Coxsackievirus-induced myocarditis in mice: A model of autoimmune disease for studying immunotoxicity
Introduction
Cardiovascular disease is the number one killer of men and women in the United States and the second most common cause of death worldwide [1], [2]. Although the true incidence of inflammation in the heart is unknown, it is estimated that 1 in 4 individuals in the United States have some form of inflammatory heart disease [1], [3]. Acute myocarditis is a principal cause of heart failure in young adults and often progresses to chronic myocarditis, DCM and congestive heart failure requiring heart transplantation [3]. CVB3 infection is believed to be a principle etiologic agent in human myocarditis, and the same virus strain that infects humans (ignoring the species barrier) induces biphasic myocarditis in genetically susceptible strains of mice [3], [4], [5], [6].
Currently, several animal models are available that closely resemble human myocarditis [7]. Murine models fall into two basic categories: acute cardiac inflammation and sudden death induced by direct viral damage (where nearly 70% of animals die at day 4–7 after infection) [8], [9], [10], [11], or inflammation triggered by adjuvant and cardiac myosin or virus and cardiac myosin (heart-passaged virus) resulting in acute myocarditis with no deaths that progresses to chronic heart disease and DCM [5], [6], [12], [13], [14], [15]. In this article, we describe a model of autoimmune myocarditis and DCM induced by inoculation with heart-passaged CVB3 [7], [15].
In the model of CVB3-induced myocarditis presented here, intraperitoneal (ip) inoculation of BALB/c mice with heart-passaged CVB3 (Nancy strain), which contains virus and cardiac myosin, induces inflammatory heart disease that is remarkably similar to disease induced by inoculation with adjuvant and cardiac myosin (experimental autoimmune myocarditis or EAM) [14], [15], [16], [17]. Acute CVB3-induced myocarditis develops in susceptible BALB/c mice from day 7 to 14 postinfection (pi) and progresses to chronic myocarditis and DCM from day 35 to at least 56 pi (Fig. 1A) [6], [15], [18], [19]. Resistant strains of mice (i.e. C57BL/6) do not develop the chronic phase of the disease (Fig. 1B). Furthermore, susceptible male mice develop significantly increased acute (Fig. 2A) and chronic (data not shown) myocarditis in response to CVB3 infection compared to female mice. Following infection with heart-passaged CVB3, mice quickly develop immunoglobulin (Ig)G autoantibodies specific for cardiac myosin, similar to those found in EAM and in patients with myocarditis and DCM [5], [6], [20], [21]. Acute CVB3-induced myocarditis is characterized by a focal cellular infiltrate with little necrosis or fibrosis, comprised primarily of macrophages, neutrophils, CD4+ T cells, and CD8+ T cells with lower numbers of B cells, mast cells, natural killer cells, and dendritic cells (Fig. 2B) [19]. In this model, virus replicates at relatively low levels in the heart during acute myocarditis (102–104 PFU) compared with other CVB3-induced models (107–109 PFU) [8], [9], [10], [11], [18], [19]. By day 14 pi, infectious virus is cleared from the heart and does not reactivate during chronic myocarditis [6], [15]. In genetically susceptible mice, a diffuse, lymphocytic infiltrate develops around day 35 pi with large areas of necrosis and fibrosis (Fig. 3A) as well as pericarditis (Fig. 3B) and DCM (Fig. 3D) [15]. Although viral genome can be detected in heart tissue during chronic myocarditis, persistent virus is found in both susceptible and resistant strains of mice [13], [22].
The development of autoimmune disease depends on a complex interaction between genetic, environmental, and endogenous factors [23], [24], [25]. Environmental agents, such as infections, diet, hormones and drugs, contribute significantly to the risk of developing and/or dying from heart disease [2], [14], [26], [27], [28]. The incidence and severity of heart disease is higher among men, but the incidence increases in women as they age [1], [29]. Young male BALB/c mice (6–8 weeks old) develop significantly greater acute and chronic myocarditis than female mice (Fig. 2), similar to humans [1], [30]. Public health measures have dramatically reduced exposure to many infections and toxic chemicals. However, the effect of xenobiotics on the immune system and the development of inflammatory heart disease following infection has not been fully evaluated. Recent studies suggest that low dose exposure to immunotoxicants, such as mercury, may exacerbate inflammatory heart disease induced by viral infection or adjuvant and cardiac myosin [31]. A clear challenge for immunotoxicologists is to better understand the mechanisms involved in the progression of inflammatory heart disease. Only then we can develop more effective prevention and intervention strategies for these diseases.
Section snippets
Reagents and equipment
Vero cells (American Type Culture Collection, ATCC, Manassas, VA, USA) Minimum essential medium (MEM), liquid and powder (Mediatech Inc., Herndon, VA, USA).
Heat-inactivated fetal bovine serum (FBS) (Invitrogen Life Technologies, Carlsbad, CA, USA).
Penicillin/streptomycin, 5000 U (Pen/Strep) (Mediatech Inc., Herndon, VA, USA).
Methyl cellulose, 4000 centipoises (Fischer Scientific, Pittsburgh, PA, USA).
Bleach (Clorox, USA).
Susceptible mice, age 6–8 weeks old (Jackson Laboratory, Bar Harbor, ME,
Tissue culture virus
Grow Vero cells until confluent at 37 °C and 5% CO2 in MEM supplemented with Pen/Strep and 10% FBS (10% MEM). Remove media and replace with MEM supplemented with Pen/Strep and 2% FBS (2% MEM) (FBS inhibits viral entry). CVB3 is infectious to people as well as mice. A 20% bleach solution or UV light will kill the virus. Always wear a protective mask and replace gloves immediately after working with the virus to prevent spread. Wash all surfaces and utensils with 20% bleach and expose the hood to
Concluding remarks
The model of CVB3-induced myocarditis presented here allows dissection of the contribution of viral infection and xenobiotics on immune dysregulation and the severity of inflammation in the heart. Because patients rarely die from acute viral myocarditis [3], autoimmune models of CVB3-induced myocarditis may more closely resemble disease as it occurs in human populations. Indeed, clinical observations closely resemble findings using this model. Little is known about the interaction of infections
Acknowledgment
Funding for this research was provided by grants from the National Institutes of Health (HL67290, HL70729, AI51835 and ES03819).
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2022, iScienceCitation Excerpt :Thus, Coxsackievirus B3 (CVB3)-induced myocarditis is commonly employed to investigate the pathogenic mechanisms of viral myocarditis. Of various rodent species, mice are highly susceptible to CVB3 infection (Fairweather and Rose, 2007), and their major histocompatibility complex (MHC) haplotypes influence the disease outcome. Although H-2 (IAb)-bearing C57Bl/6 mice develop acute infection and are resistant to the development of chronic myocarditis, A/J (IAk) and BALB/c (IAd) mice develop chronic disease, making them suitable to study the pathogenic mechanisms that may involve both viral and host factors (Blyszczuk, 2019; Fairweather and Rose, 2007; Lasrado and Reddy, 2020).