Stress-induced production of chemokines by hair follicles regulates the trafficking of dendritic cells in skin

Abstract

Langerhans cells (LCs) are epidermal dendritic cells with incompletely understood origins that associate with hair follicles for unknown reasons. Here we show that in response to external stress, mouse hair follicles recruited Gr-1(hi) monocyte-derived precursors of LCs whose epidermal entry was dependent on the chemokine receptors CCR2 and CCR6, whereas the chemokine receptor CCR8 inhibited the recruitment of LCs. Distinct hair-follicle regions had differences in their expression of ligands for CCR2 and CCR6. The isthmus expressed the chemokine CCL2; the infundibulum expressed the chemokine CCL20; and keratinocytes in the bulge produced the chemokine CCL8, which is the ligand for CCR8. Thus, distinct hair-follicle keratinocyte subpopulations promoted or inhibited repopulation with LCs via differences in chemokine production, a feature also noted in humans. Pre-LCs failed to enter hairless skin in mice or humans, which establishes hair follicles as portals for LCs.

Figure 1

MHCII⁺Lang⁻ cells of myeloid phenotype infiltrate epidermis depleted of LCs. (a) Immunofluorescence microscopy of epidermal sheets from Langerin-DTR (LDTR) mice 14 d after depletion of LCs via treatment with DT, stained to detect EpCAM and langerin. HF, hair follicle. Scale bar, 20 μm. (b) Microscopy of ear epidermis from Langerin-DTR (CD45.1⁺) mice reconstituted with congenic (CD45.2⁺) wild-type (WT) donor bone marrow (WT→LDTR) 2 weeks after DT-mediated depletion of host-derived LCs, stained to detect MHC class II and langerin. Arrowheads indicate donor-derived MHCII⁺Lang⁻ cells; asterisks indicate conventional LCs. Scale bars, 50 μm (top) or 20 μm (bottom). (c) Flow cytometry of epidermal cells from wild-type (CD45.2⁺) mice (WT) and from Langerin-DTR (CD45.1⁺) mice reconstituted with wild-type bone marrow (WT→LDTR) and treated with PBS or DT 3 weeks later, followed by analysis 2 weeks later. Numbers in quadrants indicate percent cells in each throughout. (d) Flow cytometry of epidermal cells and peripheral blood from Langerin-DTR mice (n = 6) reconstituted with Cx3cr1^gfp/gfp (CD45.2⁺) bone marrow (Cx3cr1^gfp/gfp→LDTR) and treated with DT, followed by analysis 2 weeks later, in parallel with c. Cells were gated on CD45.2⁺MHCII⁺CD11b⁺ cells in c,d. (e) Surface phenotype of LCs and MHCII⁺Lang⁻ cells from the epidermis of Langerin-DTR mice (n = 3) reconstituted with wild-type (CD45.2⁺) bone marrow, assessed by flow cytometry with gating on CD45.2⁺MHCII⁺ cells that were either Lang⁺EpCAM⁺ (LCs) or Lang⁻EpCAM⁻ for analysis of CD11b (top left), or on CD45.2⁺MHCII⁺CD11b⁺ cells (all other plots). Isotype, isotype-matched control antibody. (f) Microscopy of ear epidermal sheets from Langerin-DTR mice (n = 3) treated with DT on day −1 and painted with TNCB on day 0, collected on days 7 and 14 and stained for EpCAM, MHC class II and langerin. Scale bar (f), 20 μm. Data are from one experiment representative of five independent experiments with three to five mice per group in each (a–c) or are representative of two (d,e) or three (f) independent experiments.

Figure 2

LysM-expressing precursors give rise to a subset of LCs. (a,b) Immunofluorescence microscopy (left) of epidermal sheets from LysM-eGFP mice (a) or from Langerin-DTR (CD45.1⁺) mice reconstituted with LysM-eGFP (CD45.2⁺) bone marrow (LysM-eGFP→LDTR), treated with DT 3 weeks later, then treated with TNCB and analyzed 2 weeks later (b). Scale bars, 50 μm. Right, flow cytometry of epidermal cell suspensions assessing the expression of eGFP and langerin on cells gated for CD45.2, MHCII and CD11b. (c) Langerin expression by LysM^fm+ cells in the epidermis of Langerin-DTR mice reconstituted with LysM-eGFP bone marrow and treated with TNCB, assessed 7, 14, 28 and 56 d later. (d) Microscopy of epidermal sheets from Langerin-DTR mice reconstituted with CD115⁺Gr-1^hi LysM-eGFP bone marrow cells, then treated with DT 3 weeks later and TNCB on the next day, followed by analysis 14 d later by staining for eGFP, MHC class II and langerin. Scale bar, 25 μm. (e) Quantification of clusters of LCs (MHCII⁺Lang⁺) or pre-LCs (MHCII⁺Lang⁻) of each bone marrow origin in Langerin-DTR mice reconstituted with a mixture of equal numbers of wild-type (CD45.1⁺) and LysM-DTA (CD45.2⁺) bone marrow cells (WT + LysM-DTA→LDTR), then treated with DT and analyzed 2 weeks later. NS, not significant; *P = 0.0021 (unpaired two-tailed Student's t-test). Data are from one experiment representative of three (a,b) or two (c–e) independent experiments with three (a–c,e) or two (d) mice per group in each (mean and s.e.m. in e).

Figure 3

Dependence of LCs and pre-LCs on chemokine receptors. Flow cytometry of peripheral blood cells (PBC) and epidermal cells (LC and pre-LC) from Langerin-DTR mice reconstituted with a mixture of equal numbers of wild-type (CD45.1⁺) bone marrow cells and CD45.2⁺ bone marrow cells deficient in CCR1, CCR2 or CCR5 (a) or CCR6 or CCR8 (b), followed 3 weeks later by DT-mediated depletion of recipient mice of host LCs and analysis of the ratio of CD45.2⁺ cells to CD45.1⁺ cells among MHCII^hiCD11b^hi cells (CD45.2⁺/CD45.1⁺) 2 weeks later. *P = 0.0172 and **P < 0.006 (unpaired two-tailed Student's t-test). Data are from one experiment representative of two independent experiments with three to five mice per group in each (mean and s.e.m.).

Figure 4

Pre-LCs repopulate the epidermis via hair follicles. (a) Immunofluorescence microscopy of epidermal sheets from Langerin-DTR mice depleted of LCs and then treated with TNCB, stained 1 d later for langerin and MHC class II. (b) Immunofluorescence microscopy of lip sections from Langerin-DTR mice treated with DT, stained 2 weeks later for MHC class II. Dashed lines delineate hair follicles. IF, infundibulum; IM, isthmus. (c) Multiphoton microscopy in vivo of the infiltration of leukocytes into the ears of Langerin-DTR mice reconstituted with CAG-eGFP bone marrow (CAG-eGFP→LDTR) and treated with DT 8 weeks later, followed by stripping of the ears with adhesive tape the next day; green cells represent donor-derived leukocytes (images from Supplementary Video 1). (d) Immunofluorescence microscopy of dermal sheets from wild-type mice treated by stripping of the ears with adhesive tape, followed by staining for CD45, MHC class II and CD11b (below images) 4.5 h later. (e,f) Multiphoton microscopy of an ear from a different mouse treated as in c, assessed 18–20 h after such tape stripping (images from Supplementary Video 2); eGFP⁺ cells in hair follicles have been pseudocolored orange to facilitate visualization. Dashed lines delineate hair follicles; arrows indicate direction of pre-LC movement. Scale bars, 20 μm (a,b), 50 μm (c,d) or 10 μm (e,f). Data are representative of five (a,b) or three (d) independent experiments with three mice per group in each, or five (c) or six (e,f) experiments with one mouse per experiment.

Figure 5

Hair-follicle keratinocyte subsets are the main source of chemokines that regulate the entry of LCs. (a) Immunofluorescence microscopy of frozen sections of mouse skin stained for various molecules (above images) to delineate the interfollicular epidermis (IE), infundibulum (IF), isthmus (IM), basal bulge (BB) and suprabasal bulge (SB) in hair follicles in telogen. Arrowheads (white) and arrows (red) indicate basal bulge and suprabasal bulge, respectively. Scale bar, 20 μm. (b) Flow cytometry of epidermal keratinocytes from interfollicular epidermis and hair follicles to sort into five subsets (as in a) by their expression of surface markers. Numbers adjacent to or in outlined areas indicate percent cells in each. (c) Real-time PCR analysis of the expression of CD34, EpCAM and Sca-1 mRNA in sorted hair-follicle keratinocyte subsets (below horizontal axis), presented (in arbitrary units (AU)) relative to the expression of GAPDH mRNA (encoding glyceraldehyde phosphate dehydrogenase). (d) Real-time PCR analysis of the expression of CCL1, CCL2, CCL8 and CCL20 mRNA in sorted hair-follicle keratinocyte subsets (presented as in c). Data are from one experiment representative of three (a,b) or two (c,d) independent experiments (pooled from three mice per experiment in b; mean and s.d. in c,d).

Figure 6

S1P₁ expression identifies CCL8-producing suprabasal hair-follicle bulge cells. (a) Confocal microscopy of hair follicles in anagen and telogen in frozen sections of mouse skin stained for S1P₁. (b) Microscopy of vertical frozen skin sections of a hair follicle in anagen (top), a cross-section of a hair follicle in early anagen (middle) and a vertical section of a hair follicle in telogen (bottom), stained for S1P₁, integrin α₆ or keratin 15 (K15). (c) Microscopy of a cross-section of a hair follicle, stained for CCL8 and S1P₁. (d) Real-time PCR analysis of the expression of S1P₁ mRNA in sorted hair-follicle keratinocyte subsets (from Fig. 5c,d; presented as in Fig. 5c). (e) Immunofluorescence microscopy of epidermal ear sheets from mice given no tape stripping (left) or treated to tape stripping 18 h before collection (right), stained for CCL8. (f) Confocal microscopy of epidermal sheets from mouse ears treated to tape stripping 18 h before collection, stained for CCL8 and S1P₁. Scale bars, 100 μm (a, left), 50 μm (a, right), 20 μm (b,c,f) or 100 μm (e). Data are representative of three to five independent experiments with three mice per group in each (mean and s.d. in d).

Figure 7

Pre-LCs fail to populate hairless epidermis. (a) Immunofluorescence microscopy of epidermal sheets from nude mice given simultaneous grafting of wild-type skin (WT) and Adam17^fl/flSox9-Cre skin (ADAM17-KO) onto opposite flanks, followed 2 months later by topical application of mometasone for 2 d (for depletion of LCs from skin grafts) and then treatment with TNCB (for induction of repopulation with pre-LCs) and staining for host-derived pre-LCs (I-A-I-E positive, I-A^d positive, langerin negative) 2 weeks later. Scale bar, 100 μm. (b) Quantification of pre-LCs in epidermal sheets in a at a magnification of ×200. FOV, field of view. Each symbol represents an individual mouse; small horizontal lines indicate the mean (and s.e.m.). *P < 0.0001 (unpaired two-tailed Student's t-test). (c) Immunofluorescence microscopy of footpad epidermal sheets from Langerin-DTR mice left untreated (No DT), or treated with DT on day –1, followed by painting of PBS (DT + PBS) or TNCB (DT + TNCB) onto the footpad (FP) and the surrounding hair-bearing skin on day 0 and staining for EpCAM and MHC class II on day +1 or +3. Dashed lines delineate the border between the footpad and hair-bearing epidermis; asterisks (far right) indicate hair follicles with infiltration by pre-LCs. Scale bar, 200 μm. Data are from one experiment representative of two independent experiments with two mice per group in each.

Figure 8

Chemokine expression in human hair follicles and LCs in hairless skin. (a) Real-time PCR analysis of the expression of mRNA for various chemokines (vertical axes; presented as in Fig. 5c) in human scalp tissues (n = 2 samples from one subject) dissected into the interfollicular epidermis (IE), infundibulum (IF), bulge (Bg), bulb (Bb) and suprabulb (SBb). (b) Immunohistochemistry of scalp sections from normal humans (NHS) and from patients with alopecia areata (AA) or lichen pilanopilaris (LPP), stained for CD1a (n = 2 samples per group). Scale bar, 20 μm. (c) Quantification of LCs in sections from b (as in Fig. 7b; pool of three fields of view per section and two sections per sample). Data are from one experiment representative of two independent experiments.