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. 2016 Dec;186(12):3160-3175.
doi: 10.1016/j.ajpath.2016.08.005.

Identification of an Epitope From Adenine Nucleotide Translocator 1 That Induces Inflammation in Heart in A/J Mice

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Free PMC article

Identification of an Epitope From Adenine Nucleotide Translocator 1 That Induces Inflammation in Heart in A/J Mice

Rakesh H Basavalingappa et al. Am J Pathol. .
Free PMC article

Abstract

Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-α, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT1 encompasses multiple immunodominant epitopes (namely, ANT1 21-40, ANT1 31-50, ANT1 171-190, and ANT1 181-200). Although all four peptides induce comparable T-cell responses, only ANT1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.

Figures

Figure 1
Figure 1
Induction of myocarditis by ANT1 peptides in A/J mice. Groups of mice were immunized with or without peptide/complete Freund adjuvant (CFA) emulsions pertussis toxin (PT) twice with an interval of 7 days, and PT was administered on day 0 and day 2 after the first immunization. After 21 days, animals were euthanized and hearts were collected for histological evaluation of inflammation. A: Hematoxylin and eosin staining. Representative sections are shown for each peptide [naïve and CFA/PT groups: normal cardiac tissue with intact myocytes; ANT1 11-30: moderate inflammatory focus along the epicardium; ANT1 21-40: multiple foci near the epicardium (solid arrows) and in the myocardium (dotted arrows); ANT1 31-50 and ANT1 131-150: small focus in the endocardium; ANT1 151-170: widespread inflammatory focus along the epicardium; ANT1 161-180: isolated focus in the myocardium; and ANT1 171-190: small focus in the myocardium, all shown with solid arrows]. Boxed areas are shown at higher magnification to the right. B: Immunohistochemistry. Heart sections obtained from animals immunized with or without ANT1 21-40 were stained for the presence of CD3+ T cells, and non-T cells (CD11b+ and Iba1+) using the corresponding antibodies. After washing, sections were stained with horseradish peroxidase–conjugated, secondary antibodies to detect cells positive for each marker, shown with arrows. No CD3+ T cells are seen in the control; and rare dendritic cells were CD11b+ and Iba1+ in the controls. Inflammatory foci were also present in the cardiac valves in mice immunized with ANT1 21-40. n = 5 to 10 mice per group (A). Scale bars: 200 μm (A, main images); 50 μm (A, magnifications); 40 μm (B).
Figure 2
Figure 2
ANT1 peptides induce T-cell responses in A/J mice. Experimental autoimmune myocarditis was induced in A/J mice by immunizing with peptide/complete Freund adjuvant (CFA) emulsions, as described in Materials and Methods. Animals were euthanized on day 21 after immunization to harvest lymph nodes, and single-cell suspensions were prepared. Recall responses were tested by stimulating lymph node cells (LNCs) with immunizing peptides or RNase 43-56 as control. After 2 days, cells were pulsed with 3[H]-thymidine for 16 hours, and proliferative responses were then measured as counts per minute (cpm). A: Peptides that induce both myocarditis and T-cell responses. B: Peptides that induce negligible myocarditis but mild or no T-cell responses. C: Peptides that do not induce myocarditis but generate T-cell responses. For some peptides (ANT1 21-40, ANT1 171-190, and ANT1 181-200), proliferative responses also were measured in animals immunized with only one dose of peptide/CFA emulsions and euthanized 10 days later for preparation of LNCs. Because there were no differences between animals immunized once or twice, data were pooled. Values representing two to five individual experiments each involving three to five mice are shown, and each experiment contained three replicates. Data are expressed as means ± SEM (AC). P < 0.05, ∗∗∗P < 0.001 between indicated doses (10 μg/mL and 100 μg/mL of ANT1 peptides versus RNase 43-56).
Figure 3
Figure 3
Binding of ANT1 peptides to IAk molecules. Reaction mixtures containing thrombin-cleaved IAk monomers (0.35 μg), competitor peptides [ANT1 21-40, ANT1 171-190, and ANT1 181-200 (0.00001 to 100 μmol/L)], and biotinylated HEL 46-61 (1 μmol/L) were individually prepared and added to fluorescence plates coated with anti-IAk in duplicate. After washing, europium-labeled streptavidin was added in dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) buffer, followed by DELFIA enhancer. Fluorescence intensity was measured at excitation/emission wavelengths of 340/615 nm, and 50% inhibitory concentration (IC50) values were calculated. Representative data sets from three individual experiments with two replicates in each are shown. Data are means ± SEM. ∗∗P < 0.01, ∗∗∗P < 0.001 versus ANT1 21-40 IC50.
Figure 4
Figure 4
Cytokine analysis in the supernatants derived from ANT1 21-40–stimulated cultures. Lymph node cells obtained from ANT1 21-40–immunized mice were stimulated with or without ANT1 21-40 or RNase 43-56 (control). Supernatants collected on day 3 after stimulation were analyzed using beads conjugated with cytokine-capture antibodies and detection antibodies by cytometric bead array, as described in Materials and Methods. Values obtained from four individual experiments are shown. Data are expressed as means ± SEM. P ≤ 0.05. IFN-γ, interferon-γ; Th, T helper; TNF-α, tumor necrosis factor-α.
Figure 5
Figure 5
Induction of myocarditis by ANT1 21-40–sensitized T cells in naïve mice. Lymph node cells were prepared from animals immunized for experimental autoimmune myocarditis induction or those immunized with peptide/complete Freund adjuvant emulsions only once, and the cells were stimulated with concanavalin-A for 2 days. Viable cells were then harvested and administered i.p. or retro-orbitally into naïve mice primed with lipopolysaccharide (LPS), where LPS-primed alone, and phosphate-buffered saline (PBS) only recipients were used as controls. All animals received pertussis toxin i.p., on days 0 and 2 after transfer. At termination on day 14, hearts were collected and examined for inflammatory changes by hematoxylin and eosin staining. Representative sections are shown from two individual experiments, each involving two mice per group: normal cardiac tissues in A (PBS control) and B (LPS control), as opposed to inflammatory foci (arrows) containing infiltration by mononuclear cells in C and D (recipients of ANT1 21-40–sensitized T cells). Scale bars = 40 μm (AD).
Figure 6
Figure 6
Evaluation of ANT1 21-40–reactive antibodies by enzyme-linked immunosorbent assay. Groups of A/J mice were immunized with or without ANT1 21-40 twice with an interval of 7 days, and pertussis toxin was administered on days 0 and 2 after the first immunization. Serum samples were collected for measurement of ANT1 21-40–reactive antibodies on indicated days. Plates were coated with ANT1 21-40 or RNase 43-56 (negative control) overnight at 4°C, and after washing and blocking, 1:10 (top panel) and 1:100 (bottom panel) diluted serum samples were added. Plates were further incubated for 1 hour at 37°C, followed by addition of secondary antibody. After washing and incubating with the substrate, OD values were measured at 450 nm. Data are means ± SEM. n = 3 to 10 individual mice. ∗∗P < 0.01, ∗∗∗P < 0.001 versus control groups.
Figure 7
Figure 7
Histological evaluation of livers obtained from mice immunized with or without ANT1 21-40. Groups of A/J mice were immunized with or without ANT1 21-40 or complete Freund adjuvant (CFA) alone twice with an interval of 7 days, and the animals also received pertussis toxin (PT) on day 0 and day 2 after the first immunization. After 21 days, the indicated tissues harvested from both naive and immunized animals were evaluated for inflammatory changes by hematoxylin and eosin staining. A: Sections from naive group representing normal brain, and a small aggregate of lymphocytes in liver. Sections from CFA/PT (B), and sections from ANT1 21-40–immunized groups (C) showing unaffected brain; and liver with multiple scattered aggregates of lymphocytes (arrows). n = 5 to 14 mice per group (A–C). Scale bars: 80 μm (brain); 160 μm (liver, left column); 40 μm (liver, right column).

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