Abnormalities of collagen are detected by light microscopy in certain cutaneous diseases such as morphea and lichen sclerosus (LS). Herein we aimed to characterize collagen in normal and diseased skin using second harmonic generation (SHG) and two-photon excitation microscopy (TPE). We employed the SHG/TPE along with novel analysis approach to enable enumeration of individual collagen fibers, their spatial orientation and tissue distribution. Ninety-seven archived formalin-fixed, paraffin-embedded samples were included. Unstained 4mm sections underwent SHG/2PE imaging (Genesis Ò 100) using a 20x objective to acquire multi-tiled images. Analysis was performed using proprietary imaging software. Parameters quantified include collagen area ratio (CAR), unsaturated collagen area ratio (UCAR), collagen fiber density (CFD), unsaturated collagen fiber density (UCFD), and collagen area reticulation density (CARD). Papillary and reticular dermis in normal skin were similar by SHG/TPE in the parameters CFD, UCFD and UCAR (P > 0.99). Data from papillary dermis and reticular dermis was combined and compared to papillary and reticular dermis of morphea and LS. UCAR and UCFD were higher in morphea compared to normal skin (p < 0.001). CARD in LS was significantly higher compared to normal skin (p < 0.001). UCFD and CFD were lower in LS compared to morphea (p < 0.001). Our data demonstrated differences in spectral characteristics of collagen between morphea, LS and normal skin, and each of the studied subgroups had a specific signature. The use of SHG/TPE has the potential to quantify differences in collagen signatures in normal skin, morphea and LS, and warrant further study.

Hidradenitis suppurativa (HS) is a neutrophilic inflammatory skin disorder with an unknown etiology primarily affecting intertriginous areas.Considering the predominant cellular infiltrate, we sought to understand the role of neutrophil extracellular traps (NETs) in HS.In peripheral blood samples from HS patients, neutrophils had enhanced NETosis and WB analysis revealed that these NETs possessed proteins recognized by autoantibodies (AAbs) present in HS serum, namely antibodies against IL17B.Furthermore, serum from HS patients had significant titers of total IgG and contained AAbs against citrullinated proteins, including filaggrin, vimentin and enolase corresponding to levels detected in sera from patients with rheumatoid arthritis (p<0.05).Moreover, NETs were confirmed in HS tissue via immunofluorescent detection of citrullinated histone 4 (cit-H4). With ELISA, HS tissue homogenates revealed a positive correlation of detected cit-H3/double-stranded DNA complexes with disease stage (r 2 ¼0.7107, p¼0.0043).Finally, HS tissue displayed a significant upregulation of type I interferon (IFN) genes.Taken together, these results suggest unreported roles of autoimmunity and neutrophils in the pathogenesis of HS, identifying NETs as a source of AAbs and the type I IFN signature in HS tissue, which could impact alterations in therapeutic approaches.