Dottorato in MEDICINA SPERIMENTALE

Titolo della tesi: Extracellular vesicles and inflammatory cell microenvironment. In vitro studies on a potential pathogenetic role of EV-carried antimicrobial peptides in psoriatic disease.

Extracellular vesicles (EVs) are cell-derived lipid bilayer particles released by all cell types and involved in several physiological and pathological conditions. In different settings, EVs can play an important role in the definition of the extracellular microenvironment through the transfer of their cargo. Psoriasis is a prototypical chronic inflammatory disease characterized by several secreted mediators, among which antimicrobial peptides (AMPs) are considered pivotal in the development of the psoriatic inflammatory microenvironment. In addition, several reports highlight that psoriatic inflammatory microenvironment promotes the activation and release of Neutrophil Extracellular Traps (NETosis) by epidermis infiltrating neutrophils. Nevertheless, the role of EVs in the pathogenesis of psoriasis, as well as in psoriasis associated NETosis, has not been elucidated yet. Previous data obtained in our laboratory, using an in vitro keratinocytes cellular model (HaCaT cells), showed that IL-17A treatment modifies EVs release and EVs cargo content. Based on these observations, the aim of this work was to analyze whether the psoriasis-related cytokines (i.e., IFN-γ, TNF-α, IL-17A, IL-22 and IL-23) could affect the release of EVs by HaCaT cells and their AMPs mRNAs cargo as wells as to analyze the potential biological effect due to EVs internalization by different acceptor cells. Finally, effects of EVs on NETosis induction were also monitored. Nanoparticle tracking analysis showed that IL-17A treatment increased EVs release by HaCaT cells. Gene expression analysis showed that all the tested cytokines modulated AMPs mRNA expression both in HaCaT cells and in their respective EVs. In detail, S100A12 and hBD2 mRNAs were upregulated following IL-17A and IL-22 treatments. Upregulation of S100A12 and hBD2 mRNA was also detectable on acceptor HaCaT cells incubated with EVs derived from cells treated with psoriasis-related cytokines (henceforth defined pso-EVs). In addition, we observed that IL-17 pso-EVs appeared to be able to affect the migration rate of acceptor HaCaT cells and that all the pso-EVs, except those released following IFN-γ treatment, induced NETosis in neutrophils. The reported results highlight how extracellular vesicles can exert a role in the pathogenesis of psoriatic disease by delivering specific, cytokine-driven, molecular mediators to affect important biological functions of the disease development and spreading.