Neutrophil-derived extracellular vesicles induce endothelial inflammation and damage through the transfer of miRNAs.

The critical role of neutrophils in pathological inflammation, notably in various autoimmune disorders, is currently the focus of renewed interest. Here, we demonstrate for the first time that activation of neutrophils with various inflammatory stimuli induces the release of extracellular vesicles (EVs) that are internalized by endothelial cells (ECs), thus leading to the transfer of miR-223, miR-142-3p and miR-451 and subsequent endothelial damage. Indeed, while miR-223 has little effect on EC responses, we show that the induced expression of miR-142-3p and miR-451 in ECs results in profound cell damage, especially in inflammatory conditions, characterized by a dramatic increase in cell apoptosis, impaired angiogenic repair responses, and the induction of IL-6, IL-8, CXCL10 and CXCL11 expression. We show that the strong deleterious effect of miR-142-3p may be due in part to its ability to block the activation of ERK1/2 and eNOS-mediated signals in ECs. miR-142-3p also inhibits the expression of RAC1, ROCK2 and CLIC4, three genes that are critical for EC migration and angiogenic responses. Importantly, miR-223, miR-142-3p and miR-451 are markedly increased in kidney biopsies from patients with active ANCA-associated vasculitis, a severe autoimmune disease that is prototypical of a neutrophil-induced microvascular damage. Taken together, our results suggest that miR-142-3p and miR-451 released in EVs by activated neutrophils can target EC to trigger an inflammatory cascade and induce direct vascular damage, and that therapeutic strategies based on the inhibition of these miRNAs in ECs will have implications for neutrophil-mediated inflammatory diseases.

Inhibition of proteasome activity blocks Trypanosoma cruzi growth and metacyclogenesis.

Proteasomes are intracellular complexes that control protein degradation in organisms ranging from Archaebacteria to mammals. In some parasitic protozoa, the proteasome is involved in cell differentiation and replication. In this study, we have used proteasome inhibitors to determine the biological role of proteasomes during the replication and in vitro metacyclogenesis of Trypanosoma cruzi. We used light and transmission electron microscopy to analyze morphological data and flow cytometry to analyze changes in the cell cycle. The growth of T. cruzi epimastigote culture forms in liver infusion tryptose medium was inhibited by the presence of up to 10 microM lactacystin. Inhibition was dose-dependent, with IC50 (50% inhibitory concentration) of 4.35 microM after 24 or 72 h. The metacyclogenesis process in vitro was strongly (95%) inhibited by 5 microM lactacystin treatment. The adhesion phase was not affected, but the epimastigotes did not differentiate into metacyclic trypomastigotes. Most treated epimastigotes had replicated DNA, with swelling of the mitochondrion and an altered distribution of nuclear and kinetoplast DNA. Our findings suggest that inhibition of the ubiquitin-proteasome pathway in T. cruzi epimastigotes does not block adhesion, but disrupts cell division and affects factors triggering differentiation.