Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of MDA5 knockdown on poly(I:C)-induced gene expression.Japanese flounder were administered with PBS (control), siRC2, siMDA5, poly(I:C), or poly(I:C) in the presence siRC2 or siMDA5. 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
getmorefigures.php?uid=PMC4231074&req=5

pone-0112918-g006: Effect of MDA5 knockdown on poly(I:C)-induced gene expression.Japanese flounder were administered with PBS (control), siRC2, siMDA5, poly(I:C), or poly(I:C) in the presence siRC2 or siMDA5. 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: Since, as shown above, TLR3 knockdown reduced partly the immune effect of poly(I:C), we further examined whether other factors besides TLR3 were involved in poly(I:C)-induced response. For this purpose, we surveyed the available genes of flounder and selected MDA5 as a target. Three siRNAs targeting MDA5 were examined for interfering efficiency, and one siRNA, siMDA5, was found to be able to significantly reduce the expression of MDA5 after transfection of FG cells (Fig. S2). Consistently, when flounder was administered with siMDA5, MDA5 expression was significantly reduced (Fig. 6). Immune gene expression analysis showed that in flounder treated with siMDA5 plus poly(I:C), the expression levels of IL-1β, IL-6, IFN-I, Mx, and ISG56 were significantly lower than those in fish treated with poly(I:C) alone or with poly(I:C) plus the control siRNA, siRC2 (Fig. 6). Viral infection study showed that when megalocytivirus was inoculated into flounder pre-treated with poly(I:C) or poly(I:C) plus siRC2, the viral loads in kidney and spleen at 3 dpi, 5 dpi, and 7 dpi were significantly lower than those in the untreated control fish (Fig. 7). In contrast, when megalocytivirus was inoculated into flounder pre-treated with siMDA5 plus poly(I:C), the viral loads in kidney were significantly increased at 5 dpi and, especially, 7 dpi compared to those in flounder treated with poly(I:C). Similar results were observed with the viral burdens in spleen.


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 MDA5 knockdown on poly(I:C)-induced gene expression.Japanese flounder were administered with PBS (control), siRC2, siMDA5, poly(I:C), or poly(I:C) in the presence siRC2 or siMDA5. 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-g006: Effect of MDA5 knockdown on poly(I:C)-induced gene expression.Japanese flounder were administered with PBS (control), siRC2, siMDA5, poly(I:C), or poly(I:C) in the presence siRC2 or siMDA5. 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: Since, as shown above, TLR3 knockdown reduced partly the immune effect of poly(I:C), we further examined whether other factors besides TLR3 were involved in poly(I:C)-induced response. For this purpose, we surveyed the available genes of flounder and selected MDA5 as a target. Three siRNAs targeting MDA5 were examined for interfering efficiency, and one siRNA, siMDA5, was found to be able to significantly reduce the expression of MDA5 after transfection of FG cells (Fig. S2). Consistently, when flounder was administered with siMDA5, MDA5 expression was significantly reduced (Fig. 6). Immune gene expression analysis showed that in flounder treated with siMDA5 plus poly(I:C), the expression levels of IL-1β, IL-6, IFN-I, Mx, and ISG56 were significantly lower than those in fish treated with poly(I:C) alone or with poly(I:C) plus the control siRNA, siRC2 (Fig. 6). Viral infection study showed that when megalocytivirus was inoculated into flounder pre-treated with poly(I:C) or poly(I:C) plus siRC2, the viral loads in kidney and spleen at 3 dpi, 5 dpi, and 7 dpi were significantly lower than those in the untreated control fish (Fig. 7). In contrast, when megalocytivirus was inoculated into flounder pre-treated with siMDA5 plus poly(I:C), the viral loads in kidney were significantly increased at 5 dpi and, especially, 7 dpi compared to those in flounder treated with poly(I:C). Similar results were observed with the viral burdens in spleen.

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