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Figure 1 Analyses of in vivo and in vitro FV3 DNA loads and gene expression in tadpole and adult frog intestines. Tadpoles (N =6) and adult frogs (N = 5) were infected by water bath with 106 PFU of FV3 for 6 hrs and their intestinal (A) FV3 loads and (B) FV3 gene expression examined. Alternatively, tadpole and adult frog intestinal cells (N = 6) were infected in vitro with FV3 (0.5 MOI) and the (A) FV3 DNA loads and (B) expression of 82R (immediate early; IE), 95R (delayed early; DE) and 93L (late; L) FV3 genes, assessed by qPCR. The results are means ± SE of viral loads or viral gene expression. Asterisks above lines (*; A) denote statistical differences between the treatment groups denoted by the line and asterisks (*; B) denote statistically significant difference from in vitro expression, p < 0.05.
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Figure 2 Tadpoles and adult frogs differ in their intestinal antiviral ifn gene expression responses to FV3 infections. Tadpoles (N =6) and adult frogs (N=5) were infected by water bath with 106 PFU of FV3 for 6 hrs. Alternatively, tadpole and adult intestinal cells (N=6) were infected in vitro with FV3 (0.5 MOI). The expression of (A) intron-containing (ifn) and (B) intronless (ifnx) type I IFN genes and (C) type III intron-containing (ifnl) and intronless (ifnlx) IFN genes was examined relative to gapdh endogenous control. The results are means ± SE of gene expression. Asterisks (*) denote statistical differences between respective in vitro and in vivo expression and asterisks above lines (*) denote statistical differences between the treatment groups denoted by the line, p<0.05.
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Figure 3 FV3-infected tadpoles recruit esterase-positive myeloid cells into their intestines while adult frog intestines contain resident esterase-positive myeloid cells. Tadpoles and adult frogs were infected by water bath with 106 PFU of FV3 for 6 hrs. Their intestines were examined for the presence of specific esterase-positive cells (N=6 per treatment group) using the NASDCl- specific esterase (Leder) stain. (A) Mock-infected and (B) FV3-infected tadpole intestines. (C) Mock-infected and (D) FV3-infected adult frog intestines. Lu, lumen; Mu, mucosal; Sm, sub-mucosal r; Mus, muscularis layers. (E) Morphology of esterase-positive cells from FV3-infected tadpole intestines. These results are representative of 3 separate experiments.
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Figure 4 Myeloid cell (A) growth factor receptor and (B) growth factor gene expression in FV3-challenged tadpole and adult frog intestines. Tadpoles (N=6) and adult frogs (N=6) were infected by water bath with 106 PFU of FV3 for 6 hrs and their intestinal expression of (A) csf1r, csf3r and (B) csf1, il34 and csf3 was examined relative to gapdh endogenous control. The results are means ± SE of gene expression. Asterisks (*) denote statistical differences between mock- and FV3-infected groups and asterisks above lines (*) denote statistical differences between the treatment groups denoted by the line, p<0.05.
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Figure 5 Recombinant CSF-1 and IL-34 elicit chemotaxis of ifn-expressing cells from FV3-infected tadpole intestines. Tadpoles (N=6) were infected by water bath with 106 PFU of FV3 for 6 hrs, their intestines isolated and dispersed into cell suspensions. (A) These cells were used in chemotaxis assays, performed against medium alone (med) or 10 - 10-6 ng/ml of rCSF-1 or rIL-34 in bottom Boyden chamber wells (5 mm2; N=3 for medium, 10, 10-2, 10-6 ng/ml doses and N=9 for the 10-4 ng/ml doses of either cytokine) loaded into top wells, separated by chemotaxis filters. After incubation the bottom faces of the filters were stained with Giemsa stain and examined for numbers of migrating cells (40x objective). Chemokinesis experiments were performed using 10-4 ng/ml of rCSF-1 or rIL-34 (the most chemo-attractive concentration) in both bottom and the top chemotaxis chambers (N=3). The results are means ± SE of cells per field of view. Asterisks (*) denote statistical differences between medium alone and recombinant cytokine-induced migration (*) denote statistical differences between the treatment groups denoted by the line, p<0.05. (B) Cells chemoattracted by 10-4 ng/ml of rCSF-1 or rIL-34 (N=6) were examined for their type I and III IFN expression relative to gapdh endogenous control. The results are means ± SE of gene expression. Asterisks (*) denote statistic
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Figure 6 Frog intestines are populated my esterase positive myeloid cells during metamorphosis in a CSF1R-dependent manner. Representative images of intestines from (A) vehicle control (NaOH) and (B) T3 (10 nM final concentration)-treated tadpoles were examined by NASDCl-specific esterase (Leder) stain. Lu: lumen; Mu: mucosal; Sm: sub-mucosal r; Mus: muscularis layers. (C) Means ± SE (N=6 per treatment group) of specific-esterase positive cells (per field of view) in T3 (10 nM final concentration)-treated tadpoles administered with a vehicle control (DMSO in saline), a CSF1R inhibitor (GW-2580; 100 mg/kg body weight) or a CXCR1/2 inhibitor (reparixin; 50 mg/kg body weight). Asterisks above a line (*) denote statistical differences between the treatment groups denoted by the line, p<0.05.
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Supplementary Figure 2 | Analyses of (A) CC- and (B) CXC-motif chemokine genes in mock- and FV3-infected tadpole intestines. (C) Comparison of ccl20 expression in mock- and FV3-challenged tadpole and adult intestines. Results are means ± SE of gene expression relative to gapdh endogenous control (N=6). Asterisks above lines (∗) denote statistical differences between the treatment groups denoted by the lines, p<0.05.
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Supplementary Figure 3 | Comparison of antiviral gene expression in the intestines of tadpoles (stage NF 54), metamorphic (stage NF 62) and adult frogs. Results are means ± SE of gene expression relative to gapdh endogenous control (N=6). Asterisk (∗) denotes statistical difference from tadpole expression, p<0.05.
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Figure 1. Analyses of in vivo and in vitro FV3 DNA loads and gene expression in tadpole and adult frog intestines. Tadpoles (N =6) and adult frogs (N = 5) were infected by water bath with 106 PFU of FV3 for 6 hrs and their intestinal (A) FV3 loads and (B) FV3 gene expression examined. Alternatively, tadpole and adult frog intestinal cells (N = 6) were infected in vitro with FV3 (0.5 MOI) and the (A) FV3 DNA loads and (B) expression of 82R (immediate early; IE), 95R (delayed early; DE) and 93L (late; L) FV3 genes, assessed by qPCR. The results are means ± SE of viral loads or viral gene expression. Asterisks above lines (*; A) denote statistical differences between the treatment groups denoted by the line and asterisks (*; B) denote statistically significant difference from in vitro expression, p < 0.05.
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Figure 2. Tadpoles and adult frogs differ in their intestinal antiviral ifn gene expression responses to FV3 infections. Tadpoles (N =6) and adult frogs (N=5) were infected by water bath with 106 PFU of FV3 for 6 hrs. Alternatively, tadpole and adult intestinal cells (N=6) were infected in vitro with FV3 (0.5 MOI). The expression of (A) intron-containing (ifn) and (B) intronless (ifnx) type I IFN genes and (C) type III intron-containing (ifnl) and intronless (ifnlx) IFN genes was examined relative to gapdh endogenous control. The results are means ± SE of gene expression. Asterisks (*) denote statistical differences between respective in vitro and in vivo expression and asterisks above lines (*) denote statistical differences between the treatment groups denoted by the line, p<0.05.
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Figure 3. FV3-infected tadpoles recruit esterase-positive myeloid cells into their intestines while adult frog intestines contain resident esterase-positive myeloid cells. Tadpoles and adult frogs were infected by water bath with 106 PFU of FV3 for 6 hrs. Their intestines were examined for the presence of specific esterase-positive cells (N=6 per treatment group) using the NASDCl- specific esterase (Leder) stain. (A) Mock-infected and (B) FV3-infected tadpole intestines. (C) Mock-infected and (D) FV3-infected adult frog intestines. Lu, lumen; Mu, mucosal; Sm, sub-mucosal r; Mus, muscularis layers. (E) Morphology of esterase-positive cells from FV3-infected tadpole intestines. These results are representative of 3 separate experiments.
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Figure 4. Myeloid cell (A) growth factor receptor and (B) growth factor gene expression in FV3-challenged tadpole and adult frog intestines. Tadpoles (N=6) and adult frogs (N=6) were infected by water bath with 106 PFU of FV3 for 6 hrs and their intestinal expression of (A)
csf1r, csf3r and (B) csf1, il34 and csf3 was examined relative to gapdh endogenous control. The results are means ± SE of gene expression. Asterisks (*) denote statistical differences between mock- and FV3-infected groups and asterisks above lines (*) denote statistical differences between the treatment groups denoted by the line, p<0.05.
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Figure 5. Recombinant CSF-1 and IL-34 elicit chemotaxis of ifn-expressing cells from FV3-infected tadpole intestines. Tadpoles (N=6) were infected by water bath with 106 PFU of FV3 for 6 hrs, their intestines isolated and dispersed into cell suspensions. (A) These cells were used in chemotaxis assays, performed against medium alone (med) or 10 - 10-6 ng/ml of rCSF-1 or rIL-34 in bottom Boyden chamber wells (5 mm2; N=3 for medium, 10, 10-2, 10-6 ng/ml doses and N=9 for the 10-4 ng/ml doses of either cytokine) loaded into top wells, separated by chemotaxis filters. After incubation the bottom faces of the filters were stained with Giemsa stain and examined for numbers of migrating cells (40x objective). Chemokinesis experiments were performed using 10-4 ng/ml of rCSF-1 or rIL-34 (the most chemo-attractive concentration) in both bottom and the top chemotaxis chambers (N=3). The results are means ± SE of cells per field of view. Asterisks (*) denote statistical differences between medium alone and recombinant cytokine-induced migration (*) denote statistical differences between the treatment groups denoted by the line, p<0.05. (B) Cells chemoattracted by 10-4 ng/ml of rCSF-1 or rIL-34 (N=6) were examined for their type I and III IFN expression relative to gapdh endogenous control. The results are means ± SE of gene expression. Asterisks (*) denote statistical differences from the respective ifn expression in total intestines, denoted by line, p<0.05.
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Figure 6. Frog intestines are populated my esterase positive myeloid cells during metamorphosis in a CSF1R-dependent manner. Representative images of intestines from (A) vehicle control (NaOH) and (B) T3 (10 nM final concentration)-treated tadpoles were examined by NASDCl-specific esterase (Leder) stain. Lu: lumen; Mu: mucosal; Sm: sub-mucosal r; Mus: muscularis layers. (C) Means ± SE (N=6 per treatment group) of specific-esterase positive cells (per field of view) in T3 (10 nM final concentration)-treated tadpoles administered with a vehicle control (DMSO in saline), a CSF1R inhibitor (GW-2580; 100 mg/kg body weight) or a CXCR1/2 inhibitor (reparixin; 50 mg/kg body weight). Asterisks above a line (*) denote statistical differences between the treatment groups denoted by the line, p<0.05.
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