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???displayArticle.abstract??? Macrophage-lineage cells are indispensable to vertebrate homeostasis and immunity. In turn, macrophage development is largely regulated through colony-stimulating factor-1 (CSF1) binding to its cognate receptor (CSF1R). To study amphibian monopoiesis, we identified and characterized the X. laevis CSF1R cDNA transcript. Quantitative analysis revealed that CSF1Rtissue gene expression increased with X. laevis development, with greatest transcript levels detected in the adult lung, spleen and liver tissues. Notably, considerable levels of CSF1R mRNA were also detected in the regressing tails of metamorphosing animals, suggesting macrophage involvement in this process, and in the adult bone marrow; corroborating the roles for this organ in Xenopus monopoiesis. Following animal infections with the ranavirus Frog Virus 3 (FV3), both tadpole and adult X. laevis exhibited increased kidneyCSF1R gene expression. Conversely, while FV3-infected tadpoles increased their spleen and liverCSF1R mRNA levels, the FV3-challenged adults did not. Notably, FV3 induced elevated bone marrowCSF1R expression, and while stimulation of tadpoles with heat-killed E. coli had no transcriptional effects, bacterial stimulation of adult frogs resulted in significantly increased spleen, liver and bone marrowCSF1R expression. We produced the X. laevis CSF1R in recombinant form (rXlCSF1R) and determined, via in vitro cross-linking studies, that two molecules of rXlCSF1R bound the dimeric rXlCSF1. Finally, administration of rXlCSF1R abrogated the rXlCSF1-induced tadpolemacrophage recruitment and differentiation as well as bacterial and FV3-elicited peritoneal leukocyte accumulation. This work marks a step towards garnering greater understanding of the unique mechanisms governing amphibian macrophage biology.
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???displayArticle.pmcLink???PMC5617918 ???displayArticle.link???Int J Dev Biol ???displayArticle.grants???[+]
Fig. 1. Amino acid alignment of X. laevis CSF1R with other vertebrate CSF1R protein sequences. Protein alignments were performed using ClustalW2 server. Fully conserved residues are indicated by an asterisk (*), partially conserved and semi-conserved substitutions are represented by â:â and â.â, respectively. The X. laevis CSF1R signal peptide is in bold, the immunoglobulin-like domains are indicated as D1-D5, the evolutionarily conserved structural cysteines are in white text within black boxes and the transmembrane domain is also in bold face. As indicated overhead; the X. laevis CSF1R ATP binding and tryrosine kinase major catalystic domains are in white text within black boxes while the kinase insert domain is indicated by an overhead line.
Fig. 2. Phylogenetic analysis of teleost, amphibian, reptile, avian and mammalian colony-stimulating factor-1 receptor (CSF1R) molecules. The phylogenetic tree was constructed from multiple deduced amino acid sequence alignments using the neighbor joining method and bootstrapped 10,000 times (denoted as %). The zebrafish KitR was used as outgroup to root the tree.
Fig. 3. Quantitative colony-stimulating factor-1 receptor (CSF1R) tissue gene expression analysis of tadpoles (Stg. 54), metamorphic (Stg. 64) and adult (2 years-old) frogs. Tissues from three individuals (N =3) were used for all expression studies, the expression was performed via the delta^delta CT method using X. laevis CSF1R-specific primers. The expression examined relative to the GAPDH endogenous control and normalized against the tadpolemuscletissue expression. Results are means ± SEM, N=4 and the (*) above lines denotes significant difference between tissues indicated by the lines, P<0.05.
Fig. 4. Quantitative analysis of colony-stimulating factor-1 receptor (CSF1R) tissue gene expression in Frog Virus 3 (FV3) and heat-killed E. coli challenged tadpoles and adult X. laevis. Tadpoles (Stg. 54) and adult frogs (2 years-old) were infected by ip injections with 1x104 and 5x106 PFU of FV3, respectively. Alternatively, tadpoles and adult frogs were injected respectively with 10ml and 100ml of heat-killed (hk) E. coli. (A) FV3-infected tadpoleCSF1R gene expression. (B) FV3-infected tadult frog CSF1R gene expression. (C) Heat-killed E. coli-stimulated tadpoleCSF1R gene expression. (D) Heat-killed E. coli-stimulated adult frogCSF1R gene expression. All gene expression analysis was performed via the delta^delta CT method using X. laevis CSF1R-specific primers, the expression examined relative to the GAPDH endogenous control and normalized against respective uninfected kidney expression. Results are means ± SEM, N=4 and the (*) above lines denotes significant difference between tissues indicated by the lines, P<0.05.
Fig. 5. In vitro rXlCSF1 and rXlCSF1R cross-linking studies. One microgram each of rXlCSF1, rXlCSF1R, rXlCSF1 + rXlCSF1R and BSA + rXlCSF1R were incubated in APBS (100ml final volume) for 30 min and cross-linked for 30 min using 2.5 mM DSS, final concentration. Cross-linking reactions were terminated by addition of 50 mM Tris (final concentration). Reactions were resolved and visualized using SDS-PAGE and western blot against the V5 epitopes on the recombinant proteins.
Fig. 6. The rXlCSF1R abrogates the rXlCSF1-mediated tadpolemacrophage recruitment and differentiation. (A) Tadpoles were injected with vector control, 250ng of rXlCSF1, 1000ng of rXlCSF1R or a combination of rXlCSF1 (250ng) and rXlCSF1R (10000ng). After 24hrs peritoneal phagocytes were lavaged and enumerated. Results are means ± SEM, N=6 and the (*) above lines denotes significant difference between treatment groups indicated by the lines, P<0.05. (B) Morphological analysis of Giemsa-stained vector-control and rXlCSF1R derived cultures. Scale bar = 10mm. (C) Cultures from (A) were Giemsa-stained, enumerated for the presence of morphologically differentiated macrophages as exemplified in (B) by the rXlCSF1R-derived cells. Results are expressed as percent means ± SEM from ten random fields of view. The (*) above lines denotes significant difference between treatment groups indicated by the lines, P<0.05.
Fig. 7. The rXlCSF1R abrogates heat-killed E. coli and Frog Virus 3 elicited recruitment of tadpole peritoneal leukocytes and reduces FV3 dissemination. (A) Tadpoles were injected with vector control, heat-killed (hk) E. coli, FV3 (104 PFU), a combination of hk E. coli and rXlCSF1 (1mg) or a combination of FV3 (104 PFU) and rXlCSF1R (1mg). After 24hrs peritoneal phagocytes were lavaged and enumerated. Results are means ± SEM, N=4. (*) denotes statistical difference from the vector control and the (*) above lines denotes significant difference between treatment groups indicated by the lines, P<0.05. (B) Tadpoles were injected with 1x104 PFU of FV3 alone, or in combination with 1mg of rXlCSF1R. Following 24 hrs, tadpoles were sacrificed; their tissues were isolated and examined for infectious FV3 burdens by plaque assays. Results are means ± SEM, N=3. (*) above lines denotes significant difference between treatment groups indicated by the lines, P<0.05.
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