from the transplanted lungs of double-lung transplant recipients by experienced bronchoscopic physicians using a standardized procedure.BALF samples were immediately stored in sterile containers and transported and tested within 2 hours at room temperature（about 25°C）.Nucleic acid extraction,library preparation,and sequencing were performed to generate high-quality data by filtering out adapter sequences,low-quality,and short sequences.Human sequences were removed,then samples with less than 2000 unique microbial sequences were filtered out.Spearman correlation analysis was used to evaluate the correlation between absolute changes in lung function values and relative abundance changes in lung microbiota.A P value of<0.01 was considered significant.Results:1.Basic information:This study included a total of 45 subjects as research objects.2.Correlation between absolute changes in FEV₁ values and relative abundance changes in lung microbiotaA total of 2 different taxonomic groups were identified that showed a significant positive correlation（P<0.01）with FEV₁,with relative abundance changes in Acinetobacter baylyi and Acinetobacter piscicola of the Acinetobacter genus showing a significant positive correlation with absolute changes in FEV₁ values.3.Correlation between absolute changes in FVC values and relative abundance changes in lung microbiotaA total of 12 different taxonomic groups were identified that showed a significant positive correlation with FVC,while 3 taxonomic groups showed a significant negative correlation（P<0.01）.Relative abundance changes in Spirillum,Barnesiella,Burkholderia,Oxalobacteraceae,Ciliophora,Neisseria,Sphingomonadaceae,and Pseudomonas genera showed a significant positive correlation with absolute changes in FVC values.Conversely,relative abundance changes in Enterobacter,Haemophilus,and Streptococcus genera showed a significant negative correlation with absolute changes in FVC values.4.Correlation between absolute changes in FEV₁/FVC values and relative abundance changes in lung microbiotaA total of 41 different taxonomic groups were identified that showed a significant positive correlation with FEV₁/FVC,while 2 taxonomic groups showed a significant negative correlation（P<0.01）.Relative abundance changes in Acinetobacter,Bacillus,Clostridium,Rhizobium,Staphylococcus,Pseudomonas,Elizabethkingia,Dysgonomonas,Phormidium,Geobacillus,Lysobacter,Novosphingobium,Paracoccus,Shinella,Providencia,Burkholderia,Serratia,Lactobacillus,Shewanella,Long-tailed phages,Sphingomonas,Pedobacter,and Streptococcus genera showed a significant positive correlation with absolute changes in FEV₁/FVC values.Conversely,relative abundance changes in Micromonospora and Pedobacter genera showed a significant negative correlation with absolute changes in FEV₁/FVC values.Conclusion:Bacillus,Streptococcus,Providencia,Pseudomonas,and Neisseria are positively correlated with lung function,while Haemophilus show a negative correlation.