HPV16 E7 expression in skin induces TSLP secretion, type 2 ILC infiltration and atopic dermatitis-like lesions

Abstract

Atopic dermatitis is a common pruritic and inflammatory skin disorder with unknown etiology. Most commonly occurring during early childhood, atopic dermatitis is associated with eczematous lesions and lichenification, in which the epidermis becomes hypertrophied resulting in thickening of the skin. In this study, we report an atopic dermatitis-like pathophysiology results in a murine model following the expression of the high-risk human papillomavirus (HPV) 16 oncoprotein E7 in keratinocytes under the keratin 14 promoter. We show that HPV16 E7 expression in the skin is associated with skin thickening, acanthosis and light spongiosis. Locally, HPV16 E7-expressing skin secreted high levels of thymic stromal lymphopoietin (TSLP) and contained increased numbers of innate lymphoid cells (ILCs). High levels of circulating immunoglobulin E were associated with increased susceptibility to skin allergy in a model of cutaneous challenge, and to airway bronchiolar inflammation, enhanced airway goblet cell metaplasia and mucus production in a model of atopic march. Surprisingly, skin pathology occurred independently of T cells and mast cells. Thus, our findings suggest that the expression of a single HPV oncogene in the skin can drive the onset of atopic dermatitis-like pathology through the induction of TSLP and type 2 ILC infiltration.

Figure 1. K14.E7 transgenic mouse skin demonstrates features of atopic dermatitis

Representative histology of E7 (A) and C57 (B) ear skin stained with H&E (scale bar = 20μm). Hyperkeratosis (κ), acanthosis (α), spongiosis (*) are indicated. C) Ear skin thickness measured in naive age-matched E7 (n=8) and C57 (n=8) mice using a caliper micrometer (*** p=0.0002). Data are pooled from 3 independent experiments and analyzed using a Mann-Whitney t-test. Bars represent median values.

Figure 2. K14.E7-skin expresses and secretes increased levels of TSLP

(A) Positive control for TSLP mRNA expression in LPS-treated (n=5) or untreated (n=5) BMDCs (** p=0.0079). (B-E) Skin samples were isolated and analyzed for TSLP gene expression in (B) full thickness ear skin (E7, n=7 and C57, n=6; ** p<0.005) and (C) epidermal sheets (E7, n=4 and C57, n=4; * p=<0.05). (D) TSLP protein present in ear skin homogenates (E7, n=11 and C57, n=11; **** p<0.0001) or (E) secreted following ear skin explant culture (E7, n=12 and C57, n=12; **** p<0.0001). (F) TSLP mRNA expression in EL4 cells +/− E7 expression (n=5, **p=0.0079). Data are pooled from 2-3 independent experiments and analyzed using a Mann-Whitney t-test. ND = Not Detected (value of 0). Bars represent median values.

Figure 3. K14.E7 ear skin contains increased numbers of type 2 ILCs

Ears were collected from E7 (n=5) and C57 (n=5) mice and analyzed by flow cytometry by gating for type 2 ILCs. (A) Representative gating strategy for live type 2 ILCs; TCRβ⁻ B220⁻ CD2⁻ CD11b⁻ CD90⁺. (B) Data show the percentage (left) and absolute numbers (right, ** p<0.01) of Lin⁻ CD90⁺ type 2 ILCs in ear skin. (C) Plots show the expression of CD25 in Lin⁻ CD90⁺ type 2 ILCs. Data are pooled from 3 independent experiments and analyzed using a Mann-Whitney t-test. Bars represent median values.

Figure 4. Skin lesions occur independently of T cells and Mast cells

(A-C) Lesions are independent of T cells. (A) Representative images E7.Rag ear skin tissue stained with H&E (scale 20 μm). (B) Ear skin thickness for E7 (n=6) and E7.Rag (n=6) mice, ns = not significant. (C) TSLP gene expression in E7 (n=14) and E7.Rag (n=4) total skin, ns. (D-G) Lesions are independent of MCs. (D) TSLP gene expression in E7 (n=3), E7.Kit^W-sh/W-sh (E7.Wsh, n=6), C57 (n=5), and Kit^W-sh/W-sh (Wsh, n=3) skin (** p<0.01 and *** p<0.001). (E) Representative image of MC-deficient E7.Wsh ear skin tissue stained with H&E (scale 20 μm). (F) Ear skin thickness for K14.E7 (n=7), E7.Wsh (n=7), C57 (n=7) and Wsh (n=7) mice (*** p<0.005). Data are pooled from 2-3 independent experiments and analyzed using a Mann-Whitney t-test. Bars represent median values.

Figure 5. K14.E7 mice develop enhanced inflammatory responses

(A) E7 mice (n=11) and non-transgenic littermates (n=8) were bled and sera collected and tested by ELISA. Data show all OD values (left, * p<0.05) and total IgE concentration (right, * p<0.05). (B) Cutaneous hypersensitivity reaction to DNCB. C57 (n=8) and E7 (n=14) mice were sensitized on the shaved abdomen with 5% DNCB at day -5 and challenged at day 0 with 1% DNCB on the dorsal and ventral surface of the ear. The ear thickness was measured over time using a micrometer gauge. ΔEar swelling is the difference in thickness from baseline on day 0 (*** p=0.0001 and **** p<0.0001 between E7 and C57 treated groups).(C-D) E7 or C57 mice were sensitized i.d. at day 0 with endotoxin-free OVA (9 mice E7 and C57) or PBS (5 mice E7 and C57) and all mice were challenged at days 11, 12, 13 and 14 with OVA i.n. Representative lung tissue cross sections stained with H&E (C) to visualize cells infiltrates and (D) periodic acid – Schiff stain to visualize mucus producing goblet cells and airway inflammation; br, bronchioles; v, vessels; Scale bar = 200 μm. Data are expressed as median (A) or mean +/− SEM (B), pooled from 2-3 independent experiments and analyzed using a Mann-Whitney t-test.