Release of the pre-assembled naRNA-LL37 composite DAMP re-defines neutrophil extracellular traps (NETs) as intentional DAMP webs

Abstract

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

TLRs and other molecules signaling crosstalk in diseases

“…Thus, the signaling crosstalk between CXCL4 and TLR8 ought to contribute to inflammatory disorders. Except for CXCL4, NETassociated RNA in complex with cationic LL37 amplifies TLR8 response in peripheral blood mononuclear cells (PBMCs) [56,57]. With these examples and the evidence of charge:charge interactions between cationic chemokines and NAs, it is reasonable to deduce that chemokine CXCL10, CXCL12, CXCL14, and CCL5 might likewise contribute to TLR8 response.…”

A Biological Perspective of TLR8 Signaling in Host Defense and Inflammation

“…[72][73][74][75][76][77] Neutrophil extracellular trap-associated RNA in complex with LL37 amplifies TLR8 responses in PBMCs. 38,39 Chaperone molecules are involved in and amplify eTLR responses in inflammatory diseases. 62,[78][79][80] CXCL4, as a cationic peptide, forms nanoparticles with TLR8 ORN ligand ORN8L and promotes the uptake of ORN8L by human primary monocytes, thereby magnifying TLR8 responses.…”

“…Unlike TLR7, which senses guanosine (G)-rich ssRNAs and G-modified derivatives, 33 the natural ligands of TLR8 are uridine (U)-rich ssRNAs from microbial pathogens, 12,31,[33][34][35][36][37] and self-derived RNA such as neutrophil extracellular trap (NET)-associated RNA. 38,39 Similar to mTlr13, hTLR8 is also capable of detecting fragments derived from bacterial 23S ribosomal RNA and self-mitochondrial RNA sequence-derived oligoribonucleotides (ORNs). However, the sequence motifs recognized by hTLR8 are clearly distinct from those sensed by mTlr13, which recognizes a short sequence of 13 residues within domain V of 23S ribosomal RNA.…”

“…Sensing NET-associated RNAs by TLR8 in humans and Tlr13 in mice contributes to skin inflammation and progression of psoriasis. 38,39 IFN-Is play an important role in the pathogenesis of systemic sclerosis, a multisystem life-threatening inflammatory and fibrotic disorder. [102][103][104] TLR8 is expressed in pDCs of systemic sclerosis patients, where CXCL4 can potentiate TLR8-induced IFN-I production by pDCs.…”

BTK and MMP9 regulate NLRP3 inflammasome-dependent cytokine and NET responses in primary neutrophils

“…The latter can unleash pre-stored inflammatory mediators from pre-stored granula, but also release so-called neutrophil extracellular traps (NETs) 3,4 . NETs are an ancient antimicrobial mechanism aimed at trapping and killing microorganisms 5 , but they also contain potent DAMPs such as the newly identified NETassociated naRNA ( 6 and pre-print 7 ). Given their large abundance in the blood (~65%), PMN are a potentially pro-inflammatory cell population to be reckoned with and hence have been implicated in debilitating or even life-threatening inflammatory conditions such as psoriasis, rheumatoid arthritis and COVID-19 6,8,9 .…”