Die Bedeutung des Low Molecular Weight Protein 7 (LMP7)- spezifischen Inhibitors ONX 0914 bei der viralen Infektion der Maus

Herzinsuffizienz ist eine der häufigsten Todesursachen. Ein Auslöser hierfür kann eine entzündliche Herzerkrankung sein, die durch virale Infektionen beispielsweise mit CoxsackievirusB3 ausgelöst werden können. Dabei ist der Schaden im Herzmuskel sowohl durch direkte Viruszytotoxizität vermittelt als auch auf eine Aktivierung von immunologischen Prozessen zurückzuführen. Ein mögliches therapeutisches Ziel sind daher Modulatoren einer überschießend aktivierten Immunantwort. Diesbezüglich ist die zelluläre Proteolyse durch das Immunproteasom von Bedeutung. Das Immunproteasom wird anlagebedingt in Immunzellen exprimiert und während viraler Infektionen im Herzmuskel durch Interferonstimulus hochreguliert. Das Immunproteasom ist für die Aufrechterhaltung der Proteinhomöostase und Major Histocompatibility Complex-Klasse-I-Antigenprozessierung verantwortlich und moduliert zelluläre Signalkaskaden. Ziel dieser Studie war es, den immunmodulatorischen Effekt des Immunproteasominhibitors ONX 0914 während einer CoxsackievirusB3-Infektion zu untersuchen. Dazu benutzten wir ein Mausmodel mit genetisch-bedingt geringer (C57BL/6) oder hoher Suszeptibilität (A/J) gegenüber einer CoxsackievirusB3-Infektion. Die Mäuse wurden mit dem für die Immunproteasomuntereinheit LMP7 spezifischen Inhibitor ONX 0914 beginnend einen Tag vor CoxsackievirusB3-Infektion behandelt. Über 8 Tage wurden die Versuchsgruppen auf Anzeichen von Sepsis und Herzinsuffizienz untersucht. Ebenso wurde der Einfluss des Immunproteasoms auf die Zelldifferenzierung von Immunzellen in vivo sowie die Modulation pro-inflammatorischer Signalwege nach Aktivierung von Toll-Like Rezeptoren in Abhängigkeit von ONX 0914 in Makrophagen in vitro untersucht. Die ONX 0914-Behandlung bei niedrig-suszeptiblen C57BL/6-Mäusen resultierte in einer erhöhten Viruslast im Herzmuskel und einer gering verstärkten kardialen Pathologie. Es zeigte sich jedoch weder eine erhöhte Mortalität noch wurden andere Zeichen einer Herzinsuffizienz beobachtet. Insofern wiesen diese Mäuse unter Immunproteasom-inhibition weiterhin einen milden Phänotyp auf und bildeten eine adäquate antivirale Immunantwort aus. Im Gegensatz dazu wurde in dem hoch-suszeptiblen A/J-Stamm die kardiale Pathologie durch die ONX 0914 Behandlung aufgehoben. Der fulminante Krankheitsverlauf, der sich in der Kontrollgruppe zeigte, wurde durch eine Immunproteasominhibition verhindert. Dies zeigte sich anhand der reduzierten kardialen Inflammation und der verminderten Organschädigung. Außerdem konnte die an septische Verläufe erinnernde überschießende Aktivierung der Immunantwort deutlich reduziert werden. In dem hoch-empfindlichen A/J Stamm steigerte eine Behandlung mit ONX 0914 das bei Myokarditis reduzierte Herzzeitvolumen und bewahrte die Mäuse vor einem schweren Krankheitsverlauf mit hoher Mortalität. In vitro Untersuchungen bestätigten, dass ONX 0914 die proinflammatorische Zytokin- und Chemokinantwort auf virusgenetische molekulare Muster reduziert, was sich zumindest teilweise auf eine verringerte Mitogen-activated Proteinkinase-Aktivierung zurückführen lässt. Dies könnte die in vivo reduzierte proinflammatorische Immunantwort, die den septischen Phänotyp verhinderte, erklären. Die Ergebnisse dieser Studie zeigen, dass das Immunproteasom eine vielversprechende pharmakologische Zielstruktur bei Infektionen mit ausgeprägter Virus-induzierter Immunpathologie darstellen kann.Heart failure ranks among the leading causes for mortality. It can be caused by inflammatory heart diseases due to an infection with CoxsackievirusB3. Because the heart pathology upon virus infection is closely linked to direct viral pathology and immunopathology, it is crucial to identify targets modulating pre-disposing immune factors. A putative factor is the immunoproteasome, which is expressed by immune cells and is upregulated during viral infection by interferon signaling in cardiac tissue. The immunoproteasome is responsible for maintaining protein homeostasis and major histocompatibility complex class I processing, and modulates cell signaling. The aim of this study was to investigate the immunomodulatory effect of the immunoproteasome inhibitor ONX 0914 during CoxsackievirusB3 infection. Therefore, we utilized a murine model with genetically anchored low (C57BL/6) or high susceptibility (A/J) for CoxsackievirusB3 infection and the LMP7 immunoproteasome subunit selective inhibitor ONX 0914. An immunoproteasome inhibitor treatment was initiated prior to infection of mice with a CoxsackievirusB3 Nancy strain. Mice were analyzed for sepsis and heart failure parameters. Effects of immunoproteasome-proteolysis on immune cell differentiation were analyzed in vivo. Immunoproteasome-dependent modulation of pro-inflammatory signaling was investigated in vitro using bone marrow-derived macrophages. These cells and their progenitors represent key effector cells during CoxsackievirusB3-induced myocarditis. During viral infection in the low-grade susceptible C57BL/6-strain, ONX 0914-treatment resulted in an increased viral load of the heart with consequently slightly aggravated cardiac pathologies. However, neither increased mortality nor other signs for heart failure were detected. Conversely, in the highly susceptible A/J-strain heart pathology was reversed upon immunoproteasome inhibition. The fulminant pathology was prevented by ONX 0914-treatment as indicated by reduced heart inflammation and diminished organ damage. Furthermore, activation of a sepsis-like immune response found in vehicle-treated mice was prevented by ONX 0914. Whilst in C57BL/6 mice the immune-proteasome ameliorated viral replication in the heart, inhibitor-treated mice still showed low susceptibility to CoxsackievirusB3 infection and induced a potent antiviral immune response. However, with hereditary high susceptibility ONX 0914-treatment improved cardiac output during acute myocarditis, and saved animals from severe illness and high mortality. In vitro studies confirmed that ONX 0914-treatment reduced pro-inflammatory cytokine and chemokine production upon stimulation with viral genomic patterns partially attributed to reduced mitogen-activated protein kinase-activation. This could explain the attenuated pro-inflammatory response that prevented the septic phenotype. The results of this study suggest that the immunoproteasome might be a promising new drug target to overcome the consequences of overwhelming immune response activation during infection

Role of Proteasomes in Inflammation

The ubiquitin–proteasome system (UPS) is involved in multiple cellular functions including the regulation of protein homeostasis, major histocompatibility (MHC) class I antigen processing, cell cycle proliferation and signaling. In humans, proteasome loss-of-function mutations result in autoinflammation dominated by a prominent type I interferon (IFN) gene signature. These genomic alterations typically cause the development of proteasome-associated autoinflammatory syndromes (PRAAS) by impairing proteasome activity and perturbing protein homeostasis. However, an abnormal increased proteasomal activity can also be found in other human inflammatory diseases. In this review, we cast a light on the different clinical aspects of proteasomal activity in human disease and summarize the currently studied therapeutic approaches

Silencing the CSF-1 Axis Using Nanoparticle Encapsulated siRNA Mitigates Viral and Autoimmune Myocarditis

Myocarditis is an inflammatory disease of the heart muscle most commonly caused by viral infection and often maintained by autoimmunity. Virus-induced tissue damage triggers chemokine production and, subsequently, immune cell infiltration with pro-inflammatory and pro-fibrotic cytokine production follows. In patients, the overall inflammatory burden determines the disease outcome. Following the aim to define specific molecules that drive both immunopathology and/or autoimmunity in inflammatory heart disease, here we report on increased expression of colony stimulating factor 1 (CSF-1) in patients with myocarditis. CSF-1 controls monocytes originating from hematopoietic stem cells and subsequent progenitor stages. Both, monocytes and macrophages are centrally involved in mediating tissue damage and fibrotic scarring in the heart. CSF-1 influences monocytes via engagement of CSF-1 receptor, and it is also produced by cells of the mononuclear phagocyte system themselves. Based on this, we sought to modulate the virus-triggered inflammatory response in an experimental model of Coxsackievirus B3-induced myocarditis by silencing the CSF-1 axis in myeloid cells using nanoparticle-encapsulated siRNA. siCSF-1 inverted virus-mediated immunopathology as reflected by lower troponin T levels, a reduction of accumulating myeloid cells in heart tissue and improved cardiac function. Importantly, pathogen control was maintained and the virus was efficiently cleared from heart tissue. Since viral heart disease triggers heart-directed autoimmunity, in a second approach we investigated the influence of CSF-1 upon manifestation of heart tissue inflammation during experimental autoimmune myocarditis (EAM). EAM was induced in Balb/c mice by immunization with a myocarditogenic myosin-heavy chain-derived peptide dissolved in complete Freund's adjuvant. siCSF-1 treatment initiated upon established disease inhibited monocyte infiltration into heart tissue and this suppressed cardiac injury as reflected by diminished cardiac fibrosis and improved cardiac function at later states. Mechanistically, we found that suppression of CSF-1 production arrested both differentiation and maturation of monocytes and their precursors in the bone marrow. In conclusion, during viral and autoimmune myocarditis silencing of the myeloid CSF-1 axis by nanoparticle-encapsulated siRNA is beneficial for preventing inflammatory tissue damage in the heart and preserving cardiac function without compromising innate immunity's critical defense mechanisms

The immunoproteasome‐specific inhibitor ONX 0914 reverses susceptibility to acute viral myocarditis

Abstract Severe heart pathology upon virus infection is closely associated with the immunological equipment of the host. Since there is no specific treatment available, current research focuses on identifying new drug targets to positively modulate predisposing immune factors. Utilizing a murine model with high susceptibility to coxsackievirus B3‐induced myocarditis, this study describes ONX 0914—an immunoproteasome‐specific inhibitor—as highly protective during severe heart disease. Represented by reduced heart infiltration of monocytes/macrophages and diminished organ damage, ONX 0914 treatment reversed fulminant pathology. Virus‐induced immune response features like overwhelming pro‐inflammatory cytokine and chemokine production as well as a progressive loss of lymphocytes all being reminiscent of a sepsis‐like disease course were prevented by ONX 0914. Although the viral burden was only minimally affected in highly susceptible mice, resulting maintenance of immune homeostasis improved the cardiac output, and saved animals from severe illness as well as high mortality. Altogether, this could make ONX 0914 a potent drug for the treatment of severe virus‐mediated inflammation of the heart and might rank immunoproteasome inhibitors among drugs for preventing pathogen‐induced immunopathology

Silencing the CSF-1 Axis Using Nanoparticle Encapsulated siRNA Mitigates Viral and Autoimmune Myocarditis

Myocarditis is an inflammatory disease of the heart muscle most commonly caused by viral infection and often maintained by autoimmunity. Virus-induced tissue damage triggers chemokine production and, subsequently, immune cell infiltration with pro-inflammatory and pro-fibrotic cytokine production follows. In patients, the overall inflammatory burden determines the disease outcome. Following the aim to define specific molecules that drive both immunopathology and/or autoimmunity in inflammatory heart disease, here we report on increased expression of colony stimulating factor 1 (CSF-1) in patients with myocarditis. CSF-1 controls monocytes originating from hematopoietic stem cells and subsequent progenitor stages. Both, monocytes and macrophages are centrally involved in mediating tissue damage and fibrotic scarring in the heart. CSF-1 influences monocytes via engagement of CSF-1 receptor, and it is also produced by cells of the mononuclear phagocyte system themselves. Based on this, we sought to modulate the virus-triggered inflammatory response in an experimental model of Coxsackievirus B3-induced myocarditis by silencing the CSF-1 axis in myeloid cells using nanoparticle-encapsulated siRNA. siCSF-1 inverted virus-mediated immunopathology as reflected by lower troponin T levels, a reduction of accumulating myeloid cells in heart tissue and improved cardiac function. Importantly, pathogen control was maintained and the virus was efficiently cleared from heart tissue. Since viral heart disease triggers heart-directed autoimmunity, in a second approach we investigated the influence of CSF-1 upon manifestation of heart tissue inflammation during experimental autoimmune myocarditis (EAM). EAM was induced in Balb/c mice by immunization with a myocarditogenic myosin-heavy chain-derived peptide dissolved in complete Freund's adjuvant. siCSF-1 treatment initiated upon established disease inhibited monocyte infiltration into heart tissue and this suppressed cardiac injury as reflected by diminished cardiac fibrosis and improved cardiac function at later states. Mechanistically, we found that suppression of CSF-1 production arrested both differentiation and maturation of monocytes and their precursors in the bone marrow. In conclusion, during viral and autoimmune myocarditis silencing of the myeloid CSF-1 axis by nanoparticle-encapsulated siRNA is beneficial for preventing inflammatory tissue damage in the heart and preserving cardiac function without compromising innate immunity's critical defense mechanisms. Keywords: inflammation and immunmodulation; innate immunity, cytokines; monocytes/macrophages; RNA interference; virus; infection-immunology; myocarditi