Fasciola hepatica Extracellular Vesicles isolated from excretory-secretory products using a gravity flow method modulate dendritic cell phenotype and activity.

Anna Murphy,Mark W Robinson,Sandra M O’Neill,Lokesh Joshi,Jared Gerlach,Barry O’Connell,Krystyna Cwiklinski,Michelle Kilcoyne,John P Dalton,Richard Lalor

doi:10.1371/journal.pntd.0008626

Abstract

Parasite-released extracellular vesicles (EVs) deliver signals to the host immune system that are critical to maintaining the long-term relationship between parasite and host. In the present study, total EVs (FhEVs) released in vitro by adults of the helminth parasite Fasciola hepatica were isolated using a recently described gravity flow method that protects their structural integrity. The FhEVs molecular cargo was defined using proteomic analysis and their surface topology characterised by glycan microarrays. The proteomic analysis identified 618 proteins, 121 of which contained putative N-linked glycosylation sites while 132 proteins contained putative O-linked glycosylation sites. Glycan arrays revealed surface-exposed glycans with a high affinity for mannose-binding lectins indicating the predominance of oligo mannose-rich glycoproteins, as well as other glycans with a high affinity for complex-type N-glycans. When added to bone-marrow derived dendritic cells isolated FhEV induced a novel phenotype that was categorised by the secretion of low levels of TNF, enhanced expression of cell surface markers (CD80, CD86, CD40, OX40L, and SIGNR1) and elevation of intracellular markers (SOCS1 and SOCS3). When FhEV-stimulated BMDCs were introduced into OT-II mice by adoptive transfer, IL-2 secretion from skin draining lymph nodes and spleen cells was inhibited in response to both specific and non-specific antigen stimulation. Immunisation of mice with a suspension of FhEV did not elicit significant immune responses; however, in the presence of alum, FhEVs induced a mixed Th1/Th2 immune response with high antigen specific antibody titres. Thus, we have demonstrated that FhEVs induce a unique phentotype in DC capable of suppressing IL-2 secretion from T-cells. Our studies add to the growing immuno-proteomic database that will be an important source for the discovery of future parasite vaccines and immunotherapeutic biologicals.

Highlights

Extracellular vesicles (EVs) are small membrane-bound vesicles secreted by multiple cell types as part of normal cellular bioprocesses [1,2,3]
Since the extracellular vesicles (EVs) molecular cargo can influence host immune cells, F. hepatica EVs (FhEVs) were added to bone-marrow derived dendritic cells, inducing a novel cell phenotype that when adoptive transferred into OT-II mice inhibited IL-2 secretion from skin draining lymph nodes and spleen cells
In addition to the EV-associated/specific proteins, we identified soluble proteins associated with the cargo of the FhEVs including several peptidases

Summary

Introduction

Extracellular vesicles (EVs) are small membrane-bound vesicles secreted by multiple cell types as part of normal cellular bioprocesses [1,2,3]. EVs are important mediators of biological processes that act through an array of signalling components, including proteins, glycoproteins, lipids, and microRNAs [4,5,6] They play a significant role in immune regulation [1,7], cell-cell communication [6], tissue repair [8], protection from injury, and blood clotting [9]. Roig et al [26] showed that F. hepatica EVs modulate the activity of macrophages and DCs in addition to reducing the severity of DSS-induced colitis in mice suggesting that EVs contain potential biotherapeutic molecules Many of these vesicles are targeted by antibodies isolated form infected animals [23,27] or animals vaccinated with EVs or EV-surface proteins [23,27] making them important vaccine candidates

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