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Amphibian (Xenopus laevis) Tadpoles and Adult Frogs Differ in Their Use of Expanded Repertoires of Type I and Type III Interferon Cytokines.
Wendel ES
,
Yaparla A
,
Melnyk MLS
,
Koubourli DV
,
Grayfer L
.
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While amphibians around the globe are facing catastrophic declines, in part because of infections with pathogens such as the Frog Virus 3 (FV3) ranavirus; the mechanisms governing amphibian susceptibility and resistance to such pathogens remain poorly understood. The type I and type III interferon (IFN) cytokines represent a cornerstone of vertebrate antiviral immunity, while our recent work indicates that tadpoles and adult frogs of the amphibian Xenopus laevis may differ in their IFN responses to FV3. In this respect, it is notable that anuran (frogs and toads) tadpoles are significantly more susceptible to FV3 than adult frogs, and thus, gaining greater insight into the differences in the tadpole and adult frog antiviral immunity would be invaluable. Accordingly, we examined the FV3-elicited expression of a panel of type I and type III IFN genes in the skin (site of FV3 infection) and kidney (principal FV3 target) tissues and isolated cells of X. laevis tadpoles and adult frogs. We also examined the consequence of tadpole and adult frog skin and kidney cell stimulation with hallmark pathogen-associated molecular patterns (PAMPs) on the IFN responses of these cells. Together, our findings indicate that tadpoles and adult frogs mount drastically distinct IFN responses to FV3 as well as to viral and non-viral PAMPs, while these expression differences do not appear to be the result of a distinct pattern recognition receptor expression by tadpoles and adults.
Figure 1. Pathogen pattern recognition receptor (PRR) expression in tadpole and adult frog skin and kidney cells. Tadpoles (Nieuwkerk Faber, NF 54, N = 5) and adult frog (1 year old, N = 5) skin and kidney cells were examined for their gene expression of a panel of pathogen pattern recognition receptors. The expression analyses included (A) skin cell TLR, RIGI, and (B) cytosolic DNA sensor (CDS), genes as well as (C) kidney TLR, RIGI, and (D) CDS mRNA levels. All of the gene expressions were compared to the GAPDH control and normalized against the lowest expressed PRR (TLR7) within the respective cells. All of the results are presented as means + SEM. Significant differences between the respective experimental groups are denoted by overhead lines and asterisks (*). p < 0.05.
Figure 2. Tadpoles and adult frog skin cells mount distinct interferon (IFN) responses to PAMP stimulations. Tadpoles (NF 54, N = 5) and adult frog (1 year old, N = 5) skin cells were isolated and stimulated with a panel of pathogen-associated molecular patterns (PAMPs) for 6 h, and the IFN gene expression was examined. (A) IFN1 and IFN7; (B) IFNx2, IFNx6, IFNx11, IFNx20 (C) IFNLx1/2, IFNL3, and IFNL4 gene expression, following stimulation with TLR and RIGI-specific PAMPs. (D) IFN1 and IFN7; (E) IFNx2, IFNx6, IFNx11, and IFNx20; (F) IFNLx1/2, IFNL3, and IFNL4 gene expression, following stimulation with CDS agonists. All of the gene expressions were compared to the GAPDH control, normalized against the mock-infected expression of the respective genes within given tissues. All of the results are presented as means + SEM. Significant differences between the respective experimental groups are denoted by overhead lines and asterisks (*). p < 0.05.
Figure 3. Tadpoles and adult frog kidney cells mount distinct IFN responses to PAMP stimulations. Tadpoles (NF 54, N = 5) and adult frog (1 year old, N = 5) skin cells were isolated and stimulated with a panel of PAMPS for 6 h, and the IFN gene expression was examined. (A) IFN1 and IFN7; (B) IFNx2, IFNx6, IFNx11, and IFNx20; (C) IFNLx1/2, IFNL3, and IFNL4 gene expression following stimulation with TLR and RIGI-specific PAMPs. (D) IFN1 and IFN7; (E) IFNx2, IFNx6, IFNx11, and IFNx20; (F) IFNLx1/2, IFNL3, and IFNL4 gene expression following stimulation with CDS agonists. All of the gene expressions were compared to the GAPDH control, normalized against the mock-infected expression of the respective genes within the given tissues. All of the results are presented as means + SEM. Significant differences between the respective experimental groups are denoted by overhead lines and asterisks (*). p < 0.05.
Figure 4. Tadpoles and adult frog kidney cells mount distinct IFN responses following FV3 infections. Tadpoles (NF 54, N = 5) and adult frog (1 year old, N = 5) skin cells were isolated and infected with FV3 (0.5 MOI) for 6 h, and were examined for skin (A) and (B) kidney cell expressions of IFN genes as well as (C) skin and kidney cell expressions of FV3 genes and their (D) FV3 DNA loads. All of the gene expressions were compared to the GAPDH control, normalized against the mock-infected expression of the respective genes within given tissues. All of the results are presented as means + SEM. Significant differences between the respective experimental groups are denoted by overhead lines and asterisks (*). p < 0.05.
Figure 5. Tadpoles and adult X. laevis mount distinct IFN responses to FV3. Tadpoles (Nieuwkoop and Faber, NF stage 54, N = 5) and adult frogs (1 year old, N = 5) were challenged by water bath with 106 PFU of FV3 per ml of water or mock infected (water), and after 6 h of infection, their (A) skin and (B) kidney tissues were examined for the IFN gene expression, (C) FV3 gene expression, and (D) FV3 DNA viral loads. All of the gene expressions were compared to the GAPDH control, normalized against the mock-infected expression of the respective genes within given tissues. All of the results are presented as means + SEM. Significant differences between the respective experimental groups are denoted by overhead lines and asterisks (*). p < 0.05.
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