Microbial sensing and innate immune activation in B cells

B细胞最为人所知的是它们产生抗体的能力。然而，越来越多的文献表明，它们也可能通过各种不依赖抗体的机制参与免疫反应，如细胞因子的产生、共刺激和抗原递呈。B细胞可分为三个不同的亚群：滤泡型B细胞、先天样边缘带B细胞(MZB)和B1B细胞。滤泡B细胞是最重要的B细胞亚群，存在于第二、三级淋巴器官的滤泡中。MZB位于脾的边缘地带，具有结合免疫复合体、向脾白髓迁移、将抗原转移到滤泡树突状细胞的能力。B1B细胞以分泌天然抗体而闻名。 我们最近报道了B细胞可以捕获原生动物寄生虫杜氏利什曼原虫。一旦暴露在寄生虫中，B细胞就会形成簇，并将多诺瓦尼乳杆菌固定在细胞外富含IgM的口袋中。这种密切的相互作用导致多克隆B细胞的激活，并最终导致24-48小时后的细胞死亡。有趣的是，这种寄生虫触发内体Toll样受体(TLRs)，尽管它附着在细胞外的B细胞表面。需要激活内体TLRs来诱导IL-10和干扰素-I的表达，并促进高丙种球蛋白血症。B细胞内体TLR激活的上下游通路以及这些通路在诱导高丙种球蛋白血症中的作用尚不清楚。这些步骤的定义需要对B细胞如何与寄生虫相互作用有更深入的了解。 我们研究的总体目标是了解各种B细胞亚群如何识别微生物并对其做出反应，以及这种识别如何影响它们的功能。我们在这项应用中的目标是表征杜诺氏乳杆菌和B细胞之间的相互作用，以了解导致多克隆B细胞激活的途径。特别是，我们将研究i)杜诺瓦氏杆菌和各种B细胞亚群之间的寄生虫捕获和细胞表面相互作用；ii)内体Toll样受体和其他微生物传感器(主要是胞浆)和它们激活的下游通路对寄生虫的识别；以及iii)细胞之间的通讯(例如，隧道纳米管；受体/配体相互作用)以及这种通讯在诱导多克隆B细胞激活中的作用。 转基因寄生虫、基因敲除小鼠、共聚焦显微镜、流式细胞仪和几种生化技术将被用来在体外实验中剖析各种激活和细胞通讯途径。 这里确定的途径可能会发现新的策略，这些策略也可能被其他微生物用来激活B细胞并调节其功能。

B-cells are mostly known for their capacity to produce antibodies. However, an increasing body of literature has now shown that they may also participate in the immune response by various antibody-independent mechanisms, such as cytokine production, co-stimulation, and antigen presentation. B-cells can be divided in three different subpopulations: follicular B cells , innate-like marginal zone B-cells (MZB), and B1 B-cells. Follicular B cells are the most prominent B-cell subpopulation and reside in lymphoid follicles of secondary and tertiary lymphoid organs. MZB are located in the marginal zone of the spleen and are endowed with the capacity to bind immune complexes, migrate towards the splenic white pulp, and transfer antigen to follicular dendritic cells. B1 B-cells are known for their secretion of natural antibodies. We have recently reported that B cells can capture the protozoan parasite Leishmania donovani. Upon exposure to the parasite, B cells form clusters and hold L. donovani in extracellular IgM-rich pockets. This close interaction results in polyclonal B cell activation and eventually in cell death after 24-48h. Interestingly, the parasite triggers endosomal Toll-Like Receptors (TLRs), although it is attached extracellularly on the cell surface of the B cell. Activation of endosomal TLRs was required to induce IL-10 and IFN-I expression and to promote hypergammaglobulinemia. The pathways up- and downstream of endosomal TLR activation in B-cells and the contribution of these pathways to the induction of hypergammaglobulinemia are as yet unknown. Definition of these steps will require a deeper understanding of how B cells interact with the parasite. The overall goal of our research is to understand how various B cell subpopulations recognize and react to microorganisms, and how this recognition affects their function. Our objective in this application is to characterize the interaction between L. donovani and B cells in order to understand pathways leading to polyclonal B cell activation. Particularly, we will investigate i) parasite capture and cell surface interaction between L. donovani and various B cell subpopulations; ii) parasite recognition by endosomal toll-like receptors and other microbial sensors (mainly cytosolic) and the downstream pathways they activate; and iii) cell-to-cell communication (e.g. tunnelling nanotubules; receptor/ligand interactions) and role of this communication in the induction of polyclonal B cell activation. Transgenic parasites, knock-out mice, confocal microscopy, flow cytometry, and several biochemical techniques will be used to dissect the various activation and cellular communication pathways in in vitro experiments. The pathways identified here could uncover novel strategies that may also be used by other microorganisms to activate B cells and modulate their functions.