IRF3 and type I interferons fuel a fatal response to myocardial infarction

Abstract

Interferon regulatory factor 3 (IRF3) and type I interferons (IFNs) protect against infections and cancer, but excessive IRF3 activation and type I IFN production cause autoinflammatory conditions such as Aicardi-Goutières syndrome and STING-associated vasculopathy of infancy (SAVI). Myocardial infarction (MI) elicits inflammation, but the dominant molecular drivers of MI-associated inflammation remain unclear. Here we show that ischemic cell death and uptake of cell debris by macrophages in the heart fuel a fatal response to MI by activating IRF3 and type I IFN production. In mice, single-cell RNA-seq analysis of 4,215 leukocytes isolated from infarcted and non-infarcted hearts showed that MI provokes activation of an IRF3-interferon axis in a distinct population of interferon-inducible cells (IFNICs) that were classified as cardiac macrophages. Mice genetically deficient in cyclic GMP-AMP synthase (cGAS), its adaptor STING, IRF3, or the type I IFN receptor IFNAR exhibited impaired interferon-stimulated gene (ISG) expression and, in the case of mice deficient in IRF3 or IFNAR, improved survival after MI as compared to controls. Interruption of IRF3-dependent signaling resulted in decreased cardiac expression of inflammatory cytokines and chemokines and decreased inflammatory cell infiltration of the heart, as well as in attenuated ventricular dilation and improved cardiac function. Similarly, treatment of mice with an IFNAR-neutralizing antibody after MI ablated the interferon response and improved left ventricular dysfunction and survival. These results identify IRF3 and the type I IFN response as a potential therapeutic target for post-MI cardioprotection.

Figure 1.

Myocardial infarction activates IRF3-dependent signaling. (a) Immunoblotting for phosphorylated and total IRF3 without MI or at day 4 after MI (top) and semiquantitation of band intensities (bottom). GAPDH was used as a loading control (n=4 mice, 2 per group, from 1 experiment, 3 separate blots) AU, arbitrary units. (b-d) Levels in WT and Irf3^−/− mice at day 4 after MI of Ifnb1 (n=8 per group) and Cxcl10 (n=15 WT and n=16 Irf3^−/− mice) (b), CXCL10 protein (n=6 per group) (c), and type I interferon-stimulated genes (ISGs) (n=15 WT and n=16 Irf3^−/− mice) (d). Non-infarcted WT mice were used as a control in b and d. (e) Scatterplot of RNA-seq expression data from the infarct tissue of WT and Irf3^−/− mice at day 4 after MI (n=3 mice per group). Differentially expressed genes are shown in green and several highly differentially expressed ISGs are annotated. Inset shows the full scatterplot for all genes. (f) Single cell RNA-Seq data from cells isolated from the infarct region of WT mice at day 4 after MI (n=1858 single cells from 1 wild type mouse). The data are displayed as color-coded clusters on a t-distributed stochastic neighbor embedding (tSNE) plot. The interferon inducible cell (IFNIC) cluster shown in pink is defined based in which 8 out of 10 discriminating marker genes are interferon stimulated genes. (IFNICs = Interferon inducible cells, DCs = dendritic cells, MHCII macrophages = major histocompatibility complex molecules) (g) Top, heat map plotting the top 10 marker genes for each cluster (y axis) versus single cells grouped by clusters (x axis). The IFNIC population is indicated at the top of the heat map and selected marker genes are annotated on the right. Bottom, scatterplot showing the IRF3 Score for each cell (see Online Methods). (Monos = Monocytes, DCs = dendritic cells, P=proliferating cells, and T = T cells and natural killer cells) (h) Violin plots depicting the expression probability distribution for each single cell cluster. Ten IRF3-dependent genes are shown. Data are mean ± SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Figure 2.

Post-phagocytic macrophages initiate IRF3-dependent amplification of post-MI inflammation. (a,b) Relative Ifnb1 expression levels in CD45+ and CD45- (n=6 per group) (a) and CD11b+ and CD11b- (n=6 per group) (b) cell populations sorted from infarct tissue of WT mice on day 4 after MI. (c) Schematic of a parabiosis experiment in which Irf3^−/− mice were subjected to MI after they had been paired to either Irf3^−/− (KO→KO) or WT (WT→KO) mice (left). Expression of the indicated IRF3-dependent genes in the heart of the Irf3^−/− parabiont that underwent MI at day 4 after MI (n=4 KO->KO and n=5 WT->KO parabionts) (right and below). (d) Micrograph of the borderzone (BZ, arrow) of the heart of an Myh6Cre-mTmG reporter mouse on day 4 after MI; in these mice, cardiomyocytes express membrane EGFP (green) and all other cells express membrane tdTomato (red). IZ, infarct zone; SZ, survival zone. (e) Representative plots illustrating the gating strategy of Lin-Ly6G-CF11b+ myeloid cells used to identify post-phagocytotic macrophages that are associated with cardiomyocyte-derived DAMPs in Myh6Cre-mTmG mice. Cre negative reporters (top left) define GFP autofluorescence (0% cells above GFP threshold). Myh6Cre positive reporters (bottom left) exhibit 6% of cells above GFP fluorescence. (f) Representative backgating plot of the 6% GFP+ myeloid cells (arrow from gate to green overlay) on the GFP- myeloid population (red) from a Cre- mouse at day 4 after MI. Arrow indicates the gating strategy used to identify the GFP+ population. (g,h) Expression of Ifnb1 (g) and the indicated ISGs (h) in FACS sorted subpopulations based on CD11b and GFP status (representative of 2 experiments, n=4 mice each). (i) Fluorescence imaging of a short axis heart section from Mx1Cre-mTmG reporter mice on day 4 after MI, showing non-induced tdTomato expressing cells (red) and IFN-induced EGFP expressing cells (green). Low (left) and high (right) magnification are shown. (j) Ex vivo 2- photon autofluorescence imaging of infarct borderzone, showing surviving cardiomyocytes (green) and infiltrate (purple) in WT and Irf3^−/− mice. (k) Gating strategy (top and middle) and leukocyte subset enumeration (bottom; total leukocytes, Lin⁻CD11b⁺F4/80^highLy6C^low macrophages, and Lin⁻CD11b⁺F4/80^lowLy6C^high monocytes) from infarcts of WT and IRF3^−/− mice on day 4 after MI (representative of 2 experiments, n=5 per group). (l) Violin plots of single cell RNA-Seq data from day 4 MI hearts (n=1,585 cells) depicting the expression probability distribution of selected genes for each mononuclear cell cluster. Genes were selected based on established mononuclear cell subset marker genes. *P < 0.05, **P < 0.01.

Figure 3.

Self DNA is the dominant MI-induced IRF3-activating DAMP. (a) Schematic of the three known IRF3 activation pathways, their respective adaptor proteins (TRIF, STING, and MAVS), and their proximal activating DAMPs. (b) Expression of Cxcl10, Ifit1, and Irf7 in the infarct region at day 4 after MI in WT (n=16), Irf3^−/− (n=15), Trif^Lps2 (n=9), Mavs^−/−(n=4), and STING^gt/gt (n=12) mice, as compared to no MI in WT mice (n=4). (c) Schematic of the cytosolic DNA sensing pathway involving the DNA sensor cGAS, the transcription factor IRF3, and the type I interferon receptor IFNAR. (d) Expression of Cxcl10, Ifit1, and Irf7 in the infarct region on day 4 after MI from WT (n=16), Irf3^−/−(n=15), cGAS^−/− (n=7), and Ifnar^−/− (n=4) mice, as compared to no MI in WT mice (n=4). Data are mean ± SEM. ** P < 0.01, *** P < 0.001, **** P < 0.0001. (e) Intravital multiphoton microscopy of WT mouse after coronary ligation showing localization of the double-stranded, DNA-specific fluorescent probe SYTOX Orange (blue, left), autofluorescence of surviving cardiomyocytes (green, middle), and overlay (right) after 20 minutes of ischemia-reperfusion. (f) Quantification of dsDNA probe fluorescence and cardiomyocyte autofluorescence along the linescan (dotted line) at the level of the nucleus spanning surviving and injured cardiomyocytes. Arrows indicates nuclear DNA staining. (x axis is distance, y axis is arbitrary fluorescence units along the dotted line) (g) Experimental strategy. WT mouse cardiomyocytes were labeled in utero by EdU, such that the resulting adult mice have non-proliferative cardiomyocytes that retain EdU, whereas proliferative leukocytes dilute the EdU label. At 10 weeks of age, EdU labeled mice were subjected to MI; infarct tissue was harvested on day 4 and evaluated for EdU staining in the cytoplasm of infiltrating leukocytes. (h) EdU click-labeled to a fluorophore (purple) in autofluorescent cardiomyocytes (green) prior to MI (left) and in the borderzone of the infarcted heart, which contains EdU-positive debris (middle left). A magnified view (middle right) of the region indicated by the dashed box shows a cell (outlined with purple dots) with extranuclear EdU puncta (pink); the nucleus (outlined with white dots) is stained with a Hoechst nuclear stain (blue). EdU staining of a bone marrow derived macrophage after uptake of EdU-labeled cardiomyocyte DNA in vitro is shown for comparison (right).

Figure 4.

Genetic and pharmacologic disruption of DNA-induced IRF3 activation and type I interferon signaling protects mice from death and adverse ventricular remodeling following MI. (a) Kaplan-Meier survival curves comparing post-MI survival of WT mice (n=57) to cGAS^−/−(n=47, P=0.0133), Irf3^−/− (n=45, P<0.0001) and Ifnar^−/−(n=31, P=0.0001) mice. (b-d). M-mode quantitation of echocardiographic parameters of the indicated mouse strains at days 4 and 21 after MI: left ventricular inner diameter at end-diastole (LVIDd) (b), left ventricular inner diameter at end-systole (LVIDs) (c), and fractional shortening (FS %) (d). For each genotype, P values and n numbers (n=WT, knockout) are indicated. (e) Masson’s Trichrome stain of short axis sections taken 1 mm below the suture ligation from WT (n=5) and Irf3^−/−(n=7) mice on day 7 after MI. (f) Quantification of short axis chamber area 1 mm below the suture ligation on day 7 after MI. (g-i) Cardiac MRI of WT and Irf3^−/− mice on day 21 after MI. Short axis images are shown at the level of the papillary muscle at end diastole (g) and end systole (h), and 4-chamber long axis images are shown at end diastole (i). (j-l) Cardiac MRI-based quantification of left ventricular end-diastolic volume (LVEDV) (j), left ventricular end-systolic volume (LVESV) (k), and left ventricular ejection fraction (LVEF) (l) in WT (n=7) and Irf3^−/−(n=8) mice on day 21 after MI. (m) Experimental strategy. WT mice were treated with an IFNAR neutralizing antibody at 8–12 hr and 48 h after MI. (n) Expression of Ifnb1 and ISGs at 4 days after MI in antibody-treated or untreated mice (n=4 per group) (o) Left ventricular diameters (LVIDd, LVIDs) and fractional shortening (%FS) at 21 days after MI (n=6 per group). (p) Kaplan-Meier survival curves comparing antibody-treated to untreated (WT) mice (n=19 IFNAR Ab, n=57 WT)). Data are mean ± SEM. *P < 0.05, **P < 0.01, *** P < 0.001. (q) Working model for IRF3 activation after myocardial infarction.