INNATE IMMUNE RESPONSES OF RAINBOW TROUT INDUCED BY DOUBLE STRANDED RNA FORMULATED WITH PLANT DERIVED NANOPARTICLES
Tamiru Alkie , Wilfrid Laurier University
Tamiru Alkie1, Jondavid de Jong1,2, Kristof Jenik1, Karl Klinger2, Stephanie DeWitte-Orr1
1Department of Health Sciences, Wilfrid Laurier University, Waterloo, ON
2Mirexus Biotechnologies, Guelph, ON
The classical innate immune responses in vertebrates are initiated after the recognition of microbial molecular motifs such as double-stranded (ds)RNA by pattern recognition receptors (PRRs) that are constitutively expressed on host cells. This engagement between PRRs and pathogen-associated molecular patterns (PAMPs) triggers intracellular signaling pathways leading to the induction of effector molecules, including interferons and interferon stimulated genes (ISGs) contributing to pathogen clearance. In this study, a synthetic dsRNA, high molecular weight (HMW) polyinosinic:polycytidylic acid (polyI:C), was used as a model dsRNA molecule and was complexed with biodegradable nanoparticles (NPs). We assessed the effects of the dsRNA-NP complex at inducing antiviral effectors using a reporter cell line, RTG-P1, which expresses firefly luciferase under the control of the trout Mx1 promoter. Following this initial evaluation, innate immune responses to dsRNA NPs or the free form of dsRNA were tested using rainbow trout gut cells (RTgutGC) in three culture scenarios: (1) in a monolayer (2) in 3D cultures on a porous transwell culture system and (3) in a coculture study with rainbow trout monocyte/macrophage cell line (RTS11). The results showed that RTgutGC efficiently internalized higher amounts of dsRNA-NPs complex compared to the free form of dsRNA. The complex induced significantly higher levels of IFN (IFN1) and ISGs (Mx1 and Vig3) transcripts in all in vitro culture systems tested that correlated well with protection of RTgutGC cells from infection by viral hemorrhagic septicemia virus strain IVa (VHSV-IVa). The data in the present study suggests an increased immunostimulatory potency of dsRNA delivered by novel NPs.