B cell subsets contribute to myocardial protection by inducing neutrophil apoptosis after ischemia and reperfusion

Figures

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  Figure 1. The infiltration of B cells and Bregs into the heart after myocardial I/R. (A) Representative images for flow cytometric analysis and flow cytometry–based quantification of the number of CD19+ B cells in digested heart tissues of mice before and 1 and 3 days after myocardial I/R. (B) Gating strategies and (C) quantification of the number of B cell subsets in the heart. (D) Quantification of the percentage of circulating IL-10+ B cells before and 1, 3, and 7 days after myocardial I/R. (E) Representative images for flow cytometric analysis and flow cytometry–based quantification of percentage and the number of IL-10+ B cells in digested heart tissue. (F–H) Quantification of the number of CD39+ B cells, CD73+ B cells, and TGF-β+ B cells of digested heart tissue before and 3 days after myocardial I/R. These experiments were independently replicated at least twice. I/R, ischemia and reperfusion. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 by 1-way ANOVA test followed by Tukey’s post hoc test (A and C–E); differences between 2 groups were compared by unpaired, 2-tailed t test (F–H).

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  Figure 2. Selective B cell depletion accentuated tissue damage and deteriorated cardiac remodeling and cardiac function after myocardial I/R. (A) Representative images of H&E staining of heart slices in all groups. Scale bars: 50 μm. (B) Damage score evaluation of heart slice from mice with or without B cell depletion 1 day after myocardial I/R. (C) The measurement of serum troponin I in each group 1 day after MIRI. (D and E) Representative photos of TUNEL staining of heart slices and quantification in each group. Scale bar: 50 μm. (F) Scar size 14 days after myocardial I/R. Tissue slices were stained with Masson’s trichrome. Scale bars: 1000 μm. The infarct size was determined by calculating the ratio of the collagen area to the left ventricular area. (G) Representative M-mode echocardiography 14 days after myocardial I/R. (H) Measurement of LVIDd, LVIDs, LVEF, LVEDV, LVID, and LVFS in each group. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by unpaired, 2-tailed t test (B, E, and F) or 1-way ANOVA test followed by Tukey’s post hoc test (C and H). BD, B cell depletion; I/R, ischemia and reperfusion; LVIDd, left ventricular diastolic dimension;…

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  Figure 3. B cells attenuated MIRI via an IL-10–independent mechanism. A total of 1 × 107 splenic B cells from Il10–/– mice were transferred into μMT mice before and 2 days after myocardial I/R. WT, μMT and, μMT mice transferred with Il10–/– B cells were subjected to myocardial I/R. (A) Representative images of H&E staining of heart slice in each group 3 days after myocardial I/R. Scale bars: 100 μm. (B) Damage scores to quantify the extent of tissue injury in each group 3 days after myocardial I/R. (C) Serum cardiac troponin I level in each group. (D) Representative Masson’s trichrome staining of heart slice and quantification of infarct size after myocardial I/R in each group. Scale bars: 1000 μm. The infarct size was determined by calculating the ratio of the collagen area to the left ventricular area. (E) Representative M-mode echocardiography of each group 14 days after I/R. (F) Measurements of cardiac function and cardiac size in each group 14 days after I/R. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA test followed by Tukey’s post hoc test (n = 6–8 mice per group). cTnI, cadiac troponin I; I/R, ischemia and reperfusion; LVIDd,…

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  Figure 4. Selective B cell depletion boosted inflammation of the heart 24 hours after myocardial I/R or sham operation. Mice were injected i.v. with 200 μg anti-CD20 to deplete B cells in mice. RNA-seq of myocardial samples from mice with or without B cell depletion (BD) after MIRI or sham operation were detected (n = 3 mice per group). (A) PCA of RNA-seq expression data among the 3 groups. (B) Volcano plots of gene expression profile data in samples of the 3 groups. (C) Top 20 GO and (D) KEGG enrichment analyses of heart DEGs between mice with or without B cell depletion after MIRI. The y axis represents different pathways and the x axis represents the percentage of each functional group gene among the total genes. (E) In the Hallmark gene sets, GSEA plots with the top 25 leading targets revealed that “inflammatory response” and “TNF signaling via NF-κB” pathways were upregulated in the I/R + BD group compared with the I/R group. (F) The mRNA expression levels of inflammatory factors (Il1b, Il6, Nlrp3, Aim2, Il10, Tgfb1, Ccl2, Ccl12, Ccl7, Cxcl1, and Cxcl10) were quantified from heart tissue of B cell–depleted and control mice 1 day after MIRI. Expression was normalized to that of Actb…

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  Figure 5. Selective B cell depletion delayed acute inflammation resolution after MIRI. Mice with or without B cell depletion underwent myocardial I/R, and the hearts were harvested and digested 3 days after MIRI. (A) Flow cytometry was used to determine the levels of CD4+ and CD8+ T cells in peripheral blood and heart. (B) Ly6C+ monocytes and neutrophils in peripheral blood, and (C) neutrophils in heart tissue from B cell–depleted and control mice 3 days after MIRI. (D) Representative images of immunofluorescence of Ly6G+ neutrophils in heart sections from sham-operated, B cell–depleted, and control mice 3 days after MIRI (n = 6 mice per group). Scale bars: 50 μm. (E) Gating strategy and flow cytometry–based quantification of the number and ratio of CD45+CD11b+Ly6G–F4/80+Ly6Chi and CD45+CD11b+Ly6G–F4/80+Ly6Clo monocytes/macrophages (Mo/MΦ) on day 3 following I/R in control and B cell–depleted mice. The experiments were independently replicated twice. Data are presented as mean ± SEM. *P < 0.05, ****P < 0.001 by unpaired, 2-tailed t test. BD, B cell depletion; I/R, ischemia and reperfusion.

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  Figure 6. B cell deficiency delayed acute inflammation resolution after MIRI. (A) Representative images of flow cytometric analysis and flow cytometry–based quantification of the number of Ly6G+ neutrophils and Ly6Chi monocytes in the digested heart from WT mice, μMT mice, and μMT mice adoptively transferred with Il10–/– B cells 3 days after myocardial I/R, respectively. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01 by 1-way ANOVA test followed by Tukey’s post hoc test (n = 3 mice per group). (B) Representative images of Ly6G immunofluorescence in heart sections (n = 6 mice per group). Scale bars: 50 μm.

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  Figure 7. B cells promote macrophage polarization in the presence of neutrophils and induced neutrophil apoptosis via cell-cell interaction. (A) Bone marrow–derived macrophages (BMDMs) were incubated with B cells and/or neutrophils, or alone in the presence of 100 ng/mL LPS for 12 hours and 1 μg/mL monensin, a Golgi blocker, for the last 4 hours. Intracellular IL-10 and TNF-α of F4/80+ macrophages were detected by flow cytometry. NS, normal saline. (B) Freshly isolated neutrophils were labeled with CMFDA and cultured with B cells or alone for 12 hours. Efferocytosis assay was conducted by adding CMFDA-labeled neutrophils cocultured with or without B cells to BMDMs for 60 minutes, as described in the Methods. The percentage of BMDMs phagocytosing CMFDA-labeled neutrophils was quantified using flow cytometric analysis. (C) Confocal microscopy images show Alexa Fluor 563–conjugated phalloidin–labeled macrophages phagocytosing CMFDA-labeled neutrophils (n = 4 per group). Scale bars: 5 μm. (D) Freshly isolated neutrophils were cocultured for 12 hours with B cells or alone. In 1 group, B cells were also cocultured in Transwells. During coculture, cells were treated with 100 ng/mL LPS or vehicle.…

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  Figure 8. The protective effect of B cells against MIRI vanished in the absence of neutrophils. Neutrophil depletion (ND) was performed by i.p. injection of mAb clone 1A8 (200 μg) once every 3 days. In the condition of ND, mice with or without B cell depletion underwent MIRI. (A) Principal component analysis of RNA-seq profile of ischemic myocardium in the 2 groups (n = 4 mice per group). (B) Volcano plots of gene expression profile data in samples of the 2 groups. (C) KEGG enrichment analysis of DEGs between the 2 groups. (D) Representative images of H&E staining of heart sections from the 2 groups. Scale bars: 100 μm. (E) Apoptotic cardiac cells were detected via TUNEL staining 1 day after myocardial I/R. Scale bar: 50 μm. (F) Serum cardiac troponin I level in the 2 groups 1 day after myocardial I/R (n = 5–6 mice per group). (G) Echocardiographic measurements of cardiac function and cardiac size between B cell–depleted mice and controls 14 days after MIRI in the condition of ND. (H) Representative images of Masson’s trichrome staining of heart slice and quantification of infarct size after myocardial I/R in each group. Scale bars: 1000 μm. The infarct size was determined by calculating…

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  Figure 9. The classical death ligand–receptor pairs did not mediate the proapoptotic role of B cells against neutrophils. (A) The expression of FASL, PD-L1, and TRAIL on B cells with or without LPS (100 ng/mL) treatment. (B) The expression of FAS, PD-1, and DR5 on neutrophils. (C) Representative flow cytometry images for the detection and quantification of apoptotic neutrophils in each group. In the presence of LPS (100 ng/mL) or vehicle, freshly isolated neutrophils were cocultured for 12 hours with B cells that were preincubated with anti-FASL (10 μg/mL), anti–PD-L1 (10 μg/mL), or vehicle. The apoptotic neutrophils were detected using an annexin V/PI assay. Data are presented as mean ± SEM. *P < 0.05; **P <0.01; ***P < 0.001 by unpaired, 2-tailed t test (A and B) or 1-way ANOVA test followed by Tukey’s post hoc test (C). The experiments were independently replicated twice. FMO, fluorescence minus one; αFASL, anti-FASL antibody; αPD-L1, anti–PD-L1 antibody.

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  Figure 10. The identification of the molecule on B cells that induced neutrophil apoptosis. (A) tSNE plot from the pooled single-cell RNA sequencing of noncardiomyocyte cells from hearts at 1 and 3 days after myocardial ischemia. (B) Dot plot visualization of the top 5 different marker genes in each cluster. Dot size represents the percentage of expression per cluster; color gradient represents average expression levels per cell. (C) The chord diagram shows the significant inferred interaction from the source cell type (i.e., B cells) with the target cell type (i.e., neutrophils). The arrow thickness is proportional to the calculated communication probability. (D) FCER2A expression levels on circulating B cells of sham-operated or post-I/R mice. (E) CD18 expression levels on neutrophils in heart of sham-operated or post-I/R mice. (F) Representative flow cytometry images for the detection and quantification of apoptotic neutrophils in each group. In the presence of LPS (100 ng/mL), freshly isolated neutrophils were cultured for 12 hours with or without B cells that were preincubated with anti-CD22 (10 μg/mL), anti-FCER2A (10 μg/mL), or vehicle. (G) The violin plot shows Fcer2a expression…

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  Figure 11. Exacerbation of MIRI by in vivo FCER2A blockade. Mice were injected with anti-FCER2A antibody (150 μg per mouse, i.p.) or isotype immediately after I/R. (A) Representative M-mode echocardiography images of each group 14 days after I/R. (B) Measurements of cardiac function and size in each group 14 days after I/R. (C) Representative Masson’s trichrome staining of heart slices and quantification of infarct size after myocardial I/R in each group. Scale bars: 1000 μm. The infarct size was determined by calculating the ratio of the collagen area to the left ventricular area. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01 by unpaired t test. αFCER2A, anti-FCER2A antibody; I/R, ischemia and reperfusion; LVIDd, left ventricular diastolic dimension; LVIDs, left ventricular systolic dimension; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; I/R, ischemia and reperfusion.