Mesenchymal stromal cells but not cardiac fibroblasts exert beneficial systemic immunomodulatory effects in experimental myocarditis

Abstract

Systemic application of mesenchymal stromal cells (MSCs) in inflammatory cardiomyopathy exerts cardiobeneficial effects. The mode of action is unclear since a sufficient and long-acting cardiac homing of MSCs is unlikely. We therefore investigated the regulation of the immune response in coxsackievirus B3 (CVB3)-induced acute myocarditis after intravenous application of MSCs. Wildtype mice were infected with CVB3 and treated with either PBS, human MSCs or human cardiac fibroblasts intravenously 1 day after infection. Seven days after infection, MSCs could be detected in the spleen, heart, pancreas, liver, lung and kidney, whereby the highest presence was observed in the lung. MSCs increased significantly the myocardial expression of HGF and decreased the expression of the proinflammatory cytokines TNFα, IL1β and IL6 as well as the severity of myocarditis and ameliorated the left ventricular dysfunction measured by conductance catheter. MSCs upregulated the production of IFNγ in CD4+ and CD8+ cells, the number of IL10-producing regulatory T cells and the apoptosis rate of T cells in the spleen. An increased number of CD4+CD25+FoxP3 could be found in the spleen as well as in the circulation. In contrast, application of human cardiac fibroblasts had no effect on the severity of myocarditis and the systemic immune response observed after MSCs-administration. In conclusion, modulation of the immune response in extracardiac organs is associated with cardiobeneficial effects in experimental inflammatory cardiomyopathy after systemic application of MSCs.

Figure 1. Alu-sequences analysis for localisation of MSCs and cardiac fibroblasts in uninfected and infected mice 6 days after infection.

MSCs could be retrieved from the heart, lung, spleen, kidney, liver and pancreas. The highest amount of MSCs was found to be in the lung. Interestingly, the amount of MSCs found in the heart and lung was significantly upregulated after infection. *p<0.05 vs. Control-group.

Figure 2. Flow cytometric analysis of splenocytes 7 days after infection.

Viral infection led to a significant increase of CD4+ and CD8+ T cells in the spleen, but also induced apoptosis in both cell types. Administration of MSCs led to a significant increase of CD4+ and CD8+ T cells in the spleen in the infected animals compared to the non-treated mice. However, MSCs also significantly increased the apoptosis rate of CD4+ and CD8+ T cells in the infected mice. *p<0.05 vs PBS-CVB3, § p<0.05 vs respective control-group, # p<0.05 vs. MSC-CVB3.

Figure 3. Amount of IFNγ and IL10 measured by ELISA and intracellular staining for IFNγ and IL10 in T cells (CD4+, CD8+), dendritic cells (CD11c+) and macrophages (CD68+) in the spleen 7 days after infection.

IL10 and IFNγ were significantly upregulated in the spleen after injection of MSCs. We identified CD4+ and CD8+ T cells as the source of excess IFNγ in the MSC-treated animals (b-c). Furthermore, administration of MSCs led to a significant upregulation of induced regulatory IL10-producing T cells (g-h). *p<0.05 vs PBS-CVB3, § p<0.05 vs respective control-group, # p<0.05 vs. MSC-CVB3.

Figure 4. Flow cytometric analysis of naïve CD4+CD25+FoxP3 T cells in the spleen

(a) and expression of the FoxP3 transcription factor in the myocardium (b). a. Administration of MSCs induced a significant upregulation of naïve regulatory T cells in the spleen compared to PBS and fibroblasts. b. Expression of the transcription factor FoxP3 in the myocardium was analyzed as an indirect marker for the number of FoxP3-positive regulatory T cells in the heart. Higher expression of FoxP3 in the MSC-treated mice implies a signifcantly higher number of naïve regulatory T cells in the heart compared to PBS- and fibroblast-treated mice. *p<0.05 vs PBS-CVB3, § p<0.05 vs respective control-group, # p<0.05 vs. MSC-CVB3.

Figure 5. CD4+CD25+FoxP3 and CD8+IL10 T cells in the blood.

Similarly to the spleen, naïve and induced regulatory T cells were upregulated in the systemic circulation as an effect of the intravenous injection of MSCs. *p<0.05 vs PBS-CVB3, § p<0.05 vs respective control-group.

Figure 6. Histopathologic analysis of myocarditis (a-b) and viral replication in the heart (c).

a. Pathologic score of the severity of myocarditis assessed by hematoxylin-eosin staining and in situ hybridisation of CVB3 positive strand-RNA seven days after infection. b. Representative histological sections of extensive myocardial infiltrations (black arrows) in PBS- and fibroblasts-treated animals and detection of viral RNA in the myocardium (red arrows) (x100 magnification). c. Viral replication assessed by the number of plaque forming units from homogenized cardiac tissue on HeLa cells and viral load assessed by real-time PCR in the spleen, pancreas and liver. MSCs tended to lower not significantly the viral replication in the heart and pancreas compared to PBS and fibroblasts.

Figure 7. Gene expression of cytokines in the myocardium seven days after infection: Pro-inflammatory cytokines such as TNFα, IL6 and IL1β were significantly upregulated after infection.

Administration of MSCs led to a signicant reduction in their expression compared to PBS or fibroblasts. IFNγ, similarly to spleen, was significantly upregulated in the myocardium after treatment with MSCs (d), while IL10 was not affected either by MSCs or fibroblasts (e), Expression of the HGF in the myocardium, a well described mediator of the actions of MSCs was signifantly higher in the MSC-treated compared to the PBS- or fibroblast-treated mice (f). *p<0.05 vs PBS-CVB3, § p<0.05 vs respective control-group, # p<0.05 vs. MSC-CVB3.

Figure 8. Immune cell infiltration in the myocardium 7 days after infection: a. Macrophages (CD11b+), b. CD4+ T cells and c. CD8+ T cells.

*p<0.05 vs PBS-CVB3, § p<0.05 vs respective control-group.

Figure 9. Left ventricular function 7 days post infection, measured by conductance catheter advanced into the heart through thea apex.

Administration of MSCs led to a significant improvement of parameters of systolic and diastolic function. As a result cardiac output was also significantly improved. LVP, left ventricular pressure; dP/dt_max, derivative of left ventricular pressure rise; dP/dt_min, derivative of left ventricular pressure reduction. *p<0.05 vs PBS-CVB3, § p<0.05 vs respective control-group, # p<0.05 vs. MSC-CVB3.

Figure 10. Effects of MSCs on fibroblasts and collagen production during myocarditis.

a. Serious red analysis of collagen production by murine fibroblasts co-cultured with splenocytes from infected and healthy mice treated with PBS, MSCs or cardiac fibroblasts. Splenocytes from infected animals induced an increased production of collagen in the fibroblasts compared to those from non-infected mice. However, splenocytes from MSC-treated mice led to a significantly lower activation of murine fibroblasts as measured by their collagen production. b-c. Immunohistochemical analysis of the collagen I and III amount in the myocardium showed a significantly lower myocardial fibrosis after MSC-treatment compared to PBS and fibroblasts and confirmed our in vitro experiments. *p<0.05 vs PBS-treated mice with same infection status, § p<0.05 vs respective control-group, # p<0.05 vs. MSC-treated mice with same infection status, & p<0.05 vs. murine fibroblasts in basal conditions (cultured alone).

Figure 11. Cytokines expression and analysis of the inflammatory response in the liver and pancreas.

The amount of pro- and antiinflammatory cytokines in the liver was the same for all treatment groups. In contrast, expression of TNFα in the pancreas was significantly reduced by both MSCs and cardiac fibroblasts (f). IL1β and IFNγ in the pancreas, tended to be lower after MSCs and fibroblasts-administration but did not reach the significancy level. *p<0.05 vs PBS-CVB3, § p<0.05 vs respective control-group.