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: TLR2-GFP reporter mouse skin sections were immunostained with anti-GFP to assess TLR2 expression in the hair follicles. ( A ) Representative confocal images of P21 first telogen hair follicle immunostained for TLR2-GFP, CD34 (bulge stem cells), P-cad (secondary hair germ [sHG]), and DAPI (nuclei). The green color in the surface rendering panel represents TLR2 expression, and other surfaces show co-localization between TLR2 and specific markers. TLR2 is present in bulge, sHG, and dermal papilla (DP) cells. P represents postnatal days. Scale bar is 10 μm. ( B ) TLR2-GFP in P28 anagen was co-immunostained with CD49f of basement membrane outlining the DP. Scale bar is 10 μm. ( C ) TLR2 is co-localized to the sHG lineage (P-cad + layers), DP, and outer root sheath (ORS) lineage. Scale bar is 20 μm. ( D ) TLR2-GFP in P28 anagen was co-immunostained with CD34 in old bulge ( D ) and Ker5 in ORS ( E ) revealing TLR2 localization to the old bulge, ORS, but not inner root sheath (IRS). Scale bars are 20 μm. ( F ) Co-immunostaining of TLR2-GFP in P38 catagen hair follicle with Ker5 in ORS lineage cells showing co-localization of TLR2 with ORS and bulge. Scale bar is 20 μm. ( G ) P41 late catagen hair follicle immunostained for TLR2 and CD34 showing co-localization of TLR2 to the old bulge, new bulge, sHG, and DP. Scale bar is 20 μm. ( H ) P53 second telogen hair follicle immunostained for TLR2, CD34, and P-cad reveals co-localization of TLR2 to the bulge, sHG, and DP. Scale bar is 20 μm. ( I ) Quantification of TLR2 fluorescent intensity in bulge cells at different phases showing TLR2 upregulation in anagen. N=3 for each group. ( J ) Quantitative polymerase chain reaction (qPCR) analysis of Tlr2 mRNA expression in FACS-purified mouse HFSCs in anagen, telogen, and catagen. N=3 or 4 per group. ( K ) qPCR analysis of Tlr2 mRNA expression in mouse epidermal cells and FACS-purified HFSCs showed significantly higher Tlr2 expression in HFSCs compared with raw epidermal cells. N=6 mice per group. All bar graphs are mean ± s.e.m. Two-tailed unpaired t-test ( K ) or Kruskal-Wallis test with Dunn’s post hoc test ( I, J ) 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: Expressing, Membrane, Immunostaining, Real-time Polymerase Chain Reaction, Purification, 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: H&E staining of dorsal skin of wild-type (WT) mice. TLR2 dynamic during the second telogen. ( A ) Confocal images of dorsal skin hair follicles co-immunostained for TLR2 and GFP. Scale bars are 10 µm. ( B ) A scatter graph shows a high level of correlation between TLR2 and GFP intensity. N=3. ( C ) Representative H&E staining of the dorsal skin of WT mice at indicated time points demonstrates the typical changes in hair follicle morphology. Scale bars are 100 µm. ( D ) Confocal images of dorsal skin hair follicles from p46 and p69 mice immunostained for TLR2. Scale bars are 10 µm. ( E ) Bar graph showing an elevated level of TLR2 in hair follicle stem cell (HFSC) of p69 mice (late second telogen) compared to p46 (early second telogen). N=4. Correlation analysis and the Mann-Whitney test were 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, 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 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

Journal: eLife

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

doi: 10.7554/eLife.89335

Figure Lengend Snippet:

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: Activity Assay, Knock-In, Sequencing, Isolation, Blocking Assay, Concentration Assay, Staining, Membrane, Recombinant, Software