Center for Musculoskeletal Research, University of Rochester Medical Center, Rochester, NY, United States.
Department of Pathology & Laboratory Medicine, University of Rochester Medical Center, Rochester, NY, United States.
Front Immunol. 2023 Aug 25;14:1237498. doi: 10.3389/fimmu.2023.1237498. eCollection 2023.
Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model.
Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation.
Advanced PLN sinuses exhibited an increased expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; ) that significantly correlated with reduced talus bone volumes in the afferent ankle (R 0.54, ). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b plasma cells localized in MARCO peri-follicular medullary sinuses. A concomitant decreased expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, ) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM macrophages and CD6 T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching.
Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.
在严重类风湿关节炎（RA）患者中，炎症（Bin）关节引流淋巴结窦中的淋巴引流缺陷和 B 细胞易位是致病现象。然而，这种淋巴功能障碍的分子机制仍知之甚少。在此，我们利用多组学空间和单细胞转录组学，利用肿瘤坏死因子转基因（TNF-Tg）小鼠模型，评估 RA 发展过程中关节引流淋巴结窦中细胞组成（包括淋巴内皮细胞、巨噬细胞、B 细胞和 T 细胞）的改变及其相关表型变化和细胞-细胞相互作用。
从野生型（n=10）和 TNF-Tg 雄性小鼠的腘窝淋巴结（PLN）中采集早期（5-6 月龄；n=6）和晚期（>8 月龄；n=12）关节炎的 PLN 进行空间转录组学分析。在 TNF-Tg 队列的 PLN 中进行单细胞 RNA 测序（scRNAseq）（每个队列 6 个 PLN 混合/合并）。进行 PLN 组织病理学和 ELISPOT 以及踝关节组织学和微 CT 评估。进行人类淋巴结和滑膜的组织病理学检查以进行临床相关性分析。
晚期 PLN 窦中表达比例增加（早期 0.5±0.1 与晚期 1.4±0.5 计数/计数；），与踝关节传入的距骨骨体积减少显著相关（R=0.54，）。单细胞和空间转录组学的整合揭示了定位在 MARCO 滤泡间髓窦中的增加的 IgG2b 浆细胞。晚期 PLN 窦中表达减少（早期 2.5±0.74 与晚期 1.0±0.50 计数，）与晚期 TNF-Tg 小鼠淋巴结和滑膜中铁负荷普鲁士蓝阳性巨噬细胞的积累相关，并在 RA 临床样本中进一步验证。晚期 PLN 中的 T 细胞增加了 8 倍，生物信息学途径评估确定了 ALCAM 巨噬细胞和 CD6 T 细胞之间的相互作用是促进 IgG2b 类别转换的可能共刺激机制。
综上所述，这些数据支持慢性 TNF 诱导的关节炎发作的模型，其中受累关节引流淋巴结的淋巴流动丧失促进了流出巨噬细胞与 T 细胞之间通过 ALCAM-CD6 共刺激的相互作用，启动了扩展的 Bin 群体的 IgG2b 类别转换和浆细胞分化。有必要进一步研究免疫球蛋白的克隆性和潜在的自身免疫后果，以及抗 CD6 治疗预防这些致病事件的疗效。
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