文献学习104--IRF3 and type I interfe

17年的一篇nm

1. Myocardial infarction activates IRF3-dependent signaling

We hypothesized that the high levels of ischemic cell death during MI may disrupt DNA sequestration and liberate large quantities of self-DNA for cytosolic sensing, ultimately fueling a maladaptive IRF3- dependent innate immune response. To test this hypothesis, we studied IRF3 and the type I IFN response following permanent coronary ligation (MI) in mice.
Fig 1a： 由于 IRF3 和I型 IFNs在缺血性心脏病中的作用未知，作者首先评估了MI是否可以激活IRF3。尽管这个时候不存在感染，作者检测到了很强的p-IRF3的活化。
Fig 1b-c： IRF3 dependent cytokines Ifnb1 和 Cxcl10都在MI后4天显著高表达。而在IRF3-knockout (Irf3^−/−) 鼠，MI后I型干扰素则显著降低。
Fig 1d： I型干扰素如IFN-β1 (encoded by Ifnb1) 可以与cell-surface IFN-α receptors (encoded by Ifnar1, referred to hereafter as Ifnar)结合，放大炎症反应，引起各种ISGs的释放。与此一致，作者发现，MI 4天后，多种ISGs表达都出现了显著上调，而在Irf3^−/−鼠中这种现象消失。
Fig 1e： RNAseq的结果也显示，在心梗小鼠，多种ISGs是最显著上调的基因。
Fig 1f： 为了探究这是不是某一群细胞的特征，作者对MI后的心脏组织进行了单细胞测序。得到一群IFNICs（IRF3 score是用Ifit1, Ifit2和Ifit3做的）。
Fig 1g, h： the IFNICs were identified as cells with specialized expression of ISGs and directly IRF3-dependent genes. In contrast, we did not detect IFNICs by single-cell analysis of post-MI day 4 Irf3^−/− infarcts or non-infarcted WT hearts (Supplementary Fig. 4).

2. Post-phagocytic macrophages initiate IRF3-dependent amplification of inflammation following myocardial infarction

接着，作者使用流式探究了MI后哪群细胞account for IRF3-dependent gene expression。
Fig 2a-b： 作者从MI后4天小鼠心脏中分选了CD45 或 CD11b阳性的细胞。结果显示CD45⁺ 和 CD11b⁺ 亚群，而不是 CD45⁻ 或 CD11b⁻ 亚群，reliably 表达 Ifnb1。
Fig 2c： 随后作者进行了Parabiosis 实验，在将 Irf3^−/− 鼠paired with either WT or Irf3−/− mice后对 Irf3^−/− 鼠给予了冠脉结扎。结果显示MI后，在有外周血细胞进入心脏后IRF3-dependent genes表达显著增加。

To study how IRF3-dependent responses are initiated after MI, we created a reporter mouse (Myh6-Cre^+/–mTmG^+/+; referred to as Cre⁺) in which only cardiomyocytes express membrane EGFP (green), whereas all non-cardiomyocytes express membrane tdTomato (red). 对照鼠是Cre-deficient controls (Myh6Cre^−/−mTmG^+/+; referred to as Cre⁻)
Fig 2d-h： 在MI后4天，作者分选了根据EGFP分选了 吞噬了心肌细胞碎片的CD11b⁺ 细胞。结果显示吞噬了心肌碎片的巨噬高表达Ifnb，而两群巨噬表达ISGs的水平类似(g)。
Fig 2f： This CD11b⁺EGFP⁺ population consisted almost exclusively of F4/80^hiLy6C^lo cells (macrophages), which we term post-phagocytic macrophages.
Together, these results indicate that DAMP sensing by infarct phagocytes leads to type I IFN production, which secondarily induces ISG expression in nearby cells, regardless of their direct association with the initiating cardiomyocyte DAMP.

为了可视化interferon-responsive 细胞的空间分布，作者构建了 interferon-inducible reporter mouse (Mx1Cre mTmG)，在所有细胞中表达膜 tdTomato (red)，除了 those induced to express Cre recombinase by endogenous activation of the interferon- sensitive Mx1 promoter, which changes reporter expression to membrane EGFP (green)。
Fig 2i： 在 MI 后4天，报告鼠的梗死区存在显著的green interferon-responsive cells的聚集。
Fig 2j： 随后作者探究了是否 interferon response 影响了免疫细胞浸润的 extent 和 quality。
Fig 2k： 流式结果显示MI后，Irf3^−/−鼠的炎症浸润和WT鼠相比减轻，F4/80^loLy6C^hi 促炎性单核减少。
Fig 2l： 单细胞的结果显示IFNICs表达Adgre1 (encoding F4/80), H2.Aa (encoding major histocompatibility complex type II (MHCII)), 和 Ccr2, 但不表达 Ly6c2 (encoding Ly6C)。因此 IFNICs 是 monocyte-derived cardiac macrophages。
Taken together, these results suggest that interactions of infarct phagocytes with cardiomyocyte debris fuel IRF3-dependent leukocyte recruitment and amplify inflammation after MI.

3. Self-DNA is the dominant myocardial infarction–induced IRF3-activating DAMP

We next sought to determine the dominant DAMP responsible for MI-induced IRF3 activation.
Fig 3a： 3个adaptor 蛋白可以激活 IRF3: TIR-domain-containing adaptor inducing interferon beta (TRIF), mitochondrial antiviral signaling protein (MAVS) 和 stimulator of interferon genes (STING)。

• TRIF是MyD88非依赖途径的adaptor (被TLR激活，如识别病毒的TLR3等)
• MAVS是RIG通路的adaptor，RIG通路识别胞浆内的病毒dsRNA（TLR-TRIF识别的是内体中的病毒RNA，RIG识别的是胞浆中游离的）
• STING识别胞质中的病毒和细菌DNA

Fig 3b： 因此作者使用了三种 adaptor 的缺陷小鼠。结果显示只有Sting^gt/gt鼠在MI后存在和Irf3^-/-鼠类似的Ifnb基因表达下降。
Fig 3c： Cytosolic double-stranded DNA (dsDNA) is the only endogenous DAMP known to activate IRF3 via the adaptor STING. It does so via the DNA sensor cGAS
Fig 3d： Indeed, Mb21d1缺陷小鼠 (cGAS^−/− 鼠), 和 Irf3^−/− 以及 Sting^gt/gt 鼠一样，在MI后并没有出现显著的I型干扰素反应的活化。
类似的，缺乏I型IFN受体的 Ifnar^−/−鼠同样在MI后没有出现显著的I型干扰素反应的活化。
Taken together, these results are consistent with the concept that the cytosolic-DNA-sensing pathway is responsible for induction of an IRF3-dependent type I IFN response in the heart after MI; however, other pathways may also contribute.

为了探究是否缺血损伤 disrupts intracellular compartmentalization 并使得心肌细胞的 DNA 可以被识别，作者对小鼠进行了缺血再灌。在20min的冠脉结扎后进行了再灌注，the restoration of flow was used to allow delivery of DNA-binding- dependent fluorescent probe, PicoGreen or SYTOX Orange.
Fig 3e-f： 使用心脏 gated intravital microscopy，作者发现 DNA probes localized exclusively to the nuclei of intact surviving cells, whereas they spread throughout the cytosol of injured cells
Fig 3g： 随后作者设计实验去确定是否 infiltrating cells access cardiomyocyte DNA in the infarct in vivo. Cardiomyocyte DNA was indelibly labeled in utero by injecting pregnant mice with the modified nucleotide 5-ethynyl-2′-deoxyuridine (EdU). When the offspring grew to adults, the location of covalently DNA-bound EdU was visualized by bio-orthogonal click chemistry. 心肌细胞在子宫中快速增殖，而在成年后很少扩增，使得染色标签可以标记成年鼠，而高速增殖的造血细胞和它们的子细胞则 diluted EdU to undetectable levels。
Fig 3h： 当作者challenged the labeled mice with MI, 作者发现 EdU localized to the extranuclear space of infiltrating border- zone cells on day 4 after MI but was not detectable in circulating cells from cytospun blood.

Taken together, these results indicate that macrophages can access extracellular DNA and produce type I IFNs in a cGAS-, STING-, and IRF3- dependent fashion. Together, these findings suggest that ischemic cells in the heart release DNA that provokes infarct phagocytes to induce a cGAS- and IRF3-dependent type I IFN response.

4. Genetic and pharmacological disruption of DNA-induced IRF3 activation and type I interferon signaling protects mice from death and adverse ventricular remodeling following myocardial infarction

Fig 4a-l： 为了确定MI诱导的IRF3活化的功能，作者使用WT鼠和小鼠with impaired interferon signaling构建了MI模型，比较了其存活率，心室重塑和收缩功能差异。 结果显示和WT小鼠相比，Irf3^-/-小鼠、Ifnar^-/-小鼠和cGAS^-/-小鼠的存活率显著更高，心功能也更好。
Fig 4m-p： 为了探究这项研究的转化意义，作者在MI12和48h后给予了WT小鼠 IFNAR-neutralizing antibody治疗，显示出很好的治疗效果。死亡率和心功能都得到改善。

In summary, these findings highlight the concept that a response that is protective in the context of pathogen defense has deleterious consequences in the context of sterile ischemic injury. According to our working model (Fig. 4q), MI causes ischemic cell death in the heart, releasing debris from dying cells including danger signals such as dsDNA. Infiltrating cells such as phagocytic macrophages sense DNA via the cytosolic sensor cGAS, leading to activation of the transcription factor IRF3 and induction of the IRF3-dependent gene expression program, which includes type I IFNs. Secreted type I IFNs can then diffuse through the local microenvironment and signal to cells expressing IFNAR, in both an autocrine and paracrine fashion, to amplify the response via expression of ISGs. In comparison to WT mice after MI, mice with genetic deficiency of cGAS, IRF3, or IFNAR, or those treated with an IFNAR-neutralizing antibody, exhibit less ventricular dilation, greater preservation of contractile function, less ventricular rupture, and greater survival.

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思考：

1. IFN怎么fuel a fatal response的机制没有做。
2. 做心肌细胞来源的dsDNA诱导cGAS-STING的活化是通过label心肌细胞(带荧光)，然后检测巨噬细胞中有没有被吞噬的心肌荧光来做的。和此前看的文献学习086--TREM2hi原位巨噬细胞通过维持心肌稳态保护脓毒心和文献学习095--心脏原位巨噬细胞来源的Legumain通过促进心肌梗死后凋亡心肌细胞的清除和降解来改善心脏修复用的是同样的方法（当然这篇nm做的更早），属于做心肌吞噬的常规做法。但是作者在开篇先做了单细胞，focus到了IFNICs巨噬，后面又做的总的(外周趋化来的)巨噬，感觉有点怪。