Short-chain fatty acids (SCFAs), which are generated by the bacterial fermentation of dietary fibers, promote expansion of regulatory T cells (Tregs). Potential therapeutic value of SCFAs has been recently highlighted in the experimental models of T cell-mediated autoimmunity and allergic inflammation. These studies suggest that physiological intestinal concentrations of SCFAs within the millimolar range are crucial for dampening inflammation-mediated processes. Here, we describe opposing effects of SCFAs on T cell-mediated immune responses. In accordance with published data, lower butyrate concentrations facilitated differentiation of Tregs in vitro and in vivo under steady-state conditions. In contrast, higher concentrations of butyrate induced expression of the transcription factor T-bet in all investigated T cell subsets resulting in IFN-γ-producing Tregs or conventional T cells. This effect was mediated by the inhibition of histone deacetylase activity and was independent of SCFA-receptors FFA2 and FFA3 as well as of Na+-coupled SCFA transporter Slc5a8. Importantly, while butyrate was not able to induce the generation of Tregs in the absence of TGF-β1, the expression of T-bet and IFN-γ was triggered upon stimulation of CD4+ T cells with this SCFA alone. Moreover, the treatment of germ-free mice with butyrate enhanced the expression of T-bet and IFN-γ during acute colitis. Our data reveal that, depending on its concentration and immunological milieu, butyrate may exert either beneficial or detrimental effects on the mucosal immune system.

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.

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.

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 damageassociated 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.Keywords: Infection r Inflammation r Innate immunity r Myocarditis r Pentraxin3 r Proteasome r Stress response r Virus Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe myocardium can be injured by various pathophysiological processes. Myocarditis is an excellent model of heart muscle Correspondence: Dr. Antje Voigt e-mail: antje.voigt@charite.de injury for dissecting processes of the heart stress response to inflammation. In developed countries, myocarditis most commonly results from a virus infection, often by coxsackievirus B3 (CVB3) and adenoviruses. Fulminant injury of the heart muscle in * These authors contributed equally to this work.C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 620Anna Paeschke et al. Eur. J. Immunol. 2016. 46: 619-633 viral myocarditis is attributed to both, direct virus-mediated cytotoxic effects and destruction of the heart muscle by the immune system [1,2]. The balance of intrinsic and innate immune mechanisms determines whether there is repair of the heart muscle or progression to chronic inflammation. This process involves the engagement of pattern recognition receptors (PRRs) either with pathogen-associated molecular patterns (PAMPs) such as viral RNA, or, upon the release of endogenous material, with damageassociated molecular patterns (DAMPs) [3]. Our knowledge on disease course in patients comes mainly from the mouse model of CVB3 myocarditis. Cardiotropic CVB3 infects mice and replicates to high titers, causing acute myocarditis (ACM) that faithfully mirrors...

Protein modification with ISG15 (ISGylation) represents a major type I IFN–induced antimicrobial system. Common mechanisms of action and species-specific aspects of ISGylation, however, are still ill defined and controversial. We used a multiphasic coxsackievirus B3 (CV) infection model with a first wave resulting in hepatic injury of the liver, followed by a second wave culminating in cardiac damage. This study shows that ISGylation sets nonhematopoietic cells into a resistant state, being indispensable for CV control, which is accomplished by synergistic activity of ISG15 on antiviral IFIT1/3 proteins. Concurrent with altered energy demands, ISG15 also adapts liver metabolism during infection. Shotgun proteomics, in combination with metabolic network modeling, revealed that ISG15 increases the oxidative capacity and promotes gluconeogenesis in liver cells. Cells lacking the activity of the ISG15-specific protease USP18 exhibit increased resistance to clinically relevant CV strains, therefore suggesting that stabilizing ISGylation by inhibiting USP18 could be exploited for CV-associated human pathologies.

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.

Our results implicate Foxo3 in regulating NK cell function and suggest Foxo3 playing an important role in the antiviral innate immunity. Thus, enhanced FOXO3 activity such as in the polymorphism rs12212067 may be protective in chronic inflammation such as cancer and cardiovascular disease but disadvantageous to control acute viral infection.

Inhibition of proteasome function by small molecules is highly efficacious in cancer treatment. Other than non-selective proteasome inhibitors, immunoproteasome-specific inhibitors allow for specific targeting of the proteasome in immune cells and the profound anti-inflammatory potential of such compounds revealed implications for inflammatory scenarios. For pathogen-triggered inflammation, however, the efficacy of immunoproteasome inhibitors is controversial. In this study, we investigated how ONX 0914, an immunoproteasome-selective inhibitor, influences CoxsackievirusB3 infection in NMRI mice, resulting in the development of acute and chronic myocarditis, which is accompanied by formation of the immunoproteasome in heart tissue. In groups in which ONX 0914 treatment was initiated once viral cytotoxicity had emerged in the heart, ONX 0914 had no anti-inflammatory effect in the acute or chronic stages. ONX 0914 treatment initiated prior to infection, however, increased viral cytotoxicity in cardiomyocytes, promoting infiltration of myeloid immune cells into the heart. At this stage, ONX 0914 completely inhibited the β5 subunit of the standard cardiac proteasome and less efficiently blocked its immunoproteasome counterpart LMP7. In conclusion, ONX 0914 unselectively perturbs cardiac proteasome function in viral myocarditis of NMRI mice, reduces the capacity of the host to control the viral burden and promotes cardiac inflammation.