An adult passive transfer mouse model to study desmoglein 3 signaling in pemphigus vulgaris

Abstract

Evidence has accumulated that changes in intracellular signaling downstream of desmoglein 3 (Dsg3) may have a significant role in epithelial blistering in the autoimmune disease pemphigus vulgaris (PV). Currently, most studies on PV involve passive transfer of pathogenic antibodies into neonatal mice that have not finalized epidermal morphogenesis, and do not permit analysis of mature hair follicles (HFs) and stem cell niches. To investigate Dsg3 antibody-induced signaling in the adult epidermis at defined stages of the HF cycle, we developed a model with passive transfer of AK23 (a mouse monoclonal pathogenic anti-Dsg3 antibody) into adult 8-week-old C57Bl/6J mice. Validated using histopathological and molecular methods, we found that this model faithfully recapitulates major features described in PV patients and PV models. Two hours after AK23 transfer, we observed widening of intercellular spaces between desmosomes and EGFR activation, followed by increased Myc expression and epidermal hyperproliferation, desmosomal Dsg3 depletion, and predominant blistering in HFs and oral mucosa. These data confirm that the adult passive transfer mouse model is ideally suited for detailed studies of Dsg3 antibody-mediated signaling in adult skin, providing the basis for investigations on novel keratinocyte-specific therapeutic strategies.

Figure 1. AK23/PF IgG and AK23-treated neonatal mice show hyperproliferation, EGFR activation, c-Myc upregulation and depletion of Dsg3 from desmosomes

(a) Shown are micrographs of immunofluorescence microscopy for Ki67 and graph of total Ki67+ cells in basal keratinocytes counted on micrographs of neonatal mice injected with AK23 or control mouse IgG (mIgG), with or without a half-maximal dose of PF IgG as indicated. Counted were >1000 cells per animal. Scale bars: 25 µm. (b+c) Graphs depict average quantitative results of immunoblots from indicated proteins in Triton-X 100 soluble fractions at 24hrs. Signals on each blot were quantified, normalized to tubulin and plotted relative to mIgG set as 1. (d+e) Immunoblots and graphs of indicated proteins from Triton-X 100 soluble and insoluble fractions. Lanes indicate different animals. Blots were normalized to tubulin (soluble fractions, upper panel) or lamin B1 (insoluble fractions, lower panel; shown is lamin B1 of blots probed for d: Dsg3/Dsc3/PG/Pph1, e: Dsg3/Dsg1/2/PG). Pph1: plakophilin, DP: desmoplakin. Data are mean ± SEM. (n(mIgG/PF; mIgG) = 4, n(AK23/PF; AK23) = 5 animals), *P≤ 0.05, **P≤0.01.

Figure 2. AK23-treated 8-week-old C57Bl/6J mice exhibit typical PV lesions and epidermal hyperproliferation

(a) Blister sites and number of affected/tested animals are summarized. (b) H&E-stained paraffin sections of indicated tissues show lesions and direct immunofluorescence microscopy depicts AK23 binding to basal epidermal keratinocytes (dIF). Inserts show lesions (arrow heads) in oesophagus and snout skin and a rare lesion in the epidermis. (c) Histology and (d) percentage of HF blisters (insert, arrow head) in AK23-treated animals. Data are mean ± SDM. HF analyzed (n(24h) = 115; n(48h) = 455). (e) Hair loss by tape stripping. (f) Representative flow cytometry blots for forward scatter (FSC) and BrdU-labeled viable cells gated for BrdU-positive cells, and graph of average results. Data are mean ± SDM. (n = 2 animals/group; 2 experiments), *P≤ 0.05. (g) Representative flow cytometry histogram for BrdU-labeled cells and graph of average results. Data are mean ± SDM. (n = 4 animals/group). **P≤0.01. (h) Immunofluorescence microscopy depicting the distribution of BrdU+ and Ki67+ cells in epidermis (Inserts: close up of epidermis). Scale bars: 50 µm or as indicated.

Figure 3. EGFR is activated and Myc, cyclin D and Dsg3 are significantly increased in Triton X-100 soluble fractions of AK23-treated 8-week-old C57Bl/6J mice

(a) Immunoblots and graphs of average quantitative results are shown for indicated proteins at 24 and 48hrs. Signals were quantified, normalized to tubulin on each blot and plotted relative to mIgG set as 1. (n(2h) = 4 animals/group; n(48h, mIgG) = 4, n(48h, AK23)= 2). (b) Graphs like in a, (n(2h) = 4 animals/group, n(48h, mIgG) = 8, n(48h, AK23) = 6 animals). (c) like in a, (n(2h) = 4 animals/group, n(48h, mIgG) = 8, n(48h, AK23) = 6 animals). (a-c) Data are mean ± SEM. **P≤0.01, *P≤ 0.05. (d) Graphs show indicated mRNA steady-state levels obtained by quantitative RT-PCR. Values were normalized to cyclophilin and are plotted relative to mIgG set as 1. (n(2/24h) = 4 animals/group, n(48h, mIgG) = 8, n(48h, AK23)= 7 animals). Data are mean ± SEM.

Figure 4. Dsg3 is depleted from desmosomes in AK23-treated 8-week-old C57Bl/6J mice

Immunoblot of the Triton-X 100 insoluble fractions are shown for indicated proteins. Numbered lanes correspond to four different animals per group. Each blot was normalized with respect to lamin B1 (shown is the blot probed for Dsg3/K15 at 24hrs and for PG, K15/Pph1 at 48hrs). Signals were quantified, normalized and are plotted relative to mIgG set as 1. Pph1: plakophilin, DP: desmoplakin. Data are mean ± SEM. (n(24h) = 4 animals/group; n(48h) = 4 animals/group), *P≤ 0.05.

Figure 5. AK23 causes widening of the intercellular spaces after 2hrs and microblisters after 48hrs in basal keratinocytes of AK23-treated 8-week-old C57Bl/6J mice

(a) Histology (upper panel) and direct immunofluorescence analyses (lower panel) at 2hrs in samples processed for electron microscopy. Only one animal out of 8 showed a microblister in the hard palate (insert) but not in the epidermis. Scale bars: 25 µm. (b) Electron microscopical pictures at 2 and 48hrs. *, widened intercellular spaces; SC, stratum corneum. Tissue damage is seen in basal but not suprabasal cells or controls. (n(2h/48h) = 2 animals/group). Scale bars: as indicated.