Intracellular localization of polymyxins in human alveolar epithelial cells.

Abstract

Current inhaled polymyxin therapy is empirical and often large doses are administered, which can lead to pulmonary adverse effects. There is a dearth of information on the mechanisms of polymyxin-induced lung toxicity and their intracellular localization in lung epithelial cells. To investigate the intracellular localization of polymyxins in human lung epithelial A549 cells. A549 cells were treated with polymyxin B and intracellular organelles (early and late endosomes, endoplasmic reticulum, mitochondria, lysosomes and autophagosomes), ubiquitin protein and polymyxin B were visualized using immunostaining and confocal microscopy. Fluorescence intensities of the organelles and polymyxin B were quantified and correlated for co-localization using ImageJ and Imaris platforms. Polymyxin B co-localized with early endosomes, lysosomes and ubiquitin at 24 h. Significantly increased lysosomal activity and the autophagic protein LC3A were observed after 0.5 and 1.0 mM polymyxin B treatment at 24 h. Polymyxin B also significantly co-localized with mitochondria (Pearson's R = 0.45) and led to the alteration of mitochondrial morphology from filamentous to fragmented form (n = 3, P < 0.001). These results are in line with the polymyxin-induced activation of the mitochondrial apoptotic pathway observed in A549 cells. Accumulation of polymyxins on mitochondria probably caused mitochondrial toxicity, resulting in increased oxidative stress and cell death. The formation of autophagosomes and lysosomes was likely a cellular response to the polymyxin-induced stress and played a defensive role by disassembling dysfunctional organelles and proteins. Our study provides new mechanistic information on polymyxin-induced lung toxicity, which is vital for optimizing inhaled polymyxins in the clinic.