RNA antimicrobial peptide complexes fuel a TLR and NETosis mediated inflammatory cycle of neutrophil activation in psoriasis
Abstract Psoriasis is an inflammatory autoimmune disease characterized by skin lesions showing strong neutrophil (PMN) infiltration and high levels of the antimicrobial peptide, LL37, but the role of PMNs in this context remains unclear. We here show that primary human PMNs, especially PMNs from psoriasis patients, not only respond via TLR8 to human and bacterial RNA in complexed with LL37 by cytokine chemokine and neutrophil extracellular trap (NET) release; they also actively release additional RNA and LL37 in response to stimulation by the same complex and both RNA and LL37 were found to be highly abundant in psoriatic skin. RNA LL37 complexes can thus fuel a PMN mediated and self sustaining inflammatory loop that may represent an unexpected early initiator or amplifying event in psoriasis. Given that TLR inhibitory oligodeoxynucleotides prevent the activation of PMNs by RNA LL37 complexes in vitro, our study also highlights TLR blockade as a potential therapeutic intervention strategy in psoriasis. Psoriasis is an autoimmune disease of the skin with high incidence in Western countries (1.5 3% causing high socioeconomic and disease burden with limited but increasing treatment options 1, 2. The most common form of psoriasis, plaque psoriasis, is characterized by epidermal hyperplasia due to keratinocyte (KC) hyper proliferation, increased endothelial proliferation and an infiltrate of leukocytes, such as dendritic cells, T cells and, prominently, polymorphonuclear neutrophils (PMNs) 1. The high and early accumulation of PMNs in psoriatic plaques and micro abscesses is well documented, as well as an increase of PMNs in the circulation of psoriasis patients 34. However, it is unclear whether PMN infiltration represents a bystander phenomenon or has a causal role in the development of the disease. PMNs are known as a source of LL37, an amphipathic, positively charged 37 amino acid peptide generated from a precursor protein, the cathelicidin hCAP18, and highly abundant in psoriatic lesions 1, 5. LL37 is stored in the secondary granules of PMNs, from which it can be released upon activation 6. Apart from acting as a self antigen for CD4 helper T cells in psoriasis patients 7, LL37 was previously shown to form complexes with DNA or RNA that resisted nuclease degradation, were readily taken up by plasmacytoid dendritic cells (pDCs) and triggered high Interferon (IFN) a and low Tumor necrosis factor (TNF) or Interleukin (IL) 6 secretion from these cells 8, 9. Although type I IFNs contribute to psoriasis 10, 11, chemokines and pro inflammatory cytokines such as TNF, IL 1b or IL 23 are additionally required for immune cell infiltration and T cell polarization, respectively 12 Since pDCs are comparatively low producers of these psoriasis initiating inflammatory cytokines and do not accumulate to high numbers in psoriatic skin, this raises the question whether nucleic acid LL37 sensing by other immune cells, particularly PMNs, may contribute more prominently to initial inflammatory cytokine/chemokine milieu observed in psoriasis. In contrast to pDCs, PMNs are known producers of considerable amounts of various cytokines and chemokines 13, 14, 15. Additionally, PMNs are able to undergo neutrophil extracellular trap (NET) formation (NETosis) an activation induced process leading to the extrusion of nuclear DNA 16, as well as cellular proteins, including LL37 17. Like pDCs, PMNs express multiple pattern recognition receptors (PRRs) of the Toll like receptor (TLR) family, namely TLR2, 4, 8 18 and 9 19, but not TLR3 or TLR7 18. TLRs directly bind activating ligands, typically so called microbe associated molecular patterns (MAMPs) absent from the host 20; however, when they can gain access to endosomal compartments for example by complex formation with LL37 host nucleic acids are able to trigger the endosomal TLRs 3, 7, 8 (sensing double stranded or single stranded RNA, respectively) or TLR9 (sensing DNA) TLR7 expression in humans is restricted to pDC and (only inducibly) to B cells. TLR8 thus appears to be the primary receptor for foreign (and potentially host derived) single stranded RNA in humans 21. PMNs theoretically combine the abilities (i) to release LL37 and nucleic acids as components of immuno stimulatory ligands via NETosis; (ii) to potentially sense such ligands via TLRs; and (iii) to secrete chemo /cytokines. These mechanisms were never shown to act in concert but we speculated whether LL37 mediated DNA or RNA sensing via TLRs in PMNs might initiate and fuel inflammatory chemo /cytokine production and thus inflammation and immune cell infiltration in psoriatic skin. We here present experimental evidence that human primary PMNs readily respond to RNA LL37 complexes, but not RNA alone, leading to the release of a broad array of chemokines and cytokines a process that induces chemoattraction of other immune cells and that is elevated in psoriasis patients and can be therapeutically blocked by inhibitory oligodeoxynucleotides (iODNs) Unexpectedly, RNA LL37 also triggered the release of NETs, which contained additional LL37, RNA and DNA, thereby providing the basis for a self propagating inflammatory cycle. Key points Human and bacterial RN in complexed with LL37 activates neutrophils via TLR8 to release cytokines, chemokines and neutrophil extracellular traps (NETs) NETs and neutrophil rich areas in psoriatic skin contain RNA and LL37, suggesting RNA+LL37 may fuel a PMN mediated and self sustaining inflammatory cycle in psoriasis.