Table_4_Sphingosine-1-Phosphate Receptor 4 links neutrophils and early local inflammation to lymphocyte recruitment into the draining lymph node to facilitate robust germinal center formation.docx

The successful development of germinal centers (GC) relies heavily on innate mechanisms to amplify the initial inflammatory cascade. In addition to their role in antigen presentation, innate cells are essential for the redirection of circulating lymphocytes toward the draining lymph node (dLN) to maximize antigen surveillance. Sphingosine-1-Phosphate (S1P) and its receptors (S1PR1-5) affect various aspects of immunity; however, the role of S1PR4 in regulating an immune response is not well understood. Here we use a footpad model of localized TH1 inflammation to carefully monitor changes in leukocyte populations within the blood, the immunized tissue, and the dLN. Within hours of immunization, neutrophils failed to adequately mobilize and infiltrate into the footpad tissue of S1PR4-/- mice, thereby diminishing the local vascular changes thought to be necessary for redirecting circulating cells toward the inflamed region. Neutrophil depletion with anti-Ly6G antibodies significantly reduced early tissue edema as well as the redirection and initial accumulation of naïve lymphocytes in dLN of WT mice, while the effects were less prominent or absent in S1PR4-/- dLN. Adoptive transfer experiments further demonstrated that the lymphocyte homing deficiencies in vivo were not intrinsic to the donor S1PR4-/- lymphocytes, but were instead attributed to differences within the S1PR4-deficient host. Reduced cell recruitment in S1PR4-/- mice would seed the dLN with fewer antigen-respondent lymphocytes and indeed, dLN hypertrophy at the peak of the immune response was severely diminished, with attenuated GC and activation pathways in these mice. Histological examination of the S1PR4-/- dLN also revealed an underdeveloped vascular network with reduced expression of the leukocyte tethering ligand, PNAd, within high endothelial venule regions, suggesting inadequate growth of the dLN meant to support a robust GC response. Thus, our study reveals that S1PR4 may link early immune modulation by neutrophils to the initial recruitment of circulating lymphocytes and downstream expansion and maturation of the dLN, thereby contributing to optimal GC development during an adaptive response.

生发中心（germinal centers, GC）的成功构建高度依赖先天机制以放大初始炎症级联反应。除抗原呈递功能外，先天免疫细胞对于将循环淋巴细胞重定向至引流淋巴结（draining lymph node, dLN）以最大化抗原监视效能同样不可或缺。1-磷酸鞘氨醇（Sphingosine-1-Phosphate, S1P）及其受体（S1PR1-5）可调控免疫应答的多个环节，但目前S1PR4在免疫应答调控中的作用仍有待阐明。本研究采用局部TH1炎症足垫模型，系统性监测血液、免疫组织及引流淋巴结内白细胞群体的动态变化。免疫后数小时内，S1PR4基因敲除（S1PR4-/-）小鼠的中性粒细胞无法正常动员并浸润足垫组织，由此削弱了被认为是将循环细胞重定向至炎症区域所必需的局部血管重塑过程。使用抗Ly6G抗体耗竭中性粒细胞，可显著降低野生型（wild type, WT）小鼠的早期组织水肿，同时减少初始淋巴细胞在引流淋巴结内的重定向与初始聚集；而该效应在S1PR4-/-小鼠的引流淋巴结中则表现不明显或未出现。过继转移实验进一步证实，体内淋巴细胞归巢缺陷并非源于供体S1PR4-/-淋巴细胞本身，而是由S1PR4缺陷宿主的微环境差异所导致。S1PR4-/-小鼠的细胞招募能力减弱，将导致引流淋巴结内抗原应答淋巴细胞的定植量减少；事实上，免疫应答峰值时期的引流淋巴结增生显著受损，小鼠体内的生发中心形成与激活通路均受到抑制。对S1PR4-/-小鼠引流淋巴结的组织学检查还显示，其血管网络发育不全，高内皮微静脉区域内的白细胞拴留配体——外周淋巴结地址素（PNAd）的表达水平降低，提示引流淋巴结发育不足，无法支持强健的生发中心应答。综上，本研究揭示S1PR4可将中性粒细胞介导的早期免疫调控与循环淋巴细胞的初始招募、下游引流淋巴结的扩增与成熟相联系，从而在适应性免疫应答过程中促进生发中心的最优形成。