Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: BMP signaling in the hair bulge of wild-type (WT) and TLR2 HFSC-KO mice. ( A ) GEO2R analysis of published RNA data from sorted follicle populations in the second telogen to anagen transition demonstrates the declined expression of Bmp7 mRNA and key transcriptional target of the BMP signaling pathway in the skin, Id1 , Id2 , and Id3 mRNA. ( B ) Western blot analysis of pNFkB, NFkB, pSMAD1/5/9, SMAD1, ikBα protein level in human epidermal keratinocytes treated with/without 10 µg/ml Pam3SCK4 and 10 ng/ml BMP4. ( C ) Bar graphs show the activation effect of BMP4 treatment on the BMP signaling which was abolished in the presence of TLR2 ligand Pam3SCK4. N=3 independent experiments. ( D ) Representative microphotographs of human hair follicle stem cells (HFSCs) pre-treated with 10 µg/ml MAb-mTLR2 or DMSO and co-cultured with/without 5 µg/ml Pam3SCK4. Representative images from at least three independent assays are shown. Scale bar 50 µm. ( E ) Bar graphs show increased proliferation of HFSC in the presence of TLR2 ligand Pam3SCK4 with no effect in MAb-mTLR2 pre-treated cells compared to control. N=6 independent experiments. ( F ) Dysregulated genes were confirmed by RNA sequencing or quantitative polymerase chain reaction (qPCR) of FACS-sorted HFSCs from the first telogen dorsal skin of Tlr2 lox/lox and TLR2 HFSC-KO mice. HFSCs from four mice in each group were sorted and sequenced. Dysregulated genes in RNA sequencing were those with an adjusted p-value <0.05. ( G ) Dysregulated pathways in HFSCs lacking TLR2. ( H ) Representative confocal images of competent telogen follicles from WT or TLR2 KO mice immunostained for CD34 and pSmad1/5/9. Stars label hair shaft. Scale bars are 10 μm. ( I ) Quantification of pSmad1/5/9 + bulge stem cell number in images from H shows significantly more pSmad1/5/9 + cells in WT hair follicle bulge region compared with TLR2 KO hair follicles. N=4 for each group. ( J ) Representative confocal images of Tlr2 lox/lox and TLR2 HFSC-KO telogen hair follicles immunostained for β-catenin showed no difference between the two groups. Stars label the hair shaft. Scale bars are 20 μm. All bar graphs are mean ± s.e.m. Non-parametric Mann-Whitney test ( I ), or Kruskal-Wallis test with Dunn’s multiple comparisons test ( C ), or one-way ANOVA with Tukey’s multiple comparisons test ( E ) was used to determine statistical differences. A p-value ≤ 0.05 was considered to be statistically significant. For the western blot (WB) quantification with experiments with n=3, the minimum achievable p-value for the non-parametric tests is 0.1000. Figure 4—figure supplement 1—source data 1. Uncropped WB gels.

Article Snippet: Cell line (human) , Hair follicle stem cells , Celprogen , Cat.# 36007-08 , Human frontal region scalp extracted from hair follicle bulge.

Techniques:

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: ( A ) Dysregulated pathways in old vs young mouse HFSCs. The top pathways are labeled in bold. ( B ) Representative confocal images of telogen hair follicles from young and old mice immunostained for TLR2 and CD34 demonstrate decreased TLR2 intensity in HFSC (CD34-positive) of old mice. Scale bars are 50 μm. The middle and right panels show a magnified view of the boxed area. Scale bars are 20 μm. ( C ) Quantification of TLR2 fluorescent intensity in images from B showing significantly lower TLR2 expression in HFSCs from the old mice. N=6 for each group. ( D ) GEO2R analysis of published RNA data from sorted follicle populations in the second telogen to anagen transition demonstrates the increased level of Tlr2 mRNA accompanied by the activation of Toll-like receptors (TLRs) signaling downstream. ( E ) Representative confocal images showing TLR2 expression in hair follicles from mice fed with a normal diet (ND) or high-fat diet (HFD). CD34 is an HFSC marker. Scale bars are 50 μm. Magnified images demonstrate decreased TLR2 intensity in HFSC (CD34-positive) of mice after HFD. Scale bars are 20 μm. ( F ) Quantification of TLR2 fluorescent intensity in images from E showing significantly lower TLR2 expression in HFSCs from HFD-fed mice. N=7 and 6 for ND and HFD groups, respectively. AU, arbitrary unit. ( G ) Tlr2 mRNA expression in HFSCs from mice fed with ND or HFD for 4 days or 3 months. Data regenerated from published RNA sequencing dataset GSE131958. N=3 for each group. All bar graphs are mean ± s.e.m. Non-parametric Mann-Whitney test ( C, G ) or unpaired two-tailed t-test ( F ) was used to determine statistical difference. A p-value ≤ 0.05 was considered to be statistically significant.

Article Snippet: Human HFSCs (Celprogen cat.# 36007-08) were cultured in HFSC Un-differentiation Media with Serum (Celprogen cat.# M36007-08US) for 48 hr and then transferred into Undifferentiated ECM 96-Well Plates (Celprogen cat.# UD36007-08-96Well) for 24 hr.

Techniques: Labeling, Expressing, Activation Assay, Marker, RNA Sequencing Assay, MANN-WHITNEY, Two Tailed Test

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: ( A ) Representative confocal images of BMP7 staining in hair follicles of dorsal skin in early (P46) and late (P69) second telogen. Scale bars are 10 μm. ( B ) Quantification of BMP7 fluorescent intensity from A showing diminished BMP7 expression during the second telogen from the early to late phases. N=4 per group. ( C ) Representative confocal images of pSMAD1/5/9 staining in hair follicles of dorsal skin in early (P46) and late (P69) second telogen. Scale bars are 10 μm. ( D ) Quantification of pSMAD1/5/9 + positive cells in CD34 + bulge stem cells demonstrates a decrease of pSmad1/5/9 expression in late telogen. N=4 per group. ( E ) Quantitative polymerase chain reaction (qPCR) analysis reveals dysregulation of BMP singling molecules in hair follicle stem cells (HFSCs) lacking Tlr2 . N=4 mice for control and Bmp2 , N=3 mice for Bmp7 and Bmpr1a . ( F ) Representative confocal images of BMP7 staining in hair follicles from Tlr2 lox/lox or TLR2 HFSC-KO mice. Scale bars are 10 μm. Stars label hair shaft. ( G ) Quantification of BMP7 fluorescent intensity from F showing higher BMP7 expression in TLR2 HFSC-KO mice. N=4 per group. ( H ) P21 and P24 dorsal skin sections from Tlr2 lox/lox and TLR2 HFSC-KO mice immunostained for CD34, pSmad1/5/9, and DAPI. Scale bars are 10 μm. ( I ) Quantification of pSmad1/5/9 + cells in CD34 + bulge stem cells in P24 dorsal skin from H. N=4 and 5 for Tlr2 lox/lox and TLR2 HFSC-KO respectively. ( J ) Representative confocal images of dorsal skin sections from TLR2 HFSC-KO mice treated with BSA or noggin immunostained for CD34, pSmad1/5/9, and DAPI. Star labels the hair shaft. Scale bars are 10 μm. ( K ) Quantification of pSmad1/5/9 + cells in CD34 + bulge stem cells from images in J. N=5 per group. ( L ) Immunostaining for Ki67 and DAPI in dorsal skin sections from TLR2 HFSC-KO mice treated with BSA or noggin. Scale bars are 10 μm. ( M ) Quantification of images in L showing an increase in Ki67 + cells in secondary hair germ (sHG) of noggin-treated compared to BSA-treated TLR2 HFSC-KO dorsal skin. N=5 per group. ( N ) Representative confocal images of Ki67 and DAPI immunostaining of dorsal skin sections from TLR2 HFSC-KO mice treated with BSA or noggin. Arrows point to hair follicles with Ki67 + cells in the sHG. Scale bars are 20 μm. ( O ) Quantification of images in N showing percentages of hair follicles with Ki67 + cells in sHG. N=5 per group. ( P ) BSA- or noggin-treated TLR2 HFSC-KO mouse dorsal skin immunostained for P-cad and DAPI. The dashed line outlines the sHG. Scale bars are 10 μm. ( Q ) Bar graph showing significantly larger sHG in noggin-treated TLR2 HFSC-KO mice. N=5 per group. Mann-Whitney test was used to determine the statistical significance. All data are mean ± s.e.m. A p-value ≤ 0.05 was considered to be statistically significant.

Article Snippet: Human HFSCs (Celprogen cat.# 36007-08) were cultured in HFSC Un-differentiation Media with Serum (Celprogen cat.# M36007-08US) for 48 hr and then transferred into Undifferentiated ECM 96-Well Plates (Celprogen cat.# UD36007-08-96Well) for 24 hr.

Techniques: Staining, Expressing, Real-time Polymerase Chain Reaction, Immunostaining, MANN-WHITNEY

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: ( A ) Representative images of H&E and CEP immunostaining of consecutive skin sections from wild-type (WT) anagen mouse. Scale bars are 1 mm. ( B ) Representative confocal images of P5 WT whole-mount skin immunostained for CEP and Ker17. The merged image shows the co-localization of CEP to anagen hair follicles (Ker17 + ). Scale bar is 200 μm. ( C ) Longitudinal and cross-sections of anagen and telogen hair follicles from WT mice immunostained for CEP and Ker17. The lower left panel shows a magnified view of the boxed area. Scale bars are 100 μm for anagen, 50 μm for telogen. ( D ) Quantification of CEP fluorescent intensity at a different distance from the root of anagen hair follicles in longitudinal and cross-sections immunostaining images in images from C. A gradual decrease in CEP levels is observed from the proximal to the distal part of anagen hair follicles. N=50 follicles from 3 mice per group. ( E ) Line chart showing a sharp decrease of CEP fluorescent intensity with the distance from HF in telogen (from the lower right panel in C). N=10 follicles from 3 mice per group. ( F ) Representative confocal images of telogen hair follicles from young and old mice immunostained for CEP and Ker17. Scale bars are 20 μm. ( G ) Quantification of CEP fluorescent intensity from images in F. N=6 mice per group. ( H ) Representative photographs of dorsal skin (two left panels) and inner skin flaps (two right panels) from WT and TLR2 KO mice after irradiation and bone marrow transplantation of WT bone marrow demonstrate an increased number of pigmented hair bulbs and skin pigmentation around wounds in CEP-treated wounds compared to control in WT mice with no differences in TLR2 KO transplanted with WT bone marrow. Scale bars are 1 mm for the dorsal skin and 500 μm for the inner skin flap. ( I ) Quantitative results from H show an increased density of hair follicles upon CEP application around wounds of WT>WT transplanted mice with no changes in WT>TLR2 KO mice. N=5 for each group. ( J ) Representative photographs of dorsal skin (upper panels) and inner skin flaps (lower panels) from Tlr2 lox/lox and TLR2 HFSC-KO mice treated with CEP show a lack of pigmentation around TLR2 HFSC-KO wounds compared with Tlr2 lox/lox wounds treated with CEP. The inner skin flap of TLR2 HFSC-KO mice demonstrates an absence of pigmented hair bulbs after the CEP treatment. Scale bars are 3 mm. ( K ) Representative confocal images of skin adjacent to wound immunostained for Ker17. Scale bars are 100 μm. ( L ) Quantification of hair follicle numbers in images from K reveals a significant decrease in regenerated hair follicles in TLR2 HFSC-KO skin compared with Tlr2 lox/lox skin. N=7 for Tlr2 lox/lox . N=4 for TLR2 HFSC-KO . ( M ) Representative confocal images of skin adjacent to wound immunostained for Ki67. Scale bars are 50 μm. ( N ) Bar graph showing Ki67 fluorescent intensity in the skin adjacent to wound from images in M. N=7 for Tlr2 lox/lox . N=4 for TLR2 HFSC-KO . ( O ) Representative microphotographs of primary keratinocytes isolated from WT or TLR2 KO mouse skin co-cultured with CEP or control (PBS or BSA). Representative images from at least three independent assays are shown. Scale bar 50 µm. ( P ) Cell proliferation of primary keratinocytes in O indicates increased proliferation by CEP in WT but not in TLR2 KO keratinocytes. N=3 independent experiments. ( Q ) Quantitative polymerase chain reaction (qPCR) analyses of Nfkb2 , Il1b , and Il6 mRNA levels in FACS-purified mouse HFSCs treated with BSA control or CEP. N=3 per group. ( R ) qPCR analyses of Bmp7 mRNA levels in FACS-purified mouse HFSCs treated with BSA control or CEP. N=3 per group. ( S ) Summary of the main findings of this study. Unpaired t-test ( G, P ) or Mann-Whitney test ( I, L, N, Q, R ) was used to determine the statistical significance. All data are mean ± s.e.m. A p-value ≤ 0.05 was considered to be statistically significant.

Article Snippet: Human HFSCs (Celprogen cat.# 36007-08) were cultured in HFSC Un-differentiation Media with Serum (Celprogen cat.# M36007-08US) for 48 hr and then transferred into Undifferentiated ECM 96-Well Plates (Celprogen cat.# UD36007-08-96Well) for 24 hr.

Techniques: Immunostaining, Irradiation, Transplantation Assay, Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Purification, MANN-WHITNEY

Journal: eLife

Article Title: TLR2 regulates hair follicle cycle and regeneration via BMP signaling

doi: 10.7554/eLife.89335

Figure Lengend Snippet: The promotion of hair follicle regeneration after wound healing is dependent on TLR2. ( A ) Representative confocal images of Nile red-labeled (sebaceous gland) wild-type (WT) telogen hair follicles co-immunostained for MPO showing complete co-localization of MPO to the sebaceous gland. The isotype control panel shows the images of hair follicles stained with MPO isotype control antibody. Scale bars are 20 μm. ( B ) Representative confocal images of hair follicles from old vs young mice stained for MPO. Scale bars are 10 μm. ( C ) Quantification of MPO fluorescent intensity in B showing significantly less MPO in hair follicles from older mice. N=6 for each group. ( D ) Representative microphotographs of human hair follicle stem cells (HFSCs) pre-treated with 10 µg/ml MAb-mTLR2 or DMSO and co-cultured with/without 2.5 µM of CEP. Representative images from at least three independent assays are shown. Scale bar 50 µm. ( E ) Bar graphs show increased proliferation of HFSC in the presence of TLR2 endogenous ligand CEP compared to control, which was abolished in the presence of TLR2 blocking antibody. N=6 independent experiments. ( F ) Bar graphs show increased proliferation of human hair follicle dermal papilla cells incubated with 5 µM of CEP compared to the control. N=9 independent experiments. ( G ) Representative confocal images of Ki67 immunostaining of dorsal skin adjacent to wound of CEP-treated WT bone marrow transplanted WT and TLR2 KO mice. Scale bars are 50 μm. ( H ) Quantitative results showed increased Ki67 intensity in hair follicles around wounds of CEP-treated WT bone marrow transplanted WT mice with no differences in TLR2 KO with WT bone marrow. N=4 per group. ( I ) Representative photographs of dorsal skin (upper panels), inner skin flaps (middle panels), and representative confocal images of Ki67 immunostaining (lower panels) of vehicle- or CEP-treated WT or TLR2 KO skin. Scale bars are 1 mm for dorsal skin, 500 μm for skin flaps, and 50 μm for confocal images. ( J ) Bar graph showing quantification of hair follicle numbers of vehicle or CEP-treated skin from I. N=5 per group. ( K ) Bar graph showing quantification of Ki67 fluorescent intensity of Ki67 staining of vehicle or CEP-treated skin from I. N=4 per group. Unpaired two-tailed t-test ( C ), or non-parametric Mann-Whitney test ( H, J, K ), or Kruskal-Wallis test with Dunn’s multiple comparisons test ( F ), or one-way ANOVA with Tukey’s multiple comparisons test ( E ) was used to determine statistical differences. All bar graphs are mean ± s.e.m. A p-value ≤ 0.05 was considered to be statistically significant.

Article Snippet: Human HFSCs (Celprogen cat.# 36007-08) were cultured in HFSC Un-differentiation Media with Serum (Celprogen cat.# M36007-08US) for 48 hr and then transferred into Undifferentiated ECM 96-Well Plates (Celprogen cat.# UD36007-08-96Well) for 24 hr.

Techniques: Labeling, Staining, Cell Culture, Blocking Assay, Incubation, Immunostaining, Two Tailed Test, MANN-WHITNEY