Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan; Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Tokyo, Japan.
Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo, Japan; Faculty of International Liberal Arts, Juntendo University, Tokyo, Japan.
J Dermatol Sci. 2017 Oct;88(1):117-125. doi: 10.1016/j.jdermsci.2017.05.008. Epub 2017 May 16.
In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown.
To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism.
Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit.
Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP.
Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes.
除了具有杀菌特性外，宿主防御肽 (HDP) 还具有多种免疫调节功能，包括角质形成细胞产生细胞因子/趋化因子、增殖、迁移和伤口愈合。最近，一种新型 HDP 名为 AMP-IBP5（来源于胰岛素样生长因子结合蛋白 5 的抗菌肽）被证明具有针对多种病原体的抗菌活性，即使在与人类 β-防御素和 LL-37 相当的浓度下也是如此。然而，AMP-IBP5 在皮肤组织中的免疫调节作用尚不清楚。
研究 AMP-IBP5 是否触发角质形成细胞激活，并阐明其机制。
通过适当的 ELISA 试剂盒测定细胞因子/趋化因子和生长因子的产生。通过体外伤口闭合测定评估细胞迁移，而通过 BrdU 掺入测定和 XTT 测定分析细胞增殖。通过 Western 印迹测定 MAPK 和 NF-κB 的激活。通过 cAMP 酶免疫测定试剂盒评估细胞内 cAMP 水平。
在所测试的各种细胞因子/趋化因子和生长因子中，AMP-IBP5 选择性地增加了 IL-8 和 VEGF 的产生。此外，AMP-IBP5 显著增强了角质形成细胞的迁移和增殖。AMP-IBP5 诱导的角质形成细胞激活是通过 Mrg X1-X4 受体介导的，MAPK 和 NF-κB 途径作为下游途径起作用，这可以通过 MrgX1-X4 siRNA 和 ERK、JNK、p38 和 NF-κB 特异性抑制剂的抑制作用来证明。我们证实 AMP-IBP5 确实诱导了 MAPK 和 NF-κB 的激活。此外，AMP-IBP5 诱导的 VEGF 而不是 IL-8 的产生与细胞内 cAMP 的增加相关。
我们的研究结果表明，除了其抗菌功能外，AMP-IBP5 还可以通过激活角质形成细胞来促进伤口愈合过程。
