PDF(Pubmed)
Abstract：
Long term outcomes of lung transplantation are impacted by the occurrence of chronic lung allograft dysfunction (CLAD). Recent evidence suggests a role for the lung microbiome in the occurrence of CLAD, but the exact mechanisms are not well defined. We hypothesize that the lung microbiome inhibits epithelial autophagic clearance of pro-fibrotic proteins in an IL-33 dependent manner, thereby augmenting fibrogenesis and risk for CLAD.
Autopsy derived CLAD and non-CLAD lungs were collected. IL-33, P62 and LC3 immunofluorescence was performed and assessed using confocal microscopy. Pseudomonas aeruginosa (PsA), Streptococcus Pneumoniae (SP), Prevotella Melaninogenica (PM), recombinant IL-33 or PsA-lipopolysaccharide was co-cultured with primary human bronchial epithelial cells (PBEC) and lung fibroblasts in the presence or absence of IL-33 blockade. Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate IL-33 expression, autophagy, cytokines and fibroblast differentiation markers. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of Beclin-1.
Human CLAD lungs demonstrated markedly increased expression of IL-33 and reduced basal autophagy compared to non-CLAD lungs. Exposure of co-cultured PBECs to PsA, SP induced IL-33, and inhibited PBEC autophagy, while PM elicited no significant response. Further, PsA exposure increased myofibroblast differentiation and collagen formation. IL-33 blockade in these co-cultures recovered Beclin-1, cellular autophagy and attenuated myofibroblast activation in a Beclin-1 dependent manner.
CLAD is associated with increased airway IL-33 expression and reduced basal autophagy. PsA induces a fibrogenic response by inhibiting airway epithelial autophagy in an IL-33 dependent manner.
Autopsy derived CLAD and non-CLAD lungs were collected. IL-33, P62 and LC3 immunofluorescence was performed and assessed using confocal microscopy. Pseudomonas aeruginosa (PsA), Streptococcus Pneumoniae (SP), Prevotella Melaninogenica (PM), recombinant IL-33 or PsA-lipopolysaccharide was co-cultured with primary human bronchial epithelial cells (PBEC) and lung fibroblasts in the presence or absence of IL-33 blockade. Western blot analysis and quantitative reverse transcription (qRT) PCR was performed to evaluate IL-33 expression, autophagy, cytokines and fibroblast differentiation markers. These experiments were repeated after siRNA silencing and upregulation (plasmid vector) of Beclin-1.
Human CLAD lungs demonstrated markedly increased expression of IL-33 and reduced basal autophagy compared to non-CLAD lungs. Exposure of co-cultured PBECs to PsA, SP induced IL-33, and inhibited PBEC autophagy, while PM elicited no significant response. Further, PsA exposure increased myofibroblast differentiation and collagen formation. IL-33 blockade in these co-cultures recovered Beclin-1, cellular autophagy and attenuated myofibroblast activation in a Beclin-1 dependent manner.
CLAD is associated with increased airway IL-33 expression and reduced basal autophagy. PsA induces a fibrogenic response by inhibiting airway epithelial autophagy in an IL-33 dependent manner.
摘要：
背景：肺移植的长期结局受慢性肺移植功能障碍（CLAD）的影响。最近的证据表明，肺微生物组在CLAD的发生中起作用，但是确切的机制还没有很好的定义。我们假设肺微生物组以IL-33依赖性方式抑制促纤维化蛋白的上皮自噬清除，从而增加纤维发生和CLAD的风险。
方法：收集尸检来源的CLAD和非CLAD肺。使用共聚焦显微镜进行IL-33，P62和LC3免疫荧光并评估。铜绿假单胞菌(PsA),肺炎链球菌(SP),黑色素普氏菌（PM），在存在或不存在IL-33阻断的情况下,将重组IL-33或PsA-脂多糖与原代人支气管上皮细胞(PBEC)和肺成纤维细胞共培养。进行蛋白质印迹分析和定量逆转录（qRT）PCR以评估IL-33表达，自噬,细胞因子和成纤维细胞分化标志物。在Beclin-1的siRNA沉默和上调(质粒载体)后重复这些实验。
结果：与非CLAD肺相比，人CLAD肺显示IL-33表达显著增加，基础自噬降低。共培养的PBECs暴露于PsA，SP诱导IL-33，抑制PBEC自噬，而PM没有引起明显的反应。Further,PsA暴露增加肌成纤维细胞分化和胶原蛋白形成。这些共培养物中的IL-33阻断以Beclin-1依赖性方式恢复了Beclin-1、细胞自噬和减弱的肌成纤维细胞活化。
结论：CLAD与气道IL-33表达增加和基底自噬减少相关。PsA通过以IL-33依赖性方式抑制气道上皮自噬诱导纤维化反应。
方法：收集尸检来源的CLAD和非CLAD肺。使用共聚焦显微镜进行IL-33，P62和LC3免疫荧光并评估。铜绿假单胞菌(PsA),肺炎链球菌(SP),黑色素普氏菌（PM），在存在或不存在IL-33阻断的情况下,将重组IL-33或PsA-脂多糖与原代人支气管上皮细胞(PBEC)和肺成纤维细胞共培养。进行蛋白质印迹分析和定量逆转录（qRT）PCR以评估IL-33表达，自噬,细胞因子和成纤维细胞分化标志物。在Beclin-1的siRNA沉默和上调(质粒载体)后重复这些实验。
结果：与非CLAD肺相比，人CLAD肺显示IL-33表达显著增加，基础自噬降低。共培养的PBECs暴露于PsA，SP诱导IL-33，抑制PBEC自噬，而PM没有引起明显的反应。Further,PsA暴露增加肌成纤维细胞分化和胶原蛋白形成。这些共培养物中的IL-33阻断以Beclin-1依赖性方式恢复了Beclin-1、细胞自噬和减弱的肌成纤维细胞活化。
结论：CLAD与气道IL-33表达增加和基底自噬减少相关。PsA通过以IL-33依赖性方式抑制气道上皮自噬诱导纤维化反应。
