Mononuclear cell secretome protects from experimental autoimmune myocarditis

Abstract

Aims: Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model.

Methods and results: BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells.

Conclusion: MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases.

Keywords: Conditioned medium; Mononuclear cells; Myocarditis; Secretome.

Figure 1

Pathological evaluations of explanted hearts are depicted in (A–D). Mononuclear cell secretome applied during the sensitization process (Day 0 and Day 7) had no impact on the development of myocarditis. Treating mice with mononuclear cell secretome on Day 14, significantly reduced the lymphocytic infiltrate (mononuclear cell secretome: 0.1 ± 0.1; control medium: 2.4 ± 0.4; Mann–Whitney U test: P = 0.0089). A representative sample of a heart section is shown in (E). In the heart of a control mouse, treated with ‘unconditioned’ medium a dense inflammatory infiltrate was seen, whereas only scattered, infiltrating lymphocytes can be found in the animal treated with mononuclear cell secretome.

Figure 2

Evaluation of the autoantibody formation against MyHC-α is shown in (A). IgM and IgG1 levels were lower in mononuclear cell-treated animals, however, other immunoglobulin subtypes remained unchanged. Cytokine analysis of plasma samples obtained during scarification showed that the systemic inflammatory response was dampened in mice treated with mononuclear cell secretome when compared with control animals (B). Splenocytes obtained from mice after scarification evidenced a decreased proliferatory capacity against MyHC-α in a dose-dependent manner if animals were treated with mononuclear cell secretome (C).

Figure 3

Results of cytokine arrays of mononuclear cell secretome obtained from cultured mouse splenocytes and mononuclear cell secretome obtained from cultured human peripheral blood mononuclear cell are depicted in (A). Both preparations were comparable regarding their secreted products. (B) The proliferative response of purified human CD4+ cells in the presence or absence of mononuclear cell secretome. CD4+ cells stimulated with phytohaemagglutinin or anti-CD3 showed lower proliferation rates when treated with mononuclear cell secretome. Unstimulated, purified CD4+ cells or a commercially available T cell line (JURKAT) undergo apoptosis in the presence of mononuclear cell secretome as shown by flow cytometry (C and E) and by histone release assay (D). This effect was partially reversible by pre-incubation with a caspase-3 and a caspase-8 inhibitor, indicating an external pathway-mediated effect (F). Interestingly, known pro-apoptotic cytokines were only found in marginal concentrations in the mononuclear cell secretome (G). Blocking pathways associated with apoptosis by neutralizing antibodies had no effect on mononuclear cell secretome-induced histone release (H).

Figure 3

Results of cytokine arrays of mononuclear cell secretome obtained from cultured mouse splenocytes and mononuclear cell secretome obtained from cultured human peripheral blood mononuclear cell are depicted in (A). Both preparations were comparable regarding their secreted products. (B) The proliferative response of purified human CD4+ cells in the presence or absence of mononuclear cell secretome. CD4+ cells stimulated with phytohaemagglutinin or anti-CD3 showed lower proliferation rates when treated with mononuclear cell secretome. Unstimulated, purified CD4+ cells or a commercially available T cell line (JURKAT) undergo apoptosis in the presence of mononuclear cell secretome as shown by flow cytometry (C and E) and by histone release assay (D). This effect was partially reversible by pre-incubation with a caspase-3 and a caspase-8 inhibitor, indicating an external pathway-mediated effect (F). Interestingly, known pro-apoptotic cytokines were only found in marginal concentrations in the mononuclear cell secretome (G). Blocking pathways associated with apoptosis by neutralizing antibodies had no effect on mononuclear cell secretome-induced histone release (H).