Lung epithelial microvesicles induced by pneumolysin contain mitochondrial cargo

Streptococcus pneumoniae (S.pn.) lung infection is a major cause of acute lung injury. Previous studies demonstrate that pneumolysin (PLY), a key virulence factor of S.pn, induces the release of damage-associated molecular pattern molecules (DAMPs) from alveolar epithelial cells (AEC). Here, we hypothesized that PLY can induce AEC to release mitochondria enclosed in microvesicles (MVs). A549 (human lung AEC) were treated with live S.pn. (D39) or PLY. Ex vivo, human lung tissue was challenged with PLY (5 μg/ml) for 8 hrs. MVs were isolated from conditioned media through differential centrifugation. MVs were analyzed by flow cytometry (FACS) after staining with annexin V (MV marker) and/or EPCAM (epithelial marker). A549 MV lysates were analyzed by immunoblotting (IB) for Tom20 (mitochondrial protein). In separate experiments, A549 were incubated with MitoTracker probes that stain intact mitochondria, and then challenged with PLY. Exposure of A549 to live S.pn. or PLY was associated with increased extracellular release of annexin V positive MVs. Ex vivo, PLY challenge of human lung tissue, similarly to A549, triggers the release of annexinV/EPCAM positive MVs (~3-fold compared to control). Interestingly, MitoTracker-labeled A549 challenged with PLY shed MVs that are double positive for MitoTracker dyes and annexin V or EPCAM. The presence of mitochondrial cargo in AEC-derived MV lysates (PLY treated) was confirmed by IB using an anti-Tom20 antibody. Our results demonstrate that PLY triggers the release of MVs containing mitochondria from lung epithelial cells and suggest that these MVs may act as mitochondrial DAMPs.