Monocyte-derived extracellular vesicles, stimulated by Trypanosoma cruzi, enhance cellular invasion in vitro via activated TGF-β1

During cell invasion, large Extracellular Vesicle (lEV) release from host cells was dose-dependently triggered by Trypanosoma cruzi metacyclic trypomastigotes (Mtr). This lEV release was inhibited when IP₃-mediated Ca²⁺ exit from the ER and further Ca²⁺ entry from plasma membrane channels was blocked, but whilst any store-independent Ca²⁺ entry (SICE) could continue unabated. That lEV release was equally inhibited if all entry from external sources was blocked by chelation of external Ca²⁺ points to the major contributor to Mtr-triggered host cell lEV release being IP₃/store-mediated Ca²⁺ release, SICE playing a minor role. Host cell lEVs were released through Mtr interaction with host cell lipid raft domains, integrins, and mechanosensitive ion channels, whereupon [Ca²⁺]_cyt increased (50 to 750 nM) within 15 s. lEV release and cell entry of T. cruzi, which increased up to 30 and 60 mpi, respectively, as well as raised actin depolymerization at 60 mpi, were all reduced by TRPC inhibitor, GsMTx-4. Vesicle release and infection was also reduced with RGD peptide, methyl-β-cyclodextrin, knockdown of calpain and with the calpain inhibitor, calpeptin. Restoration of lEV levels, whether with lEVs from infected or uninfected epithelial cells, did not restore invasion, but supplementation with lEVs from infected monocytes, did. We provide evidence of THP-1 monocyte-derived lEV interaction with Mtr (lipid mixing by R18-dequenching; flow cytometry showing transfer to Mtr of R18 from R18-lEVs and of LAP(TGF-β1). Active, mature TGF-β1 (at 175 pg/×10⁵ in THP-1 lEVs) was detected in concentrated lEV-/cell-free supernatant by western blotting, only after THP-1 lEVs had interacted with Mtr. The TGF-β1 receptor (TβRI) inhibitor, SB-431542, reduced the enhanced cellular invasion due to monocyte-lEVs.