Neutrophil extracellular traps (NETs) have emerged as a key feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs), the primary leukocyte population in humans. Forming web-like structures composed of DNA, histones, and antimicrobial proteins, NETs trap and kill microbial invaders and thus enhance host defense. However, they have also been linked to inflammatory states, e.g. in atherosclerosis or psoriasis. Whilst DNA has been in focus as a primary structural component of NETs, we here characterize naRNA (NET-associated RNA), as a new canonical, abundant, and largely unexplored NET component. naRNA decorated all types of NETs in complex with the antimicrobial peptide LL37. In fact, naRNA was pre-associated with LL37 intracellularly as a "composite" danger-associated molecular pattern (DAMP) prior to neutrophil activation. Externalized, naRNA propagated NET formation in naive PMN, dependent on TLR8 in humans and Tlr13 in mice, in vitro and in vivo. naRNA-TLR8/Tlr13 signaling contributed significantly to the highly sensitive pro-inflammatory response of both tissue cells, like keratinocytes, and other immune cell types, such as macrophages. Those responses could be blocked by inhibition and genetic ablation of RNA receptors or RNase treatment. Importantly, in vivo naRNA strongly drove skin inflammation whereas genetic ablation of RNA sensing drastically ameliorated skin inflammation in the imiquimod psoriasis model. Our data highlight naRNA as a novel composite DAMP signaling and amplifying neutrophil activation. Moreover, naRNA emerges as the likely driver of inflammation in conditions previously linked to NETs and extracellular RNA, suggesting blockade of TLR-mediated RNA sensing as potential new intervention target

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IntroductionKeratinocytes form a multilayer barrier that protects the skin from invaders or injuries. The barrier function of keratinocytes is in part mediated by the production of inflammatory modulators that promote immune responses and wound healing. Skin commensals and pathogens such as Staphylococcus aureus secrete high amounts of phenol-soluble modulin (PSM) peptides, agonists of formyl-peptide receptor 2 (FPR2). FPR2 is crucial for the recruitment of neutrophils to the sites of infection, and it can influence inflammation. FPR1 and FPR2 are also expressed by keratinocytes but the consequences of FPR activation in skin cells have remained unknown.MethodsSince an inflammatory environment influences S. aureus colonization, e. g. in patients with atopic dermatitis (AD), we hypothesized that interference with FPRs may alter keratinocyte-induced inflammation, proliferation, and bacterial colonization of the skin. To assess this hypothesis, we investigated the effects of FPR activation and inhibition in keratinocytes with respect to chemokine and cytokine release as well as proliferation and skin wound gap closure.ResultsWe observed that FPR activation induces the release of IL-8, IL-1α and promotes keratinocyte proliferation in a FPR-dependent manner. To elucidate the consequence of FPR modulation on skin colonization, we used an AD-simulating S. aureus skin colonization mouse model using wild-type (WT) or Fpr2-/- mice and demonstrate that inflammation enhances the eradication of S. aureus from the skin in a FPR2-dependent way. Consistently, inhibition of FPR2 in the mouse model or in human keratinocytes as well as human skin explants promoted S. aureus colonization.DiscussionOur data indicate that FPR2 ligands promote inflammation and keratinocyte proliferation in a FPR2-dependent manner, which is necessary for eliminating S. aureus during skin colonization.

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