Huntington’s disease (HD) is caused by the expression of a mutated variant of Huntingtin (mHtt), which results in the complex pathology characterized by a defective function of the nervous system and altered inflammatory responses. While the neuronal effects of mHtt expression have been extensively studied, its effects on the physiology of immune cells have not been fully described. Mast cells (MCs) are unique tissue-resident immune cells whose activation has been linked to protective responses against parasites and bacteria, but also to deleterious inflammatory allergic reactions and, recently, to neurodegenerative diseases. Bone marrow-derived mast cells (BMMCs) were obtained from wild-type (WT-) and mHtt-expressing (R6/1) mice to evaluate the main activation parameters triggered by the high-affinity IgE receptor (FcεRI) and the Toll-like receptor (TLR) 4. Degranulation was assessed by measuring the secretion of β-hexosaminidase, MAP kinase activation was detected by Western blot, and cytokine production was determined by RT-PCR and ELISA. TLR-4 receptor and Htt vesicular trafficking was analyzed by confocal microscopy. In vivo, MC-deficient mice (c-KitWsh/Wsh) were intraperitonally reconstituted with WT or R6/1 BMMCs and the TLR4-induced production of the tumor necrosis factor (TNF) was determined by ELISA. A survival curve of mice treated with a sub-lethal dose of bacterial lipopolysaccharide (LPS) was constructed. R6/1 BMMCs showed normal β-hexosaminidase release levels in response to FcεRI, but lower cytokine production upon LPS stimulus. Impaired TLR4-induced TNF production was associated to the lack of intracellular dynamin-dependent TLR-4 receptor trafficking to perinuclear regions in BMMCs, a diminished ERK1/2 and ELK-1 phosphorylation, and a decrease in c-fos and TNF mRNA accumulation. R6/1 BMMCs also failed to produce TLR4-induced anti-inflammatory cytokines (like IL-10 and TGF-β). The detected defects were also observed in vivo, in a MCs-dependent model of endotoxemia. R6/1 and c-KitWsh/Wsh mice reconstituted with R6/1 BMMCs showed a decreased TLR4-induced TNF production and lower survival rates to LPS challenge than WT mice. Our data show that mHtt expression causes an impaired production of pro- and anti-inflammatory mediators triggered by TLR-4 receptor in MCs in vitro and in vivo, which could contribute to the aberrant immunophenotype observed in HD.

中文翻译：

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突变亨廷顿蛋白影响肥大细胞中 Toll 样受体 4 的细胞内运输和细胞因子的产生


亨廷顿病 (HD) 是由亨廷顿蛋白 (mHtt) 突变体的表达引起的，其导致以神经系统功能缺陷和炎症反应改变为特征的复杂病理学。虽然 mHtt 表达对神经元的影响已被广泛研究，但其对免疫细胞生理学的影响尚未得到充分描述。肥大细胞（MC）是独特的组织驻留免疫细胞，其激活与针对寄生虫和细菌的保护性反应有关，但也与有害的炎症过敏反应以及最近的神经退行性疾病有关。从野生型 (WT-) 和 mHtt 表达 (R6/1) 小鼠中获得骨髓源性肥大细胞 (BMMC)，以评估高亲和力 IgE 受体 (FcεRI) 和 Toll- 触发的主要激活参数。样受体(TLR) 4.通过测量β-己糖胺酶的分泌来评估脱颗粒，通过蛋白质印迹检测MAP激酶激活，并通过RT-PCR和ELISA测定细胞因子的产生。通过共聚焦显微镜分析 TLR-4 受体和 Htt 囊泡运输。在体内，用 WT 或 R6/1 BMMC 腹膜内重建 MC 缺陷小鼠 (c-KitWsh/Wsh)，并通过 ELISA 测定 TLR4 诱导的肿瘤坏死因子 (TNF) 的产生。绘制了用亚致死剂量的细菌脂多糖（LPS）治疗的小鼠的存活曲线。 R6/1 BMMC 在响应 FcεRI 时表现出正常的 β-己糖胺酶释放水平，但在 LPS 刺激下细胞因子的产生较低。 TLR4 诱导的 TNF 产生受损与 BMMC 中缺乏细胞内动力依赖性 TLR-4 受体运输至核周区域、ERK1/2 和 ELK-1 磷酸化减少以及 c-fos 和 TNF mRNA 积累减少有关。 R6/1 BMMC 也无法产生 TLR4 诱导的抗炎细胞因子（如 IL-10 和 TGF-β）。检测到的缺陷也在体内的 MCs 依赖性内毒素血症模型中观察到。与 WT 小鼠相比，用 R6/1 BMMC 重建的 R6/1 和 c-KitWsh/Wsh 小鼠显示 TLR4 诱导的 TNF 产生减少，并且 LPS 攻击的存活率较低。我们的数据表明，mHtt 表达会导致体外和体内 MC 中 TLR-4 受体触发的促炎和抗炎介质产生受损，这可能导致 HD 中观察到的异常免疫表型。