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.     

Role of mast cells in experimental anti-glomerular basement membrane glomerulonephritis

Recently, divergent reports on the role of mast cells (MC) in different glomerular diseases have brought our attention to their role in an accelerated model of anti-glomerular basement membrane (GBM) glomerulonephritis (GN). Genetically MC-deficient Kit(W)/Kit(W-v) mice, MC-reconstituted Kit(W)/Kit(W-v) mice and Kit+/+ control mice were subjected to anti-GBM GN. Kit(+/+) mice developed moderate proteinuria and glomerular damage following the induction of anti-GBM nephritis. In contrast, proteinuria and glomerular damage were dramatically increased in MC-deficient Kit(W)/Kit(W-v) mice. MC-reconstituted Kit(W)/Kit(W-v) mice showed proteinuria and glomerular damage comparable to Kit+/+ mice. A significant increase in infiltrating T cells and macrophages was detected in MC-deficient Kit(W)/Kit(W-v) mice as compared to Kit+/+ control mice and MC-reconstituted Kit(W)/Kit(W-v) mice. Accordingly, we observed an increase of TGF-beta1 mRNA in kidneys from Kit(W)/Kit(W-v) mice. Interestingly, we did not detect MC in the kidney using either Giemsa staining or RT-real-time PCR, but MC were found in the regional lymph nodes. Finally, mortality of Kit(W)/Kit(W-v) mice was significantly increased after the induction of anti-GBM GN due to uremia. Our report provides the first direct evidence that MC are protective in anti-GBM GN, possibly by modulating the influx of effector T cells and macrophages to inflammatory sites in the kidney.     

Effect of tissue fixatives on telomere length determination by quantitative PCR

Telomere length is a well established marker of cellular senescence and thus biological age. Quantitative PCR allows the determination even from very low amounts of tissue by using telomere specific and single copy gene primers. Comparing a directly processed tissue sample to a 4% formaldehyde fixed one showed a significantly reduced efficiency of PCR reactions (mainly in single copy gene experiments) in a storage time-dependent manner resulting in an artificial increase in reported relative telomere length. This effect was not seen when the tissue was stored in RNA later solution. In summary, telomere length determination from formaldehyde fixed material by quantitative PCR is not a reliable method. Unfortunately therefore, many easily accessible tissue samples from pathology laboratories are unsuitable for this technique.     

HIV-1 Tat increases the adhesion of monocytes and T-cells to the endothelium in vitro and in vivo: implications for AIDS-associated vasculopathy

HIV-1-infected patients exhibit severe damages of the aortic endothelium, develop angioproliferative lesions such as Kaposi's sarcoma (KS), and have an increased risk of cardiovascular diseases and atherosclerosis. An increased adhesion of leukocytes to the endothelium is a common pathogenic parameter of AIDS-associated vascular diseases. Here we show that the HIV-1 Tat protein, a regulatory protein of HIV-1 released by infected cells, and TNF-alpha, a cytokine increased in sera and tissues of HIV-1-infected patients, activate synergistically the adhesion of leukocytes to endothelial cells both in vitro and in vivo. This effect is selectively mediated by HIV-1 Tat, since HIV-1 Nef, another HIV-1 regulatory protein, and the HIV-1 envelope protein gp41, had no effect. In vitro adhesion assays with PBMC and quantitative cell type analysis of adherent cells by FACS demonstrated that HIV-1 Tat selectively activates the adhesion of T-cells and monocytes but not of B-cells. Intravital microscopic studies in mice confirmed the synergistic activity of HIV-1 Tat and TNF-alpha on leukocyte adhesion to the endothelium in vivo. These data indicate that HIV-1 Tat in cooperation with TNF-alpha may contribute to the vascular damage and cardiovascular diseases observed in AIDS patients but also to the prominent extravasation of T-cells and monocytes which is a key process in the formation and progression of KS lesions.     

Improving live attenuated bacterial carriers for vaccination and therapy

Live attenuated bacteria are well established as vaccines. Thus, their use as carriers for prophylactic and therapeutic macromolecules is a logical consequence. Here we describe several experimental applications of bacteria to carry heterologous macromolecules into the murine host. First, Listeria monocytogenes are described that are able to transfer eukaryotic expression plasmids into host cells for gene therapy. High multiplicities of infection are still required for efficient gene transfer and we point out some of the bottlenecks that counteract a more efficient transfer and application in vivo. Then, we describe Salmonella enterica serovar Typhimurium (S. typhimurium) as an expression plasmid transfer vehicle for oral DNA vaccination of mice. We demonstrate that the stabilization of the plasmid transformants results in an improved immune response. Stabilization was achieved by replacing the origin of replication of the original high-copy-number plasmid by a low-copy-number origin. Finally, we describe Salmonella carriers for the improved expression of heterologous proteins. We introduce a system in which the plasmid is carried as a single copy during cultivation but is amplified several fold upon infection of the host. Using the same in vivo inducible promoter for both protein expression and plasmid amplification, a substantial increase in antigen expression in vivo can be achieved. A modification of this approach is the introduction of inducible gene expression in vivo with a low-molecular-weight compound. Using P_BAD promoter and l-arabinose as inducer we were able to deliberately activate genes in the bacterial carrier. No background activity could be observed with P_BAD such that an inducible suicide gene could be introduced. This is adding an important safety feature to such live attenuated carrier bacteria.     

HIV-1 mRNA electroporation of PBMC: a simple and efficient method to monitor T-cell responses against autologous HIV-1 in HIV-1-infected patients

Efficient monitoring of HIV-1-specific T-cells is crucial for the development of HIV-1 vaccines and immunotherapies. Currently, mainly peptides and vaccinia vectors are used for detection of HIV-1-specific cytotoxic T-lymphocytes (CTL), however, as HIV-1 is a variable virus, it is unknown to what extent the T-cell response against the autologous virus is under- or overestimated by using antigens from heterologous viral strains. Therefore, we established a new method for immunomonitoring of CTL using electroporation of peripheral blood mononuclear cells (PBMC) with mRNA derived from autologous viral strains. From six HIV-1-infected patients virus derived mRNA was produced after PCR-based cloning of autologous gag (n=5) and/or nef genes (n=3) from plasma and electroporated into PBMC from patients and healthy donors. Electroporation of PBMC with mRNA resulted in efficient protein expression with good induction of γ-interferon (γ-IFN) release by specific T-cells comparable to peptide pools and better than recombinant vaccinia viruses. Three mRNA encoded autologous Gag proteins and one autologous mRNA encoded Nef protein were better recognized by autologous PBMC in comparison to heterologous mRNA encoded Gag or Nef proteins (SF2 or HXB2). However, in one case each, mRNA encoded autologous Gag or Nef, respectively, was recognized less efficiently due to the presence of CTL escape mutations. In summary, electroporation of PBMC with mRNA is a very efficient, easy and rapid method for immunomonitoring of HIV-1-specific T-cell responses against autologous viral strains. Our data demonstrate that patients' CTL responses against autologous viral strains may be under- or overestimated by using antigens from heterologous viral strains.     

Sequetyping: serotyping Streptococcus pneumoniae by a single PCR sequencing strategy

The introduction of pneumococcal conjugate vaccines necessitates continued monitoring of circulating strains to assess vaccine efficacy and replacement serotypes. Conventional serological methods are costly, labor-intensive, and prone to misidentification, while current DNA-based methods have limited serotype coverage requiring multiple PCR primers. In this study, a computer algorithm was developed to interrogate the capsulation locus (cps) of vaccine serotypes to locate primer pairs in conserved regions that border variable regions and could differentiate between serotypes. In silico analysis of cps from 92 serotypes indicated that a primer pair spanning the regulatory gene cpsB could putatively amplify 84 serotypes and differentiate 46. This primer set was specific to Streptococcus pneumoniae, with no amplification observed for other species, including S. mitis, S. oralis, and S. pseudopneumoniae. One hundred thirty-eight pneumococcal strains covering 48 serotypes were tested. Of 23 vaccine serotypes included in the study, most (19/22, 86%) were identified correctly at least to the serogroup level, including all of the 13-valent conjugate vaccine and other replacement serotypes. Reproducibility was demonstrated by the correct sequetyping of different strains of a serotype. This novel sequence-based method employing a single PCR primer pair is cost-effective and simple. Furthermore, it has the potential to identify new serotypes that may evolve in the future.