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The chytrid fungus, Batrachochytrium dendrobatidis (Bd), infects amphibian skin, causing chytridiomycosis, which is a contributing cause of worldwide declines and extinctions of amphibians. Relatively little is known about the roles of amphibian skin-resident immune cells, such as macrophages, in these antifungal defenses. Across vertebrates, macrophage differentiation is controlled through the activation of colony-stimulating factor-1 (CSF1) receptor by CSF1 and interleukin-34 (IL34) cytokines. While the precise roles of these respective cytokines in macrophage development remain to be fully explored, our ongoing studies indicate that frog (Xenopus laevis) macrophages differentiated by recombinant forms of CSF1 and IL34 are functionally distinct. Accordingly, we explored the roles of X. laevis CSF1- and IL34-macrophages in anti-Bd defenses. Enriching cutaneous IL34-macrophages, but not CSF1-macrophages, resulted in significant anti-Bd protection. In vitro analysis of frog macrophage-Bd interactions indicated that both macrophage subsets phagocytosed Bd. However, IL34-macrophages cocultured with Bd exhibited greater pro-inflammatory gene expression, whereas CSF1-macrophages cocultured with Bd showed greater immunosuppressive gene expression profiles. Concurrently, Bd-cocultured with CSF1-macrophages, but not IL34-macrophages, possessed elevated expression of genes associated with immune evasion. This work marks a step forward in our understanding of the roles of frog macrophage subsets in antifungal defenses.
Figure 1. Enrichment of cutaneous CSF1- and IL34-Mϕs and Bd challenge. Non-specific esterase (NSE) stain of epidermal skin layers from the (A) rctrl- and (B) rCSF1- and (C) rIL34-administered frogs. Arrows indicate myeloid (Mϕs) cells, and melanocytes are denoted by ‘m’. Images are representative of 4 animals per treatment group (N = 4). (D) Means ± SEMs of the number of NSE-positive cells per field of view of skin from frogs 3 days after subcutaneous administration of the rctrl or rCSF1 or rIL34 (N = 4 frogs/treatment group). (E) Bd loads (zoospore genomic equivalents, GE) in dorsal skins of frogs 3 days after control (rctrl)-, CSF1-, and IL34-Mϕ-enrichment followed by 7 days of Bd challenge (N = 7 frogs/treatment group). (F) H&E stain of Bd (arrow)-infected skin. Asterisks above bars indicate statistical significance from the rctrl groups, and asterisks above lines are indicative of statistical differences between the treatment groups denoted by the lines, p < 0.05.
Figure 2. Scanning and transmission electron microscopy images of (A–E) CSF1- and (F–J) IL34-Mϕs. CSF1- and IL34-Mϕs were incubated alone (A,B and F,G, respectively) or with developing Bd thalli (C–E and H–J, respectively) for 5 h (5 Bd/Mϕ) before preparation and electron microscopy analyses. (A) SEM of CSF1-Mϕs. (B) TEM of CSF1-Mϕs. (C) TEM of CSF1-Mϕs co-incubated with Bd. Arrows: (a) non-phagocytosed zoosporangium; (b) phagocytosed and degraded zoosporangium. (D) TEM of CSF1-Mϕs co-incubated with Bd. Arrows: phagocytosed zoosporangium. (E) Higher magnification of zoosporangium-containing phagosome in (D). (F) SEM of IL34-Mϕs. (G) TEM of IL34-Mϕs. (H) TEM of IL34-Mϕs co-incubated with Bd. Arrows: (a) phagocytosed Bd zoospore in the process of being degraded; (b) phagocytosed zoospore. (I) TEM of IL34-Mϕs co-incubated with Bd. Arrows: (a) phagocytosed, degraded zoosporangium; (b) phagocytosed zoosporangium. (J) Higher magnification of mature zoosporangium-containing phagosome.
Figure 3. Consequences of Bd exposure on CSF1- and IL34-Mϕ activation. CSF1- and IL34-Mϕs were incubated with developing Bd thalli for 5 h (5 Bd/Mϕ), and the cocultures were examined for their expression of immune genes relative to gapdh endogenous control. All data are represented relative to parallel respective CSF1- and IL34-Mϕ cultures, incubated without Bd, depicted by the horizontal line. The results are means ± SEMs of immune gene expression from 6 independent CSF1- and IL34-Mϕ cultures, derived from 6 individual frogs, N = 6. Asterisks above bars indicate statistical significance between baseline and Bd-elicited Mϕ gene expression, and asterisks above lines are indicative of statistical differences between the treatment groups denoted by the lines, p < 0.05. The examined frog Mϕ genes included noxa2 and nox2-NADPH oxidase catalytic domains; inos—inducible nitric oxide synthase; arg1—arginase-1; ido—indoleamine 2,3 dioxygenase; tnf—tumor necrosis factor, and il10—interleukin-10.
Figure 4. Consequences of Mϕ-Bd coculture on Bd gene expression. Developing Bd thalli were incubated alone or in coculture with CSF1- and IL34-Mϕs for 5 h (5 Bd/Mϕ), and Bd was examined for expression of immune evasion genes relative to the Bd gapdh endogenous control. All data are represented relative to baseline Bd gene expression, depicted by the horizontal line. The results are means ± SEMs of Bd gene expression from 6 independent CSF1- and IL34-Mϕ cocultures, with frog cells derived from 6 individual frogs, N = 6. Asterisks above bars indicate statistical significance between baseline and Mϕ-elicited Bd gene expression, and asterisks above lines are indicative of statistical differences between the treatment groups denoted by the lines, p < 0.05. The examined Bd genes included arg—arginase; amd—adenosylmethionine decarboxylase; cat—catalase; cda—chitin deacetylase; ido—indoleamine 2,3 dioxygenase; odc—ornithine decarboxylase; sdm—superoxide dismutase; and srm—spermidine synthase.
