KLK6 expression in skin induces PAR1-mediated psoriasiform dermatitis and inflammatory joint disease

Abstract

Kallikrein-related peptidase 6 (KLK6) is a secreted serine protease hypothesized to promote inflammation via cleavage of protease-activated receptor 1 (PAR1) and PAR2. KLK6 levels are elevated in multiple inflammatory and autoimmune conditions, but no definitive role in pathogenesis has been established. Here, we show that skin-targeted overexpression of KLK6 causes generalized, severe psoriasiform dermatitis with spontaneous development of debilitating psoriatic arthritis-like joint disease. The psoriatic skin and joint phenotypes are reversed by normalization of skin KLK6 levels and attenuated following genetic elimination of PAR1 but not PAR2. Conservation of this regulatory pathway was confirmed in human psoriasis using vorapaxar, an FDA-approved PAR1 antagonist, on explanted lesional skin from patients with psoriasis. Beyond defining a critical role for KLK6/PAR1 signaling in promoting psoriasis, our results demonstrate that KLK6/PAR1-mediated inflammation in the skin alone is sufficient to drive inflammatory joint disease. Further, we identify PAR1 as a promising cytokine-independent target in therapy of psoriasis and psoriatic arthritis.

Keywords: Arthritis; Dermatology; Inflammation; Kallikrein; Skin.

Figure 1. KLK6 expression is significantly elevated in psoriatic lesions and parallels disease activity.

(A) Detection of KLK transcripts by RNA-seq in healthy control skin (NN, gray; N = 90), psoriatic patient nonlesional skin (PN, yellow; N = 26), and psoriatic patient lesional skin (PP, blue; N = 99). P values were computed using Wilcoxon’s rank-sum test; false discovery rate (FDR) was used to control the multiple testing. *P < 0.05 in PP vs. PN. (B) Relative expression of select KLK transcripts by qRT-PCR in a unique cohort of healthy controls and psoriatic patients (N ≥ 6 for all). Mean is indicated by the horizontal line. Box, 25th–75th percentile. Whiskers, minimum and maximum. *P < 0.005 in PP vs. both NN and PN by ordinary 1-way ANOVA with post hoc Tukey’s multiple-comparisons test. (C) Immunostaining of KLK6 in human skin. Results are representative of 4 biological replicates. Scale bar: 100 μm. (D) Detection of KLK6 and S100A7 transcripts by RNA-seq in PN and PP at the indicated weeks’ duration of etanercept therapy in responsive patients (N = 14). Mean is indicated by the horizontal line. Box, 25th–75th percentile. For definition of whiskers and additional details, see Supplemental Methods. *P < 0.05 vs. PN at w0; ⁺P < 0.05 vs. PP at w0 by negative binomial test.

Figure 2. Epidermal Klk6 overexpression causes a severe, generalized skin rash with histologic, transcriptional, and immunological features of psoriasis.

(A) Gross images of age- and sex-matched control and Klk6+ transgenic mice. (B) H&E staining of control (N = 19) and Klk6+ (N = 38) dorsal skin. (C) Immunostaining in control and Klk6+ dorsal skin. Ki67, cell proliferation; CD4 and CD8a, T lymphocyte; GR1, granulocyte (including neutrophil); CD11c, dendritic cell; F4/80, macrophage; MECA, vascular endothelial cell. (D) Detection of IL-17A, the IL-12/23p40 subunit, the IL-23p19 subunit, and IL-6 by ELISA in control and Klk6+ skin. N ≥ 7 for all groups. P = 0.0027, P < 0.0001, P = 0.0056, and P = 0.0015, respectively, by Mann-Whitney test. Mean and SEM are indicated. (E) Phospho-STAT3 (p-STAT3) immunostaining of control and Klk6+ dorsal skin. (F) Blue-yellow heatmap showing relative expression of transcripts enriched (top panel) and depleted (bottom panel) in Klk6+ (N = 5) vs. control (N = 5) dorsal skin. Adjacent blue-red heatmaps show fold change (FC) estimates from the following 3 comparisons: Klk6+ vs. control mice, psoriatic lesional (PP) vs. nonlesional (PN) skin, and lesional skin from psoriatic arthritis patients (PsA) vs. cutaneous-only psoriatic patients (PsC). Significance by P value or false discovery rate (FDR) is indicated by solid or open arrowheads, respectively. Panels depict the 25 transcripts with the highest minimum (top) or lowest maximum (bottom) FC estimates across the 3 comparisons. For additional details, see Supplemental Methods. Scale bars: 100 μm (B, C, and E).

Figure 3. Skin-directed Klk6 overexpression leads to debilitating joint inflammation and bony abnormalities consistent with psoriatic arthritis (PsA).

(A) Gross images of control and Klk6+ transgenic forelimbs. (B) Curvature of control and Klk6+ spines visualized by micro-computed tomography (micro-CT). Data are representative of 6 animals per group. (C) Pelvic joints of control and Klk6+ mice visualized by micro-CT. Upper panels, sacroiliac (SI) joint; lower panels, pubic symphysis. Arrows, distortion of joint space in SI joints and bony erosions in pubic symphysis. Data are representative of 6 animals per group. SI joints are magnified in Figure 4B. (D) Vertebral body bone density as visualized by brightness (higher density) on micro-CT. (E) H&E staining of control and Klk6+ spine sections. Rightmost panel, misshapen vertebra at level of gross kyphosis. Data are representative of 3 animals. Scale bar: 100 μm. (F) H&E staining of Klk6+ paw sections showing synovial inflammation (black arrows) and enthesitis (white arrows). Arrowheads, bony erosions. Data are representative of 5 animals. Scale bar: 200 μm.

Figure 4. KLK6 induces psoriatic skin and joint disease through PAR1, but not PAR2.

(A) Gross images and H&E dorsal skin staining of 10-week-old mice of the indicated genotypes. (B) Micro-CT images of the sacroiliac (SI) joint, pubic symphysis, and cervico-thoracic kyphosis (spine) in mice of the indicated genotypes. (C) Quantitative analysis of micro-CT images of the SI joint, pubic symphysis (Symph), and cervico-thoracic kyphosis in mice of the indicated genotypes. Mean and SEM are indicated. N ≥ 5 per group. *P < 0.05 vs. control; ⁺P < 0.05 for Klk6+ vs. Klk6+ Par1^KO by ordinary 1-way ANOVA with post hoc Tukey’s multiple-comparisons test (SI joint and Symph) or a post hoc uncorrected Fisher’s LSD test (Spine). Control and Klk6+ data are reproduced in Supplemental Figure 4E. (D) PAR1 immunostaining in human healthy control and lesional psoriatic skin. (E) PAR1 (orange) and CD3 (green) immunofluorescence in human healthy control and lesional psoriatic skin. (F) Detection of indicated proinflammatory marker transcripts by qRT-PCR of lesional psoriatic skin explants (N = 4) treated with 0 or 100 nM of the PAR1 antagonist vorapaxar. P < 0.05 by paired t test for all. Scale bars: 100 μm (A, D, and E).