B lymphocytes trigger monocyte mobilization and impair heart function after acute myocardial infarction

Abstract

Acute myocardial infarction is a severe ischemic disease responsible for heart failure and sudden death. Here, we show that after acute myocardial infarction in mice, mature B lymphocytes selectively produce Ccl7 and induce Ly6C(hi) monocyte mobilization and recruitment to the heart, leading to enhanced tissue injury and deterioration of myocardial function. Genetic (Baff receptor deficiency) or antibody-mediated (CD20- or Baff-specific antibody) depletion of mature B lymphocytes impeded Ccl7 production and monocyte mobilization, limited myocardial injury and improved heart function. These effects were recapitulated in mice with B cell-selective Ccl7 deficiency. We also show that high circulating concentrations of CCL7 and BAFF in patients with acute myocardial infarction predict increased risk of death or recurrent myocardial infarction. This work identifies a crucial interaction between mature B lymphocytes and monocytes after acute myocardial ischemia and identifies new therapeutic targets for acute myocardial infarction.

Figure 1

The B cell–depleting CD20 mAb reduces infarct size, improves heart function and limits myocardial inflammation. (a) Representative examples (left) and quantitative analysis (right) of B220^hi IgM⁺ B cell staining in the blood of C57BL/6J mice treated with or without the CD20 mAb (anti-CD20) (n = 12–15 mice per group); ***P < 0.001. MI, myocardial infarction. (b) Echocardiographic analysis after anti-CD20 therapy. Left ventricular fractional shortening (FS), left ventricular internal diameter at end systole (LVIDs) and left ventricular posterior wall thickness at end systole (LVPWs) were measured at day 14 after myocardial infarction; *P < 0.05. (c) Representative photomicrographs (left) and quantitative analysis (right) of infarct size and myocardial fibrosis evaluation evaluated by Masson trichrome (top) and Sirius red (bottom) staining, respectively, measured at day 14 after myocardial infarction. Data are representative of 10–14 mice per group in three independent experiments. Scale bars: top, 1 mm; bottom,100 μm. (d) mRNA levels of the proinflammatory cytokines IL-1β, TNF-α and IL-18 and the anti-inflammatory cytokines IL-10 and TGF-β within the injured myocardium on day 14 after myocardial infarction (n = 8–12 mice per group). AU, arbitrary units. Mean values ± s.e.m. are represented. *P < 0.05 versus PBS.

Figure 2

B lymphocyte depletion impairs monocyte mobilization and recruitment and macrophage accumulation in the injured myocardium. (a,b) Representative examples of 7/4^hi and 7/4^lo monocyte staining (top) and quantification of their numbers (bottom) in bone marrow (a) and blood (b) of B cell–depleted mice compared to controls on day 3 after myocardial infarction (n = 8–15 mice per group). Mean values ± s.e.m. are represented. *P < 0.05 versus PBS. 7/4 (Ly6C) indicates staining with antibody clone 7/4 directed against the Ly-6B.2 alloantigen, whose staining is equivalent to staining with Ly6C. (c) Quantitative analysis of the number of CD45.1⁺CD11b⁺Ly6G⁻Ly6C⁺ cells in the cardiac tissue of anti-CD20–treated mice compared to controls on day 3 after myocardial infarction (n = 6 mice per group). Each symbol represents an individual mouse. (d) Quantitative analysis of the number of F4/80⁺ and CD68⁺ macrophages within the myocardium on day 5 after myocardial infarction (n = 5 mice per group). Mean values ± s.e.m. are represented. *P < 0.05 versus PBS.

Figure 3

B lymphocyte depletion significantly lowers Ccl7 concentrations after acute myocardial infarction and triggers Ccl7-dependent monocyte transmigration in vitro and in vivo. (a) Ccl2 and Ccl7 protein concentrations in blood after CD20 mAb or control PBS treatment, 1 and 3 d after myocardial infarction (n = 5–8 mice per group); *P < 0.05 versus PBS. (b) Ccl7 concentrations in the supernatant of nonstimulated or CpG-stimulated cultured splenic B cells isolated from WT mice. Data are representative of four independent experiments; each condition was tested in triplicate. Mean values ± s.e.m. are shown. ***P < 0.001 versus untreated B cells. (c) Representative photomicrographs (left) and histograms (right) of the transmigration of cultured monocytes in the presence of nonstimulated or activated (using IgM and CD40) B cells with or without neutralizing anti-Ccl2 or anti-Ccl7 antibodies. Data are representative of four independent experiments; each condition was tested in triplicate. Scale bar, 300 μm. Mean values ± s.e.m. are shown. ***P < 0.001 versus control condition without B cells. (d) Quantification of the number of 7/4^hi monocytes in blood (left) and their percentages in infarcted myocardium (right) of Rag1^−/− mice injected with WT splenocytes, B cell–depleted splenocytes, or B cell–depleted splenocytes resupplemented with WT or Ccl7^−/− B cells, 3 d after myocardial infarction. Data are representative of 10–12 mice per group in two independent experiments. Mean values ± s.e.m. are shown. *P < 0.05 versus WT splenocytes; ^##P < 0.01 versus anti-CD20 splenocytes; ^$P < 0.05 versus anti-CD20 splenocytes with WT B cells.

Figure 4

B lymphocytes trigger adverse ventricular remodeling and alter heart function through the production of Ccl7. (a) Echocardiographic analysis 14 d after myocardial infarction. Shown are FS, LVIDs and LVPWs of Rag1^−/− mice injected with either WT splenocytes, B cell–depleted splenocytes or B cell–depleted splenocytes resupplemented with WT or Ccl7^−/− B cells. Results are pooled from three independent experiments with 10–25 mice per group. (b,c) Representative photomicrographs (left) and quantitative analysis (right) of infarct size (b) and fibrosis and collagen content (c) in the indicated groups of mice. Results are pooled from three independent experiments with 10–25 mice per group. Mean values ± s.e.m. are shown. Scale bars: 1 mm in b and 100 μm in c. *P < 0.05 and ***P < 0.001 versus WT splenocytes; ^#P < 0.05 and ^###P < 0.001 versus anti-CD20 splenocytes; ^$P < 0.05 and ^$$$P < 0.001 versus anti-CD20 splenocytes with WT B cells. (d) Circulating Ccl7 protein concentrations at day 14 post-myocardial infarction in mice repopulated with a mixture of μMT and Ccl7^−/− bone marrow compared to the control group receiving a mixture of μMT and Ccl7^+/+ bone marrow (n = 9–13 mice per group). **P < 0.01. (e) FS at day 14 after myocardial infarction in the indicated groups of mice (n = 9–13 mice per group). Mean values ± s.e.m. are shown. *P < 0.05.

Figure 5

Blockade of Baff signaling impairs monocyte mobilization and improves heart function after acute myocardial infarction. (a) Quantification of the number of 7/4^hi monocytes in the bone marrow (left) and in the blood (right) of Baff-r deficient (Tnfrsf13c^−/−) and WT control mice compared to controls on day 3 after myocardial infarction (n = 8–11 mice per group). (b) Ccl7 protein concentration in the plasma of Tnfrsf13c^−/− mice compared to controls at day 3 after myocardial infarction (n = 8–11 mice per group). (c) FS of Tnfrsf13c^−/− mice compared to Tnfrsf13c^+/+ and Tnfrsf13c^+/− mice at day 14 after myocardial infarction. n = 8–11 mice per group. Mean values ± s.e.m. are shown. *P < 0.05 and **P < 0.01. (d,e) Numbers of B220⁺IgM⁺ B cells (d) and 7/4^hi monocytes (e) in blood of anti-Baff–treated mice compared to PBS-injected mice at day 1 after myocardial infarction (n = 8–11 mice per group). (f) FS of anti-Baff–treated mice compared to PBS-injected mice at day 14 after myocardial infarction (n = 12–15 mice per group). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate s.e.m.

Figure 6

Circulating levels of CCL7 and BAFF during the acute phase of myocardial infarction are associated with cardiovascular outcomes. (a,b) The probability of outcome events (death or recurrent myocardial infarction) as a function of baseline circulating CCL7 (a) or BAFF (b) levels in patients with acute myocardial infarction (AMI). HR, hazard ratio.