CD14 Blockade Modulates Macrophage-Mediated Immunological Injury in a Translational Model of Reperfused ST-Segment Elevation Myocardial Infarction

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  Figure 1. Characterization of the Mouse Model of Reperfused STEMI With Key Clinical Features Mice were given an anterior coronary artery occlusion, and reperfused 1 hour later. (A) Electrocardiograms were taken immediately after myocardial infarction surgery (ie, before reperfusion), demonstrating the distinct ST-segment elevation associated with occlusion of the left anterior coronary artery. (B) Representative 3-dimensional strain curves by echocardiography at baseline and 24 hours post–ST-segment elevation myocardial infarction (STEMI). (C) Echocardiographic assessment of left ventricular geometry and function from naïve and post-STEMI mice. Statistical analyses were performed using analysis of variance followed by Dunnett’s multiple comparison testing to compare baseline parameters with time elapsed after surgery. Mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. A = anterior; BL = baseline; P = posterior.

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  Figure 2. CD14 Blockade Preserves LV Systolic Function, Volumes, Biomechanics, and Hemodynamics 7 or 21 and 28 Days Post-STEMI After 1 hour left anterior coronary artery occlusion, mice were administered saline, IgG2a isotype control antibody, or anti-CD14 at the time of reperfusion (150 μL bolus, intravenous), with repeat injections at 24 hours (7-day experiment only) and weekly for up to 4 weeks. After 7 days, overt LV dysfunction, dilatation, and adverse hemodynamic changes were observed in saline- and isotype-treated mice, and these changes were significantly ameliorated with anti-CD14 treatment (Tx). Multimodality assessments of LV function and volumes by echocardiography, cardiac magnetic resonance imaging, and intracardiac pressure-volume hemodynamics revealed anti-CD14 Tx prevented progressive LV dysfunction and dilatation over 28 days post-STEMI. (Top) (A) 7-day trial design overview. (B) Echocardiographic assessment of LV geometry and function at 7 days post-STEMI. (C) Intracardiac catheter assessments of LV pressure-volume hemodynamics at 7 days post-STEMI. Single representative echocardiographic frames (parasternal long-axis view) (B), pressure-volume loops (C) are shown.…

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  Figure 3. Acute Injury Assessment and Monocyte/Macrophage Abundance and Transcriptome Response to CD14 Blockade 1 and 3 Days Post-STEMI Acute infarct size and cardiac troponin I (a marker of cardiomyocyte death) were unaffected by anti-CD14 antibody treatment. At 3 days post-STEMI, macrophage abundance was similar between all groups. However, in these macrophages, widespread down-regulation of proinflammatory genes and up-regulation of anti-inflammatory genes were observed with anti-CD14 treatment. (A) Trial design overview for 24-hour acute injury assessment study. (B) Representative images of Evans blue/tri-tetrazolium chloride-stained hearts (grid 200 μm) are shown for each group. (C) Outcome measures of acute injury at 24-hour post-STEMI. (D) Trial design overview for 3-day macrophage abundance and phenotyping study. (E) Representative images of immunohistochemistry showing CD68+ cell accumulation within the infarcted myocardium (troponin T+ cardiomyocytes). (F) Immunohistochemistry analyses of macrophage (CD68+ cell) relative abundance at 3, 7, and 28 days post-STEMI. (G) Cell sorting protocol. Cells were isolated from the infarct region at 3 days post-STEMI, and populations…

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  Figure 4. Single-Cell Analysis of Immune Cell Transcriptome Isolated From Infarcted Myocardium Following CD14 Blockade 3 Days Post-STEMI Anti-CD14 treatment blunts the monocyte proinflammatory lineage post-MI. Single-cell RNA sequencing was performed on sorted cells isolated from the infarct region at 3 days post-STEMI (described in Figure 3). (A) Uniform manifold approximation and projection (UMAP) plot displaying all 11 different populations of macrophages (>96%), dendritic cells (<3%), and neutrophils (<1%) identified in the apical region of the damaged heart at 3 days post-STEMI, and color chart (also applicable to B and C). (B) Differential expression marker genes for myeloid cell states. (C) Relative abundance histogram stack (cell composition plot), and (D) cell population density heat-map (gaussian distribution: 0-1) highlighting the reduced abundance of macrophages with high expression of Plac8/Ly6c2 (cluster 1; Ly6c2hi) in the anti-CD14 treatment group. (E) Marker expression dot-plot (including average scaled expression) and UMAP plot showing that CCR2 was highly expressed in select subpopulations in the UMAP space (including cluster 1 and cluster 2), dendritic cells (cluster 6),…

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  Figure 5. Differential Macrophage and Tissue-Level Proteomics Following CD14 Blockade at 3 Days Post-STEMI (A) Volcano plot of macrophage cell proteome of anti-CD14 treatment vs saline based on differential expression with Limma-Voom. Differential up-regulated (red) and down-regulated (blue) proteins with P < 0.05 are highlighted. (B) Lollipop plot of functional enrichment analysis of anti-CD14 treatment vs saline based on differential expression of macrophage cell proteome (Perseus 1D enrichment analysis; biological processes and cellular component). Statistics can be found within Supplemental Data 1 and 2. (C) Gene Ontology Component/Process/Function enrichments retrieved through STRING Enrichment in Cytoscape (P < 0.05). Specific proteins represented for each functional category are indicated; colors represent differential expression relative to saline, with node size representing the level of significance. (D) Volcano plot of infarcted tissue proteome of anti-CD14 treatment vs saline based on differential expression with Limma-Voom. Differential up-regulated (gold) and down-regulated (purple) proteins with P < 0.05 are highlighted. (E) Lollipop plot of functional enrichment analysis of…