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. 2018 Jun 19;115(25):6434-6439.
doi: 10.1073/pnas.1721805115. Epub 2018 Jun 4.

BP180 Dysfunction Triggers Spontaneous Skin Inflammation in Mice

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Free PMC article

BP180 Dysfunction Triggers Spontaneous Skin Inflammation in Mice

Yang Zhang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

BP180, also known as collagen XVII, is a hemidesmosomal component and plays a key role in maintaining skin dermal/epidermal adhesion. Dysfunction of BP180, either through genetic mutations in junctional epidermolysis bullosa (JEB) or autoantibody insult in bullous pemphigoid (BP), leads to subepidermal blistering accompanied by skin inflammation. However, whether BP180 is involved in skin inflammation remains unknown. To address this question, we generated a BP180-dysfunctional mouse strain and found that mice lacking functional BP180 (termed ΔNC16A) developed spontaneous skin inflammatory disease, characterized by severe itch, defective skin barrier, infiltrating immune cells, elevated serum IgE levels, and increased expression of thymic stromal lymphopoietin (TSLP). Severe itch is independent of adaptive immunity and histamine, but dependent on increased expression of TSLP by keratinocytes. In addition, a high TSLP expression is detected in BP patients. Our data provide direct evidence showing that BP180 regulates skin inflammation independently of adaptive immunity, and BP180 dysfunction leads to a TSLP-mediated itch. The newly developed mouse strain could be a model for elucidation of disease mechanisms and development of novel therapeutic strategies for skin inflammation and BP180-related skin conditions.

Keywords: TSLP; atopic dermatitis; collagen XVII; hemidesmosome; keratinocyte.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
ΔNC16A mice exhibit skin inflammation. (A) NC16A mice (WT) with NC16A-encoding exons 18 and 19 flanked by loxP sites were crossed with germ-line Cre mice, resulting in mice expressing NC16A-truncated BP180 (ΔNC16A). (B) Immunofluorescence (IF) Anti-NC16A antibody stained the BMZ of only WT skin, while anti-NC1-3 antibody stained the BMZ of both WT and ΔNC16A skin. D, dermis; E, epidermis; arrows, BMZ; n = 5 per group. (C) Immunoblotting. Anti-NC16A antibody recognized full-length BP180 in WT skin, while anti-NC1-3 antibody recognized both full-length and NC16A-truncated BP180 in both WT and ΔNC16A skin (n = 5 per group). (D) ΔNC16A showed clinical skin lesion starting around 8 wk old and became more severe at 12 wk old. Ear skin revealed minor skin inflammation with increased infiltrating immune cells at 8 wk old and minor epidermal/dermal separation at 12 wk old in ΔNC16A mice. (Original magnifications: 200×.) (Scale bars: 50 μm.)
Fig. 2.
Fig. 2.
ΔNC16A mice exhibit aberrant itch, increased TSLP level, and defective skin barrier. Compared with WT mice, ΔNC16A mice starting 8 wk old exhibited a significantly increased epidermis thickness (A), itch (scratch motion) (B), serum IgE levels (C), and TSLP level in ear skin (D) (n = 8 per group). (E) Toluidine blue penetration assay showed no barrier defect in E18.5 embryos of ΔNC16A and WT mice (n = 8 per group). Adult ΔNC16A mice started to show a significant reduction in barrier function at 8∼12 wk after birth, by Even Blue (F) (n = 6 per group) and FITC-conjugated BSA permeability assays (G). (Scale bars: 50 μm.) *P < 0.05, Student’s t test, graphs A, B, and F show mean ± SE.
Fig. 3.
Fig. 3.
SkinΔNC16A mice develop skin inflammation with increased immune cell infiltration, epidermal thickness, itch, serum IgE, TSLP, and defective barrier. (A) The ears of skinΔNC16A mice started showing skin lesions clinically and histologically at 4 wk after tamoxifen treatment compared with control mice (Cre). Immune staining of ear skin also showed that skinΔNC16A mice (Cre+) have significantly increased infiltrating immune cells at 4 wk after tamoxifen treatment. Toluidine blue (TB) staining indicated that there was a significant increase in mast cells in skinΔNC16A mice starting at 1 wk after tamoxifen treatment (A and E). (Original magnifications: 200×.) Compared with Cre mice, Cre+ mice show increased epidermis thickness (B), spontaneous itch (C), serum IgE level (D), and TSLP (F) in skin starting at 2 wk after tamoxifen treatment (n = 8 per group). (G) Evans Blue dye penetration assay showed barrier defect in skinΔNC16A but not control mice starting 1 wk after tamoxifen treatment. (H) FITC-conjugated BSA permeability assays also revealed barrier defect in skinΔNC16A but not control mice starting 2 wk after tamoxifen treatment (n = 6 per group). (Scale bars: 50 μm.) *P < 0.05, Student’s t test, graphs B, C, E, and G show mean ± SE.
Fig. 4.
Fig. 4.
Itch in ΔNC16A mice is independent on IgE and histamine but dependent on TSLP. (A) Similar to ΔNC16A mice, Rag1−/−ΔNC16A mice exhibited similar degree of itch (A), increased TSLP in skin (B), and barrier defect determined by Evans Blue penetration assay (C) and FITC-conjugated BSA permeability assays (D) (n = 6 per group). (Scale bars: 50 μm.) (E and F) B cell-deficient ΔNC16A mice exhibited a similar degree of itch and TSLP level as ΔNC16A mice. (G) Serum histamine levels in ΔNC16A, K14Cre/ΔNC16A, and WT mice were similar. (H) ΔNC16A mice at 8 wk old were administrated orally with H1R and H4R antagonist, respectively. The blockade of H1R and/or H4R had no effect on itch. (I) The ear of skinΔNC16A mice were injected with TSLP neutralizing antibody or IgG2a control antibody (20 μg per ear). Meanwhile, 8-wk-old WT mice were injected with TSLP after injection with the same amount of TSLP-neutralizing antibody or IgG2a control antibody. The mice were videotaped in the absence of antibody and 1 h after antibody injection for 15 min. TSLP blockade significantly reduced itch in skinΔNC16A mice compared with the control antibody-treated skinΔNC16A mice. *P < 0.05, Student’s t test, n = 6 per group. Graphs A, C, E, and GI show mean ± SE.
Fig. 5.
Fig. 5.
Increased TSLP is produced by keratinocytes in ΔNC16A mice and BP patients. (A) Compared with WT, ear skin from skinΔNC16A mice showed a higher expression of TSLP mainly in epidermis. (B) WT and ΔNC16A keratinocytes were stimulated with mouse TNFα for 24 h. ΔNC16A keratinocytes released significantly higher amount of TSLP in culture medium than WT keratinocytes (n = 6 per group). (CE) The serum, blister fluids, and lesional skin from BP patients exhibited an increased level of TSLP compared with normal control, blister fluids from herpes zoster, and normal control, respectively (n = 12 per group). (F) The increased expression of TSLP in BP patients is mainly seen in epidermis (n = 9 per group). (Original magnifications: A, 200×; B, 400×.) (Scale bars: A, 100 μm; F, 50 μm.) *P < 0.05. Student’s t test, graph B shows mean ± SE.

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