Exosomes Isolated from Cardiosphere‐Derived Cells Attenuate Disease Severity in Experimental Autoimmune Myocarditis

Abstract

ObjectiveMyocarditis is a leading cause of heart failure and sudden cardiac death in children and young adults. Current therapies are limited to supportive care, motivating a search for novel therapeutics. Cardiosphere‐derived cells (CDCs) are cardiac progenitor‐stromal cells that have demonstrated therapeutic bioactivity in multiple inflammatory disease models. This effect is largely mediated through exosomes secreted by CDCs. We hypothesize that CDC exosomes are salutary in experimental autoimmune myocarditis (EAM) and exert their therapeutic effect via modulation of T lymphocytes.MethodsEAM was induced by injecting Lewis rats with purified cardiac myosin and complete Freund’s adjuvant. Animals underwent randomization to receive intravenous CDC exosomes or a vehicle control. Non‐immunized littermates served as healthy controls. Phenotyping included assessment of cardiac function (left ventricular ejection fraction, LVEF) by echocardiography, electrical abnormalities by electrocardiography (EKG), and quantification of inflammation by histology and flow cytometry. In vitro polarization of naïve CD4+ T cells was conducted to assess the effect of CDC exosomes on cytokine production and proliferation.ResultsOn day 28, animals with EAM exhibited significant functional decline, compared to healthy controls (LVEF: 59.2 ± 3.6% versus 80.0 ± 0.7%, p<0.001), while animals receiving exosomes were partially protected (LVEF: 67.8 ± 2.1%, p=0.003 vs. EAM + vehicle, Figure 1B). Similarly, EAM animals receiving vehicle developed repolarization abnormalities on EKG, with significant QTc interval prolongation, compared to healthy controls (QTc: 238.7 ± 5.0 ms versus 189.1 ± 7.3 ms, p=0.004), while animals receiving exosomes had normal QTc intervals (188.2 ± 16.5 ms, p=0.004 vs. EAM + vehicle, Figure 1C). Cardiac histology revealed decreased inflammation in the exosome group (median severity: vehicle: 4, exosome: 1.5, Figure 2A). Analysis of leukocytes in the blood and spleen showed a decrease in total T lymphocytes in the exosome group, but an increase in the proportion of regulatory T cells within the T lymphocyte population (Figure 2B). In vitro, differentiated CD4+ T cells preferentially expressed IL‐10 and IFN‐γ after exosome exposure (8.5‐ and 7.3‐fold increase over control, respectively). Lastly, induced regulatory T cells proliferate more rapidly following exosome exposure (cells dividing beyond three generations: 57% in exosome group versus 18% in control group), while proliferation rates were not significantly different for conventional effector T cells.ConclusionExosomes secreted by CDCs improve the disease manifestations of EAM, with underlying upregulation of IL‐10 and IFN‐γ, factors that are known to be protective in murine models of EAM. Furthermore, exosome exposure preferentially increases proliferation of regulatory T cells, which are major producers of IL‐10. Our findings are consistent with the notion that a multiplicative amplification of IL‐10 production may explain the salutary effects of CDC exosomes in vivo.Support or Funding InformationA.A. is supported by an institutional training grant (T32HL116273)Functional and Electrocardiographic ChangesFigure 1Inflammatory Profile in Heart, Blood, and SpleenFigure 2