Interleukin-10 exhibit dose-dependent effects on macrophage phenotypes and cardiac remodeling after myocardial infarction

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  FIGURE 1. Dose-dependent effects of IL-10 on polarized macrophage phenotypes. To investigate dose-dependent effects of IL-10 on the phenotypes of polarized Mφ, naïve RAW264.7 cells were first differentiated into the classic M1 and M2 phenotypes with mIFNγ and mIL-4, respectively, followed by treatment with titrated IL-10 (0, 25, 100, 250, 500, or 1,000 ng/mL) for 24 h. We then analyzed M1 and M2 phenotypic changes by labeling cells with (A, B) anti-CD86 (classic M1 marker) and (D, E) anti-CD206 (classic M2 marker) antibodies, respectively, before subjecting cells to flow cytometry. (A) Representative graphs with averaged ratios of CD86+ cells from each IL-10 treatment group. (B) Quantification of the flow cytometry data (n = 3 per group). Only the 250 group showed significantly lower CD86 expression than the 0 group (p = 0.0148). The 500 and 1,000 groups had significantly higher CD86 expression than the 250 group (p < 0.01 and p < 0.05, respectively). (C) Detection of mTNF-α secretion by polarized M1 cells after hIL-10 treatment using ELISA. The 250 group consistently exhibited significantly lower TNF-α secretion than the 0, 100, and 1,000 groups (p < 0.0001, p = 0.0478, and p = 0.0146,…

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  FIGURE 2. Dose-dependent effects of IL-10 on macrophage polarization. To study the effect of exogenous IL-10 treatment with differential doses on the Mφ polarization process, we differentiated naïve RAW264.7 monocytes with a standard polarizing agent (mIFNγ for M1 and mIL-4 for M2) and one of the six doses of IL-10: 0, 25, 100, 250, 500, and 1,000 ng/mL simultaneously for 36 h. We analyzed the manifestation of classic M1 and M2 phenotypes with flow cytometry by labeling cells with (A, B) anti-CD86 and (D, E) anti-CD206 antibodies, respectively. (A) Representative graphs with averaged ratios of CD86+ cells from each IL-10 treatment group. (B) Quantification of the flow cytometry data (n = 3 per group). All groups had significantly lower CD86 expression than the 0 group (all p < 0.001), except the 25 group (p > 0.05). (C) Detection of mTNF-α secretion by polarized M1 cells after hIL-10 treatment using ELISA. All groups had significantly less TNF-α secretion when compared with the 0 group (all p < 0.001). (D) Representative graphs with averaged ratios of CD206+ cells from each IL-10 treatment group. (E) Quantification of the flow cytometry data (n = 3 per group). The 100 group showed…

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  FIGURE 3. Intramyocardial injection of IL-10 improves cardiac function with dose-dependent effects. Using a mouse MI model, we investigated whether intramyocardial IL-10 administration exerts any dose-dependent efficacy or potential unwanted effect on cardiac function post-MI. (A) The timeline of the animal study, including surgical procedures, cardiac functional evaluations, and tissue-based analyses. Ten minutes after the induction of MI, mice received one-time intramyocardial injection of IL-10 with one of the six doses (ng): 25 (sage), 100 (purple), 250 (green), 500 (blue), 1,000 (brown), or 2000 (red) or 0.9% saline (vehicle control, black). Echocardiography was repeatedly performed by a blinded investigator to measure left ventricular (B) fractional shortening (5D: **p < 0.01, 2000 vs saline, 25, 100, and 250; 14D: *p < 0.05, 2000 vs 250; 42D: *p < 0.05, 2000 vs 250; all other comparisons vs saline), (C) ejection fraction (5D: **p < 0.01, 2000 vs 25, 100, and 250; *p < 0.05, 1,000 vs 250; 14D: **p < 0.01, 2000 vs 250; *p < 0.05, 25 vs 250; all other comparisons vs saline), (D) end-diastolic area (all comparisons vs saline), and (E) end-systolic area (all comparisons vs saline) at 5,…

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  FIGURE 4. Early dose-dependent effects of intramyocardial IL-10 treatment on myocardial gene expression. We used qPCR to analyze the expression levels of five groups of genes in mouse LV treated with different doses of IL-10 (0, 250, or 1,000 ng) at 5 days of post-MI, including (A) pro-inflammatory and immune-activating genes (TNF-α, IL-6, IFNγ, iNOS, IL-1β, and CD64); (B) immunomodulatory and anti-inflammatory genes (mIL-10, CD206, Arg1, and CD163); (C) macrophage efferocytotic genes (Axl, Tim4, and MerTK); (D) chemotactic and angiogenic genes (CX3CL1 and VEGF-A); (E) cardiac fibrotic genes (α-SMA, Vsig4, Hyal3, PDGF, Postn, and Col1a1). Data were statistically compared using one-way ANOVA with Dunnett multiple comparisons test (n = 5 per group). The results suggest that 250 and 1,000 ng IL-10 treatment differentially activated diverse functional signaling networks, with 1,000 ng IL-10 seemingly triggering both pro-inflammatory and anti-inflammatory pathways. Note: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

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  FIGURE 5. Intramyocardial IL-10 treatment decreases chronic inflammation within the infarct region. Chronic inflammation was assessed by the number of infiltrating CD68+ phagocytic cells (green) within the infarct region at 6 weeks post-infarction. (A) Representative images of anti-CD68 immunofluorescent staining revealing phagocytic cell infiltration within the infarct region at the mid-infarct level from each group (scale bars: 50 μm). Nuclei were stained in blue. Healthy (normal) hearts served as negative controls. (B) Three IL-10 doses (100, 250, and 1,000 ng) resulted in significant decreases of CD68+ cell infiltration (n = 3 per group; p = 0.046, p = 0.007, and p = 0.006, respectively, vs saline). Note: *p < 0.05, **p < 0.01.

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  FIGURE 6. Intramyocardial IL-10 treatment reduces left ventricular myocardial fibrosis. Long-term left ventricular myocardial fibrosis was revealed by Masson’s trichrome histological staining at 6 weeks post-infarction. (A) Representative images of Masson’s trichrome-stained transverse sections of hearts at the mid-infarct level (collagen deposition stained in blue/purple, cardiac muscle stained in red; scale bars: 1 mm). Healthy (normal) hearts served as negative controls. (B) Quantitatively, only the 250 group exhibited significantly reduced LV fibrotic area fraction (n = 3 per group; *p = 0.05, vs saline).

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  FIGURE 7. Intramyocardial injection of IL-10 enhances revascularization in the ischemic myocardium. Long-term revascularization, including angiogenesis and vasculogenesis, in the ischemic myocardium was evaluated by the presence of CD31+ endothelial cells (ECs) and αSMA + peri-vascular smooth muscle cells (SMCs), respectively, at 6 weeks post-infarction (n = 3 per group). Perivascular SMCs defined as αSMA + cells around CD31+ ECs. Healthy (normal) hearts served as baseline controls (n = 3). (A) Representative images of CD31+ ECs (red) and αSMA + SMCs (green) within the infarct region at the mid-infarct level from each group (scale bars: 50 μm). Nuclei were stained in blue. (B) Quantitatively, only the 250 ng group had a significantly higher density of CD31+ ECs at the infarct (p = 0.013, vs saline). (C) No significant difference in the CD31+ EC density was noted in the peri-infarct area (all p > 0.05, vs saline). (D) Only the 1,000 group had a significantly higher density of αSMA + SMCs within the infarct region (p = 0.045, vs saline). (E) No notable difference in the αSMA + SMC density in the peri-infarct area (all p > 0.05, vs saline). Note: *p < 0.05.

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  FIGURE 8. The estimated dose-dependent effects of single IL-10 administration. The illustration summarizes the overall dose-effect relationships after single IL-10 administration in vitro and in vivo. The x- and y-axis of each horizontal bar represent the IL-10 dose ranges and the estimated levels of individual annotated effects, respectively. Note: a. u.: arbitrary unit.