Epithelialized tunnels are a source of inflammation in hidradenitis suppurativa

Abstract

Background: Hidradenitis suppurativa (HS), also known as acne inversa, is a chronic, painful, and burdensome inflammatory disease manifesting in nodules and abscesses, with progression to chronically draining tunnels in later-stage disease.

Objective: We sought to determine whether HS tunnels are immunologically active participants in disease activity.

Methods: Skin biopsy specimens were obtained by using ultrasound guidance in untreated patients with HS and those enrolled in an open-label study of brodalumab (ClinicalTrials.gov identifier NCT03960268) for patients with moderate-to-severe HS.

Results: Immunohistochemistry of HS biopsy specimens demonstrated that the epithelialized HS tunnels recapitulate the psoriasiform epidermal hyperplasia morphology of the overlying epidermis, displaying molecular inflammation, including S100A7 (psoriasin) positivity, as well as features of epidermal skin, including loricrin, filaggrin, lipocalin-2, and Melan-A positive cells. Tunnels were associated with increased infiltration of T cells, dendritic cells, and neutrophils; formation of neutrophil extracellular traps, and increased expression of psoriasiform proinflammatory cytokines. Unsupervised hierarchical clustering demonstrated a separation of HS samples based on the presence or absence of tunnels. Tunnels isolated by microdissection had higher levels of epithelium-derived inflammatory cytokines compared with the overlying epidermis and healthy controls. Clinically, the size and draining of the tunnels were decreased with treatment with the IL-17RA antagonist brodalumab.

Conclusion: These data suggest that tunnels are a source of inflammation in HS.

Keywords: Hidradenitis suppurativa; IL-17; brodalumab; neutrophils.

Figure 1:. Ultrasonography identifies deep dermal tunnels in HS.

Clinical assessment of tunnels marked by superficial ostia (white arrows) (A) Axilla (B) Axilla (C) Breast (D) Axilla. (E-H) Corresponding ultrasound images of tunnels detected by clinical examination. Red arrows highlight the hyperechoic border of the tunnel on ultrasound. (E-H) Light microscopy of the tunnel (1.2x magnification). White brackets outline the tunnel.

Figure 2:. HS tunnels recapitulate the structural properties of the overlying epidermis.

Representative biopsies from HS patients and site-matched healthy volunteers stained with (A) H&E demonstrating prominent psoriasiform lengthening of the rete ridges, thinning of the suprapapillary plate, hyper and parakeratosies as well as reduction of the granular layer in the HS epidermis compared to healthy controls. HS tunnels contain a thick stratified squamous epithelium with increasing differentiation towards the lumen (L). Scale Bar, 100μm (B) S100A7 positivity (C) Keratin-16 and (D) Ki67 identify this epithelium as composed of dividing keratinocytes with increasing differentiation towards the luminal layer compared to the more basal cells (D, black arrows). Differentiation is indicated by filaggrin (E) and loricrin (F) staining. Intermittent positive trichohyalin staining (G) is also observed. Other cell types within the tunnel include melanocytes (H) with c-Kit identifying dermal mast cells (I). (J) Lipocalin-2 staining is also increased in intensity in the luminal layers of the tunnel epithelium compared to superficial HS epithelium.

Figure 3:. Tunnels are immunologically active.

(A) Immunohistochemistry demonstrates increased infiltration of CD3, CD11c, and NE positive cells in HS compared to site-matched healthy controls. Epidermotropism and transepithelial migration towards the tunnels are also observed. Scale Bar, 100μm. “L” denotes lumen. (B) Quantitative CD3⁺, CD11c⁺ and NE⁺ cell counts highlights a significant difference in CD3, CD11c and NE positive cells between HS samples with and without tunnels. (C) Density of CD3, CD11c, and NE positive cellular infiltrate was analyzed within non-tunnel (NT) and tunnel (T) HS specimens stratified by location of cells within the biopsy (E=Epidermis; D=Dermis; T=Tunnel and depth-matched DD=Deep Dermis). There is a significant increase in inflammatory infiltration between tunnel and non-tunnel specimens when the deep dermal component of biopsies is taken into account. No significant elevation of CD3+ CD11c+ and NE+ cell density was seen between the epidermis and the superficial dermis in tunnel and non-tunnel specimens. Results are the mean ± SEM *p<0.05, **p<0.01, ***p<0.001 (D) Dense clusters of neutrophils undergoing NETosis in the tunnel epithelium adjacent to the lumen (L).

Figure 4:. HS samples cluster based on presence of tunnels.

(A) Unsupervised hierarchical clustering analysis of TLDA data based on the histological presence of tunnels demonstrates distinct clustering of tunnel and non-tunnel biopsy specimens compared to non lesional tissue (B) Heatmap of differential gene expression of HS-associated genes in HS Non-Lesional (NL) specimens (n=7), HS samples without tunnels (n=10) and HS samples containing tunnels (n=6), all confirmed by histological presence of tunnel. Results indicate FCH *p<0.05, **p<0.01, ***p<0.001. (C) Confirmatory RT-PCR of healthy controls (n=4), and actively inflamed HS lesional samples without (n=3), and with tunnels (n=5). Results are the mean ± SEM, FCH is shown. *p<0.05, **p<0.01, ***p<0.001

Figure 5:. Tunnels are active mediators of inflammation in HS and are therapeutically targetable

(A) Heatmap of supervised clustering of pro-inflammatory mediators in bisected specimens of HS skin containing healthy controls (n=6), epidermis/superficial dermis (n=8) or deep dermis containing epithelialized tunnels (n=8). Known pro-inflammatory mediators are highest in dermal (tunnel) specimens compared to epidermis/superficial dermis and normal healthy controls. FCH is shown with *p<0.05, **p<0.01, ***p<0.001 relative to healthy controls (B) RT-PCR demonstrates elevated expression of targetable cytokines in HS dermal tunnels tunnels (HS Dermis) compared to the overlying epidermis and healthy controls, relative to the total amount of RNA recovered. There is a significant elevation of cytokines in HS tunnels compared to the overlying epidermis. FCH is shown. (C) Healthy control epidermis illustrates IL-17C expression only in the basal keratinocytes. The gradient of IL-17C expression (black arrow) in epithelialized dermal tunnels also recapitulates the gradient seen in psoriasiform epithelium, with greatest expression in the basal layer with reduction of expression towards the lumen of tunnels (L). Scale Bar, 100μm. Arrow indicates direction of IL-17C gradient. (D) Tunnels in HS dermis express IL-17 family cytokines. (E) Doppler Ultrasonography representing reduction in tunnel diameter and doppler intensity following 12 weeks of treatment with IL-17 receptor antagonist. (F) There is a significant decrease in tunnel wall (G) tunnel diameter and (H) dermal doppler intensity following treatment with IL-17 receptor antagonist. Decrease in tunnel inflammation is seen as early as 4 weeks. Results are the mean ± SEM, FCH is shown. *p<0.05, **p<0.01, ***p<0.001