Endothelial Exosome Plays a Functional Role during Rickettsial Infection

Abstract

Spotted fever group rickettsioses (SFRs) are devastating human infections. Vascular endothelial cells (ECs) are the primary targets of rickettsial infection. Edema resulting from EC barrier dysfunction occurs in the brain and lungs in most cases of lethal SFR, but the underlying mechanisms remain unclear. The aim of the study was to explore the potential role of Rickettsia-infected, EC-derived exosomes (Exos) during infection. Using size exclusion chromatography (SEC), we purified Exos from conditioned, filtered, bacterium-free media collected from Rickettsia parkeri-infected human umbilical vein ECs (HUVECs) (R-ECExos) and plasma of Rickettsia australis- or R. parkeri-infected mice (R-plsExos). We observed that rickettsial infection increased the release of heterogeneous plsExos, but endothelial exosomal size, morphology, and production were not significantly altered following infection. Compared to normal plsExos and ECExos, both R-plsExos and R-ECExos induced dysfunction of recipient normal brain microvascular ECs (BMECs). The effect of R-plsExos on mouse recipient BMEC barrier function is dose dependent. The effect of R-ECExos on human recipient BMEC barrier function is dependent on the exosomal RNA cargo. Next-generation sequencing analysis and stem-loop quantitative reverse transcription-PCR (RT-qPCR) validation revealed that rickettsial infection triggered the selective enrichment of endothelial exosomal mir-23a and mir-30b, which potentially target the endothelial barrier. To our knowledge, this is the first report on the functional role of extracellular vesicles following infection by obligately intracellular bacteria. IMPORTANCE Spotted fever group rickettsioses are devastating human infections. Vascular endothelial cells are the primary targets of infection. Edema resulting from endothelial barrier dysfunction occurs in the brain and lungs in most cases of lethal rickettsioses, but the underlying mechanisms remain unclear. The aim of the study was to explore the potential role of Rickettsia-infected, endothelial cell-derived exosomes during infection. We observed that rickettsial infection increased the release of heterogeneous plasma Exos, but endothelial exosomal size, morphology, and production were not significantly altered following infection. Rickettsia-infected, endothelial cell-derived exosomes induced dysfunction of human recipient normal brain microvascular endothelial cells. The effect is dependent on the exosomal RNA cargo. Next-generation sequencing analysis revealed that rickettsial infection triggered the selective enrichment of endothelial exosomal mir-23a and mir-30b, which potentially target the endothelial barrier. To our knowledge, this is the first report on the functional role of extracellular vesicles following infection by obligately intracellular bacteria.

Extending applications of AFM to fluidic AFM in single living cell and extracellular vesicle studies

“…Using size-exclusion chromatography (SEC), small EVs (50-150 nm) were purified from Rickettsia-infected mouse plasma and culture media of primary vascular endothelial cells (ECs). Evaluation of single particle morphology using tapping mode AFM images verified the integrity of isolated exosomes from experimental specimens 35 . Using AFM, researchers are able to focus on both the biochemical assessment of an EV particle surface 36 and are able to quantify the surface biophysical characteristics of EVs at the single particle level [37][38][39][40] , beyond merely assessing the size and counting the particle number.…”

Exosomally targeting microRNA23a ameliorates microvascular endothelial barrier dysfunction following rickettsial infection

“…We recently unveiled a novel 3 functional role played by EC-derived exosomes (ECExos) during rickettsial infection [26]. We validated that ECs efficiently take up exosomes (Exos) in vivo and in vitro [26]. We found that rickettsial-infected ECExos (R-ECExos) induced disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs) in a dose-dependent manner.…”

“…Exos contain many types of biomolecules [16] and convey signals to a large repertoire of recipient cells either locally or remotely by ferrying functional cargos, thus contributing to disease pathogenesis [20; 21; 22; 23; 24; 25]. We recently unveiled a novel 3 functional role played by EC-derived exosomes (ECExos) during rickettsial infection [26]. We validated that ECs efficiently take up exosomes (Exos) in vivo and in vitro [26].…”

“…We found that rickettsial-infected ECExos (R-ECExos) induced disruption of the tight junctional (TJ) protein ZO-1 and barrier dysfunction of human normal recipient brain microvascular endothelial cells (BMECs) in a dose-dependent manner. This effect is dependent on exosomal RNA cargos [26]. However, the underlying mechnism remains unclear.…”

Host EPAC1 Modulates Rickettsial Adhesion to Vascular Endothelial Cells via Regulation of ANXA2 Y23 Phosphorylation

“…Mouse brain microvascular endothelial cells (BMECs) were isolated from wild-type, EPAC1-KO, and ANXA2-KO mice using established protocol [44,51,62].…”