Extracellular Vesicles Elicit an Immunostimulatory Response on the Murine Mammary Gland.

is a major pathogen responsible for bovine mastitis, the most common and costly disease affecting dairy cattle. naturally releases extracellular vesicles (EVs) during its growth. EVs play an important role in the bacteria-bacteria and bacteria-host interactions and are notably considered as nanocarriers that deliver virulence factors to the host tissues. Whether EVs play a role in a mastitis context is still unknown. In this work, we showed that Newbould 305 (N305), a bovine mastitis isolate, has the ability to generate EVs with a designated protein content. Purified N305-secreted EVs were not cytotoxic when tested on MAC-T and PS, two bovine mammary epithelial cell lines. However, they induced the gene expression of inflammatory cytokines at levels similar to those induced by live N305. The immune response to purified N305-secreted EVs was tested in a mouse model for bovine mastitis and their immunogenic effect was compared to that of live N305, heat-killed N305 and to lipoteichoic acid (LTA). Clinical and histopathological signs were evaluated and pro-inflammatory and chemotactic cytokine levels were measured in the mammary gland 24 h post-inoculation. Live induced a significantly stronger inflammatory response than that of any other condition tested. Nevertheless, N305-secreted EVs induced a dose-dependent neutrophil recruitment and the production of a selected set of pro-inflammatory mediators as well as chemokines. This immune response elicited by intramammary N305-secreted EVs was comparable to that of heat-killed N305 and, partly, by LTA. These results demonstrated that N305-secreted EVs induce a mild inflammatory response distinct from the live pathogen after intramammary injection. Overall, our combined and data suggest that EVs are worth to be investigated to better understand the pathogenesis and are relevant tools to develop strategies against bovine mastitis.