Cellular and genetic factors involved in the difference among rat strains to develop procainamide - induced Autoimmunity

To examine the influence cellular and genetics on the procainamide-induced autoimmune response in spleens we compared rats that are genetically Th2-predisposed (Brown Norway), Th1-predisposed (Lewis) or not genetically predisposed (Sprague Dawley). Rats were treated with procainamide three times per week. From the second week, blood samples were taken once a week for antinuclear antibody (ANA) detection. At 56d after treatment, rats were sacrificed and spleens samples were collected for microarray test and histopathology examination. Frequencies of T cell subsets and B cells in rat spleen were measured to assess the effects on splenocyte. Also, 12 serum cytokines/chemokines were determined to explored the Th1/Th2 cytokine pattern after treatment. We found that ANA were makedly elevated in BN and SD rats, while the elevated appeared earlier in BN rats, and the magnitude of its increase were much higher than that of SD rats. There was no marked change in serum ANA in Lewis rats. Histopathological analysis indicated that spleen weight increased significantly both in BN and SD rats after stimulated with procainamide. No significant changes in spleen weight and lesions were observed in Lewis rats. We also found the percentage of CD86 positive cells in spleen of BN rats was significantly increased, while the percentage of CD4+CD25+ cells was decreased in BN rats. In addition, percentage of CD11b/c positive cells were decreased, and the levels of Th-2 (IL-10), Th-1 type cytokine (IFN-γ) and chemokine (IL-1β) in serum were markedly elevated both in BN and SD rats after treatment. Th-2 type cytokine (IL-4, IL-6) in serum were markedly increased only in BN rats. Furthermore, similar immune mechanisms were found in BN and SD rat, and the altered genes were mainly associated in immune response, and inflammatory response. However, the number of differentially expressed genes (DEGs) in BN rats was higher than that in SD rats. Overall, we revealed significant differences in response to autoimmunity induced by procainamide among three strains rats, the BN rats was the most sensitive one, SD rats exhibited less sensitive while Lewis resistance to procainamide. Much more pronouncedof Th2-type responses and more complex DEGs involved in immune regulation and response in BN rats might contribute to its susceptible to drug-induced lupus erythematosus. Moreover, similar immune mechanisms were found between BN and SD rat, which suggesting that these changes would served as the potential bridge biomarkers to predict drug-induced autoimmune reactions among species. The results may also provide a scientic basis for the high sensitivity of BN rats in prediction immunotoxicity in preclinical studies.为探究细胞与遗传因素对普鲁卡因胺诱导的脾脏自身免疫应答的影响，本研究选取三类遗传背景各异的大鼠：遗传倾向于辅助性T细胞2（Th2）型免疫的布朗挪威（Brown Norway, BN）大鼠、遗传倾向于辅助性T细胞1（Th1）型免疫的刘易斯（Lewis）大鼠，以及无该免疫倾向的斯普拉格-道利（Sprague Dawley, SD）大鼠开展对照实验。所有大鼠每周接受三次普鲁卡因胺给药。自给药第二周起，每周采集一次血液样本，用于抗核抗体（antinuclear antibody, ANA）检测。给药后第56天，处死所有大鼠并采集脾脏样本，用于基因芯片（microarray）检测与组织病理学检查。同时检测大鼠脾脏中T细胞亚群与B细胞的占比，以评估给药对脾淋巴细胞的影响。此外，本研究还定量检测了12种血清细胞因子/趋化因子的表达水平，以分析给药后Th1/Th2型细胞因子的表达模式。研究结果显示，BN与SD大鼠的血清ANA水平均显著升高，其中BN大鼠的ANA升高出现更早，且升高幅度显著高于SD大鼠；而刘易斯大鼠的血清ANA水平无显著变化。组织病理学分析表明，经普鲁卡因胺刺激后，BN与SD大鼠的脾脏重量均显著增加，而刘易斯大鼠的脾脏重量与病理损伤均未出现显著改变。此外，BN大鼠脾脏中CD86阳性细胞的占比显著升高，CD4+CD25+细胞的占比则有所降低；同时其脾脏中CD11b/c阳性细胞的占比下降。给药后，BN与SD大鼠的血清Th2型细胞因子（白细胞介素10, IL-10）、Th1型细胞因子（干扰素γ, IFN-γ）以及趋化因子（白细胞介素1β, IL-1β）水平均显著升高，而血清中Th2型细胞因子IL-4、IL-6的水平仅在BN大鼠中出现显著上调。进一步分析发现，BN与SD大鼠的免疫调控机制相似，其差异表达基因（differentially expressed genes, DEGs）主要富集于免疫应答与炎症应答通路，但BN大鼠的DEGs数量显著多于SD大鼠。综上，三类大鼠对普鲁卡因胺诱导的自身免疫应答存在显著差异：BN大鼠最为敏感，SD大鼠敏感性次之，刘易斯大鼠则对该药物诱导的自身免疫具有抵抗性。BN大鼠中更为显著的Th2型免疫应答，以及参与免疫调控与应答的更为复杂的DEGs，可能是其易患药物诱导性红斑狼疮的核心原因。此外，BN与SD大鼠的相似免疫调控机制提示，上述变化可作为潜在的跨物种生物标志物，用于预测药物诱导的自身免疫反应。本研究结果还可为临床前研究中利用BN大鼠的高敏感性预测免疫毒性提供科学依据。