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.

Influenza A viruses are a threat to humans due to their ability to cross species barriers, as illustrated by the 2009 H1N1v pandemic and sporadic H5N1 transmissions. Interspecies transmission requires adaptation of the viral polymerase to importin-α, a cellular protein that mediates transport into the nucleus where transcription and replication of the viral genome takes place. In this study, we analysed replication, host specificity and pathogenicity of avian and mammalian influenza viruses, in importin-α-silenced cells and importin-α-knockout mice, to understand the role of individual importin-α isoforms in adaptation. For efficient virus replication, the polymerase subunit PB2 and the nucleoprotein (NP) of avian viruses required importin-α3, whereas PB2 and NP of mammalian viruses showed importin-α7 specificity. H1N1v replication depended on both, importin-α3 and -α7, suggesting ongoing adaptation of this virus. Thus, differences in importin-α specificity are determinants of host range underlining the importance of the nuclear envelope in interspecies transmission.

Transforming growth factor ␤ (TGF-␤) has been shown to participate in the pathophysiology of diabetic complications. As shown most recently, TGF-␤ stimulates the expression of a distinct serine͞threonine kinase (hSGK) which had previously been cloned as an early gene transcriptionally regulated by cell volume alterations. The present study was performed to elucidate transcription and function of hSGK in diabetic nephropathy. As shown by Northern blotting, an increase of extracellular glucose concentration increased hSGK mRNA levels in cultured cells, an effect qualitatively mimicked by osmotic cell shrinkage or treatment with TGF-␤ (2 g͞liter), phorbol 12,13-didecanoate (1 M), or the Ca 2؉ ionophore ionomycin (1 M) and blunted by high concentrations of nifedipine (10 and 100 M). In situ hybridization revealed that hSGK transcription was markedly enhanced in diabetic nephropathy, with particularly high expression in mesangial cells, interstitial cells, and cells in thick ascending limbs of Henle's loop and distal tubules. According to voltage clamp and tracer flux studies in Xenopus oocytes expressing the renal epithelial Na ؉ channel ENaC or the mouse thick ascending limb Na ؉ ,K ؉ ,2Cl ؊ cotransporter BSC-1, coexpression with hSGK stimulated ENaC and BSC-1 11-fold and 6-fold, respectively, effects reversed by kinase inhibitors staurosporine (1 M) and chelerythrine (1 M) and not elicited by inactive hSGK. In conclusion, excessive extracellular glucose concentrations enhance hSGK transcription, which in turn stimulates renal tubular Na ؉ transport. These observations disclose an additional element in the pathophysiology of diabetic nephropathy.protein kinase C ͉ endothelial cells ͉ kidney ͉ epithelial Na ϩ channel ͉ Na ϩ ,K ϩ ,2Cl Ϫ cotransporter

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Proteasomes recognize and degrade poly-ubiquitinylated proteins. In infectious disease, cells activated by interferons (IFNs) express three unique catalytic subunits β1i/LMP2, β2i/MECL-1 and β5i/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.

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.transcription factors | transgenic mice

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. response. Type I IFNs contribute to the suppression of viral titers and thereby ameliorate invasion of immune cells into the heart, contributing to improved survival in CVB3 infection. [6][7][8] Type I IFN-dependent processes resulted in improved cardiac function during viral cardiomyopathy 7,9 and ensured long-term survival in CVB3-positive dilated cardiomyopathy patients. 10Binding of type I IFNs to their cognate receptors results in the induction of IFN-stimulated gene of 15 kDa (ISG15), a small ubiquitin family protein consisting of 2 ubiquitinlike folds.11 ISG15 is involved in the struggle against pathogens.12-16 ISG15 modification, the process by which ISG15 is covalently attached to lysine residues of target proteins, is mediated through the sequential action of a type I IFN-induced E1-E2-E3 enzymatic cascade, 17 involving the E1-activating enzyme Ube1L, 18 E2-conjugating enzyme Ube2L6, 19 and E3 ligases Herc5 and Herc6 20 in humans and mice, respectively. The isopeptidase USP18 specifically removes ISG15 from ISG15-modified substrates. 21Pursuing the aim to define host determinants that influence the pathogenesis of viral cardiomyopathy, we provide the first evidence for the impact of the ...