Clostridioides difficile Toxin B subverts germinal center formation and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism. Clostridioides difficile Toxin B subverts germinal center formation and antibody recall responses by stimulating a drug-treatable CXCR4-dependent mechanism

Recurrent Clostridioides difficile infection (CDI) results in significant morbidity and mortality. We previously established that CDI in mice did not protect against reinfection and was associated with poor pathogen-specific B cell memory (Bmem), recapitulating our observations with human Bmem. Herein, we demonstrate that the secreted toxin TcdB2 is responsible for subversion of Bmem responses. TcdB2 from an endemic C. difficile strain delayed IgG class switch following vaccination, attenuated IgG recall to a vaccine booster, and prevented germinal center formation. The mechanism of TcdB2 action included increased B cell CXCR4 expression and responsiveness to its ligand CXCL12, accounting for altered cell migration and a failure of germinal center-dependent Bmem. These results were reproduced in a C. difficile infection model and an FDA-approved CXCR4-blocking drug rescued germinal center formation. We therefore provide mechanistic insights into C. difficile-associated pathogenesis and illuminate a target for clinical intervention to limit recurrent disease. Overall design: Female mice (n=4 per group) were given 200 µl PBS vehicle control, 10 ng D270N, or 10 ng TcdB2 in PBS by the s.c. route. Seven days post treatment, RNA was purified from aLNs and iLNs. Gene expression was quantified using the Nanostring™ nCounter SPRINT profiler platform.

复发性艰难梭菌感染（Recurrent Clostridioides difficile infection, CDI）可引发显著的发病率与死亡率。我们此前已证实，小鼠体内的CDI无法抵御再次感染，且与病原体特异性B细胞记忆（B cell memory, Bmem）功能低下相关，这与我们在人类Bmem中观察到的结果一致。本研究证实，分泌型毒素TcdB2是破坏Bmem应答的关键因子。源自流行艰难梭菌菌株的TcdB2可延缓疫苗接种后的IgG类别转换，削弱疫苗加强针诱导的IgG回忆应答，并抑制生发中心形成。TcdB2的作用机制包括上调B细胞CXCR4的表达，并增强其对配体CXCL12的响应性，这解释了细胞迁移异常以及生发中心依赖性Bmem形成失败的原因。上述结果在艰难梭菌感染模型中得到了重复验证，且一款经美国食品药品监督管理局（Food and Drug Administration, FDA）批准的CXCR4阻断药物可恢复生发中心的形成。因此，本研究为艰难梭菌相关发病机制提供了分子机制见解，并明确了可用于限制复发性疾病的临床干预靶点。
总体实验设计：将每组4只雌性小鼠经皮下（s.c.）途径注射200 μL PBS溶剂对照、10 ng D270N或10 ng TcdB2的PBS溶液。给药7天后，从腋窝淋巴结（axillary lymph nodes, aLNs）和腹股沟淋巴结（inguinal lymph nodes, iLNs）中纯化总RNA。采用Nanostring™ nCounter SPRINT基因表达分析平台对基因表达水平进行定量检测。