TF-FVIIa PAR2-ß-Arrestin Signaling Sustains Organ Dysfunction in Coxsackievirus B3 Infection of Mice.

BACKGROUND: Accumulating evidence implicates the activation of G-protein-coupled PARs (protease-activated receptors) by coagulation proteases in the regulation of innate immune responses. METHODS: Using mouse models with genetic alterations of the PAR2 signaling platform, we have explored contributions of PAR2 signaling to infection with coxsackievirus B3, a single-stranded RNA virus provoking multiorgan tissue damage, including the heart. RESULTS: We show that PAR2 activation sustains correlates of severe morbidity-hemodynamic compromise, aggravated hypothermia, and hypoglycemia-despite intact control of the virus. Following acute viral liver injury, canonical PAR2 signaling impairs the restoration process associated with exaggerated type I IFN (interferon) signatures in response to viral RNA recognition. Metabolic profiling in combination with proteomics of liver tissue shows PAR2-dependent reprogramming of liver metabolism, increased lipid droplet storage, and gluconeogenesis. PAR2-sustained hypodynamic compromise, reprograming of liver metabolism, as well as imbalanced IFN responses are prevented in ß-arrestin coupling-deficient PAR2 C-terminal phosphorylation mutant mice. Thus, wiring between upstream proteases and immune-metabolic responses results from biased PAR2 signaling mediated by intracellular recruitment of ß-arrestin. Importantly, blockade of the TF (tissue factor)-FVIIa (coagulation factor VIIa) complex capable of PAR2 proteolysis with the NAPc2 (nematode anticoagulant protein c2) mitigated virus-triggered pathology, recapitulating effects seen in protease cleavage-resistant PAR2 mice. CONCLUSIONS: These data provide insights into a TF-FVIIa signaling axis through PAR2-ß-arrestin coupling that is a regulator of inflammation-triggered tissue repair and hemodynamic compromise in coxsackievirus B3 infection and can potentially be targeted with selective coagulation inhibitors.

Biparatopic nanobodies protect mice from lethal challenge with SARS-CoV-2 variants of concern.

The ongoing COVID-19 pandemic and the emergence of new SARS-CoV-2 variants of concern (VOCs) requires continued development of effective therapeutics. Recently, we identified high-affinity neutralizing nanobodies (Nbs) specific for the receptor-binding domain (RBD) of SARS-CoV-2. Taking advantage of detailed epitope mapping, we generate two biparatopic Nbs (bipNbs) targeting a conserved epitope outside and two different epitopes inside the RBD:ACE2 interface. Both bipNbs bind all currently circulating VOCs with high affinities and are capable to neutralize cellular infection with VOC B.1.351 (Beta) and B.1.617.2 (Delta) in vitro. To assess if the bipNbs NM1267 and NM1268 confer protection against SARS-CoV-2 infection in vivo, human ACE2 transgenic mice are treated intranasally before infection with a lethal dose of SARS-CoV-2 B.1, B.1.351 (Beta) or B.1.617.2 (Delta). Nb-treated mice show significantly reduced disease progression and increased survival rates. Histopathological analyses further reveal a drastically reduced viral load and inflammatory response in lungs. These data suggest that both bipNbs are broadly active against a variety of emerging SARS-CoV-2 VOCs and represent easily applicable drug candidates.

Enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus.

The innate immune system limits viral replication via type I interferon and also induces the presentation of viral antigens to cells of the adaptive immune response. Using infection of mice with vesicular stomatitis virus, we analyzed how the innate immune system inhibits viral propagation but still allows the presentation of antigen to cells of the adaptive immune response. We found that expression of the gene encoding the inhibitory protein Usp18 in metallophilic macrophages led to lower type I interferon responsiveness, thereby allowing locally restricted replication of virus. This was essential for the induction of adaptive antiviral immune responses and, therefore, for preventing the fatal outcome of infection. In conclusion, we found that enforced viral replication in marginal zone macrophages was an immunological mechanism that ensured the production of sufficient antigen for effective activation of the adaptive immune response.

Heart-Specific Immune Responses in an Animal Model of Autoimmune-Related Myocarditis Mitigated by an Immunoproteasome Inhibitor and Genetic Ablation.

BACKGROUND: Immune checkpoint inhibitor (ICI) therapy is often accompanied by immune-related pathology, with an increasing occurrence of high-risk ICI-related myocarditis. Understanding the mechanisms involved in this side effect could enable the development of management strategies. In mouse models, immune checkpoints, such as PD-1 (programmed cell death protein 1), control the threshold of self-antigen responses directed against cardiac TnI (troponin I). We aimed to identify how the immunoproteasome, the main proteolytic machinery in immune cells harboring 3 distinct protease activities in the LMP2 (low-molecular-weight protein 2), LMP7 (low-molecular-weight protein 7), and MECL1 (multicatalytic endopeptidase complex subunit 1) subunit, affects TnI-directed autoimmune pathology of the heart. METHODS: TnI-directed autoimmune myocarditis (TnI-AM), a CD4+ T-cell-mediated disease, was induced in mice lacking all 3 immunoproteasome subunits (triple-ip-/-) or lacking either the gene encoding LMP2 and LMP7 by immunization with a cardiac TnI peptide. Alternatively, before induction of TnI-AM or after establishment of autoimmune myocarditis, mice were treated with the immunoproteasome inhibitor ONX 0914. Immune parameters defining heart-specific autoimmunity were investigated in experimental TnI-AM and in 2 cases of ICI-related myocarditis. RESULTS: All immunoproteasome-deficient strains showed mitigated autoimmune-related cardiac pathology with less inflammation, lower proinflammatory and chemotactic cytokines, less interleukin-17 production, and reduced fibrosis formation. Protection from TnI-directed autoimmune heart pathology with improved cardiac function in LMP7-/- mice involved a changed balance between effector and regulatory CD4+ T cells in the spleen, with CD4+ T cells from LMP7-/- mice showing a higher expression of inhibitory PD-1 molecules. Blocked immunoproteasome proteolysis, by treatment of TLR2 (Toll-like receptor 2)-engaged and TLR7 (Toll-like receptor 7)/TLR8 (Toll-like receptor 8)-engaged CD14+ monocytes with ONX 0914, diminished proinflammatory cytokine responses, thereby reducing the boost for the expansion of self-reactive CD4+ T cells. Correspondingly, in mice, ONX 0914 treatment reversed cardiac autoimmune pathology, preventing the induction and progression of TnI-AM when self-reactive CD4+ T cells were primed. The autoimmune signature during experimental TnI-AM, with high immunoproteasome expression, immunoglobulin G deposition, interleukin-17 production in heart tissue, and TnI-directed humoral autoimmune responses, was also present in 2 cases of ICI-related myocarditis, demonstrating the activation of heart-specific autoimmune reactions by ICI therapy. CONCLUSIONS: By reversing heart-specific autoimmune responses, immunoproteasome inhibitors applied to a mouse model demonstrate their potential to aid in the management of autoimmune myocarditis in humans, possibly including patients with ICI-related heart-specific autoimmunity.

Blocking the IL-1ß signalling pathway prevents chronic viral myocarditis and cardiac remodeling.

Coxsackieviruses of group B (CVB) are well-known causes of acute and chronic myocarditis. Chronic myocarditis can evolve into dilated cardiomyopathy (DCM) characterized by fibrosis and cardiac remodeling. Interleukin-1ß (IL-1ß) plays a decisive role in the induction of the inflammatory response as a consequence of viral replication. In this study, we analyzed the effects of IL-1ß neutralization on the transition of acute to chronic myocarditis in a mouse model of CVB3 myocarditis. Mice were treated with an anti-murine IL-1ß antibody as a surrogate for Canakinumab at different time points post CVB3 infection. Treatment was performed in the early phase (day 1-14 pi, day 3-14 pi) or at a later stage of myocarditis (day 14-28 pi). Subsequently, the hearts were examined histologically, immunohistochemically and by molecular biology. A significant reduction of viral replication, cardiac damage and inflammation was found after administration of the antibody in the early phase and in the later phase of infection. Furthermore, less collagen I deposition and a considerable reduction of fibrosis were found in antibody-treated mice. Using microarray analysis, a significant upregulation of various extracellular matrix and fibrosis-associated molecules was found in CVB3-infected mice, including TGF-ß, TIMP-1 and MMP12, as well as diverse matricellular proteins, whereas, these molecules were significantly downregulated in all IL-1ß antibody-treated infected mice. Neutralization of IL-1ß at different stages of enteroviral infection prevents the development of chronic viral myocarditis by reducing inflammation, interstitial fibrosis and adverse cardiac remodeling. These findings are relevant for the treatment of patients with acute and chronic myocarditis.

TRAF6: A player in CVB3-induced myocarditis?

Coxsackievirus B3 (CVB3) is an important inducer of myocarditis, which, in susceptible individuals, can chronify and eventually lead to the development of dilated cardiomyopathy and heart failure. The respective mechanisms are not completely understood. Here, we analyzed expression of the TRAF6 gene, encoding TNF receptor-associated factor 6 (TRAF6), a signal transduction scaffold protein that acts downstream of cytokine receptors, in heart tissue of susceptible and non-susceptible mouse strains. We found that after infection, TRAF6 expression was upregulated in both non-susceptible C57BL/6 wildtype and susceptible A.BY/SnJ and C57BL/6-TLR3 (-/-) mice, however, to different degrees. In infected HeLa cells, we also found moderately elevated TRAF6 levels after infection, in addition, activity of the transcription factor nuclear factor kappa B (NFκB), which can be activated downstream of TRAF6, was strongly enhanced in infected cells. To functionally analyze the role of TRAF6 with regard to infection progression, TRAF6 expression was knocked down in cultured HeLa cells using specific siRNAs. We found that reduction of TRAF6 expression had no effect on NFκB activation in response to infection. Taken together, our data suggest that CVB3 infection enhances TRAF6 levels, however, this induction might not be necessary for infection-induced NFκB activation.

Deletion of the activating NK cell receptor NKG2D accelerates rejection of cardiac allografts.

It has already been shown that neutralization of the activating NK cell receptor NKG2D in combination with co-stimulation blockade prolongs graft survival of vascularized transplants. In order to clarify the underlying cellular mechanisms, we transplanted complete MHC-disparate BALB/c-derived cardiac grafts into C57BL/6 wildtypes or mice deficient for NKG2D (Klrk1-/- ). Although median survival was 8 days for both recipient groups, we detected already at day 5 posttransplantation significantly greater intragraft frequencies of NKp46+ NK cells in Klrk1-/- recipients than in wildtypes. This was followed by a significantly greater infiltration of CD4+ , but a lesser infiltration of CD8+ T cell frequencies. Contrary to published observations, co-stimulation blockade with CTLA4-Ig resulted in a significant acceleration of cardiac rejection by Klrk1-/- recipients, and this result was confirmed by applying a neutralizing antibody against NKG2D to wildtypes. In both experimental setups, grafts derived from Klrk1-/- recipients were characterized by significantly higher levels of interferon-γ mRNA, and both CD4+ and CD8+ T cells displayed a greater capacity for degranulation and interferon-γ production. In summary, our results clearly illustrate that NKG2D expression in the recipient is important for cardiac allograft survival, thus supporting the hypothesis that impairment of NK cells prevents the establishment of graft acceptance.

The immunoproteasome controls the availability of the cardioprotective pattern recognition molecule Pentraxin3.

Cardiomyocyte death as a result of viral infection is an excellent model for dissecting the inflammatory stress response that occurs in heart tissue. We reported earlier that a specific proteasome isoform, the immunoproteasome, prevents exacerbation of coxsackievirus B3 (CVB3)-induced myocardial destruction and preserves cell vitality in heart tissue inflammation. Following the aim to decipher molecular targets of immunoproteasome-dependent proteolysis, we investigated the function and regulation of the soluble PRR Pentraxin3 (PTX3). We show that the ablation of PTX3 in mice aggravated CVB3-triggered inflammatory injury of heart tissue, without having any significant effect on viral titers. Thus, there might be a role of PTX3 in preventing damage-associated molecular pattern-induced cell death. We found that the catalytic activity of the immunoproteasome subunit LMP7 regulates the timely availability of factors controlling PTX3 production. We report on immunoproteasome-dependent alteration of ERK1/2 and p38MAPKs, which were both found to be involved in PTX3 expression control. Our finding of a cardioprotective function of immunoproteasome-dependent PTX3 expression revealed a crucial mechanism of the stress-induced damage response in myocardial inflammation. In addition to antigen presentation and cytokine production, proteolysis by the immunoproteasome can also regulate the innate immune response during viral infection.

Donor brain death leads to differential immune activation in solid organs but does not accelerate ischaemia-reperfusion injury.

A comparative analysis of inflammation between solid organs following donor brain death (BD) is still lacking and the detailed influence of BD accelerating ischaemia-reperfusion injury (IRI) post-transplantation remains to be addressed. Applying a murine model of BD, we demonstrated that 4 h after BD organs were characterized by distinct inflammatory expression patterns. For instance, lipocalin 2 (LCN2), a marker of acute kidney injury, was selectively induced in BD livers but not in kidneys. BD further resulted in significantly reduced frequencies of CD3(+) CD4(+) , CD3(+) CD8(+) T cells and NKp46(+) NK cells in the liver, whereas BD kidneys and hearts were characterized by significantly lower frequencies of conventional dendritic cells (cDCs). Syngeneic models of kidney (KTx) and heart transplantation (HTx) illustrated stronger gene expression in engrafted BD hearts only, but 20 h post-transplantation both organs displayed comparable intragraft lymphocyte frequencies, except for NK cells and graft function. Moreover, the complement factor C3d deposit detected in small vessels and capillaries in cardiac syngrafts did not significantly differ between BD and sham-transplanted groups. Finally, no further influence of donor BD on graft survival was detected in an allogeneic heart transplantation setting (C57BL/6 grafts into BALB/c recipients). We show for the first time that BD organs are characterized by a varying inflammatory profile; however, BD does not accelerate IRI in syngeneic KTx and HTx.

Ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) in host defense against heart failure in a mouse model of virus-induced cardiomyopathy.

BACKGROUND: Common causative agents in the development of inflammatory cardiomyopathy include cardiotropic viruses such as coxsackievirus B3 (CVB3). Here, we investigated the role of the ubiquitin-like modifier interferon-stimulated gene of 15 kDa (ISG15) in the pathogenesis of viral cardiomyopathy. METHODS AND RESULTS: In CVB3-infected mice, the absence of protein modification with ISG15 was accompanied by a profound exacerbation of myocarditis and by a significant increase in mortality and heart failure. We found that ISG15 in cardiomyocytes contributed significantly to the suppression of viral replication. In the absence of an intact ISG15 system, virus titers were markedly elevated by postinfection day 8, and viral RNA persisted in ISG15(-/-) mice at postinfection day 28. Ablation of the ISG15 protein modification system in CVB3 infection predisposed mice to long-term disease with deposition of collagen fibers, all leading to inflammatory cardiomyopathy. We found that ISG15 acts as part of the intrinsic immunity in cardiomyocytes and detected no significant effects of ISG15 modification on the cellular immune response. ISG15 modification of CVB3 2A protease counterbalanced CVB3-induced cleavage of the host cell eukaryotic initiation factor of translation eIF4G in cardiomyocytes, thereby counterbalancing the shutoff of host cell translation in CVB3 infection. We demonstrate that ISG15 suppressed infectious virus yield in human cardiac myocytes and the induction of ISG15 in patients with viral cardiomyopathy. CONCLUSIONS: The ISG15 conjugation system represents a critical innate response mechanism in cardiomyocytes to fight the battle against invading pathogens, limiting inflammatory cardiomyopathy, heart failure, and death. Interference with the ISG15 system might be a novel therapeutic approach in viral cardiomyopathy.

Interspecies differences in virus uptake versus cardiac function of the coxsackievirus and adenovirus receptor.

UNLABELLED: The coxsackievirus and adenovirus receptor (CAR) is a cell contact protein with an important role in virus uptake. Its extracellular immunoglobulin domains mediate the binding to coxsackievirus and adenovirus as well as homophilic and heterophilic interactions between cells. The cytoplasmic tail links CAR to the cytoskeleton and intracellular signaling cascades. In the heart, CAR is crucial for embryonic development, electrophysiology, and coxsackievirus B infection. Noncardiac functions are less well understood, in part due to the lack of suitable animal models. Here, we generated a transgenic mouse that rescued the otherwise embryonic-lethal CAR knockout (KO) phenotype by expressing chicken CAR exclusively in the heart. Using this rescue model, we addressed interspecies differences in coxsackievirus uptake and noncardiac functions of CAR. Survival of the noncardiac CAR KO (ncKO) mouse indicates an essential role for CAR in the developing heart but not in other tissues. In adult animals, cardiac activity was normal, suggesting that chicken CAR can replace the physiological functions of mouse CAR in the cardiomyocyte. However, chicken CAR did not mediate virus entry in vivo, so that hearts expressing chicken instead of mouse CAR were protected from infection and myocarditis. Comparison of sequence homology and modeling of the D1 domain indicate differences between mammalian and chicken CAR that relate to the sites important for virus binding but not those involved in homodimerization. Thus, CAR-directed anticoxsackievirus therapy with only minor adverse effects in noncardiac tissue could be further improved by selectively targeting the virus-host interaction while maintaining cardiac function. IMPORTANCE: Coxsackievirus B3 (CVB3) is one of the most common human pathogens causing myocarditis. Its receptor, the coxsackievirus and adenovirus receptor (CAR), not only mediates virus uptake but also relates to cytoskeletal organization and intracellular signaling. Animals without CAR die prenatally with major cardiac malformations. In the adult heart, CAR is important for virus entry and electrical conduction, but its nonmuscle functions are largely unknown. Here, we show that chicken CAR expression exclusively in the heart can rescue the otherwise embryonic-lethal CAR knockout phenotype but does not support CVB3 infection of adult cardiomyocytes. Our findings have implications for the evolution of virus-host versus physiological interactions involving CAR and could help to improve future coxsackievirus-directed therapies inhibiting virus replication while maintaining CAR's cellular functions.

The activating receptor NKG2D of natural killer cells promotes resistance against enterovirus-mediated inflammatory cardiomyopathy.

In enterovirus-induced cardiomyopathy, information regarding the detailed impact of natural killer (NK) cells on the outcome of the disease is limited. We therefore hypothesized that NK cells and certain NK cell receptors determine the different outcome of coxsackievirus B3 (CVB3) myocarditis. Here, we demonstrate in murine models that resistance to chronic CVB3 myocarditis in immunocompetent C57BL/6 mice is characterized by significantly more mature CD11b(high) NK cells, the presence of NKG2D on NK cells, and enhanced NKG2D-dependent cytotoxicity compared to CVB3-susceptible A.BY/SnJ mice. The highly protective role of NKG2D in myocarditis was further proven by in vivo neutralization of NKG2D as well as in NKG2D-deficient mice but was shown to be independent of CD8(+) T-cell-dependent immunity. Moreover, the adoptive transfer of immunocompetent C57BL/6 NK cells pre- (day -1) as well as post-infectionem (day +2) displayed the potential to prevent permissive A.BY/SnJ mice from a progressive outcome of CVB3 myocarditis reflected by significantly improved cardiopathology and heart function. Altogether, our results provide firm evidence for a protective role of NKG2D-activated NK cells in CVB3 myocarditis leading to an effective virus clearance, thus offering novel therapeutic options in the treatment of virus-induced myocarditis.

Cardiomyocyte-specific IκB kinase (IKK)/NF-κB activation induces reversible inflammatory cardiomyopathy and heart failure.

Inflammation is a major factor in heart disease. IκB kinase (IKK) and its downstream target NF-κB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-κB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2. In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b(+) cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. Upon transgene inactivation, the disease was reversible even at an advanced stage. IKK-induced cardiomyopathy was dependent on NF-κB activation, as in vivo expression of IκBα superrepressor, an inhibitor of NF-κB, prevented the development of disease. Gene expression and proteomic analyses revealed enhanced expression of inflammatory cytokines, and an IFN type I signature with activation of the IFN-stimulated gene 15 (ISG15) pathway. In that respect, IKK-induced cardiomyopathy resembled Coxsackievirus-induced myocarditis, during which the NF-κB and ISG15 pathways were also activated. Vice versa, in cardiomyocytes lacking the regulatory subunit of IKK (IKKγ/NEMO), the induction of ISG15 was attenuated. We conclude that IKK/NF-κB activation in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure by inducing an excessive inflammatory response and myocyte atrophy.

Heme oxygenase-1 mediates oxidative stress and apoptosis in coxsackievirus B3-induced myocarditis.

BACKGROUND: Heme oxygenase-1 (HO-1), which is suggested to play a role in defending the organism against oxidative stress-mediated injuries, can be induced by diverse factors including viruses and iron. As coxsackievirus B3 (CVB3)-infected SWR/J mice susceptible for chronic myocarditis were found to have a significant iron incorporation and HO-1 upregulation in the myocardium, we aimed to investigate the molecular interplay between HO-1 expression and iron homeostasis in the outcome of viral myocarditis. METHODS AND RESULTS: In susceptible SWR/J mice, but not in resistant C57BL/6 mice, we observed at later stages of CVB3 myocarditis significant iron deposits in macrophages and also in cardiomyocytes, which were spatially associated with oxidative stress, upregulation of HO-1 and caspase-3 activation. HO-1, which is also expressed in cultivated RAW 264.7 macrophages upon incubation with iron and/or CVB3, could be downregulated by inhibition of NO/iNOS using L-NAME. Moreover, specific inhibition of HO-1 by tin mesoporphyrin revealed a suppression of superoxide production in iron and/or CVB3-treated macrophages. The molecular relationship of HO-1 and caspase-3 activation was proven by downregulation with HO-1 siRNA in iron- and/or CVB3-treated cultivated cells. Importantly, iron was found to increase viral replication in vitro. CONCLUSION: These results indicate that HO-1 induces a paracrine signalling in macrophages via reactive oxygen species production, mediating apoptosis of heart muscle cells at later stages of myocarditis. Notably, in genetically susceptible mice iron potentiates the detrimental effects of CVB3 by the NO/HO-1 pathway, thus increasing cardiac pathogenicity.

Visualization of immune cell infiltration in experimental viral myocarditis by (19)F MRI in vivo.

OBJECTIVE: This paper introduces a new approach permitting for the first time a specific, non-invasive diagnosis of myocarditis by visualizing the infiltration of immune cells into the myocardium. MATERIALS AND METHODS: The feasibility of this approach is shown in a murine model of viral myocarditis. Our study uses biochemically inert perfluorocarbons (PFCs) known to be taken up by circulating monocytes/macrophages after intravenous injection. RESULTS: In vivo (19)F MRI at 9.4 T demonstrated that PFC-loaded immune cells infiltrate into inflamed myocardial areas. Because of the lack of any fluorine background in the body, detected (19)F signals of PFCs are highly specific as confirmed ex vivo by flow cytometry and histology. CONCLUSION: Since PFCs are a family of compounds previously used clinically as blood substitutes, the technique described in our paper holds the potential as a new imaging modality for the diagnosis of myocarditis in man.

Enhanced stem cell migration mediated by VCAM-1/VLA-4 interaction improves cardiac function in virus-induced dilated cardiomyopathy.

Endogenous circulation of bone marrow-derived cells (BMCs) was observed in patients with dilated cardiomyopathy (DCM) who showed cardiac upregulation of Vascular Cell Adhesion Protein-1 (VCAM-1). However, the underlying pathophysiology is currently unknown. Thus, we aimed to analyze circulation, migration and G-CSF-based mobilization of BMCs in a murine model of virus-induced DCM. Mice with coxsackievirus B3 (CVB3) induced DCM and healthy controls were analyzed regarding their myocardial homing factors by PCR. To determine cardiac VCAM-1 expression ELISA and immunohistochemistry were applied. Flow cytometry was performed to analyze BMCs. Cardiac diameters and function were evaluated by echocardiography before and 4 weeks after G-CSF treatment. In murine CVB3-induced DCM an increase of BMCs in peripheral blood and a decrease of BMCs in bone marrow was observed. We found an enhanced migration of Very Late Antigen-4 (VLA-4⁺) BMCs to the diseased heart overexpressing VCAM-1 and higher numbers of CD45⁻CD34⁻Sca-1⁺ and CD45⁻CD34⁻c-kit⁺ cells. Mobilization of BMCs by G-CSF boosted migration along the VCAM-1/VLA-4 axis and reduced apoptosis of cardiomyocytes. Significant improvement of cardiac function was detected by echocardiography in G-CSF-treated mice. Blocking VCAM-1 by a neutralizing antibody reduced the G-CSF-dependent effects on stem cell migration and cardiac function. This is the first study showing that in virus-induced DCM VCAM-1/VLA-4 interaction is crucial for recruitment of circulating BMCs leading to beneficial anti-apoptotic effects resulting in improved cardiac function after G-CSF-induced mobilization.

Impairment of immunoproteasome function by ß5i/LMP7 subunit deficiency results in severe enterovirus myocarditis.

Proteasomes recognize and degrade poly-ubiquitinylated proteins. In infectious disease, cells activated by interferons (IFNs) express three unique catalytic subunits ß1i/LMP2, ß2i/MECL-1 and ß5i/LMP7 forming an alternative proteasome isoform, the immunoproteasome (IP). The in vivo function of IPs in pathogen-induced inflammation is still a matter of controversy. IPs were mainly associated with MHC class I antigen processing. However, recent findings pointed to a more general function of IPs in response to cytokine stress. Here, we report on the role of IPs in acute coxsackievirus B3 (CVB3) myocarditis reflecting one of the most common viral disease entities among young people. Despite identical viral load in both control and IP-deficient mice, IP-deficiency was associated with severe acute heart muscle injury reflected by large foci of inflammatory lesions and severe myocardial tissue damage. Exacerbation of acute heart muscle injury in this host was ascribed to disequilibrium in protein homeostasis in viral heart disease as indicated by the detection of increased proteotoxic stress in cytokine-challenged cardiomyocytes and inflammatory cells from IP-deficient mice. In fact, due to IP-dependent removal of poly-ubiquitinylated protein aggregates in the injured myocardium IPs protected CVB3-challenged mice from oxidant-protein damage. Impaired NFκB activation in IP-deficient cardiomyocytes and inflammatory cells and proteotoxic stress in combination with severe inflammation in CVB3-challenged hearts from IP-deficient mice potentiated apoptotic cell death in this host, thus exacerbating acute tissue damage. Adoptive T cell transfer studies in IP-deficient mice are in agreement with data pointing towards an effective CD8 T cell immune. This study therefore demonstrates that IP formation primarily protects the target organ of CVB3 infection from excessive inflammatory tissue damage in a virus-induced proinflammatory cytokine milieu.

Proteomic analyses of age related changes in A.BY/SnJ mouse hearts.

BACKGROUND: A.BY/SnJ mice are used to study pathological alterations in the heart due to enteroviral infections. Since age is a well-known factor influencing the susceptibility of mice to infection, response to stress and manifestation of cardiovascular diseases, the myocardial proteome of A.BY/SnJ mice aged 1 and 4 months was comparatively studied using two dimensional-differential in-gel electrophoresis (2D-DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: Complementary analyses by 2D-DIGE and gel-free LC-MS/MS revealed 96 distinct proteins displaying age associated alterations in their levels. Proteins related to protein transport, and transport chain, lipid metabolism and fatty acid transport showed significant changes in 4 months old mouse hearts compared to juvenile hearts. Proteins involved in lipid metabolism and transport were identified at significantly higher levels in older mice and dysregulation of proteins of the respiratory transport chain were observed. CONCLUSION: The current proteomics study discloses age dependent changes occurring in the hearts already in young mice of the strain A.BY/SnJ. Besides alterations in protein transport, we provide evidence that a decrease of ATP synthase in murine hearts starts already in the first months of life, leading to well-known low expression levels manifested in old mice thereby raising the possibility of reduced energy supply. In the first few months of murine life this seems to be compensated by an increased lipid metabolism. The functional alterations described should be considered during experimental setups in disease related studies.

CYP19A1 mediates severe SARS-CoV-2 disease outcome in males.

Male sex represents one of the major risk factors for severe COVID-19 outcome. However, underlying mechanisms that mediate sex-dependent disease outcome are as yet unknown. Here, we identify the CYP19A1 gene encoding for the testosterone-to-estradiol metabolizing enzyme CYP19A1 (also known as aromatase) as a host factor that contributes to worsened disease outcome in SARS-CoV-2-infected males. We analyzed exome sequencing data obtained from a human COVID-19 cohort (n = 2,866) using a machine-learning approach and identify a CYP19A1-activity-increasing mutation to be associated with the development of severe disease in men but not women. We further analyzed human autopsy-derived lungs (n = 86) and detect increased pulmonary CYP19A1 expression at the time point of death in men compared with women. In the golden hamster model, we show that SARS-CoV-2 infection causes increased CYP19A1 expression in the lung that is associated with dysregulated plasma sex hormone levels and reduced long-term pulmonary function in males but not females. Treatment of SARS-CoV-2-infected hamsters with a clinically approved CYP19A1 inhibitor (letrozole) improves impaired lung function and supports recovery of imbalanced sex hormones specifically in males. Our study identifies CYP19A1 as a contributor to sex-specific SARS-CoV-2 disease outcome in males. Furthermore, inhibition of CYP19A1 by the clinically approved drug letrozole may furnish a new therapeutic strategy for individualized patient management and treatment.

Cyclophilin A affects inflammation, virus elimination and myocardial fibrosis in coxsackievirus B3-induced myocarditis.

Extracellular cyclophilin A (CyPA) and its receptor Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147) modulate inflammatory processes beyond metalloproteinase (MMP) activity. Recently, we have shown that CyPA and CD147 are upregulated in patients with inflammatory cardiomyopathy. Here we investigate the role of CyPA and CD147 in murine coxsackievirus B3 (CVB3)-induced myocarditis. CVB3-infected CyPA(-/-) mice (129S6/SvEv) revealed a significantly reduced T-cell and macrophage recruitment at 8 days p.i. compared to wild-type mice. In A.BY/SnJ mice, treatment with the cyclophilin-inhibitor NIM811 was associated with a reduction of inflammatory lesions and MMP-9 expression but with enhanced virus replication 8 days p.i. At 28 days p.i. the extent of lesion areas was not affected bei NIM811, whereas the collagen content was reduced. Initiation of NIM811-treatment on day 12 (after an effective virus defense) resulted in an even more pronounced reduction of myocardial fibrosis. In conclusion, in CVB3-induced myocarditis CyPA is important for macrophage and T cell recruitment and effective virus defense and may represent a pharmacological target to modulate myocardial remodeling in myocarditis.