Protection against experimental autoimmune myocarditis is mediated by interleukin-10-producing T cells that are controlled by dendritic cells

Abstract

Experimental autoimmune myocarditis (EAM) can be induced in the Lewis rat by cardiac myosin or its cryptic S2-16 peptide epitope (amino acids 1052 to 1076). To investigate cellular mechanisms and the role of antigen-presenting cells in regulation of myocarditis, we induced protection against EAM in Lewis rats by administration of S2-16 peptide in incomplete Freund's adjuvant (IFA). Protection to EAM was associated with activation of S2-16-reactive splenocytes secreting high levels of interleukin (IL)-10 and reduced levels of interferon-gamma and IL-2. Adoptive transfer of S2-16:IFA-induced splenocytes producing IL-10 suppressed myocarditis induction in syngeneic recipients, suggesting their regulatory cell nature. However, exposure of S2-16:IFA-induced cells to inflammatory cytokine IL-12 converted them to Th1 effectors that transferred EAM. Differentiated function of S2-16-reactive T cells in protected rats resulted from increased IL-10 production by dendritic cells (DCs). Purified DCs from S2-16:IFA-treated rats promoted S2-16-reactive CD4+ T cells to produce increased IL-10 and reduced interferon-gamma. In addition, adoptive transfer of IL-10-producing DCs from S2-16:IFA-treated rats also induced protection to EAM in recipient rats. These studies demonstrated DCs and key cytokines, such as IL-10 and IL-12, regulated the fate of T cells in myocarditis development in the Lewis rat.

Figure 1

S2-16:IFA-induced protection was associated with expansion of S2-16-reactive T cells producing low levels of IFN-γ and IL-2 but high levels of IL-10. Lewis rats were administered S2-16:IFA or control PBS:IFA 14 days before challenge by immunization with S2-16:CFA. Splenic lymphocytes were collected 21 days after challenge. A: Proliferative responses of splenocytes with different concentrations of S2-16 peptide. Proliferation was measured by ³H-thymidine incorporation. Results of proliferative assay were expressed as stimulation index (SI) (mean test counts per minute/mean of medium control counts per minute). B: IL-2, IL-10, and IFN-γ production by splenocytes cultured with S2-16. Supernatants were collected at 24 hours for IL-2, 48 hours for IFN-γ, and 72 hours for IL-10, and cytokine levels were measured by cytokine-specific ELISA. Error bars represent SEMs, and Student’s t-test was used to determine the significant differences between PBS:IFA-pretreated group and S2-16:IFA-pretreated group (*P ≤ 0.05, **P ≤ 0.005). Cytokine levels of cells cultured with medium alone were <20% of cytokine response to S2-16, and are not shown in figure.

Figure 2

S2-16:IFA-primed splenocytes proliferated to S2-16 and produced low levels of IFN-γ and IL-2 but high levels of IL-10. Lewis rats were treated with S2-16:IFA, and S2-16:CFA or PBS:CFA as controls. Fourteen days later, splenic lymphocytes were collected and cultured with S2-16 peptide to determine their in vitro recall response. A: Proliferative response of splenocytes after culture with different concentrations of S2-16. Proliferation was measured by ³H-thymidine incorporation. B: Cytokine production of splenocytes immunized in vivo and treated in vitro as indicated in figure and in legend above. Cell culture supernatants were collected for measurement of IFN-γ, IL-2, and IL-10 by ELISA. In some experiments, recombinant murine IL-12 or anti-rat IL-10 monoclonal antibody were added together with S2-16 to the cell culture as described in Materials and Methods. Error bars represent SEMs, and Student’s t-test was used to determine the significant differences between PBS:CFA-treated group and S2-16:IFA- or S2-16:CFA-treated group (*P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.001).

Figure 3

S2-16 and adjuvant treatment affected cytokine production of DCs. TNF-α production was increased in S2-16:CFA DCs as well as IL-12p40 mRNA, whereas increased IL-10 production was observed for S2-16:IFA DCs. Lewis rats were injected with S2-16:IFA or S2-16:CFA as a control, or left untreated. Fourteen days later, DCs were isolated from spleens by positive selection using OX62 monoclonal antibody and were stimulated by LPS or CpG-containing oligonucleotide with anti-rat CD40 antibody. TNF-α and IL-10 production from DCs in culture supernatants were measured by ELISA. IL-12p40 mRNA expression in rat DCs were measured by quantitative real-time PCR. Rat G3PDH was used as an endogenous control to allow for relative mRNA quantification. Cytokine mRNA levels are presented as fold increase in gene expression observed in triplicate wells of LPS and anti-CD40 antibody-treated DCs relative to untreated DCs. Error bars represent SEMs, and Student’s t-test was used to determine the significant differences between DCs from untreated rats and DCs from S2-16:IFA- or S2-16:CFA-treated rats (*P ≤ 0.05, **P ≤ 0.005).

Figure 4

Polarized CD4⁺ T-cell response after culture with DCs of S2-16 and adjuvant-treated rats: S2-16:IFA DCs promoted CD4⁺ T cells to produce more IL-10, whereas S2-16:CFA DCs promoted IFN-γ production from CD4⁺ T cells. Lewis rats were treated with S2-16:IFA or S2-16:CFA. Fourteen days later, CD4⁺ T cells and DCs were isolated from spleens of treated rats or untreated naïve rats, and co-cultured together with S2-16 peptide. Proliferation of cells was measured by ³H-thymidine incorporation after 96 hours. Cytokine production in cell culture supernatants was measured by ELISA. A: Proliferative response, IL-10, and IFN-γ production by CD4⁺ T cells from S2-16:CFA-treated rats after co-culture (5 × 10⁵ CD4⁺ T cells) with DCs from three groups of rats at various concentrations. B: Proliferative response and IL-10 and IFN-γ production of CD4⁺ T cells from S2-16:IFA-treated rats after co-culture (5 × 10⁵ CD4⁺ T cells) with DCs at various concentrations. C: Proliferative response and IFN-γ production of CD4⁺ T cells from naive rats after co-culture (5 × 10⁵ CD4⁺ T cells) with DCs at various concentrations. Error bars represent SEMs. Mann-Whitney test was used to determine the difference between S2-16:IFA DC or S2-16:CFA DC versus naïve DC groups (proliferation and IFN-γ), and S2-16:IFA DC versus S2-16:CFA DC groups (IL-10). *P ≤ 0.05, **P ≤ 0.005. D: Intracellular staining suggested that S2-16:IFA DCs induced higher level IL-10 production from CD4⁺ T cells than S2-16:CFA DCs. DCs were purified from spleens of S2-16:CFA- or S2-16:IFA-treated rats, and cultured with purified CD4⁺ T cells from these groups of rats, together with S2-16 peptide. Cells were removed from co-culture on day 4 and stained for CD4⁺ cells and intracellular IL-10-positive cells. Numbers denote percentage of cells in each quadrant. The data are representative of two to three independent experiments with similar results.