The Double-Stranded RNA Analogue Polyinosinic-Polycytidylic Acid Induces Keratinocyte Pyroptosis and Release of IL-36γ

Abstract

Interleukin-36 (IL-36) is the common name for the three IL-1 family members IL-36α, IL-36β and IL-36γ, formerly known as IL-1F6, IL-1F8 and IL-1F9, respectively. IL-36 appears to have pro-inflammatory activities; however, the physiological function of these cytokines remains unknown. Expression of IL-36 by keratinocytes implies its possible involvement in innate immune responses in the skin. We observed that, of the three IL-36 isoforms, human keratinocytes express high levels of IL-36γ. IL-36γ mRNA expression was dramatically induced by the Toll-like receptor ligands poly(I:C) and flagellin. Surprisingly, the IL-36γ protein was released by cells treated with poly(I:C) but remained intracellular in cells treated with flagellin only. Poly(I:C), but not flagellin, induced cell death and caspase-3/7 activation. Inhibition of caspase-3/7 and caspase-1 blocked extracellular release of IL-36γ from poly(I:C) treated cells. Furthermore, caspase-1 inhibition prevented poly(I:C)-induced caspase-3/7 activation. Interestingly, transcription of the gene IL36G was dependent upon caspase-1, but not caspase-3/7, activation. This demonstrates that the pathways leading to IL36G transcription and caspase-3/7 activation branch after caspase-1. This divergence of the pathways allows the cells to enter a state of de novo protein synthesis before committing to pyroptosis. Overall our observations suggest that IL-36γ may be an alarmin that signals the cause, e.g. viral infection, of cell death.

Autocrine and Paracrine Regulation of Keratinocyte Proliferation through a Novel Nrf2–IL-36γ Pathway

“…IL-36 cytokines are overexpressed in affected skin of patients with inflammatory skin diseases, including atopic dermatitis and psoriasis (25)(26)(27)(28). The functional importance of this upregulation is supported by several recent findings: IL-36g mediates the alarmin function of the antimicrobial peptide LL37, and it functions as an alarmin that signals pathogen infections (20,29); injection of IL-36a promotes myeloid cell infiltration and their activation in the skin (30); tg mice overexpressing IL-36a in keratinocytes develop a psoriasis-like phenotype (31); the psoriasiform dermatitis that develops in mice after treatment with imiquimod depends on an IL-36-mediated cross-talk between dendritic cells and keratinocytes (32); and IL-36RA deficiency causes generalized pustular psoriasis in humans (33). Finally, a proinflammatory function of IL-36 cytokines in tissues other than the skin is emerging (34).…”

“…Nevertheless, cultured keratinocytes from K5cre-CMVcaNrf2 mice proliferate normally (4). Therefore, the IL-36g that is produced in response to Nrf2 activation in keratinocytes is insufficient to cause hyperproliferation of these cells, most likely as a result of the strong dilution of the cytokine in the culture medium and/or the inefficient secretion of this cytokine by cultured keratinocytes, which only occurs in the presence of extracellular ATP (20). However, IL-36a and IL-36g may also stimulate keratinocyte proliferation indirectly in vivo via activation of fibroblasts.…”

Interleukin-36 cytokines may overcome microbial immune evasion strategies that inhibit interleukin-1 family signaling

“…Both in vitro and in vivo, this release is independent of caspase-1 (77,78). Extracellular IL-36g has been detected in culture medium from keratinocytes treated with ATP, the double-stranded RNA analog polyinosinic-polycytidylic acid [poly(I:C)], or the antimicrobial peptide LL-37 and from lung macrophages exposed to bacteria or bacterial components (24,26,31,79,80). Some of this released IL-36g appears to be present in extracellular vesicles (80,81); however, the purpose of such packaging is unknown.…”

“…Screens using Toll-like receptor (TLR) ligands identified a subset of agents that could induce expression of one or more IL-36s in either bronchial epithelial cells or skin keratinocytes (24,79). These ligands included double-stranded RNA [poly(I:C)], flagellin, and Mycoplasma fermentans synthetic lipopeptide (FSL-1), LPS, and zymosan and implicate the IL-36s in immunity against bacteria, fungi, and viruses (24,79,82).…”

Role of IRF6 in epithelial cell-mediated host defence and inflammation

“…The biological functions of both these cytokines are not well understood, but interestingly, HSV infection of keratinocytes elicited IL-36γ production (Kumar, McDonnell et al 2000). Poly(IC) has also been shown to induce IL-36γ expression in keratinocytes, suggesting a potential role for this cytokine downstream of TLR3 signalling in skin (Lian, Milora et al 2012). IL-37, like other members of the IL-1 family, is also produced as a precursor and must be cleaved by caspase-1 to enable regulated release (Nold, Nold-Petry et al 2010).…”