Determining the role of NLRP3 on the placement and protective potential of CD73+ BRMs post influenza infection

项目摘要 在与公共卫生相关的背景下，对抗原(Ag)特异性记忆B(BMEM)细胞知之甚少 病原体。我们的研究清楚地解析了非循环、流感特异性、居民记忆B的子集 肺中的细胞(BRM)。BRM的建立依赖于早期的CD40信号，并维持CD73 (胞外酶)+和CD73-群体，而淋巴结(LN)CD73- 人口。这就提出了CD73+和CD73在形成和功能上的内在差异的问题- BRMS。CD73是LN中生发中心(GC)迁移的标志，LN中的记忆细胞驻留在 靠近巨噬细胞捕获和转移抗原的壁龛，以及可以提供帮助的T细胞；因此， 我们假设CD73+BRM将是肺中有组织的淋巴结构区域的归宿，并 依赖T细胞的帮助。我们的RNAseq数据显示NLRP3(节点样受体蛋白3)是一种先天危险 感觉复合体，可能是CD73+BRM中表达最高的基因。据我们所知，NLRP3活动不是 与B细胞分化有关，这使得这一发现具有新颖性，并可能开启对先天 B细胞在适应性反应中的类似功能。NLRP3活性产生白介素B(IL-1B)，而IL-1B 一种众所周知的白介素6(IL-6)的上调调节剂，可促进GC和浆细胞分化。作为我们的 CD73+BRM可能有较高的NLRP3表达，我们假设NLRP3在回忆和 产生GC和ASCs，而CD73-BRM可能是肺中的哨兵，自发地转化为 ASCs，作为对返回的病毒的响应。 为了验证这些假设，我们问了以下问题：1)CD73+和CD73-BRM细胞依赖于GC吗？如果 因此，GC特异性阻断应该会减少肺中CD73+BRM，并产生低亲和力的BCR。2)做CD73+ BRMS优先驻留在iBALT？如果是这样的话，利用组织学，我们应该观察到它们聚集在 CD73-BRM散在分布于实质内。3)CD73+和CD73-BRM细胞是否有反应 在功能分析上有什么不同？我们将通过对BRM进行体外检测来回答这个问题 使用细胞因子鸡尾酒和分选的T细胞向ASCs分化。4)NLRP3复合体是否会变成 在病毒挑战时在BRMS中被激活？如果是这样的话，使用NLRP3炎症报告小鼠，我们预计 CD73+BRM细胞，而不是CD73-BRM细胞增加NLRP3活性。5)NLRP3是否调节 BRM的形成或功能？如果是这样的话，B细胞特异性NLRP3缺陷应该会改变 CD73+和CD73-BRM及其保护功能在发病率和死亡率方面有明显差异。 不管结果如何，这项研究的结果将对解决 有效疫苗设计的目标人群和潜在的分子目标。

Project Summary Little is known about antigen (Ag) specific memory B (BMEM) cells, in the context of a public health relevant pathogen. Our studies clearly resolve the subsets of non-circulating, influenza specific, resident memory B cells (BRM) in the lung. BRM establishment relied on early CD40 signaling and maintained a CD73 (ectoenzyme)+ and CD73- population, whereas the lymph node (LN) showed a decline in the CD73- population. This raised the question of the inherent differences in formation and function of CD73+ and CD73- BRMs. CD73 is a marker of germinal center (GC) emigrants in the LN and memory cells in the LN reside in niches close to macrophages that capture and transfer antigen and T cells which can provide help; therefore, we hypothesized that the CD73+BRMs will home to areas of organized lymphoid structures in the lung and be dependent on T cell help. Our RNASeq data shows NLRP3 (NOD-like receptor protein 3), an innate danger sensing complex, to be the most upregulated gene in CD73+BRMs. To our knowledge NLRP3 activity is not associated with B cell differentiation, which makes this finding novel and may inaugurate studies into innate like functions in B cells during adaptive responses. NLRP3 activity produces interleukin B (IL-1B) and IL-1B is a well-known up regulator of interleukin 6 (IL-6), which promotes GCs and plasma cell differentiation. As our CD73+BRMs may have higher NLRP3 expression, we hypothesize that NLRP3 will be active during recall and produce GCs and ASCs, while the CD73-BRMs may be sentinels in the lung which spontaneously convert to ASCs, in response to the returning virus. To test these hypotheses, we ask the following: 1) Are CD73+ and CD73- BRM cells dependent on GCs? If so, GC-specific blockade should reduce CD73+ BRMs in the lung and produce low affinity BCRs. 2) Do CD73+ BRMs preferentially reside in iBALT? If so, using histology, we should observe them congregating in the iBALT while the CD73-BRMs are scattered in the parenchyma. 3) Do CD73+ and CD73- BRM cells respond differently in functional assays? We will answer this question by performing in vitro assays for BRM differentiation to ASCs, using cytokine cocktails and sorted T cells. 4) Does the NLRP3 complex become activated in BRMs upon viral challenge? If so, using NLRP3 inflammasome reporter mice, we expect that CD73+ BRM cells, but not CD73- BRM cells to increase NLRP3 activity. 5) Does NLRP3 regulate the formation or function of BRMs? If so, B cell specific NLRP3 deficiency should alter the proportions of CD73+ and CD73-BRMs and their protective function with observable differences in morbidity and mortality. The findings from this study despite the outcome will be extremely important to allow for the resolution of a target population and potential molecular targets for an effective vaccine design.