The Programmed Death-1 Signaling Axis Modulates Inflammation and LV Structure/Function in a Stress-Induced Cardiomyopathy Model

Figure 1. Characterization of Stress Induced Cardiomyopathy Model (A) Kaplan-Meier survival curves of isoproterenol (ISO)-injected (n = 42) and diluent injected (phosphate buffer saline [PBS]) control mice (n = 33). (B) Serum troponin I levels at baseline, 1 hour, and days 1-4 in ISO-treated mice (n = 7-12 mice/time). (C) Gravimetric analysis of hearts harvested from ISO-treated mice at baseline (n = 12), 1 hour, and days 1-3 and 7 (n = 9-13 hearts/time). (D) Representative photographs of hematoxylin and eosin–stained hearts at baseline and days 2 and 7 after ISO or diluent injection (scale bar = 50 μm). (E) Representative photographs of Masson’s trichrome-stained hearts at baseline and days 2 and 7 after ISO or diluent injection (scale bar = 50 μm). (F) Inflammatory score index at baseline and days 2 and 7 after ISO or diluent injection (n = 24 fields obtained from 4 hearts/time). (G) Flow cytometry analysis of the number of CD45+ cells/mg of heart tissue at baseline (n = 11 hearts), 1 hour, and days 1-3 and 7 (n = 9-13 hearts/time) after ISO injection. Data were analyzed by repeated measures 1-way analysis of variance with Dunnett’s multiple comparisons relative to baseline (B, C, and G)…

Figure 2. LV Structure, Function, and Regional Wall Motion in a Model of Stress-Induced Cardiomyopathy (A) Representative long-axis (top) and short-axis (bottom) 2-dimensional echocardiographic images from isoproterenol (ISO)-injected mice at baseline, 1 hour, and 7 days (see Videos S1 and S2). Superimposed green lines represent speckle-tracking analysis of endocardial and epicardial contours; radially oriented vectors represent the direction and amplitude of segmental endocardial velocities. (B) Left ventricular (LV) end-diastolic volume (EDV), (C) left ventricular ejection fraction (LVEF %), (D) regional segmental wall motion from LV base to apex, and (E) global segmental wall motion score index (SWMSI) at baseline, 1 and 6 hours, and 7 days after ISO injection (n = 5-12 mice/time). The global SWMSI was determined as the average of 84 regional LV segments, where 1 = normal wall thickening, 2 = hypokinesis, and 3 = akinesis. Data were analyzed by repeated measures 1-way analysis of variance with Dunnett’s multiple comparisons relative to baseline. ∗P < 0.05; ∗∗P < 0.01.

Figure 3. Immunophenotyping of CD45+ Cells in the Heart, Blood, and Spleen Following ISO Injection Flow cytometry was performed on CD45+ cells that were isolated from the heart (cells/mg of tissue), blood (cells/20 μL) and spleen (% of CD45+ cells) of identical mice. (A) Ly6G+ neutrophils, (B) Ly6ChighCD64low monocytes, (C) CD4+ T cells, (D) CD8+ T cells, and (E) CD19+ B lymphocytes were isolated from the heart, blood, and spleen at baseline (n = 11-12 mice), 1 hour, and days 1-3 and 7 (n = 8-13 mice/time) after isoproterenol (ISO) injection. (F) CD64+Ly6Clow/− macrophages were isolated from the hearts of ISO-injected mice at baseline (n = 12 mice), 1 hour, and days 1-3 and 7 (n = 9-12 mice/time) after ISO injection. Data were analyzed by repeated measures 1-way analysis of variance with Dunnett’s multiple comparisons relative to baseline. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Figure 4. Single-Cell RNA Sequencing of CD45+ Cells Isolated From the Heart Following ISO Injection Unsupervised Uniform Manifold Approximation and Projection (UMAP) plot of 10× single-cell sequencing data from the total of 3,748 CD45+ cells (baseline: 1,104 cells; day 2: 1,457 cells; day 7: 1,187 cells) after quality control. CD45+ cells were sorted from the hearts of mice at baseline and on days 2 and 7 after isoproterenol (ISO) injection. (A) A total of 10 unique cell clusters were identified (see Methods) at each time period. (B) The proportion of cell clusters depicted in A. (C) Feature plot of CCR2+ expression in LYVE1− macrophages, dendritic cells, monocytes, and LYVE1+ macrophages. (D) Heat map of changes in gene expression for immune checkpoint inhibitors and stimulators in LYVE1− macrophages, LYVE1+ macrophages, dendritic cells, and T cells. Data represent the fold change in expression levels (following log2 normalization) on days 2 and 7 relative to baseline.

Figure 5. Effects of ISO Injury in PD-1−/− Mice and WT Mice Injected With Anti-PD-1, Anti-PD-L1, and Anti-PD-L2 Monoclonal Abs (A) Experimental protocol for isoproterenol (ISO)-injected mice treated with anti-PD-1, anti-PD-L1, anti-PD-L2 antibodies (Abs), or isotype control Abs. (B) Kaplan-Meier survival curves of ISO-injected wild-type (WT), PD-1−/− mice, or ISO-treated WT mice injected with anti-PD-1, anti-PD-L1, anti-PD-L2 Abs, or isotype control Abs. (C) Serum troponin I levels at baseline, day 2 and day 7 in ISO-injected WT, PD-1−/− mice or ISO-treated WT mice injected with anti-PD-1, anti-PD-L1, anti-PD-L2 Abs or isotype control Abs (n = 5-13 mice/group/time). (D) Flow cytometry analysis of the number of CD45+ cells/mg of heart tissue. Number (cells/mg of heart tissue) of (E) Ly6G+ neutrophils, (F) Ly6ChighCD64low monocytes, (G) CD64+Ly6Clow/− macrophages, (H) CD4+ T cells, (I) CD8+ T cells, and (J) CD19+ B lymphocytes in ISO-injected WT, PD-1−/− mice, or ISO-treated WT mice injected with anti-PD-1, anti-PD-L1, anti-PD-L2 Abs, or isotype control Abs (n = 4-13 mice/group/time). Data were analyzed by 1-way analysis of variance with Tukey’s multiple comparisons within each time point (C…

Figure 6. LV Structure, Function, and Regional Wall Motion in PD-1−/− Mice After ISO Injection (A) Regional segmental wall motion from left ventricular (LV) base to apex in wild-type (WT) mice (data from Figure 2D were reproduced here for ease of comparison) and PD-1−/− mice at baseline, 1 and 6 hours, and 7 days after isoproterenol (ISO) injection (n = 5-12 mice/group/time). (B) LV end-diastolic volume (EDV), (C) left ventricular ejection fraction (LVEF %), and (D) global segmental wall motion score index (SWMSI) at baseline, 1 and 6 hours, and 7 days after ISO injection (n = 5-12 mice/group/time). The global SWMSI was determined as the average of 84 regional LV segments, where 1 = normal wall thickening, 2 = hypokinesis, and 3 = akinesis. Data were analyzed by repeated measures 2-way analysis of variance With Sidak’s multiple comparisons (B to D). ∗P < 0.05; ∗∗P < 0.01.

Figure 7. Damage-Associated Molecular Patterns Provoke PD-1 Up-Regulation Through the NF-κB-Dependent TLR Signaling Pathway in Peritoneum-Derived Macrophages and T Lymphocytes Unfractionated peritoneum-derived inflammatory cells (PDICs) were collected following 72-hour culture and the percentage of PD-1+CD64+ macrophages, PD-1+CD4+, and PD-1+CD8+ T lymphocytes was evaluated by flow cytometry analysis. (A to F) PDICs were cultured in media alone (control) or necrotic cardiac myocyte cell extracts (NCEs) (10 μg/mL), a Toll-like receptor 2 (TLR2) agonist (Pam3CSK4; 200 ng/mL), a TLR4 agonist (lipopolysaccharide [LPS]; 100 ng/mL), or isoproterenol (ISO) (1, 10, or 100 μmol/L). PD-1 expression in (A) CD45+Aqua−CD64+ macrophages (n = 9-11 biological replicates/group), (B) CD45+Aqua−CD4+ T lymphocytes (n = 9-11 biological replicates/group), (C) CD45+Aqua−CD8+ T lymphocytes (n = 9-10 biological replicates/group), (D) CD45+Aqua−CD64+ macrophages (n = 8 biological replicates/group), (E) CD45+Aqua−CD4+ T lymphocytes (n = 8 biological replicates/group), and (F) CD45+Aqua−CD8+ T lymphocytes (n = 8 biological replicates/group). (G to I) PDICs were cultured in media alone (control) or in the presence or…