Stimulation of hair follicle stem cell proliferation through an IL-1 dependent activation of γδT-cells
- University of California, San Diego, United States
- Institute for Stem Cell Biology and Regenerative Medicine, India
- Manipal University, India
- Shanmugha Arts, Science, Technology and Research Academy (SASTRA) University, India
- The Scripps Research Institute, United States
- University of Southern California, United States
Figures
Figure 1 with 7 supplements
Impact of IL-1 signaling on epidermal stem cell proliferation.
(A) Quantification of proliferating cells in the interfollicular epidermis (IFE) of the skin from WT or IL1R-KO 8-week-old mice 3 days post-wound. The proximal area of measurement was restricted to …
Figure 1—figure supplement 1
Wound closure rate in the skin of WT and IL1R KO mice.
5 mm excisional wounds were made on 8-week-old IL1R KO and WT control males. Wound closure was determined as the percentage size reduction compared to day 0.
Figure 1—figure supplement 2
Representative image for Figure 1A.
Immunostaining of day 3 wound distal skin sections from 8-week-old WT or IL-1R KO mice for CD34 (green) and Ki67 (red) to mark proliferating HFSCs. Nuclei were stained with DAPI (blue). Scale bar = 5…
Figure 1—figure supplement 3
Proliferation of HFSCs in a distal wound region.
Immunostaining of day 3 wound distal skin sections from 8-week-old WT or IL-1R KO mice for CD34 (green) and Ki67 (red) to mark proliferating HFSCs. Nuclei were stained with DAPI (blue). Scale bar = 5…
Figure 1—figure supplement 4
Representative image of Figure 1C.
Skin sections from 8-week-old WT and IL1R KO mice were stained for keratin 5 (K5) (green), Ki67 (red) and DAPI (blue), for 1, 3, and 5 days postwounding. White dotted lines represent the basement …
Figure 1—figure supplement 5
Representative image of Figure 1D.
Skin sections from 8-week-old WT and IL1R KO mice were stained for keratin 5 (K5) (red), loricrin (Lor) (green) and DAPI (blue) 3 days postwounding. Epidermal thickness was calculated by taking …
Figure 1—figure supplement 6
Sox9 expression in wounded skin.
Skin sections from 8-week-old WT, IL1R KO, and γδT-cell KO mice were stained for Sox9 (red) to mark HFSCs and DAPI (blue) 3 days postwounding. White dotted lines denote the basement membrane …
Figure 1—figure supplement 7
In vitro migration of HFSCs.
WT or IL-1RKO HFSCs were plated on the top of a transwell chamber with conditioned media from WT, caspase-8 conditional KO (Casp8 cKO), caspase-8 conditional KO/gdTCRKO (Casp8 cKO/Tcrd-/-) or with …
Figure 2 with 7 supplements
Loss of γδT-cells phenocopies the deficiency of IL-1 signaling in C8- KO epidermis.
(A) Immunofluorescence of mouse skin sections from different genotypes. Staining for keratin 5 (K5, red) and macrophages (Mac1, green) or for granulocytes (Gr-1, green) reveals a reduction in innate …
Figure 2—figure supplement 1
Loss of γδT-cells phenocopies the proliferation deficiency of IL-1 signaling in excisional cutaneous wounds.
Immunofluorescence images of day 3 wound proximal and distal skin sections from 8-week-old WT or γδT-deficient mice. Sections were labelled with anti-SMA antibody to mark the hair-bulge region (red) …
Figure 2—figure supplement 2
Effect of γδT-cells on IFE proliferation.
The cell counts of Edu+ proliferating interfollicular epidermal (IFE) cells in skin wounds of WT and γδT-cell-deficient mice. The proximal area of measurement was restricted to the first three hair …
Figure 2—figure supplement 3
Effect of γδT-cells on HFSC proliferation.
Quantification of EdU+ hair follicle stem cell (HFSC) proliferation in the first three hair follicles adjacent to wounds in mice from the labeled genotypes.
Figure 2—figure supplement 4
Effect of γδT-cells on epidermal thickness.
The epidermal thickness was quantified by measuring the distance between the Keratin-5+ basal layer and the loricrin+ granular layer, starting from the region with keratin-5 expression in the wound …
Figure 2—figure supplement 5
Extracellular IL-1α in the genetic wound-healing model.
Unpermeabilized skin sections from wild type, caspase 8 conditional knockout, and caspase-8 conditional knockout/γδT-cell KO mice were stained for extracellular IL-1α. Scale bar = 50 μm.
Figure 2—figure supplement 6
Quantification of secreted IL-1α from excisional wounds.
Wounded 5-mm punch biopsies from WT and γδT-cell KO mice were used to condition media. ELISA for IL1α was performed on the conditioned media. Analysis was performed on 6 hr and 24 hr post-wounding sa…
Figure 2—figure supplement 7
Representative image of day one post-wound in Figure 2—figure supplement 6.
Unpermeabilized skin sections from WT or γδT-cell KO mice were stained for extracellular IL-1α. Regions shown are proximal to the wound and 2 cm distal to the wound site. The secondary control compri…
Figure 3 with 5 supplements
IL-1 signaling contributes to γδT-cell proliferation in the caspase-8 cKO skin.
(A) Quantification of γδT-cell proliferation. The numbers of γδT-cells are reported as the fold difference from WT, which was normalized to 1. **p<0.001. (B) Detection of activated γδT-cells. …
Figure 3—figure supplement 1
Proliferation of γδT-cells post wounding.
Skin sections from 8-week-old wild-type (WT) or IL1R KO mice were stained for γδT-cells (green) and Ki67 (red) 3 days post wounding. Boxed regions are shown as magnified images in the insets. White …
Figure 3—figure supplement 2
Quantification of proliferating γδT-cells.
The number of total γδT-cells and proliferating γδT-cells based on the images in Figure 3—figure supplement 1. Panel I shows quantitation of a region proximal to the wound bed. Panel II shows …
Figure 3—figure supplement 3
IL7 expression from caspase 8-deficient epidermis is not affected by IL-1α qPCR measurements of levels of IL-7 expression by keratinocytes.
Data are the fold difference ± SEM of three samples per genotype analyzed in triplicate. The p-values comparing C8CKO and C8/IL1R dKO are not statistically significant (N.S.).
Figure 3—figure supplement 4
IL-7 expression in excisional wounds.
qPCR measurements of levels of IL-7 expression by keratinocytes. Data are the fold difference ± SEM of three samples per genotype analyzed in triplicate. The levels of IL-7 in the γδT-cell KO …
Figure 3—figure supplement 5
IL-7 expression in a mouse model of wound healing as revealed by qPCR measurements of levels of IL-7 expression by keratinocytes.
Data are the fold difference ± SEM of three samples per genotype analyzed in triplicate. The levels of IL-7 in the caspase-8 cKO and the caspase-8 cKO/γδT-cell KO at postnatal day six skin are not …
Figure 4 with 3 supplements
Conditioned media from dermal fibroblasts and activated γδT-cells differentially enhance epithelial stem cell proliferation in distinct niches within the skin.
(A) Activation of dermal fibroblasts (df). df were treated with conditioned media (CM) as noted in the figure and the expression of the growth factors FGF7 and GM-CSF were assessed by qPCR. (B) …
Figure 4—figure supplement 1
IFE and HFSC proliferation invoked by activated fibroblasts and γδT-cell-conditioned media.
Proliferation rates of IFE (panel I) and HFSC cells (panel II) determined by trypan blue exclusion cell counting after treatment with CM from γδT-cells treated as indicated in the key.
Figure 4—figure supplement 2
Expression of other IL-1 family members in the dermis.
qPCR of P4 dermis from the different genotypes represented as the fold difference ± SEM. Data were collected from five mice per genotype.
Figure 4—figure supplement 3
Expression of IL-1 family members by activated γδT-cells.
qPCR of short-term γδT-cell cultures. The data shown are averagesfrom three independent experiments.
Figure 5
Proposed model of epidermal stem cell proliferation in the caspase-8 conditional knockout model of wound healing.
Upon wounding, caspase 8 levels are downregulated causing the release of IL-1α, which can stimulate the proliferation of interfollicular epidermal stem cells (IFE) through its interactions with …
Tables
Key resources table
| Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|
| Casp8 fl/fl mice (Casp8 cKO) | Jackson Laboratory | Stock No. 027002 | Animals were originally purchased from Jackson Laboratory but were bred for >5 generations at UCSD and later bred in the NCBS animal facility for >5 generations |
| IL1R KO mice | Jackson Laboratory | Stock No. 028398 | Animals were originally purchased from Jackson Laboratory (Stock No. 000664) but were bred for > 5 generations in the NCBS animal facility |
| Tcrd–/– mice (γδTCR KO) | Jackson Laboratory | Stock No. 003448 | Animals were originally purchased from Jackson Laboratory (Stock No. 000664) but were bred for > 5 generations in the NCBS animal facility |
| Krt14-Cre | Jackson Laboratory | Stock No. 018964 | Animals were originally purchased from Jackson Laboratory (Stock No. 000664) but were bred for > 5 generations in the NCBS animal facility |
| C57Bl6/JNcbs | Jackson Laboratory | Stock No. 000664 | Animals were originally purchased from Jackson Laboratory (Stock No. 000664) but were bred for > 10 generations in the NCBS animal facility |
| anti-CD34 | Ebioscience | 11-0341-82 | (1:100) |
| anti-Ki67 | abcam | ab16667 | (1:100) |
| anti-alpha-smooth muscle actin (a-SMA) | Abcam | ab5694 | (1:100) |
| anti-gamma delta TCR | BD Bioscience | GL3 | (1:100) |
| anti-CD3 | e-biosciences | 14-0032-85 | (1:100) |
| anti-JAML | eBioscience | clone eBio4E10 | (1:100) |
| anti-IL-7 inhibitory antibody (Clone M25) | BioXCell | BE0048 | (1:100) |
| anti-IL-1 alpha | Invitrogen | 14-7011-81 | (1:100) |
| anti-Keratin 5 | Jamora Lab generated | (1:500) | |
| anti-Loricrin | Jamora Lab generated | (1:500) | |
| anti-Sox9 | Abcam | ab185230 | (1:100) |
| anti-Mac1 | Thermo-fisher scientific | MA1-10080 | (1:100) |
| anti-Gr1 | R&D Systems | MAB1037-500 | (1:100) |
| anti-TNFalpha | eBiosciences | 14–7321 | (1:100) |
| Alexa 488- or 555- secondaries | Molecular Probes | (1:400) | |
| DAPI | Molecular Probes | (1:1000) | |
| rhIL-1 alpha | R&D Systems | 200-LA-010 | |
| ELISA kit for IL-1 alpha | eBiosciences | 88501986 | |
| Click-iT EdU Imaging Kit | Thermo-fischer scientific | C10340 | |
| GraphPad | Prism | For statistical analysis |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.28875.030
