Differential Interferon Responses Enhance Viral Epitope Generation by Myocardial Immunoproteasomes in Murine Enterovirus Myocarditis

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection, with host-specific outcomes ranging from complete recovery in resistant mice to chronic disease in susceptible hosts. To identify susceptibility factors that modulate the course of viral myocarditis, we show that type-I interferon (IFN) responses are considerably impaired in acute CVB3-induced myocarditis in susceptible mice, which have been linked to immunoproteasome (IP) formation. Here we report that in concurrence with distinctive type-I IFN kinetics, myocardial IP formation peaked early after infection in resistant mice and was postponed with maximum IP expression concomitant to massive inflammation and predominant type-II IFN responses in susceptible mice. IP activity is linked to a strong enhancement of antigenic viral peptide presentation. To investigate the impact of myocardial IPs in CVB3-induced myocarditis, we identified two novel CVB3 T cell epitopes, virus capsid protein 2 [285-293] and polymerase 3D [2170-2177]. Analysis of myocardial IPs in CVB3-induced myocarditis revealed that myocardial IP expression resulted in efficient epitope generation. As opposed to the susceptible host, myocardial IP expression at early stages of disease corresponded to enhanced CVB3 epitope generation in the hearts of resistant mice. We propose that this process may precondition the infected heart for adaptive immune responses. In conclusion, type-I IFN-induced myocardial IP activity at early stages coincides with less severe disease manifestation in CVB3-induced myocarditis.Myocarditis is often induced by cardiotropic viruses: in about 20% of patients, viral myocarditis leads to its sequela dilated cardiomyopathy, which is linked to chronic inflammation and persistence of cardiotropic viruses.^1,2,3,4 Dilated cardiomyopathy is the most common cause of heart failure in young patients and appears to be a major cause of sudden unexpected death in this cohort. Enteroviruses, including group-B coxsackieviruses, have been linked to the development of myocarditis and dilated cardiomyopathy associated with adverse prognosis.^5,6 Well-established murine models of coxsackievirus B3 (CVB3) myocarditis mimic the human disease progress and are valuable in delineating the underlying mechanisms that determine the divergent courses of myocarditis^7,8,9,10: resistant C57BL/6 mice eliminate the virus following mild acute myocarditis; no chronic inflammation is detected. In contrast, major histocompatibility complex (MHC)-matched A.BY/SnJ mice develop severe acute infection and ongoing chronic myocarditis, thus conferring susceptibility to chronic disease.^7,9Host responses to viral infection trigger the release of interferons (IFNs). IFNs of the α/β subtype are assigned to type I IFNs, whereas IFN-γ is the only type II IFN. IFNs exert numerous antiviral effects in innate and adaptive immunity.¹¹ Although type I IFN-receptor-deficiency was not associated with a dramatic effect on early viral replication in the heart, type I IFN signaling was found to be essential for the prevention of early death due to CVB3-infection.¹² The extraordinary impact of type I IFNs was substantiated in a recent study illustrating acute fulminant infection and chronic disease progression in IFN-β deficient mice.¹³ Deletion of type II IFN receptors was not associated with enhanced mortality in CVB3-infection.¹² IFN-γ responses were shown to be protective in cellular immunity in CVB3-infection.⁹ In addition, expression of IFN-γ conferred protection in enterovirus myocarditis, which may be linked to the activation of nitric oxide-mediated antiviral activity of macrophages.^14,15 Thus, both type I and type II IFN are active in CVB3- myocarditis.One downstream effect of IFN signaling is the induction of immunoproteasome (IP) formation in the target organ of the immune response. Particularly IFN-γ was shown to induce IP expression.^16,17,18 Efficient generation of viral epitopes that stimulate CD8⁺ T cells strongly relies on host-cell IP and, in addition, protein degradation by proteasomes is also essential in the regulation of inflammatory and stress responses, cell cyclus, and apoptosis control.¹⁹ The 20S proteasome as the catalytic core of the proteasome resembles a cylinder-shaped structure of stacked heptameric rings formed by either α or β subunits. The proteolytic function of the so-called standard proteasome is restricted to the β1, β2, and β5 subunit.²⁰ Three alternative catalytic subunits, the so-called immunosubunits β1i, β2i, and β5i, which are incorporated into 20S proteasomes, thus forming IP with altered catalytic characteristics, are expressed on cytokine stimulation.^21,22 It is highly notable that IP activity is linked to a strong enhancement of antigenic viral peptide presentation.^{23,24,25,26,27}Cardiac proteasomes contribute to the modulation of cardiac function in health and disease.²⁸ However, apart from the reported observation that IPs are expressed in the myocardium in acute CVB3 myocarditis, their functional impact has not been studied so far.¹⁰ The present study focuses on IFN-induced myocardial IP activity in CVB3 myocarditis.