Abstract：
BACKGROUND: HLA-B*35:01 has been identified as a risk allele for Polygonum multiflorum Thunb.-induced liver injury (PMLI). However, the immune mechanism underlying HLA-B*35:01-mediated PMLI remains unknown.
OBJECTIVE: To characterize the immune mechanism of HLA-B*35:01-mediated PMLI.
METHODS: Components of P. multiflorum (PM) bound to the HLA-B*35:01 molecule was screened by immunoaffinity chromatography. Both wild-type mice and HLA-B*35:01 transgenic (TG) mice were treated with emodin. The levels of transaminases, histological changes and T-cell response were assessed. Splenocytes from emodin-treated mice were isolated and cultured in vitro. Phenotypes and functions of T cells were characterized upon drug restimulation using flow cytometry or ELISA. Emodin-pulsed antigen-presenting cells (APCs) or glutaraldehyde-fixed APCs were co-cultured with splenocytes from emodin-treated transgenic mice to detect their effect on T-cell activation.
RESULTS: Emodin, the main component of PM, could non-covalently bind to the HLA-B*35:01-peptide complexes. TG mice were more sensitive to emodin-induced immune hepatic injury, as manifested by elevated aminotransferase levels, infiltration of inflammatory cells, increased percentage of CD8+T cells and release of effector molecules in the liver. However, these effects were not observed in wild-type mice. An increase in percentage of T cells and the levels of interferon-γ, granzyme B, and perforin was detected in emodin-restimulated splenocytes from TG mice. Anti-HLA-I antibodies inhibited the secretion of these effector molecules induced by emodin. Mechanistically, emodin-pulsed APCs failed to stimulate T cells, while fixed APCs in the presence of emodin could elicit the secretion of T cell effector molecules.
CONCLUSIONS: The HLA-B*35:01-mediated CD8+ T cell reaction to emodin through the P-I mechanism may contribute to P. multiflorum-induced liver injury.
OBJECTIVE: To characterize the immune mechanism of HLA-B*35:01-mediated PMLI.
METHODS: Components of P. multiflorum (PM) bound to the HLA-B*35:01 molecule was screened by immunoaffinity chromatography. Both wild-type mice and HLA-B*35:01 transgenic (TG) mice were treated with emodin. The levels of transaminases, histological changes and T-cell response were assessed. Splenocytes from emodin-treated mice were isolated and cultured in vitro. Phenotypes and functions of T cells were characterized upon drug restimulation using flow cytometry or ELISA. Emodin-pulsed antigen-presenting cells (APCs) or glutaraldehyde-fixed APCs were co-cultured with splenocytes from emodin-treated transgenic mice to detect their effect on T-cell activation.
RESULTS: Emodin, the main component of PM, could non-covalently bind to the HLA-B*35:01-peptide complexes. TG mice were more sensitive to emodin-induced immune hepatic injury, as manifested by elevated aminotransferase levels, infiltration of inflammatory cells, increased percentage of CD8+T cells and release of effector molecules in the liver. However, these effects were not observed in wild-type mice. An increase in percentage of T cells and the levels of interferon-γ, granzyme B, and perforin was detected in emodin-restimulated splenocytes from TG mice. Anti-HLA-I antibodies inhibited the secretion of these effector molecules induced by emodin. Mechanistically, emodin-pulsed APCs failed to stimulate T cells, while fixed APCs in the presence of emodin could elicit the secretion of T cell effector molecules.
CONCLUSIONS: The HLA-B*35:01-mediated CD8+ T cell reaction to emodin through the P-I mechanism may contribute to P. multiflorum-induced liver injury.
摘要：
背景：HLA-B*35:01已被鉴定为何首乌的风险等位基因。-诱导的肝损伤(PMLI)。然而,HLA-B*35:01介导的PMLI的免疫机制尚不清楚.
目的：表征HLA-B*35:01介导的PMLI的免疫机制。
方法：用免疫亲和层析法筛选与HLA-B*35:01分子结合的多花假单胞菌(PM)的组分。野生型小鼠和HLA-B*35:01转基因(TG)小鼠均用大黄素处理。转氨酶的水平，评估组织学变化和T细胞反应。分离大黄素处理的小鼠的脾细胞并在体外培养。使用流式细胞术或ELISA在药物再刺激时表征T细胞的表型和功能。将大黄素脉冲的抗原呈递细胞（APC）或戊二醛固定的APC与大黄素处理的转基因小鼠的脾细胞共培养，以检测它们对T细胞活化的影响。
结果：大黄素，PM的主要组成部分，可以非共价结合HLA-B*35:01-肽复合物。TG小鼠对大黄素诱导的免疫性肝损伤更为敏感,表现为转氨酶水平升高，炎症细胞浸润，CD8+T细胞的百分比增加和肝脏中效应分子的释放。然而,在野生型小鼠中未观察到这些效应。T细胞百分比和干扰素-γ水平的增加，颗粒酶B，在TG小鼠大黄素再刺激的脾细胞中检测到穿孔素。抗HLA-I抗体抑制大黄素诱导的这些效应分子的分泌。机械上,大黄素脉冲的APC不能刺激T细胞，而在大黄素存在下固定的APC可以引起T细胞效应分子的分泌。
结论：HLA-B*35:01通过P-I机制介导的CD8+T细胞对大黄素的反应可能有助于多花假单胞菌诱导的肝损伤。
目的：表征HLA-B*35:01介导的PMLI的免疫机制。
方法：用免疫亲和层析法筛选与HLA-B*35:01分子结合的多花假单胞菌(PM)的组分。野生型小鼠和HLA-B*35:01转基因(TG)小鼠均用大黄素处理。转氨酶的水平，评估组织学变化和T细胞反应。分离大黄素处理的小鼠的脾细胞并在体外培养。使用流式细胞术或ELISA在药物再刺激时表征T细胞的表型和功能。将大黄素脉冲的抗原呈递细胞（APC）或戊二醛固定的APC与大黄素处理的转基因小鼠的脾细胞共培养，以检测它们对T细胞活化的影响。
结果：大黄素，PM的主要组成部分，可以非共价结合HLA-B*35:01-肽复合物。TG小鼠对大黄素诱导的免疫性肝损伤更为敏感,表现为转氨酶水平升高，炎症细胞浸润，CD8+T细胞的百分比增加和肝脏中效应分子的释放。然而,在野生型小鼠中未观察到这些效应。T细胞百分比和干扰素-γ水平的增加，颗粒酶B，在TG小鼠大黄素再刺激的脾细胞中检测到穿孔素。抗HLA-I抗体抑制大黄素诱导的这些效应分子的分泌。机械上,大黄素脉冲的APC不能刺激T细胞，而在大黄素存在下固定的APC可以引起T细胞效应分子的分泌。
结论：HLA-B*35:01通过P-I机制介导的CD8+T细胞对大黄素的反应可能有助于多花假单胞菌诱导的肝损伤。
