Myocarditis Elicits Dendritic Cell and Monocyte Infiltration in the Heart and Self-Antigen Presentation by Conventional Type 2 Dendritic Cells

Figures

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  Figure 1. Transfer of bone marrow cDC2 initiates EAM. (A) Schematic overview of BMDC-induced mouse model of experimental autoimmune myocarditis (EAM). GM-CSF derived BMDCs were matured using LPS and αCD40 and loaded with heart specific self-antigen αMyHC614−629 or the harmless OVA323−339 antigen. αMyHC614−629 BMDC immunization leads to fulminant myocarditis (bottom, red arrows) as shown by heamatoxylin and eosin staining of heart sections at day 10 post-immunization, whereas OVA BMDC immunization does not (top). RV, Right Ventricle; LV, Left Ventricle. (B) Flow cytometric analysis of GM-CSF derived BMDCs harvested at day 10 of culture. Histograms display expression of the specified markers by MHCIIint GM-MCs and MHCIIhi GM-cDC2s. (C) Cell trace+ cell percentages of total living cells in mLN of i.p. injected mice with 0.5 × 106 cell trace-labeled BMDCs, GM-cDC2s, GM-MCs, or PBS 12 h before sacrifice. (D) Histograms representing CFSE dilution of TCR-M cells co-cultured with bulk BMDCs, sorted GM-cDC2s, or sorted GM-MCs with or without 15 μg/ml αMyHC614−629 (respectively, non-filled and filled histograms) in a 1/5 DC/TCR-M ratio. (E) 4 days after naïve TCR-M injection and 3 days after…

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  Figure 2. DC subsets accumulate in heart and mLN during EAM. (A) Representative flow cytometry gating strategy for cDCs, CD11c+ MCs, cDC1s, and cDC2s in the heart of OVA or αMyHC BMDC injected WT mice on day 10 post-injection. Numbers specify cell percentage inside nearby gates. (B) Absolute cell numbers of cDC1s, cDC2s, and MCs in the heart of OVA (pooled data of day 10 and day 25 post-inj.) and αMyHC BMDC (at day 10 and day 25 post-inj.) injected WT mice as assessed by flow cytometry. (C) Pie charts depicting the distribution of APC subsets in heart of OVA (pooled data of day 10 and day 25 post-inj.) and αMyHC BMDC (at day 10 and day 25 post-inj.) injected WT mice. Pie chart sizes are proportional to total absolute cardiac APC numbers. (D,E) Absolute numbers of MHCIIint and MHCIIhi DCs (D) and MHCIIint and MHCIIhi subsets (E) in the heart-draining mediastinal LN (D,E) and skin-draining inguinal LN (D) of OVA and αMyHC BMDC injected WT mice at day 10 post-injection. (F) Illustrative flow cytometry gating strategy for MHCIIint and MHCIIhi CD11c+ cells and CCR7 expression of MHCIIhi cDC1s, MHCIIhi cDC2s, MHCIIhi MCs, and MHCIIint cDCs in mLN of αMyHC BMDC injected WT mice at day 10. All bar…

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  Figure 3. Expression profiling of cardiac APCs by RNA sequencing. (A) Front view of PCA of RNA-seq data from cDC1s, cDC2s, CD11c+ MC, and CD11c- MCs in the steady state (St St) and the inflamed (EAM d10) heart. PCA was calculated using the top 15% most varying genes between cell subsets. Each dot symbolizes one independently sorted replicate of the indicated cell population and four independent sorts were performed per subset. (B) Triwise diagrams of the gene expression at EAM day 10. Red dots represent cDC1 (left) and cDC2 hallmark genes (right). (C) Heatmap showing the relative expression of MHCI- or MHCII-associated genes normalized per mean expression of each gene in cardiac cDC1s, cDC2s, and CD11c+ MCs from steady state and EAM day 10 heart. (D) Mean Fluorescence Intensity of CD40, CD80, and CD86 expression on heart DC subsets in OVA and αMyHC BMDC injected WT mice at day 10 post-injection. (E) Graphs representing absolute expression of Wash1 and Serpbinb9, two cDC2-specific DEGs between steady state and EAM day 10 assessed by RNA-seq. All bar graphs in this figure show data as the mean ± SEM; *P ≤ 0.05.

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  Figure 4. cDC2s present self-antigen in heart and draining LN in EAM. (A) CFSE dilution and CD25 expression of TCR-M cells co-cultured for 3 days with sorted heart DC subsets at steady state and at EAM day 10. Hearts were pooled from 100 steady state mice and 6 mice suffering from acute EAM. Data are representative for three independent experiments. (B) Percentage of proliferation and CD25 expression of TCR-M cells from co-cultures plotted in panel A (mean ± SEM; *P ≤ 0.05; **P ≤ 0.01). (C) After 3 days of co-culture (plotted in A), IFNγ and IL-17A (pg/ml) was measured in cell supernatant by ELISA. ELISA was performed in duplo on the same sample (mean ± SD). (D) IL-6 and IL-23p19 mRNA expression in DC subsets sorted from at least 10 pooled hearts at EAM day 10 measured by qPCR. Relative mRNA expression was calculated by normalization to housekeeping genes and qPCR reactions were performed in triplicate (mean ± SD). (E) Percentages of heart-infiltrated IL-17+, IFNγ+, IL-17+/IFNγ+, and CD25+Foxp3+ CD4+ T cells of OVA (black bars) and αMyHC BMDC injected mice (gray bars) 10 days following BMDC injection measured by flow cytometry after PMA/ionomycin restimulation (mean ± SEM; *P ≤ 0.05; **P ≤…

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  Figure 5. Loss of IRF4 in CD11c+ cells does not affect EAM severity. (A) Histograms representing IRF4 and MHCII expression on GM-cDC2s and GM-MCs from Irf4fl/fl and Irf4fl/fl.Cd11cCre BMDC cultures. (B) CD25+CFSE− TCR-M cell percentages of total TCR-M cells in co-culture with bulk BMDCs, sorted GM-cDC2s, and sorted GM-MCs from Irf4fl/fl and Irf4fl/fl.Cd11cCre BMDC cultures with OVA323−339 peptide as negative control or with αMyHC614−629 peptide. (C) Absolute CD45+ leukocyte number in the heart of WT mice immunized with OVA323−339 or αMyHC614−629 loaded BMDCs cultured from Irf4fl/fl and Irf4fl/fl.Cd11cCre BM. (D) APC subset percentages of total living cells in the heart and mLN of Irf4fl/fl and Irf4fl/fl.Cd11cCre mice immunized with OVA or αMyHC BMDCs (day 10) (E) Histograms of CFSE dilution and RoRγt expression of TCR-M cells co-cultured for 4 days with sorted migratory cDC2s from mLN of Irf4fl/fl and Irf4fl/fl.Cd11cCre mice 10 days after injection with αMyHC BMDCs. (F) CD45+ leukocyte percentage of total living cells in the heart of Irf4fl/fl and Irf4fl/fl.Cd11cCre mice immunized with OVA or αMyHC BMDCs (day 10). All bar graphs in this figure shows data as the mean ± SEM; *P ≤ 0.05; **P ≤…

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  Figure 6. Cardiac MCs display both DC and MF functions in EAM. (A) CFSE dilution and CD25 expression of TCR-M cells co-cultured for 3 days with sorted heart cDC2s and CD11c+ MCs without and with addition of dead cardiomyocytes. Data are representative for two independent experiments with at least 10 mice per experiment. (B) CathS, Lamp1 and H2DMb1 and (C) iNOS, IL-1α and CCL2 mRNA expression in cDC1s, cDC2s, and MCs sorted from at least 10 pooled hearts at EAM day 10 measured by qPCR. Relative mRNA expression was calculated by normalization to housekeeping genes and qPCR reactions were performed in triplicate. nd, not detectable. (Mean ± SD) (D) MFI of intracellular iNOS expression on heart cDC subsets and MCs in OVA and αMyHC BMDC injected WT mice at day 10 post-injection (mean ± SEM; *P ≤ 0.05). (E) Heat map of relative expression of CCL-chemokine genes in cDC1s, cDC2s, and CD11c+ MCs sorted from EAM day 10 hearts.