LL37 complexed to double-stranded RNA induces RIG-I-like receptor signalling and Gasdermin E activation facilitating IL-36γ release from keratinocytes

The Interleukin-36 (IL-36) cytokine family have emerged as important players in mounting an inflammatory response at epithelial barriers and tailoring appropriate adaptive immune responses. As members of the Interleukin-1 superfamily, IL-36 cytokines lack a signal peptide for conventional secretion and require extracellular proteolysis to generate bioactive cytokines. Although the IL-36 family plays an important role in the pathogenesis of plaque and pustular psoriasis, little is known about the release mechanisms of these cytokines from keratinocytes and the physiological stimuli involved. Nucleic acid released from damaged or dying keratinocytes initiates early inflammatory signals that result in the breaking of tolerance associated with psoriasis pathogenesis onset. Cathelicidin peptide, LL37 binds to DNA or double-stranded RNA (dsRNA) and activates a type I Interferon responses in plasmacytoid dendritic cells and keratinocytes. Here, we demonstrate that LL37 binds to dsRNA and induces IL-36 gamma release from human primary keratinocytes. LL37/dsRNA complexes activate RIG-I-like Receptor signalling, resulting in Caspase-3 and Gasdermin E (GSDME) cleavage. Subsequent GSDME pore formation facilitates IL-36 gamma release. This response is magnified by priming with psoriasis-associated cytokines, IL-17A and IFN gamma. IL-36 gamma release in this manner is largely independent of cell death in primary keratinocytes and lacked extracellular proteolysis of IL-36 gamma. Conversely, transfection of keratinocytes directly with dsRNA synthetic analogue, Poly(I:C) induces NLRP1 inflammasome activation, which facilitates IL-36 gamma expression and release in a GSDMD-dependent manner. Inflammasome-associated cell death also enables extracellular processing of IL-36 gamma by the release of keratinocyte-derived proteases. These data highlight the distinct responses triggered by dsRNA sensors in keratinocytes. Depending on the inflammatory context and magnitude of the exogenous threat, keratinocytes will release IL-36 gamma coupled with cell death and extracellular cleavage or release the inactive pro-form, which requires subsequent processing by neutrophil proteases to unleash full biological activity, as occurring in psoriatic skin.Cytoplasmic sensing of dsRNA in keratinocytes mediates IL-36 gamma release via caspase activity and GSDM pore formation Keratinocytes release IL-36 gamma upon stimulation with intracellular dsRNA alone or complexed to the psoriasis-associated cathelicidin anti-microbial peptide LL37. Left: Transfected dsRNA triggers NLRP1 inflammasome assembly and IL-1 beta release, which can enhance IL-36 gamma expression, resulting in IL-36 gamma release and extracellular cleavage by released proteases. Right: LL37/dsRNA complexes activate a MDA5-MAVS pathway facilitating the release of IL-36 gamma through Caspase-3 activation and GSDME pore formation.