Mechanisms of macrophage death co-dependent on M. tuberculosis and IFN-a,b receptor

摘要 在COVID之前，结核分枝杆菌(Mtb)是世界上唯一主要的感染死亡原因。 19.直接作用的抗分枝杆菌疗法很长，通常是有毒的，并受到出现耐药性的困扰。 辅助治疗可能会通过靶向宿主内改变的过程来加速结核病的治愈 寄主-病原菌的相互作用对寄主有利。宿主导向疗法的一种潜在形式是 以帮助巨噬细胞更好地存活于结核分枝杆菌感染。在体外，感染结核杆菌的小鼠巨噬细胞的死亡是共同的 依赖于巨噬细胞对结核分枝杆菌的反应产生的I型干扰素(干扰素-β)，以及 Mtb的补充贡献。本申请旨在确定结核分枝杆菌诱导的特定分子参与者。 I型干扰素受体下游的巨噬细胞死亡。我们已经确定了潜在的分子 参与者通过两种方法-CRISPR激活屏幕，恢复结核杆菌诱导的细胞死亡 缺乏I型干扰素受体的巨噬细胞和一种使用探针的创新生化下拉方法 基于一种能将结核杆菌感染的巨噬细胞从结核杆菌诱导的细胞死亡中拯救出来的小化合物 不影响结核分枝杆菌的生长。这个应用程序的目的是从基因上验证候选人，测试能力 已经存在的抑制剂，以概括击倒它们的效果，然后将验证过的 走在机械路线上的候选人。这将为未来的研究奠定基础，超出了本章的范围 应用，以寻找、改进或开发类药物抑制剂，用于临床前结核病模型的测试，以了解它们是否 当与直接作用的抗分枝杆菌药物联合使用时，可减轻病理，加速治愈。

ABSTRACT Mycobacterium tuberculosis (Mtb) was the world’s single leading cause of death from infection before COVID- 19. Direct-acting antimycobacterial regimens are long, often toxic and plagued by emergence of drug resistance. Adjunctive therapies could potentially speed the cure of tuberculosis by targeting a process in the host that alters the host-pathogen interaction to the advantage of the host. One potential form of host-directed therapy would be to help macrophages better survive Mtb infection. In vitro, the death of Mtb-infected mouse macrophages is co- dependent on a type I interferon (IFN), IFN-beta, that macrophages produce in response to Mtb, along with an additional contribution by Mtb. This application seeks to identify specific molecular participants in Mtb-induced macrophage death that are downstream of the type I IFN receptor. We have identified potential molecular participants by two approaches—a CRISPR activation screen that restored Mtb-induced cell death to macrophages lacking the type I IFN receptor, and an innovative biochemical pulldown approach using a probe based on a small chemical compound that rescues Mtb-infected macrophages from Mtb-induced cell death without impairing the growth of Mtb. This application aims to validate the candidates genetically, test the ability of already existing inhibitors to recapitulate the effect of knocking them out, and then place the validated candidates on a mechanistic path. That would set the stage for future studies, beyond the scope of this application, to find, improve or develop drug-like inhibitors for tests in preclinical models of TB to see if they mitigate pathology and hasten cure when used in combination with direct-acting antimycobacterial agents.