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在缺血性心臟病中的作用未知，作者首先評估了MI是否可以激活IRF3氯庆。盡管這個時候不存在感染憋沿，作者檢測到了很強的p-IRF3的活化奸远。
Fig 1b-c： IRF3 dependent cytokines Ifnb1 和 Cxcl10都在MI后4天顯著高表達份帐。而在IRF3-knockout (Irf3?/?) 鼠湾笛，MI后I型干擾素則顯著降低。
Fig 1d： I型干擾素如IFN-β1 (encoded by Ifnb1) 可以與cell-surface IFN-α receptors (encoded by Ifnar1, referred to hereafter as Ifnar)結(jié)合蝌衔，放大炎癥反應榛泛，引起各種ISGs的釋放。與此一致噩斟，作者發(fā)現(xiàn)曹锨，MI 4天后，多種ISGs表達都出現(xiàn)了顯著上調(diào)剃允，而在Irf3?/?鼠中這種現(xiàn)象消失沛简。
Fig 1e： RNAseq的結(jié)果也顯示，在心梗小鼠斥废，多種ISGs是最顯著上調(diào)的基因椒楣。
Fig 1f： 為了探究這是不是某一群細胞的特征，作者對MI后的心臟組織進行了單細胞測序营袜。得到一群IFNICs（IRF3 score是用Ifit1, Ifit2和Ifit3做的）撒顿。
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
接著，作者使用流式探究了MI后哪群細胞account for IRF3-dependent gene expression荚板。
Fig 2a-b： 作者從MI后4天小鼠心臟中分選了CD45 或 CD11b陽性的細胞凤壁。結(jié)果顯示CD45+ 和 CD11b+ 亞群吩屹，而不是 CD45? 或 CD11b? 亞群，reliably 表達 Ifnb1拧抖。
Fig 2c： 隨后作者進行了Parabiosis 實驗煤搜，在將 Irf3?/? 鼠paired with either WT or Irf3?/? mice后對 Irf3?/? 鼠給予了冠脈結(jié)扎。結(jié)果顯示MI后唧席，在有外周血細胞進入心臟后IRF3-dependent genes表達顯著增加擦盾。
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). 對照鼠是Cre-deficient controls (Myh6Cre?/?mTmG+/+; referred to as Cre?)
Fig 2d-h： 在MI后4天，作者分選了根據(jù)EGFP分選了 吞噬了心肌細胞碎片的CD11b+ 細胞淌哟。結(jié)果顯示吞噬了心肌碎片的巨噬高表達Ifnb迹卢，而兩群巨噬表達ISGs的水平類似(g)。
Fig 2f： This CD11b+EGFP+ population consisted almost exclusively of F4/80hiLy6Clo 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.
為了可視化interferon-responsive 細胞的空間分布徒仓，作者構(gòu)建了 interferon-inducible reporter mouse (Mx1Cre mTmG)腐碱，在所有細胞中表達膜 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)掉弛。
Fig 2i： 在 MI 后4天症见，報告鼠的梗死區(qū)存在顯著的green interferon-responsive cells的聚集。
Fig 2j： 隨后作者探究了是否 interferon response 影響了免疫細胞浸潤的 extent 和 quality殃饿。
Fig 2k： 流式結(jié)果顯示MI后谋作，Irf3?/?鼠的炎癥浸潤和WT鼠相比減輕，F(xiàn)4/80loLy6Chi 促炎性單核減少乎芳。
Fig 2l： 單細胞的結(jié)果顯示IFNICs表達Adgre1 (encoding F4/80), H2.Aa (encoding major histocompatibility complex type II (MHCII)), 和 Ccr2, 但不表達 Ly6c2 (encoding Ly6C)遵蚜。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個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)秒咐。
- TRIF是MyD88非依賴途徑的adaptor (被TLR激活谬晕，如識別病毒的TLR3等)
- MAVS是RIG通路的adaptor，RIG通路識別胞漿內(nèi)的病毒dsRNA（TLR-TRIF識別的是內(nèi)體中的病毒RNA携取，RIG識別的是胞漿中游離的）
- STING識別胞質(zhì)中的病毒和細菌DNA
Fig 3b： 因此作者使用了三種 adaptor 的缺陷小鼠攒钳。結(jié)果顯示只有Stinggt/gt鼠在MI后存在和Irf3-/-鼠類似的Ifnb基因表達下降。
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 Stinggt/gt 鼠一樣雷滋，在MI后并沒有出現(xiàn)顯著的I型干擾素反應的活化不撑。
類似的，缺乏I型IFN受體的 Ifnar?/?鼠同樣在MI后沒有出現(xiàn)顯著的I型干擾素反應的活化晤斩。
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.
為了探究是否缺血損傷 disrupts intracellular compartmentalization 并使得心肌細胞的 DNA 可以被識別焕檬，作者對小鼠進行了缺血再灌。在20min的冠脈結(jié)扎后進行了再灌注澳泵，the restoration of flow was used to allow delivery of DNA-binding- dependent fluorescent probe, PicoGreen or SYTOX Orange.
Fig 3e-f： 使用心臟 gated intravital microscopy实愚，作者發(fā)現(xiàn) DNA probes localized exclusively to the nuclei of intact surviving cells, whereas they spread throughout the cytosol of injured cells
Fig 3g： 隨后作者設計實驗去確定是否 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. 心肌細胞在子宮中快速增殖，而在成年后很少擴增，使得染色標簽可以標記成年鼠腊敲，而高速增殖的造血細胞和它們的子細胞則 diluted EdU to undetectable levels击喂。
Fig 3h： 當作者challenged the labeled mice with MI, 作者發(fā)現(xiàn) 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： 為了確定MI誘導的IRF3活化的功能，作者使用WT鼠和小鼠with impaired interferon signaling構(gòu)建了MI模型碰辅，比較了其存活率懂昂，心室重塑和收縮功能差異。 結(jié)果顯示和WT小鼠相比没宾，Irf3-/-小鼠凌彬、Ifnar-/-小鼠和cGAS-/-小鼠的存活率顯著更高，心功能也更好循衰。
Fig 4m-p： 為了探究這項研究的轉(zhuǎn)化意義铲敛，作者在MI12和48h后給予了WT小鼠 IFNAR-neutralizing antibody治療，顯示出很好的治療效果会钝。死亡率和心功能都得到改善原探。
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.
思考：
- IFN怎么fuel a fatal response的機制沒有做。
- 做心肌細胞來源的dsDNA誘導cGAS-STING的活化是通過label心肌細胞(帶熒光)顽素，然后檢測巨噬細胞中有沒有被吞噬的心肌熒光來做的。和此前看的文獻學習086--TREM2hi原位巨噬細胞通過維持心肌穩(wěn)態(tài)保護膿毒心和文獻學習095--心臟原位巨噬細胞來源的Legumain通過促進心肌梗死后凋亡心肌細胞的清除和降解來改善心臟修復用的是同樣的方法（當然這篇nm做的更早）徒蟆，屬于做心肌吞噬的常規(guī)做法胁出。但是作者在開篇先做了單細胞，focus到了IFNICs巨噬段审，后面又做的總的(外周趨化來的)巨噬全蝶，感覺有點怪。
- 外周趨化來的單核吞噬受損心肌后可以變成IFNIC寺枉，受損的心肌細胞損傷到什么程度可以誘導單核細胞吞噬沒有做抑淫，怎么啟動的吞噬過程沒有做。
