Spontaneous myocarditis mimicking human disease occurs in the presence of an appropriate MHC and non-MHC background in transgenic mice

Abstract

Most individuals have viral infections at some point in their life, however, only few develop autoreactivity to cardiac myosin following infection resulting in myocarditis suggesting a genetic predisposition. Most mouse models of myocarditis are induced by viral infection or by immunization with cardiac myosin. We generated HLA-DR3.Abetao and HLA-DQ8.Abetao transgenic mice in NOD and HLA-DQ8.Abetao in B10 background to study spontaneous autoimmunity. A high mortality was observed in NOD.DQ8 female mice 16 weeks or older. Echocardiography showed marked systolic dysfunction. Histopathology of various organs revealed an enlarged heart with mononuclear infiltrate consisting of CD4 and Mac-1+ cells and myocyte necrosis. The autoimmunity was associated with the presence of spontaneous autoreactive T cells and antibodies to cardiac myosin. Serologically, mice were negative for all known mouse viruses. NOD.DR3.Abetao, the transgene negative littermates, NOD, and B10.DQ8 Abetao mice had no gross or microscopic cardiac pathology. Spontaneous cellular and humoral response to cardiac myosin suggests that NOD.DQ8 may harbor autoreactive cells that can lead to spontaneous myocarditis and dilated cardiomyopathy. HLA-DQ8 is required for the predisposition to the spontaneous autoreactivity while NOD background influences onset and progression of disease. This model of myocarditis occurs predominantly in female mice and may provide insight into the pathogenesis of heart disease in women.

Figure 1

NOD.DQ8 mice develop myocarditis progressing to dilated cardiomyopathy. A) 16 week old NOD.DQ8 mice with severe edema and enlarged heart compared to healthy age and sex matched B10.DQ8 mice. B) Mononuclear infiltration observed in heart of NOD.DQ8 mouse first appeared as focal (left) which progressed to diffused with myocyte necrosis (middle) while B10.DQ8 (right) did not show any infiltration. Micrographs are at X100 magnification. C) trichrome stain of heart section shows mononuclear infiltration but no fibrosis, X400 magnification. Haematoxylin and Eosin stained sections of D) Liver E) Lungs F) Pancreas G) Salivary glands and H) Kidney. Micrographs are X400 magnification except E is at X100 magnification..

Figure 2

Echocardiographic assessment of hearts of NOD.DQ8 (n=5) and B10.DQ8 (n=5) mice. A) M-mode echocardiograms showed dilatation of both right venrticle (RV) and left ventricle (LV) with atrio-ventricular conduction block in NOD.DQ8 mice, in contrast to normal echo- and electro-cardiograms in B10.DQ8 mice. Arrows indicate diastolic (dotted) and systolic (continuous) dimensions. A and V in electrocardiograms represent P and QRS waves, respectively. B) Left ventricular (LV) dimensions (diastolic dimension; NOD.DQ8: 3.7±0.3 mm, B10.DQ8: 3.2±0.1 mm, systolic dimension; NOD.DQ8: 1.8±0.2 mm, B10.DQ8: 1.3±0.2 mm, P = 0.08). C) NOD.DQ8 hearts were significantly heavier and demonstrated reduced systolic function as demonstrated by fractional shortening (D) and, Circumferential (Cir.) shortening velocity E), compared with B10.DQ8 hearts. IVS - intra ventricular septum, PW - posterior wall.

Figure 3

Characterization of inflammatory cells in heart. A) Heart sections stained with various makers, CD4, CD8, and Mac-1 in DQ8 transgenic mice. B) Heart sections stained with anti-DQ antibody (IVD12) exhibit increased expression of class II in NOD.DQ8 mice. C) Haematoxylin & eosin (left) and eosinophil staining of consecutive section (right, bright field) of a heart from affected NOD.DQ8 mice. D) Deposition of IgG immune complex in heart sections of NOD.DQ8 (left) and B10.DQ8 transgenic mice shows deposition only in the former mice. Micrographs are at X100 magnification.

Figure 4

A) Spontaneous response to porcine cardiac myosin was observed in NOD.DQ8 (n=6) and NOD (n=4), and NOD.DR3 (n=6) mice but not in B10.DQ8 (n=6) mice. B) Only NOD.DQ8 mice respond to cardiac-myosin derived peptide, 735-747. C) Antibodies to porcine cardiac myosin in naïve DQ8 (n=10) and NOD (n=12) mice suggests high levels of antibodies in NOD.DQ8 mice (n=10), p<0.001 and NOD.DR3 mice (n=8), p<0.02. D) Anti tissue transglutaminase antibodies in male and female mice with myocarditis and controls, healthy NOD.DQ8 and B10.DQ8 mice, n=6/gp). Female NOD.DQ8 mice produce higher amounts than male mice (p<0.01). E) Spontaneous production of cytokines produced by spleen cells of NOD.DQ8 mice in response to myosin challenge in vitro (n=4/gp). TNF, myocarditis mice vs healthy NOD.DQ8 mice, p<0.02; TNF and IL-6 female myocarditis vs B10.DQ8 mice, p<0.05; IL-18 male myocarditis vs healthy NOD.DQ8 mice, p<0.04, B10.DQ8 vs healthy NOD.DQ8, IL-10, IL-6 p<0.01 . F) IFN-γ production by spleen cells from myocarditis and control NOD.DQ8 mice in the presence and absence of cardiac myosin. Myocarditis female vs male, p<0.05, myocarditis mice vs all healthy and B10.DQ8 , p<0.02 G) Subclass of anti-cardiac myosin antibodies, IgM, IgG1, IgG2b and IgE, in DQ8.NOD mice (n=10) shows O.D. ± S.D. The line in the bars depicts negative control.

Figure 5

DTH response to porcine cardiac myosin in naïve (n=4) (gray bars) and 5 days primed (black bars) (n=4) mice. *P<0.05

Figure 6

Analysis of splenic cells by FACS in control mice, B10.DQ8 (grey) and healthy NOD.DQ8 (dotted) and NOD.DQ8 mice with myocarditis (black). Upper panel shows a) expression of CD3 cells (left), CD8 cells gated on CD3+ cells (middle) and CD5+ cells (right). Lower panel shows expression of CD25 (left), CD44 (middle) and CD69 (right) gated on CD4 cell. Numbers indicate percent positive in B10.DQ8 (upper), healthy NOD.DQ8 (middle) and NOD.DQ8 myocarditis (lower). Difference in cell numbers between healthy NOD.DQ8 and sick mice was significant for CD3, CD5 and activated cells CD4+CD25 and CD69+, p<0.05.