Cutaneous ischemia‐reperfusion injury is exacerbated by IL‐36 receptor antagonist deficiency
Yasushi Tanaka, Yohei Iwata, Kenta Saito, H Fukushima, Soichiro Watanabe, Yudai Hasegawa, Masashi Akiyama, Kazumitsu Sugiura
Abstract
Abstract Background 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. Objectives We investigated the role of IL36Ra in cutaneous I/R injury. Methods 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. Results 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. Conclusions 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.