CD11b is involved in coxsackievirus B3-induced viral myocarditis in mice by inducing Th17 cells

2.1 Animal treatments

In this study, 50 male BALB/c mice (aged 6- to 8-week-old) were purchased from the Animal Experiment Center of First Affiliated Hospital of Guangxi Medical University (Guangxi, China). The mice were randomly assigned to three groups as follows: control group (n = 20), CVB3 group (n = 20) and CVB3 + CD11b knockdown group (n = 20). In the last two groups, CVB3 infection was performed as described by Li et al. [19]. Briefly, CVB3 (Nancy strain; ATCC, Manassas, VA, USA) was maintained by passage in HeLa cells (CCL-2; ATCC). The viral titer was determined using a 50% tissue culture infectious dose (TCID₅₀) assay on HeLa cell monolayers and calculated by the Reed–Muench method. Mice were infected with an intraperitoneal injection of 0.1 mL of phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA) containing 10³ TCID₅₀ of the virus. In the CVB3 + CD11b knockdown group, mice were injected with siRNA-targeting CD11b (GenePharma, Shanghai, China) and EntransterTM-in vivo transfection reagent (Engreen Biosystem Co., Ltd., Beijing, China) from the vena caudalis, according to the method previously described [20], before CVB3 injection. Half the mice in each group underwent the following tests 1 week after the treatments; the remaining mice in each group underwent the following tests 2 weeks after the treatments.

1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals and has been approved by the National Institutes of Health’s Code of Ethics the Animal Use [21] and Ethics Committee of First Affiliated Hospital of Guangxi Medical University (Guangxi, China). Mice experiment was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Medical Laboratory Animals (Ministry of Health, P. R. China, 1998).

2.2 Histopathology and myocarditis scoring

Heart tissues were collected from the mice 7 days after CVB3 infection. The tissues were fixed in 10% formalin and embedded in paraffin. Sections (5 µm thick) were cut and stained with hematoxylin and eosin (H&E). The extent of the myocardial lesions was quantified and scored for severity as follows: 0 = no inflammation; 1 = 1–5 distinct mononuclear inflammatory foci, with the involvement of 5% or less of the cross-sectional area; 2 = more than 5 distinct mononuclear inflammatory foci or the involvement of over 5% but not over 20% of the cross-sectional area; 3 = diffuse mononuclear inflammation involving over 20% of the area, without necrosis; and 4 = diffuse inflammation with necrosis. The analysis was performed in a double-blinded manner by a trained pathologist [22].

For immunohistochemistry (IHC) staining, the slides were routinely deparaffinized and hydrated. After inactivating endogenous peroxidase in 3% hydrogen peroxide, slides were then retrieved in citric acid buffer (pH 6.0) by microwave for 15 min. Slices were blocked in normal goat serum for 30 min at room temperature and incubated with anti-CD11b antibody (1:1,000; Abcam, Shanghai, China) overnight at 4°C. The slides were then washed with tris buffered saline plus tween (TBST) and incubated with appropriate secondary antibody for 2 h at 37°C. The sections were then washed with TBST and stained by using DAB Detection kit (Solarbio, Beijing, China). Finally, the sections were counterstained with hematoxylin.

2.3 Quantitative RT-PCR (qRT-PCR) analysis

Total RNA was extracted from the tissue and cell samples using the Trizol reagent (Invitrogen, Carlsbad, CA, USA). Then 2 µg of the total RNA were reversely transcribed by using the SuperScript RT kit from Invitrogen. qRT-PCR was performed using the ABI PRISM7900 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) with PowerUp™ SYBR^® Green Master Mix (Thermo Fisher Scientific). The primer information is given in Table 1. The 2^−ΔΔCt method was used to analyze the mRNA expression levels relative to that of the control (GAPDH) mRNA [23].

2.4 Flow cytometry

The percentage of Th17 cells in CD4⁺ T cells was detected according to the method reported in the previous study [24]. Splenocytes were isolated and suspended in RPMI 1640 containing 10% fetal bovine serum, and red blood cells were lysed by incubation for 3 min in ACK lysis buffer (Tiangen, Shanghai, China). The cells were collected and resuspended at a density of 1.0 × 10⁶ cells per mL. The cells were then stimulated for 4 h with 50 ng phorbol 12-myristate 13-acetate and 1 µg ionomycin per mL (Sigma-Aldrich, St. Louis, MO, USA), and cytokine secretion was blocked with 10 lg/mL brefeldin A (Sigma-Aldrich) at 37°C under 5% CO₂ in a 24-well culture plate (Corning Costar, Corning, NY, USA) in RPMI 1640 medium supplemented with 100 U penicillin and 100 µg streptomycin per mL and 10% fetal bovine serum. Surface markers were stained with PE-labeled anti-mouse CD4 antibody (Biolegend). After the cells were washed, fixed, and permeabilized according to the manufacturer’s instructions (Biolegend, Beijing, China), they were stained intracellularly with an APC-conjugated anti-mouse IL-17A antibody (eBioscience, San Diego, CA, USA). After incubation at 4°C for 30 min, the samples were washed in staining buffer and measured by flow cytometry on a FACSCalibur cell sorter. The data were analyzed using CellQuest software (BD Biosciences, Franklin Lakes, NJ, USA).

2.5 Western blot assay

Western blot was performed as previously described [25]. The heart tissues were homogenized on ice for 30 min in a RIPA buffer (Sigma-Aldrich) supplemented with protease inhibitor. The lysates were heated at 95°C for 5 min and loaded on 10% gels (Bio-Rad, San Diego, CA, USA) for sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After electrophoretic separation, the proteins were transferred onto 0.2 µm nitrocellulose membranes (Amersham, Germany), blocked with 5% nonfat milk (in Tris-buffered saline [TBS] + 0.01% Tween) and incubated overnight at 4°C with the following primary antibodies: anti-CD11b (1:1,000; Abcam) and anti-GAPDH (1:1,000; Abcam). The horseradish peroxidase (HRP)-linked secondary antibodies (1:2,000; Abcam) and ECL kit (WBLUR0100; Millipore Corporation, Billerica, MA, USA) were further used to visualize the blots in the membrane. Proteins were finally visualized by an LAS-4000 mini system (Fujifilm, Japan). The intensity of protein bands was quantified with Quantity one software. The proteins of tissue expression were standardized to GAPDH levels.

2.6 ELISA

Serum IL-17A concentration was measured using ELISA (R&D Systems, Beijing, China), according to the manufacturer’s instructions. In brief, plates were coated with the diluted capture antibody (100 mL/well) and incubated overnight at 4°C. The serum samples were added to triplicate wells and incubated at 37°C for 2 h. After the samples were washed, the biotinylated detection antibody was added for 1 h, followed by 100 mL of streptavidin-conjugated HRP. TMB substrate (eBioscience) was added to each well. The absorbance was measured at 450 nm.

2.7 Statistical analysis

The software GraphPad 8.0 was used to analyze the data. All data were repeated as an independent experiment three times and presented as mean ± standard deviation(SD). Based on ANOVA, p < 0.05 was regarded as significantly different among the experimental groups.

3.1 Increased CD11b expression during CVB3-induced mouse myocarditis

Male BALB/c mice were injected intraperitoneally with 10³ TCID₅₀ of CVB3 to construct an acute VMC model. Histological analysis showed higher myocarditis scores in cardiac tissues 1 and 2 week(s) after the CVB3 injection (p < 0.001; Figure 1a and b). The expression of CD11b was examined after CVB3 infection. As shown in Figure 1c and d, CD11b increased in the cardiac tissues in the first and second weeks (p < 0.05). These results indicated that CD11b expression increased in the cardiac tissues during CVB3-induced mouse myocarditis.

Figure 1

Expression of CD11b was increased during CVB3-induced myocarditis in mice. (a) H&E staining of heart tissue sections was prepared at 1 and 2 week(s) postinfection. Cardiac inflammation was indicated by the arrows (magnification: ×200, scan bar: 50 µm). (b) The degree of the myocardial lesions was quantified and scored according to the method described in previous literature [22]. (c and d) CD11b expression in cardiac tissues at the first and second week(s) after the infection was examined by Western blotting. Data are shown as mean ± SD. *p < 0.05 and ***p < 0.001 compared with the control group.

3.2 Expression of CD11b and its correlations with the IL-17 expression

We analyzed the frequencies of Th17 cells in spleen as well as serum levels of the proinflammatory cytokines in CVB3-infected mice. Spleen tissues showed markedly increased frequencies of Th17 cells in mice after infection with CVB3 for 1 (p < 0.05) and 2 week(s) (p < 0.01; Figure 2a and b). CVB3 infection for 1 and 2 week(s) also caused the elevation of serum IL-17 level (Figure 2c; all p < 0.01). The levels of CD11b and IL-17 mRNA were significantly increased in cardiac tissues after infection with CVB3 for 1 and 2 week(s) (Figure 2d; all p < 0.05). As shown in Figure 2e, the mRNA of CD11b expression was significantly positively correlated with IL-17 mRNA expression in cardiac tissues after CVB3-infection for 1 (r = 0.5761, p < 0.01) and 2 week(s) (r = 0.6237, p < 0.01).

Figure 2

Expression of CD11b and its correlations with the IL-17 expression. (a and b) The frequencies of Th17 cells in CD4⁺ cells in spleen in control and CVB3 mice were analyzed by flow cytometry. (c) The serum IL-17 level in mice was evaluated by ELISA at the first and second week(s) after the infection. (d) The relative IL-17 and CD11b mRNA expression in heart tissues was evaluated by qRT-PCR after CVB3 infection for 1 and 2 weeks. (e) Correlation between CD11b and IL-17 mRNA expression in heart tissues. Data are shown as mean ± SD. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the control group.

3.3 CD11b deficiency ameliorated CVB3-induced myocarditis in mice

To determine the role of CD11b in CVB3-induced myocarditis, this study knocked down CD11b in mice. IHC analysis demonstrated that siRNA-CD11b inhibited the increase in CD11b in heart tissues in CVB3-infected mice (Figure 3a). As indicated by H&E staining, the pathological scores of heart sections in the CD11b-deficient group were lower or less than those in the CVB3 group (p < 0.05 in the first week and p < 0.01 in the second week; Figure 3b). Although the levels of serum IL-17 in the CVB3 groups increased dramatically (p < 0.001), CD11b knockdown decreased the levels of serum IL-17 compared to those in CVB3 groups (p < 0.05 or p < 0.01). These data indicate that CD11b deficiency ameliorated CVB3-induced myocarditis in mice.

Figure 3

CD11b deficiency ameliorated CVB3-induced myocarditis in mice. Before CVB3 injection, mice were injected with siRNA-targeting CD11b and EntransterTM-in vivo transfection reagent from the vena caudalis to knock down CD11b in mice. (a) CD11b expression in heart tissues was assessed using IHC. CVB3: CVB3 injection, anti-CD11b: CD11b knock down. (b) H&E staining of heart tissue sections was prepared at 1 and 2 week(s) postinfection (magnification: ×200, scan bar: 50 µm). The degree of the myocardial lesions was quantified and scored according to the method described in previous literature [22]. (c) The serum IL-17 level in mice was evaluated by ELISA at the first and second week(s) after the infection. Data are shown as mean ± SD. **p < 0.01 and ***p < 0.001 compared with the control group, ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 compared with the CVB3 group.

3.4 CD11b deficiency suppressed Th17 cell responses

As shown in Figure 4a and b, CVB3 mice showed markedly increased frequencies of Th17 cells in spleen tissues (p < 0.01), while CD11b depletion inhibited the increase in Th17 cells in CVB3 mice (p < 0.01). The levels of cardiac mRNA of the IL-17, IL-23 and STAT-3 expression were elevated significantly in CVB3-infected mice as compared to the control mice (p < 0.01 or p < 0.001; Figure 4c), while the IL-17, IL-23 and STAT-3 mRNA expression markedly reduced in the CD11b-deficient mice compared with the CVB3 mice, especially in the second week (p < 0.05 or p < 0.01). These data indicate that CD11b deficiency inhibited CVB3-induced Th17 cells and inflammation in mice.

Figure 4

CD11b deficiency suppressed Th17 cell response. Before CVB3 injection, mice were injected with siRNA-targeting CD11b and EntransterTM-in vivo transfection reagent from the vena caudalis to knock down CD11b in mice. (a and b) The frequencies of Th17 cells in CD4⁺ cells in spleen in control and CVB3 mice were analyzed by flow cytometry 2 weeks after CVB3 injection. (c) The relative IL-17, IL-23 and STAT-3 mRNA expression in heart tissues was evaluated by qRT-PCR. Data are shown as mean ± SD. **p < 0.01 and ***p < 0.001 compared with the control group, ^#p < 0.05, ^##p < 0.01 and ^###p < 0.001 compared with the CVB3 group.