Anti-CTLA-4 m2a Antibody Exacerbates Cardiac Injury in Experimental Autoimmune Myocarditis Mice By Promoting Ccl5-Neutrophil Infiltration

Abstract

The risk for suffering immune checkpoint inhibitors (ICIs)-associated myocarditis increases in patients with pre-existing conditions and the mechanisms remain to be clarified. Spatial transcriptomics, single-cell RNA sequencing, and flow cytometry are used to decipher how anti-cytotoxic T lymphocyte antigen-4 m2a antibody (anti-CTLA-4 m2a antibody) aggravated cardiac injury in experimental autoimmune myocarditis (EAM) mice. It is found that anti-CTLA-4 m2a antibody increases cardiac fibroblast-derived C-X-C motif chemokine ligand 1 (Cxcl1), which promots neutrophil infiltration to the myocarditic zones (MZs) of EAM mice via enhanced Cxcl1-Cxcr2 chemotaxis. It is identified that the C-C motif chemokine ligand 5 (Ccl5)-neutrophil subpopulation is responsible for high activity of cytokine production, adaptive immune response, NF-κB signaling, and cellular response to interferon-gamma and that the Ccl5-neutrophil subpopulation and its-associated proinflammatory cytokines/chemokines promoted macrophage (Mφ) polarization to M1 Mφ. These altered infiltrating landscape and phenotypic switch of immune cells, and proinflammatory factors synergistically aggravated anti-CTLA-4 m2a antibody-induced cardiac injury in EAM mice. Neutralizing neutrophils, Cxcl1, and applying Cxcr2 antagonist dramatically alleviates anti-CTLA-4 m2a antibody-induced leukocyte infiltration, cardiac fibrosis, and dysfunction. It is suggested that Ccl5-neutrophil subpopulation plays a critical role in aggravating anti-CTLA-4 m2a antibody-induced cardiac injury in EAM mice. This data may provide a strategic rational for preventing/curing ICIs-associated myocarditis.

Keywords: CTLA‐4; Cxcl1; myocarditis; neutrophil; spatial transcriptomics.

Figure 1

Anti‐CTLA‐4 m2a antibody aggravates cardiac inflammation and dysfunction in EAM mice. a–c) Representative M‐mode view of echocardiography obtained from each indicated experimental group a). Summarized mean data of ejection fraction (EF) b) and fractional shortening (FS) c) from each experimental group (n = 5 for each group). d) The data examined by an ELISA kit demonstrating the cTnI serum levels obtained from each indicated experimental group (n = 7 for each group). e,f) Representative H&E staining of the cardiac sections (the top panel; Scale bar: 1 mm) and (the bottom panel; Scale bar: 100 µm) from each indicated group e) and the quantified data f) demonstrating the inflammation areas (%, H&E staining images analyzed by ImageJ Software) under the indicated conditions (n = 7 for each group) g,h)The representative images of Masson (upper panel) and Sirius Red (lower panel) staining of the heart sections from each indicated group g); the quantified data presenting cardiac fibrosis areas (%, Masson staining analyzed by ImageJ Software) under the different experimental conditions h) (n = 7 for each group). i–n) Representative immunostaining demonstrating infiltration of neutrophils (Ly6G positive) i), macrophages (F4/80 positive) k), and T‐cells (CD3 positive) m) in cardiac sections generated from each experimental group (scale bars equal 50 µm). Quantification data showing the absolute number of neutrophils j), macrophages l), and T‐cells n) per field (n = 7 for each group; determined by ImageJ software). Data are presented as the mean ± SEM. Statistics: One‐way ANOVA followed by Tukey's post hoc multiple comparisons test was used for analyzing b, c, d, f, h, j, l, and n. The statistics were performed between indicated groups; ^* and ^# indicate p < 0.05; ^** and ^## represent p < 0.01. Hereafter, Ctrl + anti‐IgG antibody, Ctrl + anti‐CTLA‐4 m2a antibody, EAM + anti‐IgG antibody, and EAM + anti‐CTLA‐4 m2a antibody respectively represent anti‐IgG antibody‐treated control mice group, anti‐CTLA‐4 m2a antibody‐treated control mice group, anti‐IgG antibody‐treated EAM mice group, anti‐CTLA‐4 m2a antibody‐treated EAM mice group.

Figure 2

Identification of the cell types in mouse hearts using scRNA‐seq. a) UMAP plot showing that there were nine cell types associated clusters from both the anti‐IgG antibody‐ and anti‐CTLA4 m2a antibody‐treated EAM mouse hearts and each point presenting a single cell marked with different colors according to cluster designation. b) UMAP plots demonstrating the discrepancy of cardiac cell clusters between anti‐IgG antibody‐ and anti‐CTLA4 m2a antibody‐treated EAM mice. c) Dot plots representing the expression profiles of representative marker genes of each identified cell type. Circle size indicates the percentage of cells expressing marker genes, and dot color indicates the average expression levels of the marker genes in the designated cell type. d,e) The absolute numbers of each cell type from the sorted CD45⁺ leukocytes of the hearts d) and the relative proportion of each cell type scaled by the total number of cells per condition e); the colored bars in green and in orange respectively demonstrating the alterations in each indicated cell type between anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mice. p‐value was calculated by chi‐square test (the red asterisks indicate a significant increase; the green asterisk represents a significant decrease). f) Violin plots statistically compare enrichment scores of genes annotated for relative pathways in each cardiac cell type. g) Quantitative analyses demonstrating the number of Tnf‐α⁺ neutrophils, Il‐1α⁺ neutrophils, Il‐1β⁺ neutrophils and INF‐γ⁺ neutrophils in the hearts from each indicated experimental group. (n = 6 for each group). h) The relative gene expression of TNF‐α, Il‐1α, Il‐1β, and INF‐γ analyzed by qPCR in mouse hearts from each experimental group (n = 5–8 for each group). Data are presented as the mean ± SEM. Statistics: unpaired two‐tailed Student's t‐tests and one‐way ANOVA, followed by Tukey's post hoc multiple comparisons test were respectively used for analyzing g and h. The statistics were performed between indicated groups; ^* and ^# indicate p < 0.05; ^** and ^## represent p < 0.01.

Figure 3

Anti‐CTLA‐4 m2a antibody‐mediated increase in the infiltration of Ccl5‐neutrophil subpopulation in the hearts of EAM mice. a) UMAP plot showing four neutrophil subpopulations (immature Ltf‐neutrophil, mature inflammatory Ifitm2‐neutrophil, mature inflammatory Ccl5‐neutrophil, and Hybrid Cd74‐neutrophil) colored by manually annotated clusters. b) UMAP plots demonstrating the differences of infiltrated neutrophil subpopulations in the hearts between anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mice. c) Heatmap showing the relative expression (as reflected by z score) levels of marker genes in each subpopulation of neutrophils. d) Bar graph, colored according to cluster designation, showing the proportions of each neutrophil subpopulation relative to all cardiac neutrophils in anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mice. p value was calculated by chi‐square test and the white asterisks indicated the significant differences. e) Radar plot showing GO enrichment of related proinflammatory and cytokine signaling in four neutrophil subpopulations. f) Pseudotime analyses demonstrating cell trajectories differed between anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mice and differentiated toward to cell fate 1 and cell fate 2. g) Pseudotime analyses respectively demonstrating the relative neighborhood density of the Ccl5‐neutrophil subpopulation, as labeled by the red dashed rectangular square. p‐value was calculated by chi‐square test; ^* indicates p = 1.67E‐33. h) BEAM heatmap representing the expression profiles of branch‐dependent genes over pseudotime. The branch point (in the middle of the heatmap) is the beginning of pseudotime. Both sides of the heatmap are the ends of pseudotime. Ifitm2‐and Ccl5‐neutrophil subpopulation‐related marker genes are enriched in cells on cell fate 1 trajectory (anti‐IgG antibody), and the Ccl5‐neutrophil subpopulation related marker genes are mainly enriched in cells on cell fate 2 trajectory (anti‐CTLA‐4 m2a antibody). Accordingly, genes are clustered into three modules based on expression patterns across development, in which the important GO terms are related to biological processes. i) Pseudotime analyses, from the root of the trajectory, demonstrating the dynamic alterations of genes (Ccl4, Cxcl10, Cxcl9, Il‐1α, Il‐1β, and Tnf) during differentiating to cell fate 1 (the solid line) or cell fate 2 (dashed line); the different colored dots indicating the identities of neutrophil subpopulations and each dot representing a single cell.

Figure 4

Anti‐CTLA‐4 m2a antibody promotes M1 macrophage polarization in EAM mice. a) UMAP plot showing six macrophage subpopulations (M1 Mφ, M1 Mo, M2 Mφ, MaC 3, MaC 4, and MaC 5) colored by manually annotated clusters. b) UMAP plots demonstrating the differences of infiltrated monocyte/macrophage subpopulations in the hearts between anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mice. c) Bar graph showing the proportions of each monocyte/macrophage subpopulation relative to all cardiac monocytes/macrophages in the anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mice, colored according to cluster identity. p value was calculated by chi‐square test and the white asterisks indicated the significant differences. d) Monocle pseudotime trajectories showing the differentiation of M1 Mφ, M1 Mo, M2 Mφ, MaC 3, MaC 4, and MaC 5 macrophage subpopulations from both anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mice. e) Pseudotime analysis revealed the cell trajectories within six macrophage subpopulations. f) Pseudotime analyses demonstrating cell trajectories differed between anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mice. g) BEAM heatmap representing the expression profiles of the branch‐dependent genes over pseudotime. Genes are clustered into four modules based on expression patterns across pseudotime. The branch point shown in the middle of the heatmap is the beginning of pseudotime. Both sides of the heatmap are the ends of pseudotime. The colored bar, coding from the blue to the red, indicates the relative gene expression level from low to high (the upper bar). M1 Mφ and Mac 3 macrophage subpopulation‐related marker genes are enriched in cells on cell fate 1 of the trajectory, and M2 Mφ macrophage subpopulation related marker genes are enriched in cells on cell fate 2 of the trajectory. Genes are clustered into four modules based on expression patterns across development (the lower bar). h) Pseudotime analyses, from the root of the trajectory, demonstrating the dynamic alterations of genes (Ass1, Hif1α, Ly6c2, C1qa, Cx3cr1, and Mrc1) during differentiating to cell fate 1 (the solid line) or cell fate 2 (dashed line). Each dot represents a single cell and the dot color indicates each indicated neutrophil subpopulation respectively.

Figure 5

Cardiac fibroblast‐derived Cxcl1 and Cxcr2 of neutrophils promote neutrophil infiltration in the cardiac tissues of EAM mice. a) Bar graph showing the differences in the overall information flow of the significant signaling pathways between anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mouse hearts. The top signaling pathways (the red) are more enriched in anti‐IgG antibody‐treated EAM mice, and bottom the pathways (the blue) are more enriched in anti‐CTLA‐4 m2a antibody‐treated EAM mice. CXCL signaling pathways were significantly increased by anti‐CTLA‐4 m2a antibody in EAM mice, as marked by the red solid rectangular square. b) Bubble plot showing the differentially expressed ligand‐receptor pairs between anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mouse hearts and demonstrating the strength of increased signaling from cardiac fibroblasts to B cells, dendritic cells (DCs) endothelial cells (ECs), fibroblasts, monocytes/macrophages, master cells, neutrophils, nature killer cells (NK), and T‐cells. The highlighted Cxcl‐Cxcr2 signaling was significantly increased by anti‐CTLA‐4 m2a antibody in the mouse hearts compared with that in anti‐IgG antibody‐treated EAM mouse hearts, as labeled by the red solid rectangular square. The color (from the blue to red) and size of the dots represent the communication probability and p values (shown on the right), respectively. The empty spaces indicated that the communication probability was zero. p values are computed from a two‐sided permutation test. c) Representative immunofluorescence staining of Cxcl1 (red) and vimentin (green) expression in the MZs of mice from each indicated experimental group. The white arrows represent the colocalization of Cxcl1 with the cardiac fibroblast marker vimentin. Scale bars equal 50 µm. d,e) Quantitative analyses demonstrating the fluorescence intensity intensities of Cxcl1 (n =  6 for each group) d) and mRNA expression levels of Cxcl1 (n = 10 for each group) e) in the hearts from each indicated experimental group. f) ELISA experiments showing the serum Cxcl1 protein levels of each indicated experimental group (n = 5 for each group). Data are presented as the mean ± SEM. Statistics: one‐way ANOVA, followed by Tukey's post hoc multiple comparisons test was used for analyzing (d), (e), and (f). The statistics were performed between indicated groups; ^* indicates p < 0.05; ^** and ^## represent p < 0.01.

Figure 6

Selective Cxcr2 antagonist SB225002 or Cxcl1 neutralizing antibody ameliorated anti‐CTLA‐4 m2a antibody‐induced cardiac injury in EAM mice. a–c) Representative M‐mode view of echocardiography obtained from each indicated experimental group a); summarized mean data of ejection fraction (EF) b) and fractional shortening (FS) c) from each indicated group (n = 6 for each group). d) The data examined by an ELISA kit demonstrating the CTnI serum levels obtained from each indicated experimental group (n = 6 for each group). e,f) Representative H&E staining of the cardiac sections (the top panel; Scale bar: 1 mm) and (the bottom panel; Scale bar: 100 µm) from each indicated group; f) the quantified data from the experiments shown in (e) demonstrating the inflammation areas (%; estimated by ImageJ Software) under each indicated condition (n = 6 for each group). g,h) Representative images of Sirius Red staining (upper panel) and Masson staining (lower panel) of cardiac sections from each indicated group; h) the quantified data from the experiments shown in (g) representing cardiac fibrosis areas (%; Masson staining analyzed by ImageJ Software) under the different experimental conditions (n = 6 for each group; Scale bar: 100 µm). i–n) Representative immunostaining demonstrating infiltration of neutrophils (Ly6G positive) (i), macrophages (F4/80 positive) (k), and T‐cells (CD3 positive) (m) in cardiac sections generated from each experimental group (scale bars equal 50 µm). Quantification data showing the absolute number of neutrophils j), macrophages l), and T‐cells (n) per field (n = 6 for each group; determined by ImageJ software). o,p) The relative gene expression of TNF‐α o), Il‐1α p), Il‐1β q), and IFN‐γ r) was analyzed by qPCR in mouse hearts from each experimental group (n = 5–6 for each group). Data are presented as the mean ± SEM. Statistics: one‐way ANOVA, followed by Tukey's post hoc multiple comparisons test was used for analyzing (b–d), (f,h,j,l,n,o‐r). The statistics were performed between indicated groups; ^**, ^## and ^&& indicate p < 0.01.

Figure 7

ST data revealed the dominant role of the Ccl5‐neutrophil subpopulation in the anti‐CTLA4 m2a antibody‐induced progression of myocarditis in EAM mice. a) H&E staining of cardiac tissue sections (the upper panel), the zoomed‐in views from where indicated by the white squares (the middle panel), and mapping with unbiased clustering of ST spots (Figure S8a, Supporting Information) were perfectly overlaid with the H&E images (the bottom panel) in each indicated experimental group. The cardiac MZs are circled by the dashed red lines. Scale bar: 800 µm. b) Spatial feature plots showing the relative expression levels of the marker genes Col1a1 (cardiac fibroblasts), Ptprc (immune cells), and Myh6 (cardiomyocytes) in the cardiac sections of each indicated experimental group. The colored bars shown on the bottom of each panel, coding from the blue to the red, indicate the relative gene expression levels from low to high. c) SPOTlight mapping on Visium ST from the cardiac sections demonstrating that localization of cardiac fibroblasts (upper panel), neutrophils (middle panel), and Ccl5‐neutrophil subpopulation (lower panel) within MZs was dramatically increased by anti‐CTLA‐4 m2a antibody in EAM mice. The colored bars, shown on the right of each panel, indicate the relative abundance of cardiac fibroblasts, neutrophils, and CCl5‐neutrophil subpopulation from low (the blue) to high (the red) (circled by the dashed red lines). d) ST maps showing that calculated GO terms scores for three interested proinflammatory cytokines signaling were dramatically enriched by anti‐CTLA‐4 m2a antibody in the MZs of EAM mice (circled by the dashed red lines). The colored bars shown on the right of each panel, coding from the blue to the red, indicate the signaling activity scores from low to high. e) Volcano plot showing DEGs in the MZs between anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mouse hearts; the black dashed lines show the thresholds for significantly enriched genes. The depicted blue or red solid line with arrow head shown on the top respectively indicating that the expression of genes was decreased or increased by anti‐CTLA‐4 m2a antibody in the MZs compared with those in anti‐IgG antibody treated‐EAM mice. Genes with a false discovery rate adjusted p‐value < 0.05 and average log2FC > 0.5 were considered significantly regulated by the anti‐CTLA‐4 m2a antibody. f) GO analysis showing that the interest GO terms for genes enriched in the MZs were differed between anti‐IgG antibody‐ and anti‐CTLA‐4 m2a antibody‐treated EAM mice; fatty acid oxidation, cardiac muscle contraction, ATP metabolic process, oxidative phosphorylation, and cellular respiration signaling were significantly decreased by anti‐CTLA‐4 m2a antibody in the MZs of anti‐IgG antibody treated‐EAM mice (the blue solid rectangular bars); in contrast, leukocyte migration, response to interferon‐gamma, cytokine‐mediated signaling pathway, myeloid leukocyte activation, leukocyte chemotaxis, tumor necrosis factor production, antigen processing, and presentation, regulation of T‐cell activation, regulation of adaptive immune response and interleukin‐1 production were significantly increased by anti‐CTLA‐4 m2a antibody in the MZs of anti‐IgG antibody treated‐EAM mice (the red solid rectangular bars).

Figure 8

Depletion of neutrophils mitigates anti‐CTLA‐4 m2a antibody‐induced cardiac inflammation, fibrosis, and dysfunction in EAM mice. a–h) Quantification analyses of flow cytometry, obtained from the peripheral blood of each indicated experimental group, demonstrating the percentage of CD45⁺CD11b⁺Ly6G⁺F4/80⁻ neutrophils a), CD45⁺CD11b⁺ Ly6G⁻F4/80⁺ macrophages c), CD45⁺CD11b⁺F4/80⁺MHCII⁺ M1 Mφ e), and CD45⁺CD11b⁺ F4/80⁺CD206⁺ M2 Mφ g) to total cells (n = 5 for each group). Quantification analyses of flow cytometry, generated from cardiac tissues of each indicated experimental group, demonstrating the percentage of CD45⁺CD11b⁺Ly6G⁺F4/80⁻ neutrophils b), CD45⁺CD11b⁺ Ly6G⁻F4/80⁺ macrophages d), CD45⁺CD11b⁺F4/80⁺MHCII⁺ M1 Mφ f), CD45⁺CD11b⁺ F4/80⁺CD206⁺ M2 Mφ h) to total cells (n = 5 for control groups; n = 4 for EAM or EAM + anti‐Ly6G antibody groups). i,j) Representative H&E staining of the cardiac sections (the top panel; Scale bar: 1 mm) and (the bottom panel; Scale bar: 100 µm) from each indicated group; j) the quantified data from the experiments shown in (i) demonstrating the inflammation areas (%; estimated by ImageJ Software) under each indicated conditions (n = 6 for each group). k,l) Representative images of Masson staining (upper panel) and Sirius Red staining (lower panel) of cardiac sections from each indicated group; l) the quantified data from the experiments shown in (k) representing cardiac fibrosis areas (%; Masson staining analyzed by ImageJ Software) under the different experimental conditions (n = 6 for each group; Scale bar: 100 µm). m–o) Representative M‐mode view of echocardiography obtained from each indicated experimental group m); summarized mean data of ejection fraction (EF) n) and fractional shortening (FS) o) from each indicated group (n = 5 for each group). Data are presented as the mean ± SEM. Statistics: one‐way ANOVA, followed by Tukey's post hoc multiple comparisons test was used for analyzing (a–h,j,l,n,o). The statistics were performed between indicated groups; ns represents p > 0.05; ^* and ^# indicate p < 0.05; ^**, ^## and ^&& indicate p < 0.01.