CD16+ Monocytes and Skewed Macrophage Polarization toward M2 Type Hallmark Heart Transplant Acute Cellular Rejection

Figures

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  Figure 1. Contrasting monocyte subsets in blood and tissue during heart transplant rejection. Representative monocyte subset FACS gating strategy is depicted (A). Blood: the percentage of (B) classical CD14++CD16−, (C) intermediate CD14++CD16+, and (D) non-classical CD14+CD16++ monocytes in healthy individuals (n = 33) and heart transplant recipients (n = 25) at time points no rejection (NR) and rejection (R) are presented as median ± IQR. Tissue: the absolute numbers of (E) CD14+CD16−, (F) CD14+CD16+, and (G) CD14−CD16+ monocytes in heart transplant biopsies at time points non-rejection (NR; n = 25) and R (n = 25) are given. A representative overview of double immunofluorescence stainings of rejected endomyocardial tissue is shown; (H) CD14 (green) and CD16 (red) at 10× magnification. Detailed co-localization (H) of CD14 (green) and CD16 (red) is shown at 63× magnification. Absolute numbers of cells are depicted (*p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 2. CD16+ cells in graft tissue are monocytes and accumulate upon rejection. The expression of CD14 and CD16 was analyzed in biopsies at two time points; non-rejection (NR; n = 25) and rejection (R; n = 25) and compared intra-individually. The absolute number of cells is depicted for (A) CD14+CD16−, (B) CD14+CD16+, and (C) CD14−CD16+. The expression of (D) CD56 was measured using immunohistochemistry, analyzed by ImageJ and depicted as mean % positive stained area. The absolute numbers of cells of (E,F) CD56+CD16− and CD56+16+ were determined. A representative overview of rejecting endomyocardial tissue stained for (G) CD56 (green) and CD16 (red) at 20× magnification is shown. A representative overview of rejecting endomyocardial tissue stained for (H) CD14 (green), (I) CD16 (green) and CD68 (red) at 20× magnification is given (*p < 0.05, ***p < 0.001).

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  Figure 3. Phenotypic and functional characteristics of circulating monocytes in heart transplant recipients compared with healthy individuals. Expression levels of (A) HLA-DR, (B) CD54 (ICAM-1) are increased in CD16+ monocyte pool during rejection compared to non-rejection. The percentages of IL-1β-, IL-6-, TNF-α-, IFN-γ-, IL-12-, and IL-10-producing monocytes of both healthy individual group (n = 9−14) and heart transplant recipients (n = 10) are shown at both NR and R time points after LPS stimulation (C) (mean ± SEM) (*p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 4. M2-type macrophages predominate in transplanted endomyocardial tissue and increase upon rejection. The expression of (A) CD68, (B) CD80, and (C) CD163 was measured using immunohistochemistry, analyzed by ImageJ and depicted as mean % positive stained area. The expression of (D) CD68, (E) CD80, and (F) CD163 was analyzed intra-individually at rejection and non-rejection time points. Representative histological and immunohistochemical images are shown at 20× magnification (G) HE, (H), CD68, (I) CD80, and (J) CD163. Co-localization of (K) CD68 and CD80 (M1 macrophage), and CD68 with CD163 (M2 macrophages) is shown at 40× magnification (***p < 0.001).

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  Figure 5. Severe fibrosis at rejection which is irreversible over time. Sirius Red staining was used to assess fibrosis. Representative histological images for non-rejection time point (NR; first time point), rejection time point (R; second time point), and 1-year post-rejection time point (1-year post-rejection, third time point) are shown at 20× magnification (A). Fibrosis is quantified as mean % positive stained Sirius Red area (B) (mean ± SEM) (***p < 0.001).

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  Figure 6. A model for contrasting findings between blood and tissue monocyte–macrophage lineage cells during heart transplant rejection. The putative scenario of events based on our findings is described in Section “Discussion.” Briefly, step 1: subendothelial migration of monocytes, step 2: migration of pro-inflammatory CD16+ monocytes toward the tissue, and step 3: macrophage polarization in to high numbers of anti-inflammatory M2-macropghages contributing to remodeling and fibrosis of the damaged heart.