Regulation of IkB kinase family crosstalk by an N4BP1-caspase-8 axis [scRNA-seq]

The cell death protease caspase-8 plays an essential role in controlling inflammation, as severe immunodeficiency results from its loss. We previously found that caspase-8 promotes inflammatory responses by cleaving NEDD4-binding protein 1 (N4BP1), a suppressor of cytokine production, but the underlying mechanisms remained unclear. Here we find that N4BP1 curtails the duration, rather than initial induction, of proinflammatory signaling through a mechanism involving noncanonical IKK (ncIKK)-mediated inhibition of the canonical IkB kinase (IKK) complex, a crosstalk event among the IKK family facilitated by N4BP1. Accordingly, co-deletion of the ncIKKs or their adaptor protein TANK largely phenocopied deletion of N4BP1, augmenting cytokine responses by macrophages upon engagement of TRIF-independent toll-like receptors (TLR) 1/2, TLR7, or TLR9. Like N4BP1, TANK was largely prevented from inhibiting the TRIF-dependent TLR4 response due to caspase-8. Biochemically, N4BP1 binds both the canonical and noncanonical IKK complexes, in a manner promoted by linear and/or K63-linked polyubiquitin chain binding by N4BP1 and independent of its RNAse activity. Consistent with this, a knock-in mutant of N4BP1 with diminished ubiquitin chain-binding capacity led to increased proinflammatory cytokine responses. These findings thereby unveil a mechanism of late-phase inflammatory gene control, whereby N4BP1 prevents persistent IKK activity through ncIKK-mediated inhibition. This molecular crosstalk among caspase-8, N4BP1, and the IKKs and ncIKKs may have implications for our understanding of genetic immune diseases caused by mutations in caspase-8 or TBK1 and suggest a novel ‘guarding’ mechanism against pathogens that attempt to subvert the ncIKKs. Single cell transcriptome on wildtype and N4bp1 mouse bone marrow derived macrophages (BMDMs) that were co-cultured with GFP expressing BMDMs, with and without TLR7 treatment

细胞死亡蛋白酶半胱天冬酶-8（caspase-8）在炎症调控中发挥核心作用，其缺失会引发严重的免疫缺陷。我们此前的研究证实，半胱天冬酶-8可通过切割细胞因子产生的抑制因子NEDD4结合蛋白1（NEDD4-binding protein 1, N4BP1）来促进炎症应答，但其具体分子机制始终未明。本研究发现，N4BP1并非调控促炎信号的初始诱导过程，而是通过一种涉及非经典IKK（ncIKK）介导抑制经典IKK复合物的机制，限制促炎信号的持续时长——这是由N4BP1介导的IKK家族成员间的串扰事件。实验结果显示，共敲除非经典IKK或其接头蛋白TANK，其表型在很大程度上模拟了N4BP1的敲除：当巨噬细胞接触TRIF非依赖型Toll样受体（Toll-like receptor, TLR）1/2、TLR7或TLR9时，会显著增强细胞因子的应答水平。与N4BP1的作用一致，半胱天冬酶-8的存在可使TANK无法有效抑制TRIF依赖型的TLR4应答。生化实验表明，N4BP1可同时结合经典与非经典IKK复合物，该结合过程依赖于N4BP1对线性及/或K63位连接的多泛素链的结合能力，且不依赖于其核糖核酸酶活性。与此一致的是，携带泛素链结合能力减弱的N4BP1敲入突变体，会导致促炎细胞因子的应答水平升高。综上，本研究揭示了一种炎症基因的晚期调控机制：N4BP1通过非经典IKK介导的抑制作用，阻断IKK活性的持续存在。半胱天冬酶-8、N4BP1与IKK及非经典IKK之间的这种分子串扰，或有助于我们理解由半胱天冬酶-8或TBK1突变引发的遗传性免疫疾病，并为阐释机体对抗试图破坏非经典IKK通路的病原体的新型‘防御’机制提供了新的视角。本数据集包含野生型与N4bp1敲除小鼠骨髓来源巨噬细胞（bone marrow derived macrophages, BMDMs）的单细胞转录组数据，这些巨噬细胞分别与表达GFP的巨噬细胞共培养，并施加或不施加TLR7激动剂处理。