Bacteria induce skin regeneration via IL-1β signaling

Abstract

Environmental factors that enhance regeneration are largely unknown. The immune system and microbiome are attributed roles in repairing and regenerating structure but their precise interplay is unclear. Here, we assessed the function of skin bacteria in wound healing and wound-induced hair follicle neogenesis (WIHN), a rare adult organogenesis model. WIHN levels and stem cell markers correlate with bacterial counts, being lowest in germ-free (GF), intermediate in conventional specific pathogen-free (SPF), and highest in wild-type mice, even those infected with pathogenic Staphylococcus aureus. Reducing skin microbiota via cage changes or topical antibiotics decreased WIHN. Inflammatory cytokine IL-1β and keratinocyte-dependent IL-1R-MyD88 signaling are necessary and sufficient for bacteria to promote regeneration. Finally, in a small trial, a topical broad-spectrum antibiotic also slowed skin wound healing in adult volunteers. These results demonstrate a role for IL-1β to control morphogenesis and support the need to reconsider routine applications of topical prophylactic antibiotics.

Keywords: bacteria; hair; regeneration.

Figure 1.. Germ-free (GF) mice have significantly lower wound induced hair follicle neogenesis (WIHN) than Specific pathogen-free (SPF) mice.

(A) GF mice exhibit significantly less WIHN than SPF mice as detected by Confocal Scanning Laser Microscopy (CLSM) (right top), hematoxylin and eosin (H&E) staining (right bottom), and quantification (left). The red dotted line frames the regenerative hair follicles. (n=6–7 independent animals per group). (B) Microbiota α-diversity based on the Shannon and Observed species of GF mice were significantly lower than SPF mice and roughly equivalent to air control. (n=4–14 independent animals per group). (C) Principal component analysis (PCA) clustering based on Microbiota β-diversity shows clustering of GF mice with air controls and separation from SPF mice. (n=4–14 independent animals per group). (D) Morphogenesis and differentiation categories are included in Gene ontology (GO) enrichment analysis of the top and bottom 500 differentially expressed genes between SPF and GF mice near wound day (WD) 12 wound beds, the day of scab detachment (SD0). (n=3 independent animals per group). (E) mRNA expression of stem cell marker (Krt15), hair regeneration marker (Wnt7b, tgfb1) are higher in SPF mice, while keratinocyte differentiation markers (Flg, Krt1) are higher in GF mice. (n=3 independent animals per group). (F) β-Catenin expression of SD0 wound bed epidermis as normalized to adjacent normal skin were higher in SPF mice than in GF mice as detected by immunofluorescence staining (up) and quantification (down). The dotted white line demarcates the boundary between the wound bed and normal tissue, and the white scale bar is 200um. (n=3 independent animals per group). (G) The wound size of GF mice was significantly bigger than the SPF mice at WD5 as per photos (top) and quantification (bottom) (n=5–6 independent mice per group). (H) WIHN is elevated in GF mice after co-housing with SPF mice as detected by CLSM (right top), H&E staining (right bottom), and quantification (left). (n=6 independent animals per group). Representative data from 2~3 independent experiments are shown. Boxplot graphs indicated the value of minimum, first quartile, median, third quartile, and maximum. Scatter plots and histogram graphs indicated means ± SEM; Paired Student t-test was used to compare statistical difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. “NS” indicates no significant difference.

Figure 2.. Reducing skin commensal microbiota inhibits WIHN.

(A) WIHN of bedding change every day caged (CED) mice was significantly less than bedding never change caged (NC) mice as detected by CLSM (right top), H&E staining (right bottom), and quantification (left). (n=4 independent animals per group). (B) Among CED mice, microbiota α-diversity (Shannon and Observed species) was significantly lower than in NC mice. (n=4 independent animals per group). (C) PCA based on microbiota β-diversity shows separate clustering of CED mice and NC mice. (n=4 independent animals per group). (D) Absolute value of taxonomic classifications at genus level of 16S rDNA sequence from skins of CED and NC mice. (n=4 independent animals per group). (E) WIHN is lower after topical Neosporin treatment compared to control mice as detected by CLSM (top left), H&E staining (bottom left), and quantification (right). The red dotted line frames regenerative hair follicles. (n=5 independent animals per group). Representative data from 2~3 independent experiments are shown. Boxplot graphs indicated the value of minimum, first quartile, median, third quartile, and maximum. Scatter plots and histogram graphs indicated means ± SEM; Paired Student t-test was used to compare statistical difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 3.. Staphylococcus aureus induce WIHN.

(A) Elevated WIHN in Rnasel^−/− mice (left) correlates with S. aureus gene ontology category of gene expression in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the top 500 differentially expressed genes of Rnasel^−/− versus WT mice SD0 wound beds (right). (B) As in A, except comparing wild type (higher WIHN) versus Rarα^−/− (lower WIHN) (C) Schematic of hair neogenesis preferential localization to wound center (high WIHN) rather than edge (low WIHN) (left). Proteomic KEGG enrichment analysis of SD0 wound center versus wound edge in wild type mice (right), also showing the S. aureus category. (D) S. aureus treatment of mouse skin induces entry to the anagen hair growth phase more than vehicle treated mice after 14 days (left). Anagen hair follicle surface area is marked by red lines and quantified (right). (n=5 independent animals per group). (E) WIHN in S. epidermidis and S. aureus treated mice was significantly more than control mice as detected by CLSM (right top), H&E staining (right bottom), and quantification (left). (n=6–7 independent animals per group). (F) The de novo hair follicles induced by S. aureus mimic embryonic hair follicle development pattern with high Krt17 in hair germs. After morphogenesis, follicles appropriately express β-catenin, and Wnt7b in the bulge and bulb regions, with intact sebaceous gland (Oil red O). Abbreviations: BU: bulge, SG: sebaceous gland, HS: hair shaft. (G) S. aureus persisted in the skin longer than S. epidermidis (right) as quantified by in vivo bioluminescent signals total flux (p/s) (left up) and normalized flux (p/s) (left bottom). (n=6 independent animals per group). (H) Krt5 and Krt15 expression in epidermis of S. aureus treated mice, as normalized to adjacent normal skin were significantly higher than in control mice as detected by immunofluorescence staining and quantification. The dotted white line marks the boundary between the wound and normal tissue, and the white scale bar is 200um. (n=3 independent animals per group). (I) Higher mRNA expression of hair regeneration signaling (Wnt7b, Shh, tgfb1), inflammatory cytokines (Tnf) with lower expression of keratinocytes differentiation markers (Flg, Krt1) in S. aureus versus control treated mice. (n=3 independent animals per group). (J) GO enrichment analysis of the top and bottom 500 differentially expressed genes illustrate higher immune and decreased differentiation categories in S. aureus versus control treated mice SD0 wound beds. Inset are selected genes from notable categories (n=3 independent animals per group). (K) WIHN in WT mice co-housed with S. aureus infected mice are significantly higher than control mice as detected by CLSM (right top), H&E staining (right bottom), and quantification (left). (n=4–7 independent animals per group). (L) Microbiota α-diversity based on Shannon and Observed species of mice co-housed with S. aureus infected mice and S. aureus infected mice themselves were lower than in control mice. (n=3–14 independent animals per group). (M) PCA based on Microbiota β-diversity shows clustering of mice co-housed with S. aureus infected mice and S. aureus infected mice apart from control mice. (n=3–14 independent animals per group). (N) Taxonomic classifications at genus level of 16S rDNA sequence from the skins of C57/B6j control mice, C57/B6j co-housed with S. aureus infected mice, and S. aureus infected mice. (n=3–14 independent animals per group). Representative data from 2~3 independent experiments are shown. Boxplot graphs indicated the value of minimum, first quartile, median, third quartile, and maximum. Scatter plots and histogram graphs indicated means ± SEM; Paired Student t-test was used to compare statistical difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. “NS” indicates no significant difference.

Figure 4.. Bacteria induced WIHN is mediated by MyD88 signaling in keratinocytes.

(A) WIHN in Myd88^−/− mice was significantly less than in WT mice (top) and could not be induced by S. aureus treatment (bottom). (n=4–7 independent animals per group). (B) The wound closure speed of Myd88^−/− mice was significantly slower than the WT mice as per photos (top) and quantification (bottom) (n=4–7 independent mice per group). (C) WIHN in S. aureus treated or untreated WT, K14-Myd88^−/−, LysM-Myd88^−/−, LysM-Myd88^+/−, and Myd88^−/− mice (top) with quantification (bottom). WIHN in K14-Myd88^−/− mice was significantly less than WT mice and unresponsive to S .aureus. (n=4–10 independent animals per group). (D) Microbiota α-diversity (Shannon and Observed species) was significantly lower than in K14-Myd88^−/− and Myd88^−/− mice versus WT mice. (n=4 independent animals per group). (E) Percentage of taxonomic classifications at genus level of 16S rDNA sequence from skins of WT, K14-Myd88^−/−, and Myd88^−/− mice demonstrate that Pseudomonas or Streptococcus increased while other bacteria, including Staphylococci (yellow), decreased in Myd88 deficient mice (n=4 independent animals per group). Representative data from 2~3 independent experiments are shown. Scatter plots and histogram graphs indicated means ± SEM; Paired Student t-test was used to compare statistical difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. “NS” indicates no significant difference.

Figure 5.. IL-1β-IL-1R signaling in keratinocytes is the primary MyD88 stimulus to promote WIHN.

(A) WIHN in S. aureus treated or untreated WT, Tlr2^−/−, Il-1r^−/−, and Il-36r^−/− mice (top) with quantification (bottom). WIHN in Il-1r^−/− mice was significantly less than WT mice and unresponsive to S. aureus. (n=3–7 independent animals per group). (B) Among Il-1r^−/− mice, microbiota α-diversity (Shannon and Observed species) was not significantly different from WT mice. (n=4 independent animals per group). Percentage of taxonomic classifications at genus level of 16S rDNA sequence from skins of WT and Il-1r ^−/− mice demonstrate that Pseudomonas and Streptococcus increased while other bacteria including Staphylococcus (yellow) decreased in IL-1 deficient mice (n=4 independent animals per group). (C) Differentially expressed genes presented by volcano plot demonstrate that Il-1β is one of the most significantly up-regulated genes shared by SPF and S. aureus treated mice. (n=3 independent animals per group). (D) Relative microarray mRNA expression of interleukins and chemokines in GF, SPF, PBS treated, and S. aureus treated mice SD0 wound bed tissues demonstrate that Il-1β correlates with WIHN. (n=3 independent animals per group). (E) qRT-PCR mRNA expression of Il-1α and Il-1β of GF, SPF, PBS treated, and S. aureus treated mice at baseline (unwounded), WD3, and WD10 confirm that Il-1β correlates with WIHN. (n=3 independent animals per group). (F) qRT-PCR mRNA expression show increases of hair regeneration markers (Wnt7B, Shh, Lef1) and stem cell marker (Krt7) with decreases in keratinocyte differentiation markers (Flg, Krt1) in rhIL-1β treated human foreskin keratinocytes (HFKC) compared to vehicle. (n=2–4 independent human samples per group). (G) WIHN in rmIL-1β treated mice is significantly more than control, heat killed S. aureus treated, and rmIL-1α treated mice. (n=3–6 independent animals per group). (H) WIHN in S. aureus treated or untreated WT, K14-Il-1r^−/−, Il-1α^−/−, and Il-1β^−/− mice (top) with quantification (bottom). WIHN in K14-Il-1r^−/− mice and Il-1β^−/− mice was significantly less than WT mice and unresponsive to S. aureus. (n=3–7 independent animals per group). (I) The wound closure speed of Il-1β ^−/− mice was significantly slower than the WT mice as per photos (top) and quantification (bottom) (n=4–7 independent mice per group). (J) There is no significant difference in WD28 wound bed tensile strength between WT and Il-1β^−/− mice, both of which are lower than their respective healthy skin (n=6–9 independent mice per group). (K) Relative mRNA expression of hair regeneration markers (Wnt7b, Shh) and keratinocytes differentiation markers (Flg, Krt1) in rmIl-1β treated, untreated control, Il-1r^−/−, and Myd88^−/− mice keratinocytes. (n=3 independent animals per group). (L) WIHN in rmIl-1β treated K14-Myd88^−/− mice was the same as control mice (n=8–9 independent animals per group). Representative data from 2~3 independent experiments are shown. Scatter plots and histogram graphs indicated means ± SEM; Paired Student t-test was used to compare statistical difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. “NS” indicates no significant difference.

Figure 6.. Antibiotics Inhibit Human Wound Healing.

(A) After identical full thickness punch wounds in the same individual, the size of Neosporin treated human wounds were significantly larger than the Vaseline treated control wounds as per photos (top), quantification (left bottom), and intra-individual normalization (right bottom). (n=6 independent human samples per group). (B and C) Absolute value (B) and percentage (C) of taxonomic classifications at genus level of 16S rDNA sequence from skins of Vaseline and Neosporin treated patients demonstrate that Staphylococcus (yellow) is the bacteria with the biggest difference after antibiotic treatment. (n=6 independent human samples per group). (D) Neosporin delayed wound healing as visualized by hematoxylin and eosin (H&E) staining (left). Neosporin also decreased the expression of KRT5 and IL-1β (indicated by arrow) in the wound bed epidermis as detected by immunofluorescence staining (left) and quantification (right). (n=4 independent human samples per group). (E) qRT-PCR mRNA expression analysis of Vaseline and Neosporin treated human wounded skin. (n=3 independent human samples per group). (F and G) Microarray mRNA analysis demonstrates that wound associated keratins and chemokines are more highly expressed in Vaseline compared to Neosporin treated skin. (n=3 independent human samples per group). (H and I) Gene ontology analysis shows S. aureus and Epidermal Development signatures are elevated in Vaseline compared to Collagen categories in Neosporin treated samples. (n=3 independent human samples per group). Representative data from 2 independent experiments are shown. Scatter plots and histogram graphs indicated means ± SEM; Paired Student t-test was used to compare statistical difference, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.