Background

Mitochondria play a key role in immune defense pathways, particularly for macrophages. We and others have previously demonstrated that cystic fibrosis (CF) macrophages exhibit weak autophagy activity and exacerbated inflammatory responses. Previous studies have revealed that mitochondria are defective in CF epithelial cells, but to date, the connection between defective mitochondrial function and CF macrophage immune dysregulation has not been fully elucidated. Here, we present a characterization of mitochondrial dysfunction in CF macrophages.

Methods

Mitochondrial function in wild-type (WT) and CF F508del/F508del murine macrophages was measured using the Seahorse Extracellular Flux analyzer. Mitochondrial morphology was investigated using transmission electron and confocal microscopy. Mitochondrial membrane potential (MMP) as well as mitochondrial reactive oxygen species (mROS) were measured using TMRM and MitoSOX Red fluorescent dyes, respectively. All assays were performed at baseline and following infection by Burkholderia cenocepacia, a multi-drug resistant bacterium that causes detrimental infections in CF patients.

Results

We have identified impaired oxygen consumption in CF macrophages without and with B. cenocepacia infection. We also observed increased mitochondrial fragmentation in CF macrophages following infection. Lastly, we observed increased MMP and impaired mROS production in CF macrophages following infection with B. cenocepacia.

Conclusions

The mitochondrial defects identified are key components of the macrophage response to infection. Their presence suggests that mitochondrial dysfunction contributes to impaired bacterial killing in CF macrophages. Our current study will enhance our understanding of the pathobiology of CF and lead to the identification of novel mitochondrial therapeutic targets for CF.

中文翻译：

---

囊性纤维化巨噬细胞中的免疫代谢途径缺陷

背景

线粒体在免疫防御途径中发挥关键作用，特别是对于巨噬细胞。我们和其他人之前已经证明，囊性纤维化 (CF) 巨噬细胞表现出较弱的自噬活性并加剧了炎症反应。先前的研究表明，线粒体在 CF 上皮细胞中存在缺陷，但迄今为止，线粒体功能缺陷与 CF 巨噬细胞免疫失调之间的联系尚未完全阐明。在这里，我们提出了 CF 巨噬细胞线粒体功能障碍的特征。

方法

使用 Seahorse 细胞外通量分析仪测量野生型 (WT) 和 CF F508del/F508del 小鼠巨噬细胞的线粒体功能。使用透射电子和共聚焦显微镜研究线粒体形态。分别使用 TMRM 和 MitoSOX Red 荧光染料测量线粒体膜电位 (MMP) 以及线粒体活性氧 (mROS)。所有测定均在基线时和新洋葱伯克霍尔德菌感染后进行，该菌是一种导致 CF 患者有害感染的多药耐药细菌。

结果

我们已经确定 CF 巨噬细胞的耗氧量受损，而没有和有B. cecepacia感染。我们还观察到感染后 CF 巨噬细胞线粒体碎片增加。最后，我们观察到在感染B. cenocepacia 后，CF 巨噬细胞中 MMP 增加和 mROS 产生受损。

结论

确定的线粒体缺陷是巨噬细胞对感染的反应的关键组成部分。它们的存在表明线粒体功能障碍导致 CF 巨噬细胞中的细菌杀伤受损。我们目前的研究将增强我们对 CF 病理生物学的理解，并导致识别 CF 的新线粒体治疗靶点。