Ingenol mebutate (IM) is highly effective in the treatment of human papillomavirus (HPV)-induced anogenital warts (AGW) leading to fast ablation within hours. However, the exact mode of action is still largely unknown. We performed dermoscopy, in vivo confocal microscopy (CLM), histology, immunohistochemistry, and immunofluorescence to gain insights in mechanisms of IM treatment in AGW. In addition, we used in vitro assays (ELISA, HPV-transfection models) to further investigate in vivo findings. IM treatment leads to a strong recruitment of neutrophils with thrombosis of small skin vessels within 8 h, in a sense of immunothrombosis. In vivo and in vitro analyses showed that IM supports a prothrombotic environment by endothelial cell activation and von Willebrand factor (VWF) secretion, in addition to induction of neutrophil extracellular traps (NETosis). IM superinduces CXCL8/IL-8 expression in HPV-E6/E7 transfected HaCaT cells when compared to non-infected keratinocytes. Rapid ablation of warts after IM treatment can be well explained by the observed immunothrombosis. This new mechanism has so far only been observed in HPV-induced lesions and is completely different from the mechanisms we see in the treatment of transformed keratinocytes in actinic keratosis. Our initial findings indicate an HPV-specific effect, which could be also of interest for the treatment of other HPV-induced lesions. Larger studies are now needed to further investigate the potential of IM in different HPV tumors.

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Neutrophils are key players of the immune system and possess an arsenal of effector functions, including the ability to form and expel neutrophil extracellular traps (NETs) in a process termed NETosis. During NETosis, the nuclear DNA/chromatin expands until it fills the whole cell and is released into the extracellular space. NETs are composed of DNA decorated with histones, proteins or peptides and NETosis is implicated in many diseases. Resolving the structure and dynamics of the nucleus in great detail is essential to understand the underlying processes but so far super-resolution methods have not been applied. Here, we developed an expansion microscopy-based method and determined the spatial distribution of chromatin/DNA, histone H1, and nucleophosmin (NPM1) with a 4.9-fold improved resolution (< 40 nm) and increased information content. It allowed us to identify the punctate localization of NPM1 in the nucleus and histone-rich domains in NETotic cells with a size of 54 nm. The technique could also be applied to components of the nuclear envelope (lamins B1 and B2) and myeloperoxidase (MPO) providing a complete picture of nuclear dynamics and structure. In conclusion, expansion microscopy enables super-resolved imaging of the highly dynamic structure of nuclei in immune cells.