TLR7 inhibition limits cardiac ischemic injury by disrupting ITGAM-dependent immune–endothelial interaction

Figures

• 

  Figure 1.. Plasma small RNA-seq and identification of proinflammatory miRNAs in a mouse model of MI. A, Plasma RNA quantifications 24 hours after Sham or I/R procedure (n=5–10). B, RNA-seq 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-seq. Four miRNAs contain at least one of the five proinflammatory motifs (.uuc; u..uu.; .a…..uu.; g.uu.; uu…u.). D, MIP-2 production in bone marrow-derived macrophages (BMDM) treated with miRNA mimics. I/R, ischemia-reperfusion. * Indicates miRNAs containing proinflammatory motifs in their sequences (see Table S1).

• 

  Figure 2.. Enpatoran reduces infarct size and preserves cardiac function after I/R. A, Study diagram for intraperitoneal administration of enpatoran in cardiac I/R model and experimental endpoints (n = 6–10 mice per group). For each protocol (PP, TP, ERP, or LRP), two doses of enpatoran (5 mg/kg) were administered 2 hours apart; the diagram shows the 1st dose. B, Representative TTC-stained images of MI. C-E, AAR/LV, MI/LV, and MI/AAR 24h after I/R. F, Plasma cTnI following I/R (PP protocol). G, M-mode images of echocardiography. H-K, Cardiac output (CO) (H), left ventricular ejection fraction (LVEF) (I), GLS (J), and GRS (K). L, B-mode images showing LA diameter (left panel), E waves (middle panel), and e’ sr waves (right panel). M-O, Quantification of LA diameter (M), E wave velocity (N), and E/e’ sr (O). Data shown are means ± SEM. One-way and two-way ANOVA were used to test statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Enpa, enpatoran; NS, normal saline; I/R, ischemia-reperfusion; PP, prevention protocol; TP, treatment protocol; ERP, early rescue protocol; LRP, late rescue protocol; ECHO, echocardiography; AAR, area-at-risk; MI, myocardial infarction; RV,…

• 

  Figure 3.. snRNA-seq reveals expansion of myeloid and endothelial cells with distinct transcriptomic signatures in ischemic MI. A, UMAP visualizing cardiac cell clusters (upper) and the percentage of CM, EC8, NE, and Mo among total cells (lower right) from three groups of male mice (n=3 per group). B, UMAP of Itgam+ cells with Itgam expression levels shown by a light grey-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. scRNA-seq analysis of monocyte ITGAM expression from healthy controls and STEMI patients before and after coronary reperfusion. Dot plots represent the percentage of ITGAM+ monocytes among total mononuclear cells analyzed (n=38 patients per group). Data shown are means ± SEM. A one-way ANOVA was used…

• 

  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 qRT-PCR. Relative expression of Itgam in the R837-treatment group was calculated with the non-treated control (NC). C, IL-6 production by BMDMs as measured by ELISA. D & F, The representative images of ITGAM expression in BMDMs (D) and HCAECs (F). E & G, Quantification of the ITGAM+ cell total integrated intensity. Data shown are means ± SEM. A one-way ANOVA was used to test for statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, nontreatment control; DAPI, 4′,6-diamidino-2-phenylindole; BMDMs, bone-marrow-derived macrophage; HCAEC, human coronary artery endothelial cell; FC, fold-change; lipo, lipofectamine; integrated intensity, the sum of pixel intensities within a designated area of a cell.

• 

  Figure 5.. TLR7 activation increases leukocyte-endothelial adhesion. A, C, E, Microfluidic adhesion assay of THP-1 (Mo) or human fresh neutrophil adhesion to HCAEC under different treatment conditions as indicated (R848/enpatoran: 3h for Mo, 18h for EC; ITGAM Ab: 1h). B, D, & 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 means ± SEM. A one-way ANOVA was used to test for statistical significance. *P < 0.05, ***P < 0.001, ****P < 0.0001. HCAEC, Human coronary artery endothelial cell; NE, neutrophil; Mo, monocyte; ITGAM Ab, ITGAM neutralizing antibody.

• 

  Figure 6.. ITGAM neutralization attenuates leukocyte infiltration and limits ischemic MI. A, Study diagram for intraperitoneal administration of ITGAM neutralizing antibody 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 (TTC) staining. C, Quantification of AAR/LV, MI/LV, and MI/AAR 24 hours after I/R. D-E, Cardiac output (CO) (D) and left ventricular ejection fraction (LVEF) (E). F, Echocardiography speckle tracking imaging showing global longitudinal strain (GLS). G-H, Echocardiographic data depict 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 & L, Quantification of the cardiac infiltrated NE and Mo after I/R injury. Data shown are means ± SEM. Two-way ANOVA was used to test statistical significance. **P < 0.01, ***P < 0.001, ****P < 0.0001. I/R, ischemia-reperfusion; Enpa, enpatoran; ECHO, echocardiography; CO, cardiac output; LVEF, left ventricular ejection fraction; GRS, global radial…

• 

  Figure 7.. Myocardial I/R 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, STED 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. I/R, ischemia-reperfusion; Mac-1, Itgam + Itgb2; NS, normal saline; Enpa, enpatoran; EC, Endothelial cell; Mo, monocyte; NE, neutrophil; WGA, Wheat Germ Agglutinin.

• 

  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. 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: 3h for Mo, 18h for EC; ICAM-1 Ab: 1h. Adherent CD45+ cells appear as discrete green puncta, while continuous green streaks reflect perfused monocytes under flow conditions. Data shown are means ± SEM. A one-way ANOVA was used to test for statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. NC, nontreatment control; DAPI, 4′,6-diamidino-2-phenylindole; BMDMs, bone-marrow-derived macrophages; HCAECs, human coronary artery endothelial cells; Integrated intensity, the sum of pixel intensities within a designated area of a cell; ICAM1 Ab, ICAM1 neutralizing antibody; Mo, monocyte; NE, neutrophil.