Deciphering the function of the atypical inhibitor of NF-kB, IkBNS/Nfkbid, in macrophages and neutrophils

活化B细胞核因子-kappa型轻链增强子(NF-kB)是一种可诱导转录因子，受kB抑制因子(IKB)蛋白的调节。除了胞质中的IKB蛋白外，还存在另一类核(或非典型)IKB蛋白，它可以调节NF-kB的转录活性。IkBNS是一种非典型的IkB蛋白，由NfkBid基因编码，在调节性T细胞的发育以及Th1和Th17效应细胞的分化中起着至关重要的作用。尽管它在T细胞中发挥作用，但IkBNS在B细胞和巨噬细胞中的关键功能已被描述。我们利用一种新的报告小鼠，在内源Nfkid启动子的控制下表达LacZ，以筛选Nfkid基因在不同细胞类型中的启动子活性。我们证实了在巨噬细胞中的表达，并在中性粒细胞中检测到了高启动子活性。为了研究IkBNS在这些髓系细胞中的功能，我们产生了在两种细胞类型或中性粒细胞中缺乏IkBNS表达的条件性基因敲除小鼠。此外，我们从野生型和IkBNS缺陷的骨髓细胞中产生了Hoxb8转导的祖细胞，可以分化为巨噬细胞或中性粒细胞。利用这些系统，我们将分析IkBNS在巨噬细胞和中性粒细胞中的吞噬功能、亚群极化、抗原呈递、脱颗粒、网络形成、活性氧生成以及体内外细菌杀灭等方面的功能。我们将使用金黄色葡萄球菌作为感染源，这是一种机会性病原体，是医院感染的主要原因，通常具有多重耐药性。对这些细菌的免疫反应主要依赖于中性粒细胞和巨噬细胞，金黄色葡萄球菌是中性粒细胞网状形成的主要刺激因素。因此，这种病原体非常适合于破译中性粒细胞和巨噬细胞中IkBNS的功能。

The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) is the paradigm of an inducible transcription factor, being regulated by inhibitor of kB (IkB) proteins. In addition to the cytosolic IkB proteins, another class of nuclear (or atypical) IkB proteins exists that modulates the transcriptional activity of NF-kB. IkBNS, which is encoded by the Nfkbid gene, is one of these atypical IkB proteins and we previously demonstrated that it is crucial for the development of regulatory T cells and the differentiation of Th1 and Th17 effector cells. Despite its role in T cells, crucial functions in B cells and macrophages have been described for IkBNS. Using a novel reporter mouse that expresses lacZ under the control of the endogenous Nfkbid promoter, we screened for promoter activity of the Nfkbid gene in different cell types. We confirmed expression in macrophages and detected high promoter activity in neutrophils. To investigate the function of IkBNS in these myeloid cells, we generated conditional knockout mice lacking IkBNS expression either in both cell types or specifically in neutrophils. Moreover, we generated Hoxb8-transduced progenitor cells from wildtype and IkBNS-deficient bone marrow cells, which can be differentiated into macrophages or neutrophils. Using these systems, we will analyze the function of IkBNS in macrophages and neutrophils with respect to phagocytosis, polarization into subsets, antigen presentation, degranulation, NET formation, generation of reactive oxygen species, and bacterial killing in vitro and in vivo. We will use Staphylococcus aureus, an opportunistic pathogen that is the leading cause of nosocomial infections and often multidrug-resistant, as an infectious agent. The immune response against these bacteria crucially depends on neutrophils and macrophages with S. aureus being a prime stimulus of NET formation in neutrophils. Thus, this pathogen is perfectly suited to decipher the functions of IkBNS in neutrophils and macrophages.