Cutaneous ischemia-reperfusion injury is exacerbated by IL-36 receptor antagonist deficiency.

Abstract

Loss-of-function homozygous or compound heterozygous mutations in IL36RN, which encodes interleukin-36 receptor antagonist (IL-36Ra), has been implicated in the pathogenesis of skin disorders. However, the pathogenic role of IL-36Ra in cutaneous ischemia-reperfusion (I/R) injury remains unclear. We investigated the role of IL36Ra in cutaneous I/R injury. We examined I/R injury in Il36rn-/- mice. The area of wounds, numbers of infiltrated cells, apoptotic cells and neutrophil extracellular trap (NET) formation were assessed. The expression levels of various genes were analysed using real-time RT-PCR. The expression of high mobility group box 1 (HMGB1), an endogenous toll-like receptor (TLR) 4 ligand, was confirmed using immunohistology, and serum HMGB1 levels were measured by ELISA. Cytokine production by stimulated cultured J774A.1 and HaCaT cells was examined. IL-36Ra deficiency resulted in significantly delayed wound healing and increased neutrophil and macrophage infiltration into the wound tissues. Il36rn-/- mice had increased mRNA expression levels of CXCL1, CXCL2, CCL4, TNF-α, TGF-β, IL-1β, IL-6 and IL-36γ relative to wild-type mice. Apoptosis was identified in keratinocytes by TUNEL assay. HMGB1 expression in the I/R site was decreased in both keratinocytes and adnexal cells, while serum HMGB1 levels were significantly elevated after reperfusion. The mRNA levels of various cytokines, including IL-1β, were elevated in J774A.1 cells through TLR4 signalling by HMGB1 stimulation. In addition, HaCaT cells stimulated with IL-1β showed significantly increased CXCL1, TNF-α, IL-6, IL-36β and IL-36γ mRNA expression. Furthermore, NET formation was increased by IL-36Ra deficiency. Finally, either the blockade of TLR4 signalling by TAK-242 or inhibition of NET formation by Cl-amidine normalized exacerbated I/R injury in Il36rn-/- mice. This study indicated that IL-36Ra deficiency exacerbates cutaneous I/R injury due to excessive inflammatory cell recruitment, NET formation, and excessive cytokine and chemokine production via the TLR4 pathway by HMGB1 released from epidermal apoptotic cells.