TNF-α induced extracellular release of keratinocyte high-mobility group box 1 in Stevens-Johnson syndrome/toxic epidermal necrolysis: Biomarker and putative mechanism of pathogenesis

Abstract

Decreased epidermal high-mobility group box 1 (HMGB1) expression is an early marker of epidermal injury in Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN). Etanercept, an anti-tumor necrosis factor therapeutic, is effective in the treatment of SJS/TEN. The objective was to characterize antitumor necrosis factor-alpha (TNF-α)-mediated HMGB1 keratinocyte/epidermal release and etanercept modulation. HMGB1 release from TNF-α treated (± etanercept), or doxycycline-inducible RIPK3 or Bak-expressing human keratinocyte cells (HaCaTs) was determined by western blot/ELISA. Healthy skin explants were treated with TNF-α or serum (1:10 dilution) from immune checkpoint inhibitor-tolerant, lichenoid dermatitis or SJS/TEN patients ± etanercept. Histological and immunohistochemical analysis of HMGB1 was undertaken. TNF-α induced HMGB1 release in vitro via both necroptosis and apoptosis. Exposure of skin explants to TNF-α or SJS/TEN serum resulted in significant epidermal toxicity/detachment with substantial HMGB1 release which was attenuated by etanercept. Whole-slide image analysis of biopsies demonstrated significantly lower epidermal HMGB1 in pre-blistered SJS/TEN versus control (P < 0.05). Keratinocyte HMGB1 release, predominantly caused by necroptosis, can be attenuated by etanercept. Although TNF-α is a key mediator of epidermal HMGB1 release, other cytokines/cytotoxic proteins also contribute. Skin explant models represent a potential model of SJS/TEN that could be utilized for further mechanistic studies and targeted therapy screening.

Keywords: Stevens-Johnson syndrome; high-mobility group box 1; toxic epidermal necrolysis; tumor necrosis factor alpha.

Figure 1

A combination of TNF‐α and BV‐6‐induced HMGB1 release in HaCaTs which is modulated by inhibitors of both necroptosis (necrostatin) and apoptosis (Z‐VAD‐FMK). (a) HaCaT cell viability determined by MTT assay, (b) representative flow cytometric scatter plot of AV‐ and PI‐stained cells with (c) percentage cells positive stained and (d) extracellular HMGB1 following 24 h TNF‐α exposure ±40 μM necrostatin, 50 μM ZVAD (exemplar western blot image selected from n = 3). Densitometric analysis was performed on three separate blots and statistical analysis was undertaken using the Mann–Whitney U test (*P ˂ 0.05 and **P ˂ 0.01). (e) Supernatant HMGB1 levels determined by ELISA and (f) corresponding HaCaT viability by MTT assay (with BV‐6 pre‐treatment). Data represent mean normalized to untreated control (±SE) of three separate experiments conducted in triplicate (*P ˂ 0.05, **P ˂ 0.01, ***P < 0.001 and # < 0.05 vs. TNF‐a/BV‐6 treated cells).

Figure 2

Effect of doxycycline‐induced RIPK3 and Bak induction on (a) HaCaT cell viability (data represent mean normalized to untreated control ±SE, n = 3, *P ˂ 0.05, **P ˂ 0.01), (b) corresponding induction of RIPK3, Bak, and full‐length MLKL expression by doxycycline, (c) effect of RIPK3 and Bak expression on necroptotic and apoptotic markers, and (d) modulation of HMGB1 extracellular release following induction of RIPK3, MLKL, and Bak. HaCaT cells were treated for 6 h with 1 μg/mL doxycycline. Exemplar western blot images selected from n = 3 (b–d). Images have been rearranged to aid clarity. Densitometric analysis was performed on three separate blots and statistical analysis was undertaken using the Mann–Whitney U test (*P ˂ 0.05 and **P ˂ 0.01).

Figure 3

Effect of 72 h of 10 ng/mL TNF‐α or cutaneous ADR patient serum (control, lichenoid dermatitis or SJS/TEN) exposure ±1 μg/mL etanercept on healthy skin explant morphology (H&E stained). Images show HMGB1, immunohistochemical expression, and localization. Images are representative of n = 3 skin sample. For H&E images: black horizontal scale bar = 100 μm (400× magnification). For HMGB1: black horizontal bar =60 μm (zoomed 40× whole slide scanned image). Dashed lines represent the dermal epidermal junction.

Figure 4

Digital whole slide image analysis of epidermal and dermal HMGB1 cell positivity in healthy, maculopapular exanthem, and SJS/TEN skin samples from the Cleveland cohort. (a) Exemplar cell detection‐visual deployment of the result. The border of the cells represents the following: blue, negative; yellow, weakly positive; orange, moderately positive; red, strongly positive (400× magnification). (b) Epidermal and (c) dermal cellular staining for HMGB1 cutaneous ADRs. Data represent percentage cells with positive HMGB1 staining (as visualized by immunohistochemistry) for healthy, rash, and SJS/TEN. Horizontal lines represent mean values. *P < 0.05, ns = not significant.