Programmed Death-Ligand 2 Deficiency Exacerbates Experimental Autoimmune Myocarditis in Mice

Figures

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  Figure 1. Dynamics of inflammatory cells in cardiac myosin peptide-induced autoimmune myocarditis. Quantification of inflammatory cells in the hearts obtained from mice with experimental autoimmune myocarditis, represented as a percentage of live cells at the indicated time points (n = 10 at each time point). The values are expressed as the mean ± standard error of the mean. * p < 0.05 vs. day 0.

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  Figure 2. Expression levels of programmed cell death protein 1 (PD-1) and programmed cell death ligands (PD-Ls) in the experimental autoimmune myocarditis (EAM) hearts. (A) mRNA expression of PD-1, PD-L1, and PD-L2 in the hearts obtained from mice with EAM (days 14 and 28) and control mice (n = 6–7). (B) Flow cytometric analysis of the expression of PD-1, PD-L1, or PD-L2 on infiltrating CD4+ T cells, CD8+ T cells, and CD11c+MHC II+ dendritic cells in the hearts obtained from mice with EAM on days 0, 7, 14, 21, and 35 after first immunization with cardiac myosin peptide (n = 6 at each time point). Results are presented as the mean ± standard error of the mean. * p < 0.05 vs. day 0.

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  Figure 3. PD-L2 deficiency exacerbates myocardial inflammation in experimental autoimmune myocarditis. F1 hybrids of PD-1−/−, PD-L2−/−, and wild-type (WT) mice on the C57BL/6J background and BALB/c mice (PD-1+/−, PD-L2+/−, and WT, respectively) were immunized twice, on days 0 and 7, with cardiac myosin epitope peptide. (A) Representative H&E-stained sections of the hearts on day 14. Scale bars, 1 mm or 50 μm. (B) Myocarditis severity in heart sections (n = 6–7 per group). (C) Heart-to-body weight ratios (HW/BW) of the mice (n = 10 per group). Results are presented as the mean ± standard error of the mean. * p < 0.05 vs. WT.

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  Figure 4. PD-L2 deficiency increases inflammatory cell infiltration in the experimental autoimmune myocarditis (EAM) heart. (A) Representative flow cytometric plots showing CD45+ leukocytes, CD3e+ lymphocytes, CD8+ T cells, CD4+ T cells, CD11b+Ly6G+ neutrophils, and CD11b+CD11c+ DCs from WT, PD-1+/−, and PD-L2+/− hearts on day 14 after EAM induction. The orange squares indicate CD3e+CD45+ lymphocytes and the green squares indicate CD11b+Ly6Gdull cells. (B) Quantification of inflammatory cells in the hearts obtained from WT mice as well as PD-1+/− and PD-L2+/− mice with EAM. Results are presented as the mean ± SEM, n = 7–9 per group. * p < 0.05 vs. WT.

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  Figure 5. PD-L2 deficiency promotes proinflammatory cytokine expression in the experimental autoimmune myocarditis heart. mRNA expression of inflammatory markers in the hearts of wild-type (WT), PD-1+/−, and PD-L2+/- mice at 14 days after immunization. Results are presented as the mean ± standard error of the mean. n = 3–6, * p < 0.05 vs. control, # p < 0.05 vs. WT, and † p < 0.05 vs. PD-1+/−.

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  Figure 6. PD-L2 deficiency in dendritic cells (DCs) promoted CD4+ T cell proliferation. (A,B) Carboxyfluorescein succinimidyl ester (CFSE)-labeled WT CD4+ T cells were co-cultured with bone marrow-derived dendritic cells (BMDCs) generated from wild-type (WT) or PD-L2−/− mice and stimulated with anti-CD3 and anti-CD28 for 72 h. Proliferation of CD4+ T cells was visualized by observing the incremental loss of CFSE fluorescence. Representative histograms (gated on CD4+ T cells) and the frequency of the proliferated CD4+ T cells among all CD4+ cells are shown. n = 3 per group. IL-2 concentrations in the culture supernatants were assessed by ELISA. n = 6 per group. Results are presented as the mean ± SEM. * p < 0.05 vs. WT. (C,D) CD4+ T cells were isolated from WT or PD-L2−/− mice and labeled with CFSE. Then, they were co-cultured with WT BMDCs and stimulated with anti-CD3 and anti-CD28 for 72 h. Proliferation of CD4+ T cells was visualized by observing the incremental loss of CFSE fluorescence. Representative histograms and the frequency of the proliferated CD4+ T cells among all CD4+ cells are shown. n = 3 per group. IL-2 concentrations in the culture supernatants were assessed by…