Immunomodulatory Effects of Amblyomma variegatum Saliva on Bovine Cells: Characterization of Cellular Responses and Identification of Molecular Determinants.

Valérie Rodrigues,Bernard Fernandez,Martial Seveno,Frédéric Stachurski,Léo Chamayou,Alexandre Andersen,Etienne Loire,Nathalie Vachiéry,Arthur Vercoutere,Edith Demettre,Ken Giraud-Girard,Oana Vigy,Rosalie Aprelon,Philippe Holzmuller

doi:10.3389/fcimb.2017.00521

Abstract

The tropical bont tick, Amblyomma variegatum, is a tick species of veterinary importance and is considered as one of major pest of ruminants in Africa and in the Caribbean. It causes direct skin lesions, transmits heartwater, and reactivates bovine dermatophilosis. Tick saliva is reported to affect overall host responses through immunomodulatory and anti-inflammatory molecules, among other bioactive molecules. The general objective of this study was to better understand the role of saliva in interaction between the Amblyomma tick and the host using cellular biology approaches and proteomics, and to discuss its impact on disease transmission and/or activation. We first focused on the immuno-modulating effects of semi-fed A. variegatum female saliva on bovine peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages in vitro. We analyzed its immuno-suppressive properties by measuring the effect of saliva on PBMC proliferation, and observed a significant decrease in ConA-stimulated PBMC lymphoproliferation. We then studied the effect of saliva on bovine macrophages using flow cytometry to analyze the expression of MHC-II and co-stimulation molecules (CD40, CD80, and CD86) and by measuring the production of nitric oxide (NO) and pro- or anti-inflammatory cytokines. We observed a significant decrease in the expression of MHC-II, CD40, and CD80 molecules, associated with decreased levels of IL-12-p40 and TNF-α and increased level of IL-10, which could explain the saliva-induced modulation of NO. To elucidate these immunomodulatory effects, crude saliva proteins were analyzed using proteomics with an Orbitrap Elite mass spectrometer. Among the 336 proteins identified in A. variegatum saliva, we evidenced bioactive molecules exhibiting anti-inflammatory, immuno-modulatory, and anti-oxidant properties (e.g., serpins, phospholipases A2, heme lipoprotein). We also characterized an intriguing ubiquitination complex that could be involved in saliva-induced immune modulation of the host. We propose a model for the interaction between A. variegatum saliva and host immune cells that could have an effect during tick feeding by favoring pathogen dissemination or activation by reducing the efficiency of host immune response to the corresponding tick-borne diseases.

Highlights

Amblyomma variegatum is among the most important and widely distributed ticks of tropical livestock, and is the subject of veterinary and public health concerns in Africa and islands in the Indian Ocean and the Caribbean (Stachurski et al, 2010; Bournez et al, 2015)
In order to link the molecular data with the biological data obtained in the cell experiments, we focused on the characterization of A. variegatum saliva proteins with immunomodulatory properties already described in the literature in other tick species
We demonstrated high immunomodulation of bovine immune cells, lymphocytes and macrophages by A. variegatum saliva

Summary

Introduction

Amblyomma variegatum is among the most important and widely distributed ticks of tropical livestock, and is the subject of veterinary and public health concerns in Africa and islands in the Indian Ocean and the Caribbean (Stachurski et al, 2010; Bournez et al, 2015). In tick-host interactions, tick saliva is the source of biologically active molecules that target a wide spectrum of host physiological mechanisms, mainly inhibiting host defense reactions to the benefit of the feeding ticks (Kazimírová and Štibrániová, 2013; Stibrániová et al, 2013). The immediate inflammatory response to the skin injury induces rapid infiltration of leukocytes at the tick bite site, where both resident and infiltrated cells (keratinocytes, endothelial cells, mast cells, dendritic cells, macrophages, and lymphocytes) are activated by direct contact with tick saliva (Brossard and Wikel, 2004; Francischetti et al, 2009; Wikel, 2013). Omics analyses have enabled the comprehensive characterization of the molecular determinants of tick saliva, with a functional involvement of these bioactive molecules in host immune suppression (Kotál et al, 2015; Chmelar et al, 2016a,b)

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