The Shapiro Family Laboratory of Viral Oncology and Aging Research, Los Angeles, CA, 90095, USA; Section of Restorative Dentistry, UCLA School of Dentistry, Los Angeles, CA, 90095, USA.
The Shapiro Family Laboratory of Viral Oncology and Aging Research, Los Angeles, CA, 90095, USA; Section of Restorative Dentistry, UCLA School of Dentistry, Los Angeles, CA, 90095, USA; UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA, 90095, USA.
Biochem Biophys Res Commun. 2022 May 21;605:56-62. doi: 10.1016/j.bbrc.2022.02.088. Epub 2022 Mar 9.
The epithelium is an integral part of barrier tissues, and plays a critical role in the initiation of the innate immune responses. The pro-inflammatory cytokine IL-36α has been previously reported to be strongly expressed during oral mucosal wound healing, but regulation of IL-36α expression and secretion in the oral mucosa are not well known. The objective of this study was to determine the types of stimuli that lead to expression and secretion of IL-36α in epithelial cells. Maxillary tissues from C57BL/6J mice during wound healing were utilized to identify endogenous expression of IL-36α, β, and γ in oral epithelial tissue. Immortalized HaCaT cells and primary normal human oral keratinocytes were subjected to Escherichia coli derived lipopolysaccharide (LPS), Poly(I:C), heat killed Candida albicans (HKCa), and mechanical damage. IL-36α and IL-1β levels in supernatant were assessed by sandwich ELISA, and expression of pro-inflammatory cytokines and IL-36 family genes were assessed by quantitative real-time PCR in HaCaT cells. Migration ability of keratinocytes was assessed with or without functional IL-36 signaling. IL-36α but not IL-36β or γ levels in the oral epithelium were elevated during wound healing. Treatment of epithelial cells with LPS, Poly(I:C), HKCa and mechanical damage revealed little to no soluble IL-36α in the media supernatant. However, sonication of the supernatant to disrupt the membranes of extracellular vesicles revealed a dose-dependent increase in IL-36α for each of the tested conditions. IL-1 superfamily genes were upregulated following mechanical damage in keratinocytes. Abrogation of IL-36 signaling led to severe inhibition of migration. Our data show for the first time that IL-36α is released primarily in extracellular vesicles by oral keratinocytes. Additionally, we show that IL-36α - but not IL-36β or γ - is upregulated in keratinocytes following mechanical damage, and that IL-36 signaling is important for keratinocyte migration.
上皮组织是屏障组织的重要组成部分，在启动先天免疫反应中起着关键作用。先前有研究报道，促炎细胞因子 IL-36α 在口腔黏膜伤口愈合过程中强烈表达，但 IL-36α 在口腔黏膜中的表达和分泌调控尚不清楚。本研究旨在确定导致上皮细胞表达和分泌 IL-36α 的刺激类型。利用 C57BL/6J 小鼠上颌组织在伤口愈合过程中鉴定口腔上皮组织中 IL-36α、β 和 γ 的内源性表达。将永生化 HaCaT 细胞和原代正常人口腔角质形成细胞分别用大肠杆菌来源的脂多糖（LPS）、聚肌胞（Poly(I:C)）、热灭活白色念珠菌（HKCa）和机械损伤处理。通过夹心 ELISA 测定上清液中 IL-36α 和 IL-1β 的水平，用定量实时 PCR 测定 HaCaT 细胞中促炎细胞因子和 IL-36 家族基因的表达。在有或没有功能性 IL-36 信号的情况下评估角质形成细胞的迁移能力。伤口愈合过程中，口腔上皮组织中 IL-36α 而非 IL-36β 或 γ 的水平升高。用 LPS、Poly(I:C)、HKCa 和机械损伤处理上皮细胞后，上清液中的可溶性 IL-36α 很少或没有。然而，用超声处理上清液破坏细胞外囊泡的膜后，每种检测条件下的 IL-36α 均呈剂量依赖性增加。机械损伤后，IL-1 超家族基因在角质形成细胞中上调。阻断 IL-36 信号导致迁移严重抑制。我们的数据首次表明，IL-36α 主要由口腔角质形成细胞通过细胞外囊泡释放。此外，我们表明机械损伤后角质形成细胞中 IL-36α 上调，而不是 IL-36β 或 γ 上调，并且 IL-36 信号对于角质形成细胞迁移很重要。
