Interferon gamma (IFN-γ) restricts the intracellular replication of many pathogens, but the mechanism by which IFN-γ confers cell-intrinsic pathogen resistance remains unclear. For example, intracellular replication of the bacterial pathogen Legionella pneumophila in macrophages is potently curtailed by IFN-γ. However, consistent with prior studies, no individual genetic deficiency that we tested completely abolished IFN-γ-mediated control. Intriguingly, we observed that the glycolysis inhibitor 2-deoxyglucose (2DG) partially rescued L. pneumophila replication in IFN-γ-treated macrophages. 2DG inhibits glycolysis and triggers the unfolded protein response, but unexpectedly, it appears these effects are not responsible for perturbing the antimicrobial activity of IFN-γ. Instead, we found that 2DG rescues bacterial replication by inhibiting the expression of two key antimicrobial factors, inducible nitric oxide synthase (iNOS) and immune-responsive gene 1 (IRG1). Using immortalized and primary macrophages deficient in iNOS and IRG1, we confirmed that loss of both iNOS and IRG1, but not individual deficiency in either gene, partially reduced IFN-γ-mediated restriction of L. pneumophila Further, using a combinatorial CRISPR/Cas9 mutagenesis approach, we found that mutation of iNOS and IRG1 in combination with four other genes (CASP11, IRGM1, IRGM3, and NOX2) resulted in a total loss of L. pneumophila restriction by IFN-γ in primary bone marrow macrophages. Our study defines a complete set of cell-intrinsic factors required for IFN-γ-mediated restriction of an intracellular bacterial pathogen and highlights the combinatorial strategy used by hosts to block bacterial replication in macrophages.IMPORTANCELegionella pneumophila is one example among many species of pathogenic bacteria that replicate within mammalian macrophages during infection. The immune signaling factor interferon gamma (IFN-γ) blocks L. pneumophila replication in macrophages and is an essential component of the immune response to L. pneumophila and other intracellular pathogens. However, to date, no study has identified the exact molecular factors induced by IFN-γ that are required for its activity. We generated macrophages lacking different combinations of IFN-γ-induced genes in an attempt to find a genetic background in which there is a complete loss of IFN-γ-mediated restriction of L. pneumophila We identified six genes that comprise the totality of the IFN-γ-dependent restriction of L. pneumophila replication in macrophages. Our results clarify the molecular basis underlying the potent effects of IFN-γ and highlight how redundancy downstream of IFN-γ is key to prevent exploitation of macrophages by pathogens.

中文翻译：

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IRG1 和诱导型一氧化氮合酶与其他干扰素-γ 诱导因子共同作用以限制嗜肺军团菌的细胞内复制。

干扰素 γ (IFN-γ) 限制了许多病原体的细胞内复制，但 IFN-γ 赋予细胞内在病原体抗性的机制仍不清楚。例如，细菌病原体嗜肺军团菌在巨噬细胞中的细胞内复制被 IFN-γ 有效抑制。然而，与先前的研究一致，我们测试的任何个体遗传缺陷都没有完全消除 IFN-γ 介导的控制。有趣的是，我们观察到糖酵解抑制剂 2-脱氧葡萄糖 (2DG) 部分挽救了 IFN-γ 处理的巨噬细胞中嗜肺军团菌的复制。2DG 抑制糖酵解并触发未折叠蛋白反应，但出乎意料的是，这些作用似乎与干扰 IFN-γ 的抗菌活性无关。反而，我们发现 2DG 通过抑制两种关键抗菌因子的表达来拯救细菌复制，诱导型一氧化氮合酶 (iNOS) 和免疫反应基因 1 (IRG1)。使用 iNOS 和 IRG1 缺陷的永生化和原代巨噬细胞，我们证实 iNOS 和 IRG1 的缺失，但不是任何一个基因的个体缺陷，部分减少了 IFN-γ 介导的嗜肺军团菌限制。此外，使用组合 CRISPR/Cas9 诱变通过方法，我们发现 iNOS 和 IRG1 的突变与其他四个基因（CASP11、IRGM1、IRGM3 和 NOX2）相结合，导致原发性骨髓巨噬细胞中 IFN-γ 对嗜肺军团菌的限制完全丧失。我们的研究定义了 IFN-γ 介导的细胞内细菌病原体限制所需的一整套细胞内在因素，并强调了宿主用于阻断巨噬细胞中细菌复制的组合策略。重要提示嗜肺军团菌是许多致病细菌中的一个例子在感染期间在哺乳动物巨噬细胞内复制。免疫信号因子干扰素 γ (IFN-γ) 可阻断嗜肺军团菌在巨噬细胞中的复制，并且是对嗜肺军团菌和其他细胞内病原体的免疫反应的重要组成部分。然而，迄今为止，还没有研究确定 IFN-γ 诱导其活性所需的确切分子因子。我们产生了缺乏 IFN-γ 诱导基因的不同组合的巨噬细胞，以试图找到完全丧失 IFN-γ 介导的嗜肺军团菌限制的遗传背景 我们确定了构成 IFN 全部的六个基因-γ依赖性限制嗜肺军团菌在巨噬细胞中的复制。我们的研究结果阐明了 IFN-γ 有效作用的分子基础，并强调了 IFN-γ 下游的冗余如何是防止病原体利用巨噬细胞的关键。