Department of Dermatology, Ehime University Graduate School of Medicine, Ehime, Japan.
Department of Dermatology, Ehime University Graduate School of Medicine, Ehime, Japan.
J Dermatol Sci. 2018 May;90(2):154-165. doi: 10.1016/j.jdermsci.2018.01.007. Epub 2018 Feb 2.
High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes DNA and facilitates gene transcription. Additionally, cell stress or death induces the release of HMGB1 outside the cell membrane, where HMGB1 functions as an alarmin, causing an inflammatory response in combination with other cytokines, damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs).
To evaluate the effect of reduced-HMGB1 (previously termed chemoattractive-HMGB1) on polyinosine-polycytidylic acid [poly(I:C)]-induced inflammation in normal human keratinocytes (NHKs).
We focused on downstream components of the poly(I:C)-Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) pathways, including IκBα, nuclear factor (NF)-κB p65, mitogen-activated protein kinase (MAPK), and interferon regulatory factor 3 (IRF3), and assessed whether these pathways are involved in the suppression of poly(I:C)-induced inflammation in NHKs by HMGB1. An immunoprecipitation was performed to know whether HMGB1 could bind to poly(I:C), and immunofluorescence staining and flow cytometric analysis were performed to check whether reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis).
Application of exogenous HMGB1 before, but not after, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs. In addition, reduced-HMGB1, but not disulfide-HMGB1, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs, suggesting the importance of the redox status of exogenous HMGB1. Pre-treatment with reduced-HMGB1 inhibited the phosphorylation of IκBα, NF-κB p65, and IRF3 induced by poly(I:C) stimulation in NHKs; however, phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was unaffected. Disulfide-HMGB1 formed a complex with poly(I:C), as did reduced- and oxidized-HMGB1, albeit to a lesser extent. Immunofluorescence staining and flow cytometric analysis indicated that reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis).
These findings suggest that pre-treatment with reduced-HMGB1 ameliorates poly(I:C)-mediated inflammation in NHKs.
高迁移率族蛋白 B1（HMGB1）是一种稳定 DNA 并促进基因转录的核蛋白。此外，细胞应激或死亡会诱导 HMGB1 从细胞膜外释放，HMGB1 在此作为警报素发挥作用，与其他细胞因子、损伤相关分子模式（DAMPs）和病原体相关分子模式（PAMPs）一起引起炎症反应。
评估低 HMGB1（以前称为趋化性-HMGB1）对正常人角质形成细胞（NHKs）中多聚肌苷酸-多聚胞苷酸[poly(I:C)]诱导的炎症的影响。
我们专注于 poly(I:C)-Toll 样受体 3（TLR3）、视黄酸诱导基因-I（RIG-I）和黑色素瘤分化相关蛋白 5（MDA5）途径的下游成分，包括 IκBα、核因子（NF）-κB p65、丝裂原活化蛋白激酶（MAPK）和干扰素调节因子 3（IRF3），并评估这些途径是否参与 HMGB1 抑制 NHKs 中 poly(I:C)诱导的炎症。进行免疫沉淀以了解 HMGB1 是否可以与 poly(I:C)结合，进行免疫荧光染色和流式细胞术分析以检查还原 HMGB 是否干扰 poly(I:C)的细胞摄取和易位（可能通过内吞作用）。
外源性 HMGB1 在施加之前而非之后施加对 NHKs 中的 poly(I:C)诱导的炎症具有抑制作用。此外，还原 HMGB1 而不是二硫键 HMGB1 对 NHKs 中的 poly(I:C)诱导的炎症具有抑制作用，表明外源性 HMGB1 的氧化还原状态很重要。还原 HMGB1 预处理抑制了 poly(I:C)刺激诱导的 NHKs 中 IκBα、NF-κB p65 和 IRF3 的磷酸化；然而，p38、细胞外信号调节激酶（ERK）和 c-Jun N-末端激酶（JNK）的磷酸化不受影响。二硫键 HMGB1 与 poly(I:C)形成复合物，还原 HMGB1 和氧化 HMGB1 也是如此，尽管程度较轻。免疫荧光染色和流式细胞术分析表明，还原 HMGB 干扰 poly(I:C)易位的细胞摄取（可能通过内吞作用）。
这些发现表明，还原 HMGB1 预处理可改善 NHKs 中的 poly(I:C)介导的炎症。
