Biopsy-proven myocarditis: peripheral immunophenotypes correlate with histology, etiology, immunosuppressive therapy

Figures

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  Fig. 1. Immunophenotypic features of acquired immune system in autoimmune myocarditis Data on immunophenotype were obtained by flow-cytometry and are reported in box plot graphs as populations percentage ratio or absolute count ratio (mathematically derived from blood cell count and expressed as cells x 103/μL, A, B), or as percentages (C, D Left) or as absolute count (D Right). Mann-Whitney (B, C) or Kruskall-Wallis (reported in the bottom of each graph) and Dunn post-hoc tests with Benjamini-Hochberg for multiple comparisons correction (A, D) were used for the statistical evaluation. Only the significant group comparisons are reported. These results showed that autoimmune myocarditis had higher Th1/Th2 and Th17/Treg ratios (A and B). Indeed, Th1, Th17 and CD4+/IL-17+ cells were increased (C Left, Middle and D), while a subtype of Th17 cells, i.e. IL-17/IL-23Receptor(R)/CD4/TCRγδ(GD) + cells, was reduced (C Right).

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  Fig. 2. Immunophenotypic features of innate immune system in autoimmune myocarditis Data on immunophenotype were obtained by flow-cytometry and are reported in box plot graphs as percentages (A Left, B Left, C) or as absolute count (mathematically derived from blood cell count and expressed as cells x 103/μL, A Right, E), or as mean fluorescence intensity (MFI, i.e. expression level, B Right, D Left). Mann-Whitney (D) or Kruskall-Wallis (reported in the bottom of each graph) and Dunn post-hoc tests with Benjamini-Hochberg for multiple comparisons correction (A, B, C, E) were used for the statistical evaluation. Only the significant group comparisons are reported. Plasmacytoid dendritic cells (pDCs, A), non classic monocytes (B, Left) and CD3-/CD16+ NK cells were reduced in autoimmune myocarditis (E). On the other hand, NK cells over-expressed CD62L in autoimmune forms (D); this marker evidences less mature NK cells and indeed immature NK cells (CD56b/CD62L+) are increased in myocarditis and in particular for viral forms (C).

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  Fig. 3. Immunophenotypic features in different myocarditis histologic types Data on immunophenotype were obtained by flow-cytometry and are reported in box plot graphs as percentages (A Left, C, D Left), or as absolute count (mathematically derived from blood cell count and expressed as cells x 103/μL, A Right, D Right), or as ratio of populations percentage or count (B). Kruskall-Wallis (reported in the bottom of each graph) and Dunn post-hoc tests with Benjamini-Hochberg for multiple comparisons correction were used for the statistical evaluation. Only the significant group comparisons are reported. Lymphocytic myocarditis had reduced plasmacytoid dendritic cells (pDCs, A) and Treg cells (C), leading to and increased Th17/Treg ratio (B). Eosinophilic/Polymorphic (Eosinophil/Polymorph) myocarditis had higher the pro-inflammatory dendritic cells, named DC3, in comparison to lymphocytic forms (D).

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  Fig. 4. Immunophenotypic features in autoimmune myocarditis with or without AHA and other autoimmune diseases. Data on immunophenotype were obtained by flow-cytometry and are reported in box plot graphs as percentages (A Left and Right panels, B Left, C, D and E), or as absolute count (mathematically derived from blood cell count and expressed as cells x 103/μL, A Middle panel, B Left). Kruskall-Wallis (reported in the bottom of each graph) and Dunn post-hoc tests with Benjamini-Hochberg for multiple comparisons correction were used for the statistical evaluation. Only the significant group comparisons are reported. Treg cells were reduced in autoimmune myocarditis with organ specific anti-heart antibodies (AHA) and without other autoimmune diseases (AD, A). Autoimmune myocarditis with an organ specific disease had more Th17 cells equipped with a TCRγδ+ (TCR-GD, B) and reduced plasmacytoid dendritic cells (pDCs, C). AHA + patients had less CD8+ cells (D), while patients without AHA had increased mature NK cells with a high expression of CD16 (CD16h) positive for CD62L and negative for CD57 (E).

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  Fig. 5. Immune cells distribution in responders vs non responders patients to immunosuppression Data on cell blood count and data on immunophenotype (obtained by flow-cytometry) are reported in box plots graphs as percentages (A, B, D Left, F Left), or as absolute count (mathematically derived from blood cell count and expressed as cells x 103/μL, C, D Right, F Right). Kruskall-Wallis (reported in the bottom of each graph) and Dunn post-hoc tests with Benjamini-Hochberg for multiple comparisons correction were used for the statistical evaluation. Only the significant group comparisons are reported. Patients unresponsive to immunosuppression (IS) were characterized by an increase of neutrophils (A), a reduction of eosinophils (B, C), of cells expressing Human Leukocyte Antigen type DR (HLA-DR, D), and of T lymphocytes equipped with a TCRγδ+ (TCR-GD, E). Patients who never required IS, because of spontaneous healing, could be identified by the reduction in plasmacytoid dendritic cells (pDCs, F).