Extracellular vesicles released from the filarial parasite Brugia malayi downregulate the host mTOR pathway

Abstract

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.

Fig 1. Characterization of mf-derived EVs.

Live B. malayi mf were cultured for 24 hours at 37°C in 5% CO₂. EVs were then purified from the mf culture media. The profile of these isolated EVs was assessed using a NanoSight NS300 and NTA software. The majority of the EVs in the sample fall within the size range of exosomes.

Fig 2. Proteomic characterization of mf-derived EVs.

Four mf-derived EV samples were prepared for liquid chromatography- tandem mass spectrometry. The proteomic analysis revealed several parasite proteins. The total number of identified filarial proteins differed slightly between the EV preparations (A). Proteomics results for each EV sample was assessed in order to determine overlapping protein hits (B).

Fig 3. Internalization of mf-derived EVs by human DC.

THP-1 cells were incubated for 10 minutes, 60 minutes, or 24 hours with mf-derived EVS that were stained with Exo-Red dye. EV uptake was assessed by flow cytometry (A). Human monocyte derived dendritic cells were co-cultured with B. malayi mf-derived EVs for 72 hours at 37°C in 5% CO₂. The dendritic cells were labelled with PKH26 (red) and counterstained with VECTASHIELD Hardset Antifade Mounting Media with DAPI (blue) to visualize the nuclei. The parasite EVs were labelled with PKH67 (green). The merged image demonstrates that no parasite-derived EVs remain bound on the surface of the cell (B). The internalized vesicles appear diffused throughout the cytoplasm of the cells (C), with some EV products potentially translocating into the nucleus (D). All images were captured using Zeiss 780. The assay was performed three times.

Fig 4. Overview of the differential expression of miRNAs between mf EVs and secreted products.

Microarray analysis was used to assess differential miRNA expression between mf-derived EVs and mf secreted products. The miRNAs differentially expressed in mf secreted products were compared to those of mf EVs using log₂-fold changes on the x-axis and adjusted P values (log₁₀) on the y-axis (A). The heat map illustrates the hierarchical clustering of the normalized expression of miRNAs from mf EVs and secreted products with blue to red indicating low to high expression (B). The parallel coordinate plots of miRNAs demonstrate the downregulation or upregulation when comparing mf EVs and mf secreted product samples (C). Throughout the figures, brown/tan lines and dots represent downregulation in EVs, green lines and dots represent slight differential expression between EVs and secreted products, and blue/red lines and dots represent enrichment in EVs.

Fig 5. Presence of miRNAs predicted to target the mTOR pathway found in mf-derived EVs.

cDNA was synthesized from small RNA that was extracted from mf-derived EVs. RT-PCR was then performed in order to screen EV nucleic acid cargo for miRNAs potentially targeting the mTOR pathway. The miRNA of interest included mir-100, mir-7, mir-71, let-7, and mir-155 as they are predicted to target genes relating to the mTOR pathway.

Fig 6. Mf-derived EVs downregulate mTOR phosphorylation in human monocytes.

Human THP-1 cells were cultured in the presence of media alone, rapamycin, live mf, or mf-derived EVs for 1 hour at 37°C in 5% CO₂. Following the stimulation, fixation, permeabilization, and intracellular staining for phosphorylated mTOR was performed on the cells. The samples were analyzed by flow cytometry; a representative plot demonstrates the assessment of phosphorylated mTOR (Phospho-mTOR (Ser2448)) (A). The results are presented as both frequencies (B) and MFI values (C). n = 10 for rapamycin condition, n = 9 for both live mf and EV conditions. *: ≤ 0.05, **: ≤ 0.01.