Divergent antiviral roles of amphibian (Xenopus laevis) macrophages elicited by colony-stimulating factor-1 and interleukin-34

Abstract

Macrophages are integral to amphibian immunity against RVs, as well as to the infection strategies of these pathogens. Although CSF-1 was considered to be the principal mediator of macrophage development, the IL-34 cytokine, which shares no sequence identity with CSF-1, is now believed to contribute to vertebrate monopoiesis. However, the respective roles of CSF-1- and IL-34-derived macrophages are still poorly understood. To delineate the contribution of these macrophage populations to amphibian immunity against the RV FV3, we identified the Xenopus laevis IL-34 and transcriptionally and functionally compared this cytokine with the previously identified X. laevis CSF-1. The X. laevis CSF-1 and IL-34 displayed strikingly nonoverlapping developmental and tissue-specific gene-expression patterns. Furthermore, only CSF-1 but not IL-34 was up-regulated in the kidneys of FV3-challenged tadpoles. Intriguingly, recombinant forms of these cytokines (rXlCSF-1, rXlIL-34) elicited morphologically distinct tadpole macrophages, and whereas rXlCSF-1 pretreatment decreased the survival of FV3-infected tadpoles, rXlIL-34 administration significantly prolonged FV3-challenged animal survival. Compared with rXlIL-34-elicited macrophages, macrophages derived by rXlCSF-1 were more phagocytic but also significantly more susceptible to in vitro FV3 infections. By contrast, rXlIL-34-derived macrophages exhibited significantly greater in vitro antiranaviral activity and displayed substantially more robust gene expression of the NADPH oxidase components (p67(phox), gp91(phox)) and type I IFN. Moreover, FV3-challenged, rXlIL-34-derived macrophages exhibited several orders of magnitude greater up-regulation of the type I IFN gene expression. This marks the first report of the disparate roles of CSF-1 and IL-34 in vertebrate antiviral immunity.

Keywords: CSF-1; FV3; IL-34; immunity; ranavirus.

Figure 1.. Characterization of X. laevis IL-34 (A) phylogeny and (B) CSF-1 and IL-34 quantitative tissue gene expression.

(A) The phylogenetic tree was constructed from multiple deduced protein sequence alignments using the neighbor-joining method and bootstrapped 10,000 times (denoted as percent). (B) Outbred premetamorphic (stage 54) tadpoles and metamorphic (stage 64) and adult (2 years old) frog tissues were assessed. Tissues from three individuals of each stage were examined (n=3). *P < 0.05, statistical difference between indicated groups. Tissues examined: S, spleen; L, liver; I, intestine; M, muscle; Sk, skin; K, kidney; Lu, lung; BM, bone marrow. RQ, Relative quantification.

Figure 2.. The rXlCSF-1 and rXlIL-34 cytokines elicit morphologically distinct tadpole peritoneal macrophages.

Stage 54 tadpoles were i.p. injected with vector control, 1, 10, 100, or 1000 ng rXlCSF-1 or rXlIL-34. Three days postinjections, peritoneal macrophages were isolated. (A) rXlCSF-1 and rXlIL-34 concentration-dependent elicitation of tadpole peritoneal macrophages. *P < 0.05, statistical difference compared with the vector-treatment group. (B) Giemsa-stained peritoneal phagocytes, isolated from tadpoles, 3 days postinjection, with vector control; 1 μg rXlCSF-1; 1 μg rXlIL-34; a combination of rXlCSF-1 and rXlIL-34 (1 μg each); or 1 μg rXlCSF-1, followed 36 h later by 1 μg rXlIL-34.

Figure 3.. Tadpoles pretreated with rXlCSF-1 and rXlIL-34 and peritoneal macrophages derived from these animals display distinct susceptibility to FV3.

(A) Administration of rXlCSF-1 to tadpoles before FV3 challenge decreases their survival time, whereas preinjection with rXlIL-34 increases tadpole survival. Stage 50 tadpoles (10/treatment group; n=10) were injected with rXlCSF-1, rXlIL-34 (1 μg/tadpole), or equal volume of vector control. Three days postinjection, animals were infected i.p. with 1 × 10⁴ PFU of FV3 (in APBS) or sham infected with equal volumes of APBS. Animal survival was monitored over the course of 16 days. *P < 0.05, statistical difference relative to vector controls. (B) Tadpole rXlCSF-1-pMϕ infected in vitro with FV3 exhibit enhanced FV3 susceptibility, higher viral loads, and increased viral gene expression. Tadpole rXlIL-34-pMϕ and rXlCSF-1-pMϕ were infected at a MOI of 0.5 with FV3 for 24 h, and then viral loads and gene expression were assessed by qPCR. *P < 0.05, statistical difference between indicated groups. IE, Immediate-early; DE, delayed-early; L, late.

Figure 4.. Tadpole rXlCSF-1-pMϕ are more phagocytic, whereas rXlIL-34-pMϕ are more antiviral.

(A) rXlCSF-1-pMϕ, rXlIL-34-pMϕ, and vector control-elicited tadpole peritoneal phagocytes were incubated overnight (16 h) with 1 μm FITC latex beads and analyzed by flow cytometry. Cells from two individuals were pooled immediately before analysis, with six separate pools assessed per treatment group (n=6). (B) Phagocytic indexes of rXlCSF-1-pMϕ, rXlIL-34-pMϕ, and control phagocyte cultures. (C) rXlCSF-1-pMϕ and rXlIL-34-pMϕ exhibit similar FV3 internalization, whereas rXlIL-34-pMϕ eliminate a significant proportion of the virus. rXlCSF-1-pMϕ, rXlIL-34-pMϕ, and control phagocyte cultures were infected in vitro at a MOI of 0.5 and cells harvested for plaque assay analyses at 2 and 24 h. (D) rXlCSF-1 + rXlIL-34-pMϕ are as susceptible to FV3 as rXlCSF-1-pMϕ, whereas macrophages from tadpoles injected with rXlCSF-1 and 36 h later, with rXlIL-34 (rXlCSF-1, rXlIL-34-pMϕ) are significantly more resistant to in vitro FV3 infection. rXlCSF-1-pMϕ, rXlIL-34-pMϕ, rXlCSF-1 + rXlIL-34-pMϕ, and rXlCSF-1, as well as rXlIL-34-pMϕ cultures, were infected in vitro at a MOI of 0.5 for 24 h before harvesting the cells for plaque assay analyses. (B–D) Results are means ± sem. *P < 0.05, significant difference from vector controls; *P < 0.05 (when over bars), significant differences between treatment groups denoted by the bars.

Figure 5.. Quantitative immune gene-expression analysis of rXlCSF-1-pMϕ and rXlIL-34-pMϕ.

Three days after administration of recombinant cytokines, rXlCSF-1-pMϕ and rXlIL-34-pMϕ, and vector-control peritoneal phagocytes were isolated and assessed by qRT-PCR for immune genes expression of: (A) IL-10, TNF-α, IFN, and Mx1 (Mx1 relative quantification [RQ] values × 10) (B) p67^phox, gp91^phox, iNOS, and IDO; (C) MHC class I and II, β2m, and XNC10. Gene expression was examined relative to GAPDH control and normalized against respective vector-control gene expression (horizontal dashed lines). (D) Type I IFN gene expression in uninfected and FV3-infected, vector-control leukocytes (vector), rXlCSF-1-pMϕ (rXlCSF1), and rXlIL-34-pMϕ (rXlIL34). All FV3-infected cell populations possessed significantly greater IFN expression than respective uninfected controls. Results are means±sem. *P < 0.05, significant difference from vector controls; *P < 0.05 (when over bars), significant differences between treatment groups denoted by the bars.

Figure 6.. Quantitative gene expression of CSF-1 and IL-34 in kidneys of FV3-infected tadpole and adult X. laevis.

Tadpoles and adult frogs were infected i.p. with 1 × 10⁴ and 5 × 10⁶ PFU of FV3, respectively. At indicated times, animals were euthanized and kidneys collected from six individuals/treatment group (n=6). Gene expression was performed relative to the GAPDH endogenous control and results presented as mean relative quantification ± sem. *P < 0.05, significantly different from the uninfected controls.