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

Zhou ZX, Zhang BC, Sun L - PLoS ONE (2014)

Bottom Line: 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.These results provide insights to the working mechanism of poly(I:C), TLR3, and Myd88 in fish.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.

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.

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Effect of TLR3 knockdown on poly(I:C)-induced gene expression.Japanese flounder were administered with PBS (control), siRC, siTLR3, poly(I:C), or poly(I:C) in the presence siRC or siTLR3. The expression of immune genes in spleen was then examined by quantitative real time RT-PCR. Data are presented as means ± SE (N = 3). N, the number of experimental repeat. *P<0.05; **P<0.01.
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pone-0112918-g003: Effect of TLR3 knockdown on poly(I:C)-induced gene expression.Japanese flounder were administered with PBS (control), siRC, siTLR3, poly(I:C), or poly(I:C) in the presence siRC or siTLR3. The expression of immune genes in spleen was then examined by quantitative real time RT-PCR. Data are presented as means ± SE (N = 3). N, the number of experimental repeat. *P<0.05; **P<0.01.

Mentions: To examine whether the above observed effect of poly(I:C) was mediated through TLR3 signaling pathway, the expression of TLR3 was knocked down by siRNA. For this purpose, two siRNAs were used, i.e., siTLR3, a TLR3-specific siRNA, and siRC, a nonspecific siRNA. The effect of siTLR3 was determined in flounder FG cells, which showed that in cells transfected with siTLR3, the expression of TLR3 was significantly reduced compared to that in un-transfected cells and in cells transfected with siRC (Fig. S1). To examine the effect of TLR3 knockdown on poly(I:C)-induced gene expression, flounder were pre-administered with siTLR3 or siRC before being treated with poly(I:C), and the expression of immune genes involved in TLR3 signaling pathway was subsequently determined by qRT-PCR. The results showed that in fish treated with siTLR3, TLR3 mRNA level was reduced by 55.7% (Fig. 3), suggesting that siTLR3 effectively interfered with TLR3 expression. In fish treated with poly(I:C) alone and with siRC plus poly(I:C), the expression levels of TLR3, IL-1β, IL-6, IFN-I, and Mx were comparable and significantly higher than those in the untreated control fish (Fig. 3). In contrast, in fish treated with siTLR3 plus poly(I:C), the expression levels of TLR3, IL-1β, IFN-I, Mx, and ISG56 were significantly reduced compared to those in fish treated with poly(I:C) alone.


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

Zhou ZX, Zhang BC, Sun L - PLoS ONE (2014)

Effect of TLR3 knockdown on poly(I:C)-induced gene expression.Japanese flounder were administered with PBS (control), siRC, siTLR3, poly(I:C), or poly(I:C) in the presence siRC or siTLR3. The expression of immune genes in spleen was then examined by quantitative real time RT-PCR. Data are presented as means ± SE (N = 3). N, the number of experimental repeat. *P<0.05; **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231074&req=5

pone-0112918-g003: Effect of TLR3 knockdown on poly(I:C)-induced gene expression.Japanese flounder were administered with PBS (control), siRC, siTLR3, poly(I:C), or poly(I:C) in the presence siRC or siTLR3. The expression of immune genes in spleen was then examined by quantitative real time RT-PCR. Data are presented as means ± SE (N = 3). N, the number of experimental repeat. *P<0.05; **P<0.01.
Mentions: To examine whether the above observed effect of poly(I:C) was mediated through TLR3 signaling pathway, the expression of TLR3 was knocked down by siRNA. For this purpose, two siRNAs were used, i.e., siTLR3, a TLR3-specific siRNA, and siRC, a nonspecific siRNA. The effect of siTLR3 was determined in flounder FG cells, which showed that in cells transfected with siTLR3, the expression of TLR3 was significantly reduced compared to that in un-transfected cells and in cells transfected with siRC (Fig. S1). To examine the effect of TLR3 knockdown on poly(I:C)-induced gene expression, flounder were pre-administered with siTLR3 or siRC before being treated with poly(I:C), and the expression of immune genes involved in TLR3 signaling pathway was subsequently determined by qRT-PCR. The results showed that in fish treated with siTLR3, TLR3 mRNA level was reduced by 55.7% (Fig. 3), suggesting that siTLR3 effectively interfered with TLR3 expression. In fish treated with poly(I:C) alone and with siRC plus poly(I:C), the expression levels of TLR3, IL-1β, IL-6, IFN-I, and Mx were comparable and significantly higher than those in the untreated control fish (Fig. 3). In contrast, in fish treated with siTLR3 plus poly(I:C), the expression levels of TLR3, IL-1β, IFN-I, Mx, and ISG56 were significantly reduced compared to those in fish treated with poly(I:C) alone.

Bottom Line: 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.These results provide insights to the working mechanism of poly(I:C), TLR3, and Myd88 in fish.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China.

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.

Show MeSH
Related in: MedlinePlus