Division of Neuroimmunology and Neuroinfectious Disease, Department of Neurology, Massachusetts General Hospital (MGH), Boston, United States.
Genetics and Aging Research Unit, McCance Center for Brain Health, Department of Neurology, Massachusetts General Hospital (MGH), Boston, MA, United States.
Front Immunol. 2023 Jul 12;14:1177672. doi: 10.3389/fimmu.2023.1177672. eCollection 2023.
Encephalitis is a devastating neurologic disorder with high morbidity and mortality. Autoimmune causes are roughly as common as infectious ones. N-methyl-D-aspartic acid receptor (NMDAR) encephalitis (NMDARE), characterized by serum and/or spinal fluid NMDAR antibodies, is the most common form of autoimmune encephalitis (AE). A translational rodent NMDARE model would allow for pathophysiologic studies of AE, leading to advances in the diagnosis and treatment of this debilitating neuropsychiatric disorder. The main objective of this work was to identify optimal active immunization conditions for NMDARE in mice.
Female C57BL/6J mice aged 8 weeks old were injected subcutaneously with an emulsion of complete Freund's adjuvant, killed and dessicated , and a 30 amino acid peptide flanking the NMDAR GluN1 subunit N368/G369 residue targeted by NMDARE patients' antibodies. Three different induction methods were examined using subcutaneous injection of the peptide emulsion mixture into mice in 1) the ventral surface, 2) the dorsal surface, or 3) the dorsal surface with reimmunization at 4 and 8 weeks (boosted). Mice were bled biweekly and sacrificed at 2, 4, 6, 8, and 14 weeks. Serum and CSF NMDAR antibody titer, mouse behavior, hippocampal cell surface and postsynaptic NMDAR cluster density, and brain immune cell entry and cytokine content were examined.
All immunized mice produced serum and CSF NMDAR antibodies, which peaked at 6 weeks in the serum and at 6 (ventral and dorsal boosted) or 8 weeks (dorsal unboosted) post-immunization in the CSF, and demonstrated decreased hippocampal NMDAR cluster density by 6 weeks post-immunization. In contrast to dorsally-immunized mice, ventrally-induced mice displayed a translationally-relevant phenotype including memory deficits and depressive behavior, changes in cerebral cytokines, and entry of T-cells into the brain at the 4-week timepoint. A similar phenotype of memory dysfunction and anxiety was seen in dorsally-immunized mice only when they were serially boosted, which also resulted in higher antibody titers.
Our study revealed induction method-dependent differences in active immunization mouse models of NMDARE disease. A novel ventrally-induced NMDARE model demonstrated characteristics of AE earlier compared to dorsally-induced animals and is likely suitable for most short-term studies. However, boosting and improving the durability of the immune response might be preferred in prolonged longitudinal studies.
脑炎是一种具有高发病率和死亡率的严重神经系统疾病。其病因大致可分为感染性和自身免疫性两类。N-甲基-D-天冬氨酸受体（NMDAR）脑炎（NMDARE）的特征是血清和/或脑脊液中存在 NMDAR 抗体，是最常见的自身免疫性脑炎（AE）形式。一种转化型啮齿动物 NMDARE 模型将允许对 AE 的病理生理学进行研究，从而推动对这种使人衰弱的神经精神疾病的诊断和治疗。本研究的主要目的是确定 NMDARE 在小鼠中进行主动免疫的最佳条件。
8 周龄雌性 C57BL/6J 小鼠经皮下注射完全弗氏佐剂乳剂、处死并干燥后，用针对 NMDARE 患者抗体靶向的 NMDAR GluN1 亚基 N368/G369 残基的 30 个氨基酸肽进行免疫。使用皮下注射肽乳剂混合物的 3 种不同诱导方法对小鼠进行免疫：1）腹部表面、2）背部表面或 3）背部表面，在第 4 周和第 8 周（加强）进行再免疫。每两周采集一次血样，在第 2、4、6、8 和 14 周时处死小鼠。检测血清和 CSF NMDAR 抗体滴度、小鼠行为、海马细胞表面和突触后 NMDAR 簇密度以及脑免疫细胞浸润和细胞因子含量。
所有免疫小鼠均产生血清和 CSF NMDAR 抗体，血清中的抗体滴度在第 6 周达到峰值，CSF 中的抗体滴度在第 6 周（腹部和背部加强）或第 8 周（背部未加强）达到峰值，免疫后 6 周海马 NMDAR 簇密度下降。与背部免疫的小鼠相比，腹部诱导的小鼠表现出具有翻译相关性的表型，包括记忆缺陷和抑郁行为、大脑细胞因子的变化以及 T 细胞在第 4 周进入大脑。只有当背部免疫的小鼠进行连续加强时，才会出现类似的记忆功能障碍和焦虑表型，且这也会导致更高的抗体滴度。
本研究揭示了 NMDARE 疾病主动免疫小鼠模型中诱导方法的差异。一种新的腹部诱导 NMDARE 模型比背部诱导的动物更早表现出 AE 的特征，可能更适合大多数短期研究。然而，在长期纵向研究中，加强免疫反应并提高其耐久性可能更有优势。
