336 NET-associated DLL4 activates notch-γ secretase signaling in macrophages and fibroblasts and promotes pro-fibrotic responses in hidradenitis suppurativa

Hidradenitis suppurativa (HS) is an incapacitating inflammatory skin disorder of unknown etiology and characterized by profound inflamed abscess-like nodules and boils that result in sinus tract formation and tissue scarring. Notch signaling, in particular γ-secretase, has been associated with HS pathogenesis, but the precise mechanisms remain unknown. Western blot and quantitative PCR analyses were performed in HS skin lesions and showed elevated levels of Notch ligands, delta-like ligand 4 (DLL4) and jagged 2 (JAG2). Levels of DLL4, JAG2 and γ-secretase activity correlated with disease severity, as measured by Hurley staging. Additionally, levels of Notch ligands and γ-secretase activity were particularly increased in dissected sinus tracts when compared to the rest of HS tissue. Immunofluorescence microscopy performed in HS skin lesions showed activation of Notch 1 signaling in macrophages and skin fibroblasts. DLL4 was also elevated in purified neutrophil extracellular traps (NETs) from HS patients. Macrophages and skin fibroblasts isolated from HS patients displayed activation of Notch signaling when compared to healthy controls. HS-NETs activated the Notch pathway in control macrophages and dermal fibroblasts. HS skin fibroblasts displayed elevated levels of profibrotic genes and increased migratory capacity when compared to control fibroblasts. NETs from HS patients increased γ-secretase activity, migratory capacity, and the release of profibrotic molecules in control fibroblasts while pharmacologic inhibition of γ-secretase decreased migratory capacity of HS fibroblasts. These data support a pathogenic role of NETs in the activation of Notch/ γ-secretase signaling promoting dysregulation of macrophages and skin fibroblasts in this disease.