Autoimmune skin inflammation is dependent on plasmacytoid dendritic cell activation by nucleic acids via TLR7 and TLR9

Abstract

Recognition of endogenous DNA and RNA by cells expressing TLR7 and TLR9 is an important contributor to the pathogenesis of systemic lupus erythematosus and has been suggested to contribute to cutaneous lupus and to a group of related inflammatory skin diseases termed interface dermatitis. We have developed a mouse model of TLR7- and TLR9-dependent skin inflammation using tape stripping. In normal mice, this resulted in a rapid but transient inflammatory cell infiltration accompanied by induction of type I IFN production by plasmacytoid dendritic cells (PDCs) and release of extracellular traps and proinflammatory cytokines by neutrophils. These responses were strongly reduced in MyD88-deficient mice and in mice treated with a bifunctional inhibitor of TLR7 and TLR9. In contrast, in lupus-prone (NZBxNZW)F(1) mice, tape stripping induced the development of chronic lesions characterized by a persistent type I IFN gene signature and many clinical and histological features of cutaneous lupus. Depletion of PDCs before injury prevented the development of skin lesions, whereas treatment with a bifunctional TLR7/9 inhibitor before tape stripping or after the initial lesion was established led to a significant reduction of the disease. These data suggest that inhibitors of TLR7 and TLR9 signaling have potential therapeutic application for the treatment of interface dermatitis.

Figure 1.

Skin injury provokes leukocyte infiltration and activation, including production of IFN-α by PDCs and secretion of NETs by neutrophils. (A and B) Cellular infiltrate in the skin of 129 mice was characterized before (A) and 24 h after inflammation via tape stripping (B) by flow cytometry. PDCs were identified as CD11C⁺PDCA1⁺120G8⁺, conventional DCs as CD11c⁺PDCA1⁻120G8⁻, neutrophils as Ly-6G⁺(1A8) F480⁻, skin macrophages as F480⁺ Ly-6G low, and T cells as CD4⁺CD3⁺ and CD8⁺CD3⁺. Representative FACS plots of at least 10 mice processed from three independent experiments are shown. (C) 129 mice were tape stripped, and 24 h later, PDC infiltrating cells were assessed for IFN-α production by flow cytometry analysis. Neutrophils (Ly-6G⁺) and T cells (CD3⁺) were used as a negative control. Cultured BM-derived PDCs stimulated for 3 h with CpG-ISS were used as a positive control. A representative of three independent experiments (10 mice per experiments) with similar results is shown. (D and E) The ability of neutrophils to form NETs when isolated from BM (as source of inactivated neutrophils; D) or the skin of mice 24 h after tape stripping (E) was determined by immunostaining using Ly-6G to detect neutrophils and the SYTOX dye to stain DNA. (F and G) The presence of LL37-containing DNA (F) or RNA (G) NET fiber was detected by immunostaining using specific dyes. (D) Data from two independent experiments are shown. (E–G) Representative data from five fields from the skin of 10 mice from two independent experiments are shown. Bars, 20 µm.

Figure 2.

The MyD88 signaling pathway is necessary for the up-regulation of both type I IFN–regulated and proinflammatory genes. (A) MyD88^−/− (white bars) and age-matched WT C57BL/6 mice (black bars) were either left untreated (naive) or tape stripped to provoke inflammation. 24 h later, skin biopsies were isolated, and the levels of proinflammatory genes were evaluated by TaqMan. (B) IFNAR^−/− mice (white bars) and age-matched WT 129 mice (black bars) were either left untreated (naive) or tape stripped to provoke inflammation. 24 h later, skin biopsies were isolated, and the levels of proinflammatory genes were evaluated by TaqMan. Naive (untreated) groups are shown for C57BL/6 and 129 mice only. Cumulative data from at least two independent experiments (n = 15–20) are shown (mean ± SEM; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Figure 3.

Stimulation of TLR7 and TLR9 is required for the induction of skin inflammation but not for the cellular infiltration after skin injury. 129 mice were left untreated (naive; white bars), tape stripped (TAPE; black bars), or tape stripped immediately after treatment (s.c.) with 100 µg of the dual TLR7 and TLR9 inhibitor IRS 954. (A) 24 h later, skin-infiltrating cells were isolated, and PDCs and neutrophils were identified as in Fig. 1 A. Histograms show total cell number obtained from 2 × 2–cm skin biopsies (n = 10 mice) from two independent experiments (mean ± SEM). (B) Gene expression levels were evaluated by TaqMan. Cumulative data from at least two independent experiments (n = 10–15 mice) are shown (mean ± SEM; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Figure 4.

The activation of both PDCs and neutrophils is critical for the burst of inflammatory genes after tape-stripping injury. 129 mice were either left untreated or tape stripped. PDCs and/or neutrophils were depleted before tape stripping using specific antibodies (250 µg given i.p. at day −2, day 0, and 8 h before tape stripping). Anti-120G8 antibody was used for depletion of PDCs and anti-GR1–Ly-6G antibody for depletion of neutrophils (Neut). Over 95% cellular depletion was achieved in both blood stream and skin infiltrate. 24 h after tape stripping, gene expression levels were measured in skin biopsies. Naive groups are shown for untreated mice only. Cumulative data from three independent experiments (n = 14 mice) are shown (mean ± SEM; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Figure 5.

Lupus-prone (NZBxNZW)F₁ mice develop a severe and chronic skin disease resembling human CLE after tape stripping. (A) Lupus-prone mice (NZBxNZW)F₁ and age-matched 129 and C57BL/6 mice were tape stripped, skin biopsies were collected 24 h, 4 d, and 20 d later, and gene expression was evaluated. The levels of expression at 24 h were set as 100 and compared with levels obtained at 4 d and 20 d after tape stripping. Cumulative data from three independent experiments are shown (n = 10; mean ± SEM; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) Macroscopic lesions in (NZBxNZW)F₁ at 15–23 d after tape stripping as compared with normal 129 mice. (C) Quantification of area with open lesions 15–23 d after tape stripping in (NZBxNZW)F₁, 129, and C57BL/6 mice. Cumulative data from at least two independent experiments (n = 15; mean ± SEM) are shown. (D–I) Representative sections of skin from untouched (NZBxNZW)F₁ mice (D) or from the skin of 129 (E), C57BL/6 (F), or (NZBxNZW)F₁ mice (G–I) 15–23 d after tape stripping. (D–I) Representative sections from >20 mice are shown. Bars, 200 µm.

Figure 6.

PDCs and TLR7 and TLR9 signaling are required for cutaneous disease formation in lupus-prone mice. (A–C) Macroscopical skin lesions 15–23 d after tape stripping in (NZBxNZW)F₁ mice (A), (NZBxNZW)F₁ treated with weekly injection of IRS 954 (B), and (NZBxNZW)F₁ mice in which PDCs were depleted during the course of the experiment (C; see Fig. S8). (D) Quantification of area with open lesions 15–23 d after tape stripping in mice as in A–C. Cumulative data from at least two independent experiments (n = 12; mean ± SEM; *, P ≤ 0.05; **, P ≤ 0.01) are shown. (E–J) Representative sections of skin from untouched (NZBxNZW)F₁ mice (naive; E) or from skin isolated 15–23 d after tape stripping from (NZBxNZW)F₁ mice left untreated (F) or treated with IRS 954 (G and H) or depleted of PDCs (I and J). Representative sections from ∼15 mice are shown. Bars, 200 µm.

Figure 7.

Therapeutic treatment of lupus-prone mice with chronic skin inflammation using IRS 954 significantly ameliorates CLE-like phenotype. (A and B) Gross appearance of skin lesions isolated 15–23 d after tape stripping from (NZBxNZW)F₁ mice left untreated or (NZBxNZW)F₁ mice treated from day 4–20 with IRS 954 in a therapeutic setting (scheme of treatment in Fig. S8). (C) Quantification of area with open lesions 15–23 d after tape stripping in mice as in A and B. Cumulative data from two independent experiments (n = 12; mean ± SEM; **, P ≤ 0.01) are shown. (D–G) Representative sections of skin from (NZBxNZW)F₁ mice either left untreated (D and E) or treated from day 4 with IRS 954 (F and G). Data show representative sections of ∼12 mice. Bars, 200 µm.