Banday Mudassir M, Kumar Archit, Vestal Grant, Sethi Jaskaran, Patel Kapil N, O'Neill Edward B, Finan Jon, Cheng Feng, Lin Muling, Davis Nicole M, Goldberg Hilary, Coppolino Antonio, Mallidi Hari R, Dunning John, Visner Gary, Gaggar Amit, Seyfang Andreas, Sharma Nirmal S
Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts.
University of South Florida/Tampa General Hospital,Tampa, Florida.
J Heart Lung Transplant. 2021 Jun;40(6):447-457. doi: 10.1016/j.healun.2021.02.014. Epub 2021 Mar 2.
Recent evidence suggests a role for lung microbiome in occurrence of chronic lung allograft dysfunction (CLAD). However, the mechanisms linking the microbiome to CLAD are poorly delineated. We investigated a possible mechanism involved in microbial modulation of mucosal response leading to CLAD with the hypothesis that a Proteobacteria dominant lung microbiome would inhibit N-myc-interactor (NMI) expression and induce epithelial to mesenchymal transition (EMT).
Explant CLAD, non-CLAD, and healthy nontransplant lung tissue were collected, as well as bronchoalveolar lavage from 14 CLAD and matched non-CLAD subjects, which were followed by 16S rRNA amplicon sequencing and quantitative polymerase chain reaction (PCR) analysis. Pseudomonas aeruginosa (PsA) or PsA-lipopolysaccharide was cocultured with primary human bronchial epithelial cells (PBEC). Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate NMI expression and EMT in explants and in PsA-exposed PBECs. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of EMT regulator NMI.
16S rRNA amplicon analyses revealed that CLAD patients have a higher abundance of phyla Proteobacteria and reduced abundance of the phyla Bacteroidetes. At the genera level, CLAD subjects had an increased abundance of genera Pseudomonas and reduced Prevotella. Human CLAD airway cells showed a downregulation of the N-myc-interactor gene and presence of EMT. Furthermore, exposure of human primary bronchial epithelial cells to PsA resulted in downregulation of NMI and induction of an EMT phenotype while NMI upregulation resulted in attenuation of this PsA-induced EMT response.
CLAD is associated with increased bacterial biomass and a Proteobacteria enriched airway microbiome and EMT. Proteobacteria such as PsA induces EMT in human bronchial epithelial cells via NMI, demonstrating a newly uncovered mechanism by which the microbiome induces cellular metaplasia.
近期证据表明肺微生物群在慢性肺移植功能障碍（CLAD）的发生中起作用。然而，微生物群与CLAD之间的联系机制尚不清楚。我们研究了微生物调节黏膜反应导致CLAD的一种可能机制，假设以变形菌门为主的肺微生物群会抑制N - myc相互作用蛋白（NMI）表达并诱导上皮 - 间质转化（EMT）。
收集CLAD患者、非CLAD患者及健康非移植者的肺组织外植体，以及14例CLAD患者和配对的非CLAD患者的支气管肺泡灌洗样本，随后进行16S rRNA扩增子测序和定量聚合酶链反应（PCR）分析。将铜绿假单胞菌（PsA）或PsA - 脂多糖与人原代支气管上皮细胞（PBEC）共培养。进行蛋白质印迹分析和定量逆转录（qRT）PCR以评估外植体和暴露于PsA的PBEC中的NMI表达和EMT。在对EMT调节因子NMI进行小干扰RNA（siRNA）沉默和上调（质粒载体）后重复这些实验。
16S rRNA扩增子分析显示，CLAD患者中变形菌门的丰度较高，拟杆菌门的丰度较低。在属水平上，CLAD患者中假单胞菌属的丰度增加，普雷沃菌属减少。人类CLAD气道细胞显示N - myc相互作用蛋白基因下调并存在EMT。此外，人原代支气管上皮细胞暴露于PsA导致NMI下调并诱导EMT表型，而NMI上调导致这种PsA诱导的EMT反应减弱。
CLAD与细菌生物量增加、富含变形菌门的气道微生物群和EMT相关。像PsA这样的变形菌门通过NMI在人支气管上皮细胞中诱导EMT，揭示了微生物群诱导细胞化生的一种新发现机制。
