A New Mouse Model of Chronic Myocarditis Induced by Recombinant Bacille Calmette–Guèrin Expressing a T-Cell Epitope of Cardiac Myosin Heavy Chain-α

Figures

• 

  Figure 1. Construction and characterization of rBCG-MyHCα. (a) Oligonucleotides that encode a MyHCα CD4 epitope were cloned into an XhoI site in the coding region of the Ag85B gene from Mycobacterium kansasii cloned into pSO246 to be fused with the gene, and the plasmid was introduced into BCG by electroporation, giving rise to rBCG-MyHCα. (b) Western blot analysis of recombinant Ag85 protein or M. kansasii, cell lysates of BCG and rBCG-MyHCα, and culture supernatants of BCG and rBCG-MyHCα.

• 

  Figure 2. A single injection of rBCG-MyHCα induced chronic but weak myocarditis. (a) Mice were immunized with rBCG-MyHCα or rBCG-pSO246 on day 0. (b) Representative histology (H&E staining and TN-C immunostaining) of the heart sections on days 60 and 120 after rBCG immunization. Arrows indicate myocardial inflammation.

• 

  Figure 3. rBCG-MyHCα immunization induced heart failure in mice. (a) Mice were immunized twice with 10 μg of MyHCα peptide/CFA on days 14 and 21 before rBCG immunization for priming. rBCG-MyHCα, rBCG-pSO246, or PBS were injected subcutaneously on day 0. (b) Representative histology (H&E staining and TN-C immunostaining) of the heart sections on day 0 (21 days after first immunization with MyHCα). (c,d) HW/BW (c) and serum troponin I concentrations (d) at indicated time points are shown (n = 10 each). Results are presented as mean ± SEM. * p < 0.05 vs. PBS and # p < 0.05 vs. rBCG-pSO246 by one-way ANOVA with Tukey’s post hoc test. (e) Representative M mode images of echocardiography 12 weeks after rBCG immunization. Bar graphs represent echocardiographic parameters. Results are presented as mean ± SEM, n = 9–12, * p < 0.05 vs. PBS and rBCG-pSO246 by Kruskal-Wallis analysis with a post hoc Steel-Dwass test. (f) Bar graphs represent hemodynamic parameters at 12 weeks after rBCG immunization. Results are presented as mean ± SEM, n = 9 each, * p < 0.05 vs. PBS and rBCG-pSO246 by Kruskal-Wallis analysis with a post hoc Steel-Dwass test.

• 

  Figure 4. rBCG-MyHCα immunization induced chronic heart inflammation in mice. Immunization protocol is shown in Figure 1a. (a) Representative flow cytometric plots showing CD45+ leukocyte infiltration in hearts from mice at 12 weeks after rBCG immunization. Bar graph shows the quantification of CD45+ leukocytes as a percentage of live cells. Results are presented as mean ± SEM, n = 6 each, * p < 0.05 vs. PBS and # p < 0.05 vs. rBCG-pSO246 by Kruskal-Wallis analysis with a post hoc Steel-Dwass test. (b) Representative histology (H&E and Sirius red staining and TN-C immunostaining) of the heart sections at 12 weeks after rBCG immunization. (c) Cytokine and chemokine secretion in homogenized hearts obtained from indicated mice at 12 weeks after rBCG immunization was assessed by an ELISA. n = 6 per group. * p < 0.05 by Kruskal-Wallis analysis with a post hoc Steel-Dwass test.

• 

  Figure 5. A model of prolonged chronic myocarditis following acute myocarditis. (a) Mice were immunized twice with 100 μg of MyHCα peptide/CFA on days 14 and 21 before rBCG immunization to induce EAM. rBCG-MyHCα, rBCG-pSO246, or PBS were injected subcutaneously on day 0. (b) Representative histology of the heart sections on day 0 (21 days after first immunization with MyHCα). (c) Representative histology of the heart sections at 90 days after rBCG immunization. (d) Prevalence of active myocarditis, n = 5–6 at each time point for each group.

• 

  Figure 6. Assessment of CD4+ T-cell function. Immunization protocol is shown in Figure 5a. (a) Flow cytometric analysis of the heart-infiltrating CD4+ T cells at 12 weeks after rBCG immunization. Graphs show the percentage of the effector (CD44highCD62Llow) and naïve (CD44lowCD62Lhigh) T cells in CD4+ T cells (n = 5 per group). * p < 0.05 vs. PBS and # p < 0.05 vs. rBCG-pSO246 by Kruskal-Wallis analysis with a post hoc Steel-Dwass test. (b) Bar graphs show MyHCα-specific cytokine production of CD4+ T cells isolated at 12 weeks after rBCG immunization. n = 5 per group. * p < 0.05 vs. PBS, # p < 0.05 vs. ovalbumin (OVA) stimulation by Kruskal-Wallis analysis with a post hoc Steel-Dwass test. (c) CD4+ T cells were isolated from rBCG-immunized mice and stimulated with MyHCα for 48 h. After stimulation, the CD4+ T cells were transferred into SCID mice. (d) Representative H&E-stained section of the hearts at 10 days after adoptive transfer. (e) Myocarditis severity in heart sections stained with H&E (n = 6 mice/group). * p < 0.05 vs. PBS and # p < 0.05 vs. rBCG-pSO246. (f) CD4+ T cells were separated into CD62L+ cells and CD62L− cells, and both cell populations were transferred into SCID mice.…

• 

  Figure 7. rBCG-MyHCα promoted cytokine and chemokine production in DCs. BMDCs were infected with rBCG-MyHCα at the indicated MOIs for 72 h. Cytokine and chemokine concentrations in the culture supernatant were assessed by ELISA. The values are expressed as means of pentuiplicate wells. * p < 0.05 vs. MOI 0 by one-way ANOVA with Tukey’s post hoc test.

• 

  Figure 8. Culture supernatant of rBCG-MyHCα-infected DCs promoted Th1 and Th17 differentiation. Naïve CD4+ T cells were cultured in the supernatant of rBCG-MyHCα-infected BMDCs for 48 h. The gene expression of Tbx21 and Rorc were assessed by qRT-PCR and cytokine concentrations in the culture supernatant were assessed by ELISA. The values are expressed as means of pentuiplicate wells. * p < 0.05 vs. MOI 0 by one-way ANOVA with Tukey’s post hoc test.

• 

  Figure 9. rBCG-MyHCα-infected DCs induced MyHCα-specific CD4+ T-cell proliferation. (a) EAM was induced by immunization with MyHCα peptide emulsified in CFA. CD4+ T cells were isolated from spleens of EAM or naïve mice and co-cultured with rBCG-infected BMDCs, MyHCα-loaded BMDCs, or non-stimulated BMDCs (PBS). (b) T-cell proliferation was assessed by measurement of [3H]-thymidine incorporation. The values are expressed as means ± SEM of pentuiplicate wells. * p < 0.05 vs. naive by one-way ANOVA with Tukey’s post hoc test.

• 

  Figure 10. rBCG-MyHCα-infected DCs induced chronic myocarditis. (a) After priming with 10 μg of MyHCα peptides on days-30 and -23, BMDCs infected with rBCG-MyHCα or rBCG-pSO246 were injected into mice on days 0, 2, and 4. (b) Representative histology (H&E and Sirius red staining and TN-C immunostaining) of the heart sections on 90 days after DC injection.