Abstract (within 200 words)
Inflammatory dilated cardiomyopathy (iDCM) is a potentially lethal disorder characterized by progressively impaired cardiac function due to heart inflammation (myocarditis). Despite ongoing advances made in the treatment of iDCM, improvement of outcome remains problematic. Although many different environmental agents can trigger myocarditis including viral or bacterial infections, toxins and drugs, autoimmunity is considered to play an important role in myocarditis as well as contributes to its progression to iDCM. Animal models have greatly advanced our knowledge of the pathogenesis of myocarditis and inflammatory cardiomyopathy. Experimental autoimmune myocarditis (EAM) induced by cardiac myosin immunization is a model of postinfectious myocarditis. In this model, the myocardial inflammation peaks on 14 to 21 days after immunization, gradually subsides and disappears around day 25. We need more long-lasting heart inflammation model for better understanding the mechanism that leads to iDCM.
Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only licensed vaccine that has substantially helped controlling tuberculosis. This vaccine can be engineered to express foreign molecules in a functional form, and this has driven the development of BCG as a recombinant vector. Micobacteria are intracellular bacteria that persist within antigen presenting cells (APCs) for a long time. The MHC class II on the professional APCs present antigenic peptides derived from Micobacteria residing in the phagosome, which leads to the stimulation of antigen-specific CD4 T cells. For this reason, we hypothesized that the recombinant BCG (rBCG) designed to express cardiac antigen may induce the cardiac antigen-specific T-cell response on a long-term basis, which could lead chronic autoimmune myocarditis in mice.
Since a single injection of rBCG expressing a CD4 epitope of myosin heavy chain-α induced only weak inflammation in the heart, mice were immunized with cardiac myosin peptide twice before rBCG injection for priming. The immunized mice showed chronic heart inflammation and cardiac dysfunction. The cardiac myosin-specific CD4 T cells were readily elicited over a long period of time, and the adoptive transfer of the CD4 T cells induced myocarditis in the recipient mice. These results indicated that the auto-cardiac antigen-expressing rBCG immunization effectively induced the myocarditis-inducible CD4 T-cells in the mice.
Introduction
Inflammatory dilated cardiomyopathy (iDCM) is a potentially lethal disorder characterized by progressively impaired cardiac function due to heart inflammation (myocarditis) 1. Despite ongoing advances made in the treatment of iDCM, improvement of outcome remains problematic. Although many different environmental agents can trigger myocarditis including viral and bacterial infections, toxins and drugs 2, autoimmunity is considered to play an important role in myocarditis as well as contributes to its progression to iDCM 3. Long-term follow-up studies in patients with acute myocarditis have documented the development of iDCM in 21% of patients over a mean follow-up period of 3 years 4. The reason why some people recover and others progress to iDCM is an area of active investigation 5. Animal models have greatly advanced our knowledge of the pathogenesis of myocarditis and inflammatory cardiomyopathy. Experimental autoimmune myocarditis (EAM) induced by cardiac myosin immunization is a model of postinfectious myocarditis 6. In this model, the myocardial inflammation peaks on 14 to 21 d after immunization, gradually subsides and disappears around d 25 7. We need more long-lasting heart inflammation model for better understanding the mechanism that leads to iDCM.
Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only licensed vaccine that has substantially helped controlling tuberculosis for more than 80 yrs. The vaccine is immunogenic and is stable and cheap to produce. Additionally, the vaccine can be engineered to express foreign molecules in a functional form, and this has driven the development of BCG as a recombinant vector to protect against infectious diseases, such as tuberculosis and HIV, and cancer 8. Principally, Micobacteria are intracellular bacteria that persist within macrophages and dendritic cells for a long time. The MHC class II on the professional APCs present antigenic peptides derived from Micobacteria residing in the phagosome, which leads to the stimulation of CD4 T cells. For this reason, recombinant BCG (rBCG) designed to express autoantigen may induce the autoantigen-specific T-cell response on a long-term basis, which could lead autoimmune disease.
In the present study, rBCG was assessed as a vehicle for inducing chronic autoimmune myocarditis and iDCM in mice. We demonstrated that cardiac myosin-specific CD4 T cells are readily elicited in mice through immunization with a rBCG expressing a CD4 epitope of myosin heavy chain-α.
Results and Discussion (figure 8つまで)
To achieve expression of the CD4 T-cell epitope of cardiac myosin heavy chain-α (MyHCα), we used α-antigen (Ag85B) gene from Mycobacterium kansasii as a carrier protein gene 9 for fusion with the DNA fragment encoding the corresponding CD4 T-cell epitope (Figure 1A). The resulting recombinant clones (pSO246-MyHCα and pSO246) were designated as rBCG-MyHCα and rBCG-pSO246, respectively. Transformation of cells with rBCG-MyHCα and analysis of the cell lysates and culture supernatant by Western blot revealed a single band corresponding to a slightly larger molecular weight than that of Ag85B (Figure 1B). This indicated that the T-cell epitope along with the Ag85B carrier was secreted from the rBCG. Next we investigated the ability of rBCG-MyHCα for antigen presentation. Cardiac specific CD4 T cells were proliferated when cultured with rBCG-MyHCα-infected DCs as well as with MyHCα peptide-loading DCs, but not with rBCG-pSO246 (Figure 1C and 1D). This indicated that APCs infected with rBCG-MyHCα presented CD4 T-cell epitope to CD4+ T cells effectively.
Since a single injection of rBCG-MyHCα induced only weak inflammation in the heart (Supplementary Figure 1), mice were immunized with small amount of cardiac myosin peptide to prime immunological responses before rBCG-MyHCα immunization (Figure 2A). The priming did not induce inflammatory cell infiltration in the heart (Supplementary Figure 2) as we reported before 10. The heart to body weight ratio of the rBCG-MyHCα-immunized mice gradually increased during follow-up and significantly elevated than that of PBS- or rBCG-pSO246-injected mice. The histological analyses revealed mononuclear cell infiltration and severe fibrosis in rBCG-MyHCα-immunized heart in the chronic phase (Figure). Echocardiography of mice 90 days after rBCG-MyHCα immunization showed increased left ventricular (LV) diastolic dimensions (LVDd) and LV systolic dimensions (LVSd) indicative of DCM (Figure 3A). Furthermore, mice immunized with rBCG-MyHCα developed severe cardiac dysfunction, as determined by decreased fractional shortening (FS), LV end-systolic pressure (LVESP) and maximum and minimum rates of LV pressure development (±dP/dt) (Figure 3A and 3B). Progressively impaired cardiac contractility, inflammation, fibrosis and dilation of heart chambers characterize iDCM phenotypically 1. Our established new mice model demonstrated those features of iDCM.
A flow cytometric analysis of the heart infiltrates revealed a significant increase of inflammatory cells (CD45+ leukocytes) in rBCG-MyHCα-immunized mice compared with that in PBS- or rBCG-pSO246-injected mice (Figure 4A). We found no differences in the distribution of monocytes (CD11b), granulocytes (Gr-1), macrophages (F4/80), DCs (CD11c) and CD4+ T cells (CD4) within the CD45+ heart-infiltrating cell subsets among PBS-, rBCG-pSO246 and rBCG-MyHCα-injected mice (Figure 4B). Autoimmune myocarditis is a CD4+ T-cell–mediated disease 11. We found that effector memory CD4+ T cells (CD44highCD62Llow) were increased and naïve T-cell populations (CD44lowCD62Lhigh) were reduced in total CD4+ infiltrating T cells of rBCG-MyHCα-immunized mice (Figure 4C). After priming, CD4+ T cells produced small amounts of IFN-γ and IL-17 in response to restimulation with MyHCα pulsed irradiated splenocytes (Figure 4D). In the chronic phase, CD4+ T cells from rBCG-MyHCα-immunized mice produced much greater amounts of IFN-γ and IL-17 (Figure 4E). On the other hand, they were uniformly low in supernatants of CD4+ T cells from both PBS- and rBCG-pSO246-injected mice (Figure 4E). We confirmed, using adoptive transfer studies, that CD4+ T cells from rBCG-MyHCα-immunized mice mediate inflammation in this chronic myocarditis model (Figure 4F and Table 1). These results indicated that the cardiac antigen-expressing rBCG immunization effectively induced myocarditis-inducible CD4 T-cells in the mice.
iDCM contains two types of chronic myocarditis; the one is a latent chronic inflammation-induced iDCM and the other is that acute apparent myocarditis evolves into a chronic immune-inflammatory process, which results in iDCM. The model we showed above represents the former iDCM. Then we tried to create another model that represents the latter one. First, we induced EAM by immunization with regular amount of MyHCα. In EAM, we previously reported that myocardial inflammation peaks on 14 to 21 d after immunization, gradually subsides and disappears around d 25 7. As shown in Figure 5B, severe inflammatory cell infiltration was observed in the myocardium 14 days after MyHCα immunization. Twenty-one days after EAM induction, we injected rBCG-MyHCα to create chronic inflammation (Figure 5A). Ninety days later, rBCG-MyHCα-immunized mice still had severe mononuclear cell infiltration with a high amount of tenascin-C (TN-C) expression that represented active inflammatory process was exist 12 (Figure 5E-J). In the PBS- or rBCG-pSO246-injected mice, inflammatory cell infiltration and apparent TN-C expression were no longer observed; only collagen deposition existed then (Figure 5B-D and 5K-M). Thus rBCG-MyHCα immunization prolonged inflammatory responses of EAM mice, and this could be a useful model of iDCM patients with a history of acute myocarditis.
TN-Cと炎症。拡張型心筋症患者。
recombinant BCGの有効性
Animal models have greatly advanced our knowledge of the pathogenesis of myocarditis and inflammatory cardiomyopathy. Experimental autoimmune myocarditis (EAM) induced by cardiac myosin immunization is a model of postinfectious myocarditis 6. In this model, the myocardial inflammation peaked on 14 to 21 days after immunization, gradually subsided and disappeared around 25 days after immunization (Machino-Ohtsuka et al., 2014).
BCG is only efficacious as a live vaccine; killed BCG does not provide protection in animal models, and killed Mycobacterium tuberculosis in human trials provided only weak and transient protection. In clinical trials of BCG vaccination, the observed efficacy has ranged from no protection to 80% fewer cases of TB 13. Survival and persistence of the live BCG vaccine are necessary to elicit protective immunity. It has been shown that early treatment of infected mice with isoniazid, which inhibits BCG growth, prevents the development of effective acquired resistance 14. Similarly, killed BCG strains elicit an immune response that is weak and transient 15. A recent study also showed that, in a mouse model of infection, prior exposure to live environmental mycobacteria blocked the multiplication of BCG, and consequently, BCG failed to provide protective immunity against a TB challenge 16. The continuous secretion of many different antigens by live BCG is likely important for the induction of protective immunity and is a key advantage over subunit vaccines or DNA vaccines that transiently produce a few antigens.
The reasons why some people recover and others do not is an area of active investigation. Most patients with myocarditis resolve the disease relatively quickly. Only certain susceptible individuals progress from acute to chronic myocarditis and dilated cardiomyopathy 5. Research has shown that susceptibility to develop “chronic myocarditis” depends on the type of immune response an individual has in response to infections, chemicals or physical damage to the heart 17–21. Examination of heart tissue under a microscope is the only way to prove the diagnosis of chronic myocarditis. Up to 40% of patients with chronic dilated cardiomyopathy who have symptoms of heart failure despite standard medical care can have myocarditis when special techniques are used to study heart tissue. Magnetic resonance imaging (MRI) of the heart is under evaluation to aid in diagnosis of chronic myocarditis. The sensitivity and specificity of MRI for the diagnosis of myocarditis varies with duration of illness and the sequences used. Blood tests that measure damage to heart muscle cells (e.g. troponin T) can also be elevated in chronic myocarditis, but the levels may be normal or only minimally elevated. The electrocardiogram has nonspecific changes in the majority of myocarditis cases. There are also no specific echocardiographic features of chronic myocarditis. Research in animal models has shown that myocarditis can be a biphasic disease, meaning that acute myocarditis can resolve and then several weeks or months later it can reappear 5. Researchers have found that proteins called cytokines that are released from inflammatory cells during acute myocarditis begin to cause changes in heart structure that only appear several weeks or months later 17,18,22,23. This process is called “remodeling” and leads to fibrosis and dilated cardiomyopathy. However, it is important to note that only some individuals progress from acute to chronic myocarditis, while most patients with acute myocarditis do not develop the chronic phase of disease. In most cases, chronic myocarditis involves autoimmunity 24. An autoimmune response occurs when the immune system attacks the cells and tissues of our body. The immune system does this in an attempt to heal the damage induced by infections or chemicals, for example, but can end up causing damage itself. This results in chronic inflammation and may involve deposition of proteins called autoantibodies on cardiac tissue that can adversely affect heart function and further promote inflammation 24–26. Most patients with chronic myocarditis also have dilated cardiomyopathy, or an enlarged heart 27. Having an enlarged heart places the patient at a greater risk for developing heart failure. Because of this, and the difficulty of treating dilated cardiomyopathy, patients may need a heart transplant. However, there are other causes of dilated cardiomyopathy and heart failure besides myocarditis. There is no established treatment for chronic myocarditis. However, studies in patients with chronic heart failure that did not respond to usual care suggest a limited role for immunosuppression. Randomized trials of patients with chronic myocarditis (diagnosed by special stains of heart biopsy tissue) immunosuppression with azathioprine and prednisone resulted in an improvement in quality of life and left ventricular ejection fraction. This treatment strategy is now under evaluation in well-designed, multicenter trials. Other studies seek to evaluate antiviral therapy in patients with evidence of chronic viral heart infections.