Innate enteric immunity during induced Paneth cell deficiency

描述（由申请人提供）：所提出的研究将检验以下假设：Wnt/β-连环蛋白信号传导中的诱导失衡导致潘氏细胞耗竭、常驻微生物群的生态失调、炎症、对病原菌感染的易感性增强，并且这些作用可以使用Wnt/β-连环蛋白信号传导途径的特异性调节剂逆转或改善。在解剖学上局限于小肠，潘氏细胞释放防御素和宿主防御分子，维持潘氏细胞的稳态对小肠的健康和完整性至关重要。Wnt信号级联在肠道个体发育中具有关键作用，并且由β-连环蛋白p300共激活因子复合物介导的Wnt信号对于潘氏细胞分化至关重要。â-连环蛋白/CBP介导的转录维持干细胞效能;相比之下，â-连环蛋白/p300转录复合物启动增殖能力更有限的分化程序。第一次…- 连环蛋白共激活剂CBP和p300具有广泛的相似性，但通过其独特的N末端与-连环蛋白相互作用，这使得 开发特异性抑制它们与β-连环蛋白的结合和参与β-连环蛋白介导的转录的小分子拮抗剂。例如，ICG-001通过特异性结合CBP而不是p300来促进Wnt依赖性分化，阻断CBP/<$-连环蛋白转录而不1）干扰p300，2）改变<$-连环蛋白的水平，但仍然允许CBP参与其他转录复合物。相反，IQ-1拮抗β-catenin-p300相互作用，但不拮抗CBP相关性，为诱导选择性潘氏细胞缺陷提供了一种手段。因此，我们将测试以下假设：在IQ-1小鼠中，<$-连环蛋白/p300信号传导的拮抗作用诱导潘氏细胞耗竭和功能障碍。将通过监测IQ-1诱导的帕氏细胞缺乏症的时间进程以及帕氏细胞特异性标志物表达的同步变化和诱导的炎症（如骨髓和淋巴细胞的浸润和易位）来确定破坏帕氏细胞稳态的影响。将表征潘氏细胞缺乏对原核和真核微生物群的组成以及对从体内肠道细菌移位推断的屏障完整性的影响。在建立潘氏细胞缺陷后，将确定停药和ICG-001施用对纠正共激活因子失衡和恢复潘氏细胞分化和屏障完整性的作用，并将在离体隐窝类器官上确定潘氏细胞对IQ-1和ICG- 001的自主反应。还将确定重建潘氏细胞谱系分配和隐窝稳态对逆转原核和真核生态失调的影响。我们预期IQ-1诱导的潘氏细胞耗竭将引起促炎反应，诱导生态失调，并增强对细菌易位的易感性，并且ICG-001将选择性地使p300/β-连环蛋白驱动的转录能够促进Wnt依赖性潘氏细胞分化并加速小肠稳态的恢复。

DESCRIPTION (provided by applicant): The proposed studies will test the hypothesis that induced imbalance in Wnt/¿-catenin signaling results in Paneth cell depletion, dysbiosis of the resident microbiota, inflammation, enhanced susceptibility to infection by pathogenic bacteria, and that these effects can be reversed or ameliorated using a specific modulator of the Wnt/¿-catenin signaling pathway. Anatomically restricted to the small bowel, Paneth cells release ¿-defensins and host defense molecules, and maintenance of Paneth cell homeostasis is essential to small intestinal health and integrity. The Wnt signaling cascade has a critical role i gut ontogeny, and Wnt signaling mediated by ¿- catenin p300 coactivator complexes is critical for Paneth cell differentiation. ¿-catenin/CBP-mediated transcription maintains stem cell potency; in contrast, ¿-catenin/p300 transcription complexes initiate differentiation programs of more limited proliferative capacity. The primary ¿-catenin co-activators CBP and p300 share extensive similarity but interact with ¿-catenin via their distinctive N termini, which has enabled development of small molecule antagonists that specifically inhibit their association with ¿-catenin and participation in ¿-catenin-mediated transcription. For example, ICG-001 promotes Wnt-dependent differentiation by binding to CBP specifically, but not to p300, blocking CBP/¿-catenin transcription without 1) interfering with p300, 2) altering levels of ¿-catenin, but still ) allowing CBP to participate in other transcription complexes. Conversely, IQ-1 antagonizes the ¿-catenin-p300 interaction but not CBP associations, providing a means for inducing selective Paneth cell deficiency. Thus, we will test the hypothesis that antagonism of ¿- catenin/p300 signaling in mice with IQ-1 induces Paneth cell depletion and dysfunction. The impact of disrupting Paneth cell homeostasis will be determined by monitoring the time course of IQ-1 induced Paneth cell deficiency and coincident changes in expression of Paneth cell specific markers and induced inflammation evident as infiltration and translocation of myeloid and lymphoid cells. Effects of Paneth cell deficiency on the composition of the prokaryotic and eukaryotic microbiota and on barrier integrity inferred from translocation of gut bacteria in vivo will be characterized. After establishing Paneth cell deficiency, the effects of drug withdrawal and of ICG-001 administration to correct co-activator imbalance and restore Paneth cell differentiation and barrier integrity will be determined, and Paneth cell autonomous responses to IQ-1 and ICG- 001 will be determined on crypt organoids ex vivo. The effect of reestablishing Paneth cell lineage allocation and crypt homeostasis on reversing prokaryotic and eukaryotic dysbiosis also will be determined. We anticipate that IQ-1-induced Paneth cell depletion will evoke proinflammatory responses, induce dysbiosis, and enhance susceptibility to bacterial translocation and that ICG-001 will selectively enable p300/¿-catenin driven transcription to promote Wnt-dependent Paneth cell differentiation and accelerate recovery of small bowel homeostasis.