The Case of the Missing Macrophages: Investigating the role of NKR-P1B:Clr-b self-recognition on tissue-resident myeloid cells.

先天免疫细胞功能是通过复杂的模式识别受体系统来调节的。调节先天免疫反应的受体系统的一个例子是NKR-P1家族的C型凝集素样受体及其结合配体，即C型凝集素相关(CLR)表面蛋白家族。NKR-P1B是该受体家族的抑制性成员，识别ClR-b，表达于自然杀伤细胞。使用缺乏NKR-P1B表达的转基因小鼠，我们观察到肺泡巨噬细胞(AM)数量显著减少，这是一种完全意想不到的表型。我们还发现NKR-P1B在AM上表达。我们现在试图了解NKR-P1B：CLR-b识别系统如何调节肺居民髓系免疫细胞的发育和功能。我们提出了以下两个目标：*1.发现NKR-P1B的表达对AM和DC的影响。我们将充分研究野生型和NKR-P1B缺陷的肺泡巨噬细胞和树突状细胞(DC)之间的功能和表型差异。使用共聚焦显微镜，我们将分析不同年龄小鼠的肺组织，以确定AM的分布和表型。我们将进行几项功能分析，包括细胞因子释放、吞噬和在NKR-P1B交联抗体存在下的抗原呈递分析，以模拟NKR-P1B参与对正常髓系细胞功能的影响。最后，利用骨髓嵌合体在野生型环境中研究NKR-P1B缺陷的髓系细胞，反之亦然，我们将探讨NKR-P1B对髓系细胞群体的作用是细胞内的还是外在的。*2.揭示NKR-P1B对AM和DC的作用机制。我们观察到NKR-P1B缺陷的AM代谢过程受损的证据。我们将比较WT和NKR-P1B缺陷AM的代谢谱，以及通过RNA序列(RNAseq)分析获得的它们的转录本，以确定差异表达的基因，以及涉及的细胞和代谢途径。我们还将使用邻近生物素化BioID系统研究NKR-P1B在巨噬细胞中的相互作用和细胞信号伙伴。*了解髓系免疫细胞的发育和功能加强了我们对细胞的总体理解。我们偶然发现了肺泡巨噬细胞生物学中一个以前没有被认识到的-而且显然是关键的-成分，我们希望探索NKR-P1B：CLR-b在这些细胞中的参与-包括总体作用和特定的信号通路-将教给我们关于髓系细胞发育和功能的基本教训。

Innate immune cell functions are regulated through sophisticated systems of pattern-recognition receptors. One example of a receptor system that regulates innate immune responses is the NKR-P1 family of C-type lectin-like receptors, and its binding ligands, the C-type lectin-related (Clr) family of surface proteins. NKR-P1B is an inhibitory member of this receptor family, which recognizes Clr-b, and is expressed on natural killer cells. Using genetically modified mice lacking NKR-P1B expression, we have observed a marked impairment in lung alveolar macrophage (AM) numbers, a completely unexpected phenotype. We have also found the NKR-P1B is expressed on AMs. We now seek to understand how NKR-P1B:Clr-b recognition system regulates lung-resident myeloid immune cell development and function. We have proposed the follow two objectives:***1. Discover the impact of NKR-P1B expression on AMs and DCs. We will fully characterize the functional and phenotypic differences between wild-type and NKR-P1B-deficient lung-resident AM and dendritic cells (DC). Using confocal microscopy, we will analyze lungs of mice at various ages to determine AM distribution and phenotype. We will perform several functional assays, including cytokine release, phagocytosis, and antigen-presenting assays in the presence of NKR-P1B cross-linking antibodies to simulate the effects of NKR-P1B engagement on normal myeloid cell function. Finally, using bone-marrow chimeras to study NKR-P1B-deficient myeloid cells in wild-type environments and vice versa, we will investigate whether NKR-P1B effects on the myeloid cell populations are cell-intrinsic or extrinsic.***2. Discover the mechanism through which NKR-P1B exerts its effects on AMs and DCs. We have observed evidence for impaired metabolic processing in NKR-P1B-deficient AMs. We will compare the metabolic profiles of WT and NKR-P1B-deficient AMs, as well as their transcriptomes obtained by RNA sequence (RNAseq) analysis to determine differentially expressed genes, and the cellular and metabolic pathways that are involved. We will also investigate NKR-P1B interacting and cell signaling partners in macrophages using the proximity-biotinylation BioID system.***Understanding myeloid immune cell development and function strengthens our understanding of cells in general. We have fortuitously stumbled into a previously unappreciated—and apparently critical—component of alveolar macrophage biology, and it is our hope that exploring the involvement—both overall role and specific signaling pathways—of NKR-P1B:Clr-b interactions in these cells will teach us fundamental lessons about myeloid cell development and function.