Recombinant cardiac myosin fragment induces experimental autoimmune myocarditis via activation of Th1 and Th17 immunity

Abstract

The specificity and function of T helper (Th) immune responses underlying the induction, progression, and resolution of experimental autoimmune myocarditis (EAM) in A/J mice are unclear. Published data suggest involvement of both Th1 and Th2 responses in EAM; however, the previous inability to assess antigen-specific in vivo and in vitro T-cell responses in cardiac myosin-immunized animals has confounded our understanding of this important model of autoimmune myocarditis. The goal of our study was to develop an alternative model of EAM based on a recombinant fragment of cardiac myosin, in hopes that the recombinant protein will permit measurement of functional T-cell responses that is not possible with purified native protein. A/J mice immunized with a recombinant fragment of cardiac myosin spanning amino acids 1074-1646, termed Myo4, developed severe myocarditis characterized by cardiac hypertrophy, massive mononuclear cell infiltration and fibrosis, three weeks post-immunization. The mice also developed an IgG1 dominant humoral immune response specific for both Myo4 and purified cardiac myosin. The in vitro stimulation of splenocytes harvested from Myo4-immunized animals with Myo4 resulted in cellular proliferation with preferential production of the Th1- and Th17-associated cytokines, IFN-gamma, IL-17, and IL-6, respectively. Production of IL-4 was negligible by comparison. This study describes a new model of EAM, inducible by immunization with a specific fragment of cardiac myosin, from which antigen-specific analyses reveal an importance for both Th1 and Th17 immunity.

Figure 1. Myo4 expression, purification, and concentration

Myo4 is expressed as an abundant 68 kDa protein in E. coli found in both the pellet (Lys Pellet) and supernatant (Lys Sup) of a bacterial lysate. Myo4 is easily purified by nickel chromatography (Pur Myo4) and can be concentrated (Conc Myo4) to approximately 5 mg/ml for use in experiments. Myo4 can be detected by western blotting with anti-his antibodies that detect the C-terminal hexahistidine tag used for protein purification (Anti-His).

Figure 2. Immunization of A/J mice with cardiac myosin, purified Myo4 or Allen peptide induces similar cardiac hypertrophy, inflammation, and fibrosis 21 days post immunization

A/J mice were immunized with 300 μg of cardiac myosin/CFA, 250 μg of Myo4/CFA, 50–150 nmols of Allen peptide/CFA, or PBS/CFA and sacrificed 21 days post immunization. (A) Cardiac hypertrophy was assessed by determination of the heart weight to body weight ratio. (B) Cardiac fibrosis and inflammation were assessed by staining with Masson’s trichrome or hematoxylin and eosin. Bars = 100 μm. (C) Quantitation of inflammation and fibrosis. Error bars represent standard error of the mean (SEM). n = 10 mice per group. * p ≤ 0.05 vs PBS control; ¥ p = 0.057 vs PBS control.

Figure 2. Immunization of A/J mice with cardiac myosin, purified Myo4 or Allen peptide induces similar cardiac hypertrophy, inflammation, and fibrosis 21 days post immunization

A/J mice were immunized with 300 μg of cardiac myosin/CFA, 250 μg of Myo4/CFA, 50–150 nmols of Allen peptide/CFA, or PBS/CFA and sacrificed 21 days post immunization. (A) Cardiac hypertrophy was assessed by determination of the heart weight to body weight ratio. (B) Cardiac fibrosis and inflammation were assessed by staining with Masson’s trichrome or hematoxylin and eosin. Bars = 100 μm. (C) Quantitation of inflammation and fibrosis. Error bars represent standard error of the mean (SEM). n = 10 mice per group. * p ≤ 0.05 vs PBS control; ¥ p = 0.057 vs PBS control.

Figure 2. Immunization of A/J mice with cardiac myosin, purified Myo4 or Allen peptide induces similar cardiac hypertrophy, inflammation, and fibrosis 21 days post immunization

A/J mice were immunized with 300 μg of cardiac myosin/CFA, 250 μg of Myo4/CFA, 50–150 nmols of Allen peptide/CFA, or PBS/CFA and sacrificed 21 days post immunization. (A) Cardiac hypertrophy was assessed by determination of the heart weight to body weight ratio. (B) Cardiac fibrosis and inflammation were assessed by staining with Masson’s trichrome or hematoxylin and eosin. Bars = 100 μm. (C) Quantitation of inflammation and fibrosis. Error bars represent standard error of the mean (SEM). n = 10 mice per group. * p ≤ 0.05 vs PBS control; ¥ p = 0.057 vs PBS control.

Figure 3. Myo4-immunization induces dominant anti-Myo4 and anti-myosin IgG1 antibody production

Serum obtained from Myo4-immunized animals at days 7, 14, and 21 post immunization was analyzed by endpoint antibody titration for anti-myosin and anti-Myo4 antibodies by ELISA. In addition to total IgG production, serum was specifically analyzed for both IgG1 and IgG2a isoforms. Error bars represent SEM from at least 5 mice per group. Values without error bars were at the maximum detection capacity for the assay.

Figure 4. Antibodies from Myo4-immunized mice react with multiple cardiac proteins

Sera from mice immunized with cardiac myosin, Myo4, Allen peptide, or PBS were obtained 21 days post-immunization and was pooled for western blot analysis. Heart homogenate (H), cardiac myosin (C), Myo4 (M), and BSA (B) were resolved by SDS-PAGE and either stained with GelCode Blue or transferred to nitrocellulose and incubated with 1:200 dilutions of sera. Secondary antibody incubations were performed using a 1:2000 dilution of alkaline phosphatase-conjugated goat anti-mouse IgG and blots were developed using 5-bromo-4-chloro-3-indolyl phosphate and p-nitroblue tetrazolium phosphate.

Figure 5. Myo4-immunization induces strong delayed-type hypersensitivity responses specific for Myo4, purified cardiac myosin, and Allen peptide

Cellular immune responses to Myo4, myosin, and Allen peptide were measured by a standard 24 hr ear swelling, delayed-type hypersensitivity (DTH) assay, in mice immunized with myosin, Myo4, Allen peptide or PBS. Right ears were injected with 10 μg of test antigen and left ears were injected with 10 μg BSA and net ear swelling (right minus left) was determined. Error bars, when shown, represent SEM. The absence of an error bar indicates that the SEM could not be calculated due to an insufficient number of measurements. * p ≤ 0.05.

Figure 6. Myo4-induced myocarditis is mediated by Th1 and Th17 immunity in the acute phase of disease

(A) Myo4-specific production of IFN-γ (Th1), IL-4 (Th2), IL-6 and IL-17 (Th17) was analyzed using a standard ELISPOT assay. Following the isolation of splenocytes from Myo4-immunized animals at days 7, 10, 14, 17 and 21 days post immunization, cells were stimulated with purified Myo4 (10 μM) for 24 hrs for IFN-γ, IL-6, and IL-17 or 48 hrs for IL-4. (B) For a more detailed investigation of cytokine production, intracellular staining with antibodies specific for IFN-γ, IL-4 and IL-17 was performed. CD90.2+ T cells were analyzed from splenocyte (SP) and lymphocyte (LN) preparations from either Myo4- or PBS-immunized mice. All experiments were repeated at least three times. For ELISPOT analysis, error bars represent SEM.

Figure 6. Myo4-induced myocarditis is mediated by Th1 and Th17 immunity in the acute phase of disease

(A) Myo4-specific production of IFN-γ (Th1), IL-4 (Th2), IL-6 and IL-17 (Th17) was analyzed using a standard ELISPOT assay. Following the isolation of splenocytes from Myo4-immunized animals at days 7, 10, 14, 17 and 21 days post immunization, cells were stimulated with purified Myo4 (10 μM) for 24 hrs for IFN-γ, IL-6, and IL-17 or 48 hrs for IL-4. (B) For a more detailed investigation of cytokine production, intracellular staining with antibodies specific for IFN-γ, IL-4 and IL-17 was performed. CD90.2+ T cells were analyzed from splenocyte (SP) and lymphocyte (LN) preparations from either Myo4- or PBS-immunized mice. All experiments were repeated at least three times. For ELISPOT analysis, error bars represent SEM.