Neutrophil extracellular traps impair regeneration

Abstract

Fibrosis is a major health burden across diseases and organs. To remedy this, we study wound-induced hair follicle neogenesis (WIHN) as a model of non-fibrotic healing that recapitulates embryogenesis for de novo hair follicle morphogenesis after wounding. We previously demonstrated that TLR3 promotes WIHN through binding wound-associated dsRNA, the source of which is still unclear. Here, we find that multiple distinct contexts of high WIHN all show a strong neutrophil signature. Given the correlation between neutrophil infiltration and endogenous dsRNA release, we hypothesized that neutrophil extracellular traps (NETs) likely release nuclear spliceosomal U1 dsRNA and modulate WIHN. However, rather than enhance regeneration, we find mature neutrophils inhibit WIHN such that mice with mature neutrophil depletion exhibit higher WIHN. Similarly, Pad4 null mice, which are defective in NET production, show augmented WIHN. Finally, using single-cell RNA sequencing, we identify a dramatic increase in mature and activated neutrophils in the wound beds of low regenerating Tlr3-/- mice. Taken together, these results demonstrate that although mature neutrophils are stimulated by a common pro-regenerative cue, their presence and NETs hinder regeneration.

Keywords: Neutrophils; fibrosis; regeneration.

FIGURE 1

Neutrophil signatures correlate with high Wound‐induced Hair Neogenesis (WIHN) in multiple models. (A) Schematic of hair neogenesis preferential localization to wound centre (high WIHN) rather than wound edge (low WIHN); image duplicated from Figure 3F for clarity. (B) Proteomic gene ontology (GO) analysis of the top 100 genes wound centre vs. wound edge in wild‐type (WT) mice shows a predominance of innate immune response pathways and neutrophil signatures in the wound centre. (C) Abundance ratios of genes from select GO terms highlighted in b. show enrichment in the wound centre of antimicrobial and granular proteins, labelled in red. (D) GO analysis of the top 200 genes from high WIHN Rnasel^−/− vs. low WIHN WT mice shows a predominance of neutrophil and innate immune cell chemotaxis pathways in Rnasel^−/− mice. Inset graphs show the gene fold expression changes for genes present in that category. (E) GO enrichment analysis of the top vs. bottom 500 differentially expressed genes between specific pathogen‐free (SPF; high WIHN) and germ‐free (GF; low WIHN) mice demonstrates higher neutrophil chemotaxis and innate immune categories in SPF mice (n = 3 independent animals per group). Inset graphs show the gene fold expression changes for genes present in that category

FIGURE 2

Neutrophils persist in wound bed after the acute inflammatory phase, producing extracellular traps. (A) Neutrophils are present in the wound beds of C57BL/6J mice at early time points, as visible in representative haematoxylin and eosin (H&E) staining. Arrows indicate regions of interest, and dashed line demarcates boundaries. Black scale bar = 50 µm. (B) Neutrophils predominate throughout the wound beds of C57BL/6J mice on wound day (WD) 1 and WD3, visible in prominent myeloperoxidase (MPO) immunofluorescence (IF) staining (green). Few macrophages are present (Red, F4/80). White scale bar = 200 µm. (C) Per cent neutrophil (Ly6G+ cells from total CD45+ cells) levels are consistent in the blood throughout the wound time course but drop in the wound bed at WD11, as measured by FACS. ****p < 0.0001, as calculated by two‐way ANOVA. N = 2 vs. 4. Results are representative of at least two independent experiments. (D) Macrophage (F4/80) levels are largely absent from the blood and low in the wound bed during the early phase of healing but increase dramatically at WD11, as measured by FACS. ***p < 0.004, as calculated by two‐way ANOVA. n.s., not significant. N = 2 vs. 4. Results are representative of at least two independent experiments. (E) Citrullinated histone H3 (H3Cit, red) co‐localized with Ly6G+ neutrophils (green), beginning at WD3 in the wound beds of IF‐stained C57BL/6J mice, indicating the formation of extracellular traps. (F) Neutrophil extracellular trap‐positive cells (MPO+, SYTOX green +) are present at late wound time points, but are absent in the wound beds of PAD4−/− mice, as measured by FACs. ****p < 0.0001, as calculated by two‐way ANOVA. N = 7 vs. 4. Results are representative of at least two independent experiments. (G) Cytoplasmic U1 snRNA is present in the wound bed of C57BL/6J mice, while it localized exclusively in the nuclei of unwounded controls, as visualized by representative FISH. The solid white line delineates a hair follicle. White scale bar = 80 µm

FIGURE 3

Mature neutrophils inhibit WIHN. (A) Schematic of neutrophil depletion via diphtheria toxin (DT, 250 ng) injection in heterozygous ROSA26iDTR/MRP8‐Cre‐ires mice (PMN^DTR and PMN^WT). (B) Diphtheria toxin injection in heterozygous ROSA26iDTR/MRP8‐Cre‐ires mice (PMN^DTR and PMN^WT), following the injection scheme in (A), successfully depletes neutrophils from the blood in mice with large wounds. Fold = −4.33. **p = 0.0098. N = 3 vs. 3. Results are representative of at least two independent experiments. (C) Mice treated as in (A) are depleted of neutrophils in their wound beds at WD2. Fold = −4.27. **p = 0.0069. N = 3 vs. 3. Results are representative of at least two independent experiments. (D) PMN^DTR mice IP injected with diphtheria toxin (DT, 250 ng) on WD‐1 and WD1 exhibit increased WIHN (CSLM, images; fold = 3.23, ***p = 0.0010, N = 14 vs. 6). In each image, the dash red box indicates the area of hair follicle regeneration. (E) Pad4^−/− mice defective in extracellular traps exhibit increased WIHN (CSLM, images; fold = 2.47, p = 0.026, N = 10 vs. 19 (F) Tlr3^−/− mice exhibit decreased WIHN (fold = −3.75 p = 0.0218, N = 8 vs. 10). (G) The presence of increased mature neutrophils correlates with decreased WIHN in TLR3^−/− mice. scRNA‐seq t‐SNE plot shows differences between Tlr3−/− (blue, 4843 cells) and WT (red, 3172 cells) wound beds at WD 10. The plots were generated via Seurat. The neutrophil cluster is circled in red. Per cent of neutrophils is significantly different in Tlr3−/− vs. WT mice as graphed to bottom right. ****p < 0.0001, as calculated by chi‐square test. Neutrophils have the greatest fold change (fold = 6.98) between Tlr3−/− and WT mice, graphed to bottom left. (H) Neutrophil‐associated gene expression is more pronounced within the neutrophils of Tlr3−/− mice, compared to WT. Generated in Seurat with RidgePlot function. (I) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway gene ontology analysis of the top 50 differentially of the mature neutrophil cluster demonstrates categories associated with neutrophil activation preferentially in Tlr3 −/− mice compared with WT