Extracellular Vesicles Released From The Filarial Parasite Brugia Malayi Downregulate The Host Mtor Pathway

We have previously shown that the microfilarial (mf) stage of Brugia malayi can inhibit the mammalian target of rapamycin (mTOR; a conserved serine/threonine kinase critical for immune regulation and cellular growth) in human dendritic cells (DC) and we have proposed that this mTOR inhibition is associated with the DC dysfunction seen in filarial infections. Extracellular vesicles (EVs) contain many proteins and nucleic acids including microRNAs (miRNAs) that might affect a variety of intracellular pathways. Thus, EVs secreted from mf may elucidate the mechanism by which the parasite is able to modulate the host immune response during infection. EVs, purified from mf of Brugia malayi and confirmed by size through nanoparticle tracking analysis, were assessed by miRNA microarrays (accession number GSE157226) and shown to be enriched (>2-fold, p-value<0.05, FDR = 0.05) for miR100, miR71, miR34, and miR7. The microarray analysis compared mf-derived EVs and mf supernatant. After confirming their presence in EVs using qPCR for these miRNA targets, web-based target predictions (using MIRPathv3, TarBAse and MicroT-CD) predicted that miR100 targeted mTOR and its downstream regulatory protein 4E-BP1. Our previous data with live parasites demonstrated that mf downregulate the phosphorylation of mTOR and its downstream effectors. Additionally, our proteomic analysis of the mf-derived EVs revealed the presence of proteins commonly found in these vesicles (data are available via ProteomeXchange with identifier PXD021844). We confirmed internalization of mf-derived EVs by human DCs and monocytes using confocal microscopy and flow cytometry, and further demonstrated through flow cytometry, that mf-derived EVs downregulate the phosphorylation of mTOR in human monocytes (THP-1 cells) to the same degree that rapamycin (a known mTOR inhibitor) does. Our data collectively suggest that mf release EVs that interact with host cells, such as DC, to modulate host responses.Author summaryLymphatic filariasis, a neglected tropical disease caused by parasitic worms which affects millions of individuals, represents an important public health concern due to its high morbidity that significantly diminishes quality of life. The parasite is able to establish a chronic infection by manipulating its host's immune responses. The larval mf stage of the parasite is of particular interest as the parasites are present in the peripheral blood and in constant contact with the host's immune cells. We decided to investigate the role of mf-derived EVs in the modulation of human antigen presenting cells during infection. We showed that mf release EVs that are similar to exosomes, and these parasite vesicles are readily internalized by human DC. The mf-derived EVs possess a unique miRNA profile and are enriched in miRNAs that can target the mTOR pathway. We have also demonstrated that purified mf-derived EVs are capable of inhibiting mTOR signaling in human monocytes. Collectively, our results suggest that mf release exosome-like vesicles that modulate the immune metabolism of host antigen presenting cells.