Garvey Griffith CN et al. (2025), The contribution of amphibian macrophage subset...

XB-ART-61650

Front Immunol 2025 Dec 11;16:1713361. doi: 10.3389/fimmu.2025.1713361.

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The contribution of amphibian macrophage subsets to scarless regeneration of skin wounds.

Garvey Griffith CN, Hauser KA, Abramson B, Chapkin ER, Jones EJ, Duttargi AN, Grayfer L.

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Xenopus laevis juvenile frogs regenerate wounded skin without scarring, yet the underlying mechanisms driving this process remain poorly defined. Macrophages are critical to wound repair across vertebrates, and our results indicate a transient influx of macrophages into regenerating frog wounds. The colony stimulating factor-1 (CSF1) and interleukin-34 (IL34) growth factors control macrophage development. Through RNA in situ hybridization studies, we found that csf1 gene expression peaked early during juvenile frog wound responses, whereas il34 expression increased later in the repair process. Our past studies indicate that X. laevis CSF1- and IL34-differentiated macrophages are functionally distinct. Presently, we treated frog wounds with recombinant (r)CSF1 and rIL34 to determine the roles of the corresponding macrophage subsets in wound repair. Using a combination of RNA in situ hybridization, RNA sequencing and histology, we demonstrated that wounds skewed towards greater proportions of rCSF1-macrophages exhibited greater infiltration of leukocytes, chiefly amongst them neutrophils. These wounds also possessed robust expression of inflammatory genes and transcripts associated with granulation and fibrosis. By contrast, rIL34-treated frog wounds exhibited greater fibroblast activation concurrent with greater type I/III collagen ratios and expression of genes typically seen at later phases of wound repair. Together, we propose that while CSF1-macrophages are likely more prominently involved in the inflammatory phase of X. laevis wound repair, IL34-macrophages predominate the later reparative phase of these responses.

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Species referenced: Xenopus laevis
Genes referenced: acta2 ccn2 col1a1 col1a2 col2a1 csf1 csf1r edn1 fgf9 il34 mmp1 mmp13 mmp14 mmp24 mmp25 mmp28 mmp7 mmp9.2 mpo pdgfra sparc tgfb1 tgfbr2 timp2
GO keywords: macrophage differentiation [+]

???displayArticle.gses??? GSE306422: NCBI
Phenotypes: Xla wt juvenile + skin wound + rCSF1(Fig. 4. BEH) [+]

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	Figure 1. Juvenile X. laevis skin regeneration over time. (AF) Hematoxylin and eosin (H&E) stained skin sections from (A) unwounded controls (ctrl) and at (B) 7, (C) 14, (D) 21, (E) 28 and (F) 60 days post wounding (dpw). Open arrowheads mark wound edges. Control skin showing epidermis (E), dermis (D), and well-defined mucus (M) and serous (S) glands. WE, wound epidermis. (GN) Top-down images of (G) control skin and wounds at (H) 0, (I) 3, (J) 7, (K) 14 (L) 21, (M), 28 and (N) 60 dpw. Contrast and brightness adjusted uniformly across images.
	Figure 2. Csf1r+ macrophages accumulate transiently during wound repair. (AD') RNA in situ hybridization detecting csf1r+ cells (teal) in (A, A') unwounded skin and at (B, B) 7, (C, C') 14 and (D, D') 21 days post wounding (dpw). Open arrowheads mark wound edges, boxed regions indicate higher magnification in AD. (E) Quantification of csf1r+ cell density (cells/cm2). Data are mean +/-SEM (n = 4-5 biological replicates per group). One-way ANOVA with Tukeys post hoc test; p < 0.05,**p < 0.001.
	Figure 3. Expression of csf1 and il34 in regenerating skin wounds. (AD) RNA in situ hybridization detecting csf1 (magenta) and il34 (blue/teal) transcripts in (A) unwounded skin, and skin at (B) 7, (C) 14 and (D) 21 days post wounding (dpw; counterstained with hematoxylin). WE, wound epidermis; E, epidermis; D, dermis. (E) Quantification of csf1 and il34 transcript density (transcripts/cm2. Data represent mean +/- SEM (n = 4-7 biological replicates per group). One-way ANOVA with Tukeys post hoc test; p<0.05. Asterisks () indicate statistical significance from ctrl. Asterisks () above bar denotes significance between the groups indicated by the bar. p < 0.05, ** p < 0.01.
	Figure 4. Analyses of histology and neutrophil infiltration of rCSF1- and rIL34-administered wounds. Histology, 7 days post wounding (dpw) of (A, A') rctrl-, (B, B') rCSF1-, and (C, C') rIL34-administered wounds (cytokines administered 3 dpw). Inset panels (A'C') depict higher magnification. Lymphocytes (black arrows), macrophages (red arrows), and neutrophils (blue arrows) are indicated. (DF) RNA in situ hybridization for mpo (magenta), visualizing neutrophil localization 7 days post wounding (dpw) in (D) rctrl-, (E) rCSF1- and (F) rIL34-administered wounds (cytokines administered 3 dpw). Open arrowheads mark wound edges. (G) Quantification of mpo+ cell density (cells/cm2) at 7 and 14 dpw. Data are mean +/- SEM (n = 3-7 biological replicates per group). Statistical comparisons were performed using one-way ANOVA with Tukeys post hoc test; *p < 0.05. (H) Expression analysis of inflammatory response genes at 7 dpw across treatments. Boxplots represent medians with interquartile range; letters indicate statistically distinct groups (p < 0.05, pairwise Wilcox test).
	Figure 5. Myofibroblast accumulation in rCSF1- and rIL34-administered wounds. (A-C') Immunofluorescence staining of (red) acta2 and (blue) DAPI in (A, A') rctrl, (B, B') rCSF1, and (C, C') rIL34 wounds at 7 dpw (cytokines administered 3 dpw). Open arrowheads mark wound edges, boxed regions indicate sections of higher magnification in (A'-C'). (D) Quantification of acta2 area (% total wound region, excluding gland-adjacent expression). Data represent mean +/- SEM (n = 4-7 biological replicates per group). One-way ANOVA with Tukeys post hoc test; *p < 0.05. Images were created using fluorescent microscopy and inverting uniformly in ImageJ.
	Figure 6. Collagen I and III deposition in rCSF1- and rIL34-administered wounds. (A-C') Picrosirius red (PSR) staining of (A, A') rctrl, (B, B') rCSF1, and (C, C') rIL34-administered wounds at 7 days post wound (dpw; cytokines administered 3 dpw), imaged with polarized light microscopy. Collagen I (red/orange/yellow) and collagen III (green) birefringence is shown; corresponding binary masks are in A-C'. (D-F') PSR staining of (D, D) rctrl, (E, E') rCSF1, and (F, F') rIL34 wounds at 14 dpw with corresponding masks (DF). (G) Quantification of collagen I/III ratios at 7 and 14 dpw. Data represent mean +/- SEM (n = 7-12 biological replicates per group). One-way ANOVA with Tukeys post hoc test; p < 0.05, *p < 0.01.
	Figure 7. Gene expression in rCSF1- and rIL34-administered wounds. (A) RNA-seq expression analysis of ECM- and fibrosis-associated genes at 7 and 14 dpw across treatments. (B) RNA-seq expression analysis of matrix metalloproteinases (MMPs) and their regulators (TIMPs) at 7 and 14 dpw across treatments. Boxplots show median, interquartile range, and statistical groupings, letters denote significance; p < 0.05.
	Juvenile X. laevis skin section, Hematoxylin and eosin (H&E) stained epidermis (unwounded ) with Inset A' depicting higher magnification. 3 key cell types, Lymphocytes (black arrows), macrophages (red arrows), and neutrophils (blue arrows) are indicated.