TLR7-expressing cells comprise an interfollicular epidermal stem cell population in murine epidermis

Abstract

Normal interfollicular epidermis (IFE) homeostasis is maintained throughout the entire life by its own stem cells that self-renew and generate progeny that undergo terminal differentiation. However, the fine markers of the stem cells in interfollicular epidermis are not well defined yet. Here we found that TLR7 identified the existence of progenitors and interfollicular epidermal stem cells in murine skin. In vitro, TLR7-expressing cells comprised of two subpopulations that were competent to proliferate and exhibited distinct differentiation potentials. Three-dimensional (3D) organotypic culture and skin reconstitution assays showed that TLR7-expressing cells were able to reconstruct the interfollicular epidermis. Finally, TLR7-expressing cells maintained the intact interfollicular epidermal structures revealed in serial transplantation assays in vivo in mice. Taken together, our results suggest that TLR7-expressing cells comprise an interfollicular epidermal stem cell population.

Figure 1. Activation of TLR7 by imiquimod promoted epidermis thickening.

(A–G) Histological skin sections of the mice were used in hematoxylin-eosinstaining (HE). Imiquimod treated skin(A); Imiquimod and IRS661 treated skin (B); Imiquimod and control ODN treated skin (C); DMSO treated skin (D); DMSO and IRS661 treated skin (E); DMSO and control ODN treated skin (F); Untreated skin (G). (H) Quantification of the average nucleated cell number per 10 μm thickness of the IFE. ns: no significance.

Figure 2. TLR7 activated by imiquimod induced ectopic expression of K17 and stimulated interfollicular cell proliferation.

(A–F) Immunohistochemistry staining for K17 highlighted its expression pattern in epidermis. Arrowheads indicated the ectopic expression of K17 in A and C. (G–L) Immunohistochemistry with the proliferation associated marker PCNA showed the proliferative epidermal cells. Black dashed lines outlined the epidermal region. Imiquimod treated skin (G); Imiquimod and IRS661 treated skin (H); Imiquimod and control ODN treated skin (I); DMSO treated skin (J); DMSO and IRS661 treated skin (K); DMSO and control ODN treated skin (L). IFE, interfollicular epidermis. (M) Quantification of the proliferation of interfollicular cells in G–L. **: P < 0.01, t-test.

Figure 3. Imiquimod induced the proliferation of TLR7 positive keratinocytes in vitro.

(A) Cells were treated with imiquimod at the indicated concentrations. EdU highlighted the proliferation of TLR7 positive cells. 2.4% DMSO in a, 10 ug/ml imiquimod (dissolved in 100% DMSO) in b, 10 ug/ml imiquimod (dissolved in 100% DMSO) and 0.03 mg/ml IRS661 in c, 0.24% DMSO in d, 1 ug/ml imiquimod in e, 1 ug/ml imiquimod and 0.03 mg/ml IRS661 in f, 0.03 mg/ml IRS661 in g, untreated group as blank group in h. (B) Quantification of the proliferation of interfollicular cells in A. These data are representative of at least three independent experiments with similar findings. **: P < 0.01, t-test. ns: no significance.

Figure 4. TLR7 expression in mouse epidermis.

(A–F) Immunofluorescence staining of TLR7 (red) in skin obtained from wild-type mice at P1 (A and B) and in telogen (C and D), catagen (E) and anagen (F). Isotype control at P1 (B) and in telogen (D). Arrowheads indicate positively stained calls in infundibulum (D). The asterisksin (C–E) mark the red staining of the hair shaft caused by non-specific stainingwhich was verified by isotype control (D). White dashed lines represent the boundary between epidermis and dermis or edge of the hair follicles. IFE, interfollicular epidermis; HP, hair peg; IFD, infundibulum; SG, sebaceous gland; Bu, bulge.

Figure 5. Characterization of TLR7-positive population.

(A, B) Immunofluorescence was performed on dorsal skin sections of wild-type mice at P1. Co-staining of TLR7 and CD34 (A), TLR7 and ITGA6 (B). (C) FACS analysis of P1 epidermis from wild-type micelabelled with antibodies to ITGA6 and TLR7. (D) FACS analysis of P1 epidermis from wild-type micelabelled with antibodies to CD34 and TLR7. (E–H) Analysis of the differentiation potential of different populations from P1 skin. Four distinct populations: TLR7⁺/CD34⁺, TLR7⁺/CD34⁻, TLR7⁻/CD34⁺ and All (the unfractionated population) in E and F; TLR7⁺/ITGA6⁺, TLR7⁺/ITGA6⁻, TLR7⁻/ITGA6⁺ and All (the unfractionated population) in G and H. AE13: follicular marker; PPARγ: sebaceous gland marker; K1: epidermal marker. **: P < 0.01, t-test.

Figure 6. Three-dimensional organotypic cultures in vitro.

(A) Various developmental stages of organotypic cultures viewed with an inverted fluorescent microscope (D3, D12, D21, D30 and D35). (a1–a5) The unfractionated population of epidermis without dermal cells (All), (b1–b5) the unfractionated population of epidermis mixed with dermal cells (All + dermal), (c1–c5) TLR7-positive cells in the absence of dermal cells (TLR7⁺), (d1–d5) TLR7-positive cells with dermal cells (TLR7⁺ + dermal), (e1–e2) TLR7-negative cells without dermal cells (TLR7⁻), (f1–f2) TLR7-negative cells with dermal cells (TLR7⁻ + dermal). The white arrowhead indicates two bright elevated spheroids in epidermal equivalent. (B–E′) Paraffin section of epidermal equivalent on PCF membranes labeled with hematoxylin stain. Structures like cutaneous appendages are indicated by the black arrowhead.Data are means of triplicate determinations from a single representative experiment.

Figure 7. The reconstituted skin inepidermal reconstitution assay.

(A–E) Sections of the fist grafts were processed for immunofluorescence staining. Epifluorescent EGFP was employed in conjunction with antibodies against the epidermal marker K1 (A, A′) or the hair follicular marker K17 (B, B′) to examine the contribution to skin lineages.Isotype control (C), section of graft from mice transplanted with TLR7-negative cells (D), section from un-transplantation mice (E). (F) Section of graft stained with GFP antibody (brown) and hematoxylin counterstain (blue) showing the contribution of TLR7-positive cells to the graft. IFE, interfollicular epidermis; SG, sebaceous gland; HF, hair follicle; DE, dermis. (G–J′) Sections of the second graft were stained with antibodies against the epidermal marker K1 (G, G′) or the hair follicular marker K17 (I, I′, J, J′) to show the contribution of EGFP-positive cells. EGFP-positive cells contributed to the sebaceous gland (H). The region of reconstituted IFE is demarcated by white dashed lines in G. The asterisk marks thehair follicles that did notreconstitute by EGFP-positive cells (J). White dashed lines represent the boundary between epidermis and dermis or edge of the hair follicles in (H–J). IFE, interfollicular epidermis; SG, sebaceous gland; Bu, bulge.