Langerhans cell antigen capture through tight junctions confers preemptive immunity in experimental staphylococcal scalded skin syndrome

Abstract

Epidermal Langerhans cells (LCs) extend dendrites through tight junctions (TJs) to survey the skin surface, but their immunological contribution in vivo remains elusive. We show that LCs were essential for inducing IgG(1) responses to patch-immunized ovalbumin in mice that lacked skin dendritic cell subsets. The significance of LC-induced humoral responses was demonstrated in a mouse model of staphylococcal scalded skin syndrome (SSSS), a severe blistering disease in which the desmosomal protein Dsg1 (desmoglein1) is cleaved by Staphylococcus aureus-derived exfoliative toxin (ET). Importantly, ET did not penetrate TJs, and patch immunization did not alter epidermal integrity. Nevertheless, neutralizing anti-ET IgG(1) was induced after patch immunization and abolished upon LC depletion, indicating that antigen capture through TJs by LCs induced humoral immunity. Strikingly, the ET-patched mice were protected from developing SSSS after intraperitoneal ET challenge, whereas LC-depleted mice were susceptible to SSSS, demonstrating a vital role for LC-induced IgG(1) in systemic defense against circulating toxin in vivo. Therefore, LCs elicit humoral immunity to antigens that have not yet violated the epidermal barrier, providing preemptive immunity against potentially pathogenic skin microbes. Targeting this immunological process confers protection with minimal invasiveness and should have a marked impact on future strategies for development of percutaneous vaccines.

Figure 1.

LCs are essential for the induction of IgG₁ responses to OVA captured through TJs. (A) Patch immunization protocol. 4 mg/ml OVA in PBS was used to patch immunize mice via an occlusive dressing on the gently tape-stripped ears (yellow); the patch was removed after 24 h. In DC depletion experiments, DT was administered i.p. to mice 24 h before each patch immunization. (B) OVA-specific IgG₁ responses were determined by ELISA of sera (1:500 dilution) from mice that received OVA i.p. (n = 13), OVA-patched WT mice (n = 10), Langerin-DTR mice without DT i.p. (n = 10), Langerin-DTR mice with DT i.p. (n = 17), and PBS-patched (n = 8) WT mice. ***, P = 0.0006 (second lane vs. fourth lane) and P = 0.0008 (third lane vs. fourth lane) by Student’s t test. Shown are results from a single experiment representative of three experiments. (C) Lethally irradiated recipient mice were reconstituted with BM from the indicated donor mice. After complete chimerism, the mice were treated with DT i.p. to generate mice that harbored (top; WT → WT) or lacked (bottom; CD11c-DTR → L-DTR) all skin DCs and mice that lacked only langerin⁺ dDCs (middle; L-DTR → WT). BMT, BM transfer. (D) OVA-specific IgG₁ responses in sera (1:500 dilution) of mice from C. n = 10 WT → WT, n = 8 L-DTR → WT, and n = 9 CD11c-DTR → L-DTR mice from a single experiment that was reproduced in a similar, independent experiment. **, P = 0.0035 (first lane vs. third lane) and P = 0.0017 (second lane vs. third lane) by Student’s t test. (B and D) Error bars represent mean value ± SEM.

Figure 2.

Patch-immunized ETA does not alter epidermal integrity. (A) Development of SSSS 3 h after i.p. injection of ETA. (B) Histological features of superficial acantholysis in experimental SSSS. The asterisk denotes blister formation. (C) Visualization of Dsg1 and TJs in ear skin after intradermal (i.d.) injection of PBS or ETA. Dsg1 (red) and TJs (shown via ZO-1 staining in green; arrows) were visualized in the periphery of the blister in ear skin denoted by the asterisk (bottom) that received ETA intradermally (right). (D) Histological features of ear skin that received PBS or ETA patch immunization for 1, 3, or 24 h (right). Each experiment was performed three times with n = 3. Bars: (A) 1 cm; (B, C [left], and D) 25 µm; (C, right) 10 µm.

Figure 3.

ACT by LCs confers protective and systemic humoral immunity against experimental SSSS. (A) The anti-ETA IgG₁ response in WT mice that received ETA i.p., PBS patch, or ETA patch, as well as Langerin-DTR mice that were treated with DT 1 d before each ETA patch immunization. Sera were diluted 1:500. **, P = 0.0024. (B) Cleavage analysis of hrDsg1. ETA was preincubated with purified IgG from mice patch immunized with either PBS (PBS-IgG) or ETA (ETA-IgG) and then further incubated with hrDsg1. Each lane represents IgG purified from a single mouse. The arrow denotes hrDsg1, ∼75 kD in size. (C) Dose dependency of the neutralizing activity of ETA-IgG. (D) PBS patch-immunized WT mice (n = 16), ETA patch-immunized WT mice (n = 14), and DT-treated, ETA patch-immunized Langerin-DTR mice (n = 15) from A were challenged i.p. with 200 µg ETA, and images were taken 3 h later. Shown are representative data from a single experiment. Bar, 1 cm. Experiments in A–C were performed in two other independent experiments and D in one other experiment with similar results. (E) Erosion area index of ventral trunk skin in mice from D. *, P = 0.0432; ***, P = 0.0003. (A and E) Error bars represent mean value ± SEM.