慢性阻塞性肺疾病小鼠肠道免疫球蛋白A包被微生物组成及基因功能分析

Composition and gene functions of intestinal IgA-coated microbiota in the mouse model of chronic obstructive pulmonary disease

摘要:

【背景】小气道免疫球蛋白A (immunoglobulin A,IgA)在慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)的病理生理学中发挥着重要作用。然而肠黏膜来源的IgA在COPD病程中的作用及包被微生物群尚不明确。【目的】探讨COPD小鼠肠来源IgA包被肠道微生物组成、丰度及菌群基因功能变化。【方法】采用鼻腔滴入脂多糖和熏香烟法相结合建立COPD小鼠模型。收集COPD小鼠和野生型小鼠粪便样品各12份，IgA磁珠分选IgA包被的肠道微生物菌群，16S rRNA基因高通量测序分析其组成及功能。【结果】通过比较两组肺组织切片染色、平均内衬间隔(mean linear intercept,mLI)和肺泡灌洗液炎症因子浓度证实COPD模型鼠建模成功。OTU和主成分分析(principal component analysis,PCA)均发现两组间粪便样品中肠来源IgA包被微生物群落差异大，具有可比性。α多样性分析显示两组间物种多样性无显著统计学差异(P>0.05)。物种组成分析显示：两组肠来源IgA包被的菌群结构和菌群差异具有统计学意义(P<0.05)。COPD组的菌群结构中显著富集的菌目是：蛭弧菌目(Bdellovibrionales)、梭菌目(Clostridiales)和双歧杆菌目(Bifidobacteriales)；科层面分类中富集的主要是：普雷沃氏菌科(Prevotellaceae)、梭菌科(Clostridiaceae)、类芽孢杆菌科(Paenibacillaceae)、蛭弧菌科(Bdellovibrionaceae)及双歧杆菌科(Bifidobacteriaceae)；菌属层面分类上主要富集拟普雷沃氏菌属(Alloprevotella)、短芽孢杆菌属(Brevibacillus)、狭义梭菌属(Clostridium-sensu-stricto)、苏黎世杆菌属(Turicibacter)、粪杆菌属(Faecalibacterium)、吸血弧菌属(Vampirovibrio)和双歧杆菌属(Bifidobacterium)。菌群差异基因通过京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)数据库通路富集分析结果显示COPD组细胞生长与死亡、核苷酸代谢以及消化系统相关通路明显上调，而膜运输相关通路显著下调。【结论】COPD小鼠肠来源IgA包被肠道微生物存在紊乱及基因功能失调。

关键词:慢性阻塞性肺疾病;免疫球蛋白A包被肠道微生物;16S rRNA基因测序

Abstract:

[Background] Immunoglobulin A (IgA) secreted by small airways plays a key role in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the role of IgA derived from intestinal mucosa in the pathogenesis of COPD and the IgA-coated microbiota remain unclear. [Objective] To investigate the composition, abundance, and differential gene functions of intestinal-derived IgA-coated gut microbiota in the mouse model of COPD. [Methods] A mouse model of COPD was established by intranasal instillation of lipopolysaccharide combined with cigarette smoking. Twelve fecal samples from COPD mice and 12 fecal samples from wild-type mice were collected. IgA magnetic beads were used to separate IgA-coated gut microbiota, followed by 16S rRNA gene high-throughput sequencing. [Results] We examined the modeling of COPD in mice by comparing the staining of lung tissue sections, mean linear intercept (mLI), and inflammatory cytokine concentrations in the alveolar lavage fluid between the two groups. Both OTU and PCA showed that the intestinal IgA-coated microbiota in fecal samples were different and comparable between the two groups. The alpha diversity analysis showed no statistically significant difference in the species diversity between the two groups (P>0.05). The structure of intestinal-derived IgA-coated microbiota had differences between the two groups (P<0.05). In the COPD group, the bacterial orders that were significantly enriched were Bdellovibrionales, Clostridiales, and Bifidobacteriales; the mainly enriched bacterial families were Prevotellaceae, Clostridiaceae, Paenibacillaceae, Bdellovibrionaceae, and Bifidobacteriaceae; and the mainly enriched bacterial genera were Alloprevotella, Brevibacillus, Clostridium-sensu-stricto, Turicibacter, Faecalibacterium, Vampirovibrio, and Bifidobacterium. The results of KEGG pathway enrichment analysis of differentially expressed genes showed that the cell growth and death, nucleotide metabolism, and digestive system-related pathways in the COPD group were significantly up-regulated, while the membrane transport pathway was significantly down-regulated. [Conclusion] The CODP mice present altered intestinal-derived IgA-coated gut microbiota and gene dysfunction.

Key words:chronic obstructive pulmonary disease;IgA-coated gut microbiota;16S rRNA gene sequencing