TLR7 Inhibition Limits Ischemic Cardiac Injury by Disrupting ITGAM-Dependent Immune-Endothelial Interactions

Figures

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  Figure 1. Plasma Small RNA Sequencing and Identification of Proinflammatory miRNAs in a Mouse Model of Ischemia-Reperfusion (A) Plasma RNA quantifications 24 hours after sham or I/R procedure (n = 5-10). Data are shown as mean ± SEM. (B) RNA sequencing analysis with mapped read counts and relative percentage of different RNA biotypes in the plasma (n = 6-8). (C) Upregulated plasma miRNAs between I/R vs sham identified by RNA sequencing. Four miRNAs contain at least 1 of the 5 proinflammatory motifs (.uuc; u..uu.; .a.....uu.; g.uu.; uu...u.). (D) MIP-2 production in bone marrow–derived macrophages (BMDMs) treated with miRNA mimics. ∗Indicates miRNAs containing proinflammatory motifs (see Supplemental Table 1). I/R = ischemia-reperfusion.

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  Figure 2. Enpatoran Reduces Infarct Size and Preserves Cardiac Function After Ischemia-Reperfusion (A) Study diagram illustrating enpatoran administration (intraperitoneal) and experimental endpoints in murine models of I/R injury (n = 6-10 mice per group). For each dosing regimen (prevention protocol [PP], treatment protocol [TP], early rescue protocol [ERP], and late rescue protocol [LRP]), 2 doses of enpatoran (5 mg/kg) were administered 2 hours apart, with the first dose shown. (B) Representative triphenyl tetrazolium chloride–stained images of myocardial infarction. (C to E) AAR/LV, MI/LV, and MI/AAR 24 hours after I/R. (F) Plasma cardiac troponin I (cTn1) following I/R (prevention protocol). (G) M-mode images of echocardiography. (H) Cardiac output (CO), (I) left ventricular ejection fraction (LVEF), (J) global longitudinal strain (GLS), and (K) global radial strain (GRS). (L) B-mode images showing left atrial (LA) diameter (left panel), E waves (middle panel), and e′ sr waves (right panel). (M to O) Quantification of LA diameter (M), E-wave velocity (N), and E/e′ sr (O). Data shown are mean ± SEM. Statistical significance was analyzed using 1-way (F) or ordinary 2-way ANOVA (C to E…

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  Figure 3. Single-Nucleus RNA Sequencing Reveals Expansion of Myeloid and Endothelial Cells With Distinct Transcriptomic Signatures in Ischemic Myocardial Infarction (A) UMAP (uniform manifold approximation and projection) visualizing cardiac cell clusters (upper) and the percentage of cardiomyocytes (CM), EC8, neutrophils (NE), and monocytes (Mo) among total cells (lower right) from 3 groups of male mice (n = 3 per group). (B) UMAP of Itgam+ cells with Itgam expression levels shown by a light gray–to–dark purple gradient (log2-normalized expression; left). Quantification of Itgam+ EC8, NE, and Mo was shown on the right. (C) Density-expression plot of Itgam in EC8 (left), NE (middle), and Mo (right). (D) Representative immunofluorescent images of sarcomeric actin (red), CD31/Ly6G/CCR2 (magenta), and Itgam (green) of heart sections. Nuclei were stained with blue DAPI. The bar graph illustrates quantification of Itgam+ EC, NE, and Mo (n = 5-6 mice per group). (E) UMAP of human blood mononuclear cells showing ITGAM expression. Single-cell RNA sequencing analysis of monocyte ITGAM expression from healthy controls and STEMI patients before and after coronary reperfusion. Dot plots represent the…

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  Figure 4. TLR7 Activation Increases Itgam Expression in Macrophages and Endothelial Cells (A) Experimental diagram of cell treatment with TLR7 agonists. (B) Expression level of Itgam in BMDMs was examined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Relative expression of Itgam in the R837-treatment group was calculated with the nontreated control. (C) IL-6 production by BMDMs as measured by ELISA. (D and F) The representative images of ITGAM expression in BMDMs (D) and HCAECs (F). (E and G) Quantification of the ITGAM+ cell total integrated intensity (the sum of pixel intensities within a designated area of a cell). Data shown are mean ± SEM. Statistical significance for independent samples collected at different time points in panels (B) and (C) was analyzed using ordinary two-way ANOVA followed by Sidak's multiple-comparisons test. Panels E and G were analyzed using ordinary one-way ANOVA followed by Tukey's multiple-comparisons test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. BMDM = bone marrow–derived macrophage; ELISA = enzyme-linked immunosorbent assay; FC = fold-change; HCAEC = human coronary artery endothelial cell; IF = immunofluorescence; lipo =…

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  Figure 5. TLR7 Activation Increases Leukocyte-Endothelial Adhesion (A, C, and E) Microfluidic adhesion assay of THP-1 (Mo) or human fresh neutrophil adhesion to HCAEC under different treatment conditions as indicated (R848/enpatoran: 3 hours for Mo, 18 hours for EC; ITGAM Ab: 1 hour). (B, D, and F) Quantification of THP-1 or human fresh neutrophil adhesion under different treatment conditions. CD45+ immune cells are shown in green. Adherent CD45+ cells appear as discrete green puncta, while continuous green streaks reflect perfused monocytes under flow conditions. Data shown are mean ± SEM. Statistical significance was analyzed using ordinary 1-way ANOVA followed by Tukey's multiple-comparisons test. ∗P < 0.05, ∗∗∗P < 0.001. EC = endothelial cell; Enpa = enpatoran; HCAEC = human coronary artery endothelial cell; ITGAM Ab = ITGAM neutralizing antibody; Mo = monocyte; NE = neutrophil.

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  Figure 6. ITGAM Neutralization Attenuates Leukocyte Infiltration and Limits Ischemic Myocardial Infarction (A) Study diagram for intraperitoneal administration of ITGAM-neutralizing antibody (ITGAM Ab) or control isotype in a mouse model of MI and various experimental endpoints (n = 9 mice per group). (B) Representative images of MI areas after triphenyl tetrazolium chloride staining. (C) Quantification of AAR/LV, MI/LV, and MI/AAR 24 hours after I/R. (D) Cardiac output (CO) and (E) left ventricular ejection fraction (LVEF). (F) Echocardiography speckle tracking imaging showing global longitudinal strain (GLS). (G to H) Echocardiographic data depict left atrial (LA) diameter (G), and E wave–to–reversed longitudinal strain rate ratio (E/e′ sr) (H). (I and K) Immunofluorescence staining of heart sections to show sarcomeric actin (red), and Ly6G/CCR2 (magenta). DAPI was stained in blue. (J and L) Quantification of the cardiac infiltrated neutrophils (NE) and monocytes (Mo) after I/R injury. Data shown are mean ± SEM. Statistical significance in panel C was analyzed using ordinary 2-way ANOVA followed by Tukey's multiple-comparisons test. Longitudinal echocardiographic measurements in panels D…

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  Figure 7. Myocardial Ischemia-Reperfusion Enhances ICAM1-ITGAM Interaction (A) In silico ligand-receptor (L-R) interaction analysis of the snRNA-seq dataset. The 24 listed L-R pairs are associated with inflammatory and immune-related functions. Communication probabilities via L-R pair interaction between endothelial cells (ECs) and neutrophils (NEs) or monocytes (Mos) across sham, NS-I/R, and Enpa-I/R groups are shown. Each dot represents an individual L-R pair, with color indicating communication probability and dot size reflecting statistical significance (P value). Key endothelial-to-immune interactions, Icam1-Itgam, are bolded, revealing the dynamic changes in communication in response to I/R and to enpatoran treatment. (B) Stimulated emission depletion super-resolution immunofluorescent images of heart sections to show ITGAM (red), ICAM1 (green), and cell membranes (WGA, magenta). ICAM1-ITGAM colocalization was observed within endothelial junctional membrane protrusions in I/R mice. ECM = extracellular matrix; Enpa = enpatoran; I/R = ischemia-reperfusion; Mac-1 = Itgam + Itgb2; NS = normal saline; WGA = wheat germ agglutinin.

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  Figure 8. TLR7 Activation Promotes ICAM1 Expression and Leukocyte-Endothelial Interaction (A) The representative images of ICAM1 expression in BMDMs and HCAECs. (B) Quantification of the ICAM1+ cell total integrated intensity (HCAECs: NV vs R848, P = 0.053). (C-D) Microfluidic adhesion assay of THP-1 (Mo) adhesion to HCAEC (C) and quantification (D) under different treatment conditions. (E-F) Microfluidic adhesion assay of human fresh neutrophil adhesion to HCAEC (E) and quantification (F) under different treatment conditions. Treatment time for R848/enpatoran: 3 hours for Mo, 18 hours for EC; ICAM1 Ab: 1 hour. Adherent CD45+ cells appear as discrete green puncta, while continuous green streaks reflect perfused monocytes under flow conditions. Data shown are means ± SEM. Statistical significance was analyzed using ordinary 1-way ANOVA followed by Tukey's multiple-comparisons test. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. BMDM = bone marrow–derived macrophage; NC = nontreated control; other abbreviations as in Figure 5.