Center for Biomedical Science The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY, 11030, USA.
Center for Molecular Innovation, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, NY, 11030, USA.
Mol Med. 2019 Apr 11;25(1):13. doi: 10.1186/s10020-019-0081-6.
Extracellular high mobility group box 1 protein (HMGB1) serves a central role in inflammation as a transporter protein, which binds other immune-activating molecules that are endocytosed via the receptor for advanced glycation end-products (RAGE). These pro-inflammatory complexes are targeted to the endolysosomal compartment, where HMGB1 permeabilizes the lysosomes. This enables HMGB1-partner molecules to avoid degradation, to leak into the cytosol, and to reach cognate immune-activating sensors. Lipopolysaccharide (LPS) requires this pathway to generate pyroptosis by accessing its key cytosolic receptors, murine caspase 11, or the human caspases 4 and 5. This lytic, pro-inflammatory cell death plays a fundamental pathogenic role in gram-negative sepsis. The aim of the study was to identify molecules inhibiting HMGB1 or HMGB1/LPS cellular internalization.
Endocytosis was studied in cultured macrophages using Alexa Fluor-labeled HMGB1 or complexes of HMGB1 and Alexa Fluor-labeled LPS in the presence of an anti-HMGB1 monoclonal antibody (mAb), recombinant HMGB1 box A protein, acetylcholine, the nicotinic acetylcholine receptor subtype alpha 7 (α7 nAChR) agonist GTS-21, or a dynamin-specific inhibitor of endocytosis. Images were obtained by fluorescence microscopy and quantified by the ImageJ processing program (NIH). Data were analyzed using student's t test or one-way ANOVA followed by the least significant difference or Tukey's tests.
Anti-HMGB1 mAb, recombinant HMGB1 antagonist box A protein, acetylcholine, GTS-21, and the dynamin-specific inhibitor of endocytosis inhibited internalization of HMGB1 or HMGB1-LPS complexes in cultured macrophages. These agents prevented macrophage activation in response to HMGB1 and/or HMGB1-LPS complexes.
These results demonstrate that therapies based on HMGB1 antagonists and the cholinergic anti-inflammatory pathway share a previously unrecognized molecular mechanism of substantial clinical relevance.
细胞外高迁移率族蛋白 B1 (HMGB1)作为一种转运蛋白,在炎症中起着核心作用,它结合其他通过晚期糖基化终产物受体(RAGE)内化的免疫激活分子。这些促炎复合物被靶向到内溶酶体隔室,在那里 HMGB1 使溶酶体通透。这使得 HMGB1 伴侣分子能够避免降解,漏入细胞质,并到达同源免疫激活传感器。脂多糖(LPS)需要通过进入其关键的细胞质受体,即鼠 caspase 11 或人 caspase 4 和 5,来生成细胞焦亡。这种裂解的、促炎的细胞死亡在革兰氏阴性菌败血症中起着根本的致病作用。本研究的目的是鉴定抑制 HMGB1 或 HMGB1/LPS 细胞内化的分子。
在存在抗 HMGB1 单克隆抗体(mAb)、重组 HMGB1 盒 A 蛋白、乙酰胆碱、烟碱型乙酰胆碱受体亚型α7(α7 nAChR)激动剂 GTS-21 或内吞作用的特异性抑制剂 dynasore 的情况下,用 Alexa Fluor 标记的 HMGB1 或 HMGB1 和 Alexa Fluor 标记的 LPS 复合物研究培养的巨噬细胞中的内吞作用。通过荧光显微镜获取图像,并使用 NIH 的 ImageJ 处理程序进行量化。使用学生 t 检验或单向方差分析(ANOVA),然后是最小显著差异或 Tukey 检验分析数据。
抗 HMGB1 mAb、重组 HMGB1 拮抗剂盒 A 蛋白、乙酰胆碱、GTS-21 和内吞作用的特异性抑制剂抑制了培养的巨噬细胞中 HMGB1 或 HMGB1-LPS 复合物的内化。这些药物阻止了巨噬细胞对 HMGB1 和/或 HMGB1-LPS 复合物的反应性激活。
这些结果表明,基于 HMGB1 拮抗剂和胆碱能抗炎途径的治疗方法具有以前未被认识到的、具有重要临床相关性的分子机制。