Eosinophils protect pressure overload- and β-adrenoreceptor agonist-induced cardiac hypertrophy

Figure 1. Increased EOS in hypertrophic hearts are the major source of cardiac IL4 and mEar1. (A) Linear regression analysis of blood EOS counts vs. LV mass and LV mass index from 644 patients with hypertension and chest pain after exclusions. (B) Immunofluorescent double staining of GFP and IL4 detected GFP-positive and GFP+IL4+ double-positive EOS, and IL4-positive areas in hearts from eoCRE+/-GFP+/– mice at 14 days after sham or TAC. Scale: 100 µm, inset: 35 µm. (C) FACS quantified heart EOS number in WT mice at 14 days after sham or TAC. Gating strategies are included. (D/E) FACS determined heart EOS, monocyte, macrophage, and neutrophil counts and percentages in CD45+ cells (D) and immunoblot analysis detected heart IL4 and mEar1 levels (E) from WT and ΔdblGATA mice at 0, 1, 2, and 4 weeks after TAC. (F) ELISA determined culture medium IL4 and mEar1 levels from WT EOS treated with different doses of mEar1 or IL4, or their antibodies. Data are mean ± SEM. Representative images or immunoblots in B and E are shown. The numbers of samples or tests are indicated. (F) Results from three experiments. P < 0.05 was considered statistically significant. Non-parametric Mann–Whitney U test…

Figure 2. EOS and EOS-derived IL4 protect mice from TAC-induced cardiac hypertrophy. (A) Schematic outline of experimental procedures. (B) Representative M-mode echocardiographic images, time stamp: 100 ms, vertical bar: 2 mm. (C) Echocardiogram results from WT and ΔdblGATA mice and those received reconstitution of EOS from WT and Il4–/– mice at 14 days after sham or TAC. (D) Heart frozen section WGA immunofluorescent staining and measurement, bar: 40 µm. (E) Heart frozen section TUNEL staining, bar: 200 µm. Arrows indicate apoptotic cells. (F) Heart frozen section cleaved caspase-3 immunofluorescent double staining with β-myosin or α-SMA, bar: 200 µm, inset bar: 75 µm. (G) Heart frozen section Sirius red staining, bar: 400 µm. Mouse genotypes, treatments, and numbers in each group are indicated. Representative images in panels (D), (E), and (G) are shown to the right. Data are mean ± SEM. P < 0.05 was considered statistically significant. One-way ANOVA followed by a post hoc Tukey’s test.

Figure 3. EOS and EOS-derived IL4 and mEar1 reduce ISO-induced cardiomyocyte hypertrophy. (A) RT-PCR determined the c-Fos, ANF, and β-myosin expression in cardiomyocytes treated without or with ISO, IL4, mEar1, WT EOS, or Il4–/– EOS with or without IL4 for different times as indicated. Length, width, and area of cardiomyocytes were also shown. (B) Representative images of cardiomyocytes after different treatments for 6 h as indicated. Bar: 50 µm. (C) Immunoblot analyses of total and phosphorylated ERK1/2, p38, and JNK from cardiomyocytes pre-treated with mEar1 and different types of EOS for 12 h and then ISO for 30 min. Representative immunoblots are shown to the left. All data are mean ± SEM from three independent experiments. P < 0.05 was considered statistically significant. One-way ANOVA followed by a post hoc Tukey’s test.

Figure 4. EOS, mEar1, and IL4 activities in mouse cardiomyocyte apoptosis and cardiac fibroblast TGF-β signalling and fibrosis. (A) Cardiomyocyte death percentage (bar: 100 µm) after cells were pre-treated with or without mEar1 or different EOS lysates for 12 h, followed by ISO for 6 h. (B) Immunoblot analysis of Bcl-2 and cleaved caspase-3 from cardiomyocytes with the same treatments as in (A). (C) Immunoblot analysis of p-Smad2/3 after cardiac fibroblasts were pre-treated with or without mEar1, IL4, or different EOS lysates with or without IL4 for 12 h, followed by TGF-β for 30 min. (D) Immunoblot analysis of α-SMA, Collagen I and III after cardiac fibroblasts were pre-treated with or without mEar1 or different EOS lysate for 12 h, followed by TGF-β for 24 h. (E) RT-PCR determined the mRNA levels of α-SMA, Collagen I and III in cardiac fibroblasts after the same treatment as in (D). GAPDH was used as protein loading control for all immunoblots. Representative images for panels (A–D) are shown. All data are mean ± SEM from six experiments per group. P < 0.05 was considered statistically significant. One-way ANOVA followed by a post hoc Tukey’s test.

Figure 5. EOS survival in response to ISO infusion. FACS analysis of CD45+CD11b+Siglec-F+ EOS in heart (A), blood (B), and spleen (C) at different days after ISO infusion. Representative images are shown to the left of each panel. Data were mean ± SEM from 6 to 9 mice per group. (D) FACS detection of annexin V+propidium iodide (PI)+ apoptotic EOS (CD11b+Siglec-F+) at 14 days after differentiation with and without 10 or 50 µM ISO starting from Day 4 during the course of differentiation. (E) FACS analysis of total CD11b+Siglec-F+ EOS during the differentiation of 1 × 107 bone marrow cells from WT mice, when 10–50 µM ISO was added to the differentiation media starting from Day 4 and onward. Data were mean ± SEM from three experiments per treatment. (F) Echocardiogram results from WT and ΔdblGATA mice or those received adoptive transfer of WT or Il4–/– EOS at 14 days after mice were infused with PBS (sham) or ISO. The number of mice in each group is indicated. (G) FACS analysis determined donor EOS changes in the blood. (H) Anti-mouse CD45.1 antibody immunostaining determined CD45.1-positive donor EOS in the heart at different days after adoptive transfer. Scale: 200 µm, inset scale: 50 µm.…

Figure 6. mEar1 protects mouse heart from ISO-induced cardiac hypertrophy. (A/B) Echocardiogram results and cardiac cell hypertrophy (WGA staining) of ΔdblGATA (A) and WT mice (B) treated with ISO infusion from implanted minipumps and daily i.p. injection of PBS or mEar1 (2 µg/day) for 7 and 14 days. The number of mice in each group is indicated. All data are mean ± SEM. P < 0.05 was considered statistically significant. One-way ANOVA followed by a post hoc Tukey’s test.

Figure 7. ECP activities on human cardiomyocytes, cardiac fibroblasts, and ECs. (A) Human ECP32–41, human EDN32–41, and mouse mEar132–41 amino acid sequence alignment, corresponding to the CPP region in ECP. (B) Crystal structure of the CPP region in human ECP (left) and corresponding regions in EDN structure (middle) and mouse mEar1 model (right). Adenosine 2′-5′-diphosphate molecule co-crystalized with EDN was transferred into the ECP structure and mEar1 model to show the location of the catalytic active site. (C) Human ECP, human EDN, and mouse mEar1 molecule surface charges. (D) TUNEL staining detected human cardiomyocyte death after cells were exposed to 10 µM ISO with or without different cationic proteins or human EOS lysates. (E) Human β-myosin immunofluorescent staining detected human cardiomyocyte hypertrophy after the same treatments as in (D). Scale: 200 µm. (F) Immunoblot analyses of p-Smad2/3 from human cardiac fibroblasts treated with and without 10 ng/mL TGF-β and different doses of ECP, EDN, and mEar1. (G) Immunoblot detected the expression of ICAM-1 and VCAM-1 in human aortic ECs treated with or without 1 ng/mL TNF-α, ECP, EDN, human EOS lysate, and mEar1 as indicated.…