Poly(I:C) induces antiviral immune responses in Japanese flounder (Paralichthys olivaceus) that require TLR3 and MDA5 and is negatively regulated by Myd88.

Zhi-Xia Zhou,Li Sun,Bao-Cun Zhang,Roland Lang

doi:10.1371/journal.pone.0112918

Abstract

Polyinosinic:polycytidylic acid (poly(I:C)) is a ligand of toll-like receptor (TLR) 3 that has been used as an immunostimulant in humans and mice against viral diseases based on its ability to enhance innate and adapt immunity. Antiviral effect of poly(I:C) has also been observed in teleost, however, the underling mechanism is not clear. In this study, we investigated the potential and signaling mechanism of poly(I:C) as an antiviral agent in a model of Japanese flounder (Paralichthys olivaceus) infected with megalocytivirus. We found that poly(I:C) exhibited strong antiviral activity and enhanced activation of head kidney macrophages and peripheral blood leukocytes. In vivo studies showed that (i) TLR3 as well as MDA5 knockdown reduced poly(I:C)-mediated immune response and antiviral activity to significant extents; (ii) when Myd88 was overexpressed in flounder, poly(I:C)-mediated antiviral activity was significantly decreased; (iii) when Myd88 was inactivated, the antiviral effect of poly(I:C) was significantly increased. Cellular study showed that (i) the NF-κB activity induced by poly(I:C) was upregulated in Myd88-overexpressing cells and unaffected in Myd88-inactivated cells; (ii) Myd88 overexpression inhibited and upregulated the expression of poly(I:C)-induced antiviral genes and inflammatory genes respectively; (iii) Myd88 inactivation enhanced the expression of the antiviral genes induced by poly(I:C). Taken together, these results indicate that poly(I:C) is an immunostimulant with antiviral potential, and that the immune response of poly(I:C) requires TLR3 and MDA5 and is negatively regulated by Myd88 in a manner not involving NK-κB. These results provide insights to the working mechanism of poly(I:C), TLR3, and Myd88 in fish.

Highlights

Polyinosinic:polycytidylic acid (poly(I:C)) is a structural analogue of double-stranded RNA
Upon binding to double-stranded RNA (dsRNA), TLR3 signaling is activated, which leads to three major outcomes in inflammation and innate immunity: (i) development of antiviral response mediated by activation of IFN regulatory transcription factor (IRF) 3 and IRF7 and by production of type I IFN [6]; (ii) generation of a pro-inflammatory environment by the activation of pro-inflammatory and prosurvival transcription factors, nuclear factor-kB (NF-kB), and activator protein 1 (AP-1) [7]; (iii) induction of cytopathic effect or cell death in a caspase-8-dependent fashion via receptor interacting protein 1 (RIP1) [2]
It has been shown that for some viruses, such as A/H1N1/2009 influenza virus, herpes simplex virus, influenza A virus, hepatitis B virus, and HIV-1, TLR3-mediated immune response is important for protection against viral infection [37,38,39,40,41,42,43], while for other viruses such as rotavirus, tick-borne encephalitis virus, and hepatitis C virus, TLR3 contributes to a detrimental inflammatory response [44,45,46]

Summary

Introduction

Polyinosinic:polycytidylic acid (poly(I:C)) is a structural analogue of double-stranded RNA (dsRNA). It has been used widely in the study of immune responses associated with viral infection. Upon binding to dsRNA, TLR3 signaling is activated, which leads to three major outcomes in inflammation and innate immunity: (i) development of antiviral response mediated by activation of IFN regulatory transcription factor (IRF) 3 and IRF7 and by production of type I IFN [6]; (ii) generation of a pro-inflammatory environment by the activation of pro-inflammatory and prosurvival transcription factors, nuclear factor-kB (NF-kB), and activator protein 1 (AP-1) [7]; (iii) induction of cytopathic effect or cell death in a caspase-8-dependent fashion via receptor interacting protein 1 (RIP1) [2]. TLR3 signaling can upregulate the expression of positive and negative co-stimulatory molecules on DCs and influence the magnitude of CD8+ T cell responses [10]

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