Mediastinal Lymphadenopathy, Class-Switched Auto-Antibodies and Myocardial Immune-Complexes During Heart Failure in Rodents and Humans

Abstract

Mediastinal lymphadenopathy and auto-antibodies are clinical phenomena during ischemic heart failure pointing to an autoimmune response against the heart. T and B cells have been convincingly demonstrated to be activated after myocardial infarction, a prerequisite for the generation of mature auto-antibodies. Yet, little is known about the immunoglobulin isotype repertoire thus pathological potential of anti-heart auto-antibodies during heart failure. We obtained human myocardial tissue from ischemic heart failure patients and induced experimental MI in rats. We found that anti-heart autoimmunity persists during heart failure. Rat mediastinal lymph nodes are enlarged and contain active secondary follicles with mature isotype-switched IgG2a B cells. Mature IgG2a auto-antibodies specific for cardiac antigens are present in rat heart failure serum, and IgG and complement C3 deposits are evident in heart failure tissue of both rats and human patients. Previously established myocardial inflammation, and the herein provided proof of B cell maturation in lymph nodes and myocardial deposition of mature auto-antibodies, provide all the hallmark signs of an established autoimmune response in chronic heart failure.

Keywords: adaptive immune system; anti-heart; auto-antibodies; autoimmunity; chronic heart failure; myocardial infarction.

FIGURE 1

Persistent activation of heart-draining lymph nodes at CHF stage after infarction. (A) Example of 1.5 T cardiac MRI images of mediastinal lymphadenopathy in a patient with ischemic cardiomyopathy 2 months after transmural myocardial infarction in the circumflex artery territory. (a) 3-chamber cine view of the heart before gadolinium injection showing the left ventricle (LV), left atrium (LA) and aorta (Ao). (b) 3-chamber view of the heart showing late gadolinium enhancement (LGE) and transmural myocardial infarction in the infero-lateral wall of the LV. (c,d) Cardiac MRI transverse (c) and coronal (d) HASTE sequences showing mediastinal lymphadenopathy (arrows) in a patient with ischemic cardiomyopathy. (B–D) Myocardial infarction was induced by surgical ligation of the LAD. Mediastinal (heart draining) lymph nodes were obtained from healthy rats (ctrl), 2 weeks (acute post-MI) and 16 weeks (CHF stage) post-MI. (B) Histology (H&E) sections of representative lymph nodes. Post-MI rats show distinct lymphoid follicles containing active germinal centers (black arrows). Hemorrhage (gray arrow) is present in the medullary sinuses of the week 2 post-MI lymph nodes, indicating that they drain an area of hemorrhage. (C) Lymph node size and quantification of the total number of follicles and the percent of follicles with germinal centers. Data are expressed as mean +/–SEM for n = 3 (ctrl) and 4 (week 2, 16) animals, *p < 0.05, **p < 0.005, ***p < 0.001 (ANOVA with Dunnett’s multiple comparisons post hoc test). (D) qPCR Array testing a range of genes involved in B cell activation. Bars represent expression change over baseline, with healthy baseline values set to 0. Experiment performed in triplicates on 3 pooled spleens of ctrl (healthy baseline), week 2 and week 16 post-MI rats.

FIGURE 2

Levels of IgG2a antibodies in the circulation and IgG2a+ cells in the heart-draining lymph nodes are elevated during CHF. Myocardial infarction was induced by surgical ligation of the LAD. Serum was collected 2 weeks (acute post-MI) and 16 weeks (chronic heart failure stage) post-MI and analyzed by ELISA for relative concentration of (A) immunoglobulin (Ig) light chains (IgKappa and IgLambda) and (B,C) heavy chains (immature IgM (B), and mature class-switched IgG1, IgG2a, b and c (C). n = 5 (ctrl, week 2) and 11 (week 16) / group (D) Immunofluorescence staining of mediastinal lymph nodes with anti-rat IgM (green) and anti-rat IgG2a (red). n = 4 (ctrl, week 2) and 6 (week 16) / group, Data are expressed as mean +/–SEM, **p < 0.001, ***p < 0.0001 (ANOVA with Dunnett’s multiple comparisons post hoc test).

FIGURE 3

CHF serum contains anti-heart auto-reactive antibodies of mature class-switched isotypes. Myocardial infarction was induced by surgical ligation of the LAD. Serum was collected 2 weeks (acute post-MI) and 16 weeks (chronic heart failure stage) post-MI and analyzed by ELISA and immunofluorescence microscopy for the presence of heart specific auto-antibodies. (A,B) ELISA using post-MI serum against rat heart lysate (protein fraction) showing a progressive increase in the levels of (A) total IgG (n = 5 / group) and (B) IgG1, IgG2a, and IgG2b isotype auto-antibodies reactive against the heart (n = 5 (ctrl, week 2) and 10 (week 16) / group). (C) Representative images and quantification of immunofluorescence staining using post-MI serum on frozen sections of healthy rat hearts to assess binding of auto-antibodies to cardiac structures. Staining intensity was quantified by measuring mean fluorescence intensity (MFI) using FIJI/ImageJ. (D,E) ELISA using post-MI serum against recombinant pig myosin (D) and mouse troponin I (E). Dotted line represents threshold for calculation of % of positive animals. Data are expressed as mean +/–SEM, *p < 0.05, **p < 0.001, ***p < 0.0001 [ANOVA with Dunnett’s multiple comparisons post hoc test (A–C), one-tailed Student’s t-test with Welch correction (D)].

FIGURE 4

Immunoglobulin G and complement C3 deposit in rodent and human CHF heart tissue. MI was induced by surgical ligation of the LAD and heart tissue was collected during CHF after 16 weeks of MI. Human left ventricular tissue sections were obtained from end-stage heart failure patients during transplant surgery. Control samples for comparison were obtained from organ donor hearts assessed to be unsuitable for donation. Representative images and corresponding quantification (mean fluorescence intensity) of immunofluorescence staining against IgG and complement C3 on frozen sections of CHF rat hearts (A,B) and end-stage human HF heart (C,D) to assess in vivo IgG and C3 deposition on cardiac structures, respectively. rat n = 3 (control), 5 (CHF); human n = 5 (donors), 7 (CHF), 3 (myocarditis). Staining intensity was quantified by measuring mean fluorescence intensity (MFI) using FIJI/ImageJ. Data are expressed as mean +/–SEM, *p < 0.05, ***p < 0.001 (one-tailed Student’s t-test with Welch correction).