Genome-wide consequences of deleting any single gene.

Loss or duplication of chromosome segments can lead to further genomic changes associated with cancer. However, it is not known whether only a select subset of genes is responsible for driving further changes. To determine whether perturbation of any given gene in a genome suffices to drive subsequent genetic changes, we analyzed the yeast knockout collection for secondary mutations of functional consequence. Unlike wild-type, most gene knockout strains were found to have one additional mutant gene affecting nutrient responses and/or heat-stress-induced cell death. Moreover, independent knockouts of the same gene often evolved mutations in the same secondary gene. Genome sequencing identified acquired mutations in several human tumor suppressor homologs. Thus, mutation of any single gene may cause a genomic imbalance, with consequences sufficient to drive adaptive genetic changes. This complicates genetic analyses but is a logical consequence of losing a functional unit originally acquired under pressure during evolution.

Whi2 is a conserved negative regulator of TORC1 in response to low amino acids.

Yeast WHI2 was originally identified in a genetic screen for regulators of cell cycle arrest and later suggested to function in general stress responses. However, the function of Whi2 is unknown. Whi2 has predicted structure and sequence similarity to human KCTD family proteins, which have been implicated in several cancers and are causally associated with neurological disorders but are largely uncharacterized. The identification of conserved functions between these yeast and human proteins may provide insight into disease mechanisms. We report that yeast WHI2 is a new negative regulator of TORC1 required to suppress TORC1 activity and cell growth specifically in response to low amino acids. In contrast to current opinion, WHI2 is dispensable for TORC1 inhibition in low glucose. The only widely conserved mechanism that actively suppresses both yeast and mammalian TORC1 specifically in response to low amino acids is the conserved SEACIT/GATOR1 complex that inactivates the TORC1-activating RAG-like GTPases. Unexpectedly, Whi2 acts independently and simultaneously with these established GATOR1-like Npr2-Npr3-Iml1 and RAG-like Gtr1-Gtr2 complexes, and also acts independently of the PKA pathway. Instead, Whi2 inhibits TORC1 activity through its binding partners, protein phosphatases Psr1 and Psr2, which were previously thought to only regulate amino acid levels downstream of TORC1. Furthermore, the ability to suppress TORC1 is conserved in the SKP1/BTB/POZ domain-containing, Whi2-like human protein KCTD11 but not other KCTD family members tested.

Sca-1+ cardiac fibroblasts promote development of heart failure.

The causative effect of GM-CSF produced by cardiac fibroblasts to development of heart failure has not been shown. We identified the pathological GM-CSF-producing cardiac fibroblast subset and the specific deletion of IL-17A signaling to these cells attenuated cardiac inflammation and heart failure. We describe here the CD45- CD31- CD29+ mEF-SK4+ PDGFRα+ Sca-1+ periostin+ (Sca-1+ ) cardiac fibroblast subset as the main GM-CSF producer in both experimental autoimmune myocarditis and myocardial infarction mouse models. Specific ablation of IL-17A signaling to Sca-1+ periostin+ cardiac fibroblasts (PostnCre Il17rafl/fl ) protected mice from post-infarct heart failure and death. Moreover, PostnCre Il17rafl/fl mice had significantly fewer GM-CSF-producing Sca-1+ cardiac fibroblasts and inflammatory Ly6Chi monocytes in the heart. Sca-1+ cardiac fibroblasts were not only potent GM-CSF producers, but also exhibited plasticity and switched their cytokine production profiles depending on local microenvironments. Moreover, we also found GM-CSF-positive cardiac fibroblasts in cardiac biopsy samples from heart failure patients of myocarditis or ischemic origin. Thus, this is the first identification of a pathological GM-CSF-producing cardiac fibroblast subset in human and mice hearts with myocarditis and ischemic cardiomyopathy. Sca-1+ cardiac fibroblasts direct the type of immune cells infiltrating the heart during cardiac inflammation and drive the development of heart failure.

Macrophages and cardiac fibroblasts are the main producers of eotaxins and regulate eosinophil trafficking to the heart.

Cardiac manifestations are a major cause of morbidity and mortality in patients with eosinophil-associated diseases. Eosinophils are thought to play a pathogenic role in myocarditis. We investigated the pathways that recruit eosinophils to the heart using a model of eosinophilic myocarditis, in which experimental autoimmune myocarditis (EAM) is induced in IFNγ-/- IL-17A-/- mice. Two conditions are necessary for efficient eosinophil trafficking to the heart: high eotaxin (CCL11, CCL24) expression in the heart and expression of the eotaxin receptor CCR3 by eosinophils. We identified cardiac fibroblasts as the source of CCL11 in the heart interstitium. CCL24 is produced by F4/80+ macrophages localized at inflammatory foci in the heart. Expression of CCL11 and CCL24 is controlled by Th2 cytokines, IL-4 and IL-13. To determine the relevance of this pathway in humans, we analyzed endomyocardial biopsy samples from myocarditis patients. Expression of CCL11 and CCL26 was significantly increased in eosinophilic myocarditis compared to chronic lymphocytic myocarditis and positively correlated with the number of eosinophils. Thus, eosinophil trafficking to the heart is dependent on the eotaxin-CCR3 pathway in a mouse model of EAM and associated with cardiac eotaxin expression in patients with eosinophilic myocarditis. Blocking this pathway may prevent eosinophil-mediated cardiac damage.

Pathogenic IL-23 signaling is required to initiate GM-CSF-driven autoimmune myocarditis in mice.

Using a mouse model of experimental autoimmune myocarditis (EAM), we showed for the first time that IL-23 stimulation of CD4(+) T cells is required only briefly at the initiation of GM-CFS-dependent cardiac autoimmunity. IL-23 signal, acting as a switch, turns on pathogenicity of CD4(+) T cells, and becomes dispensable once autoreactivity is established. Il23a(-/-) mice failed to mount an efficient Th17 response to immunization, and were protected from myocarditis. However, remarkably, transient IL-23 stimulation ex vivo fully restored pathogenicity in otherwise nonpathogenic CD4(+) T cells raised from Il23a(-/-) donors. Thus, IL-23 may no longer be necessary to uphold inflammation in established autoimmune diseases. In addition, we demonstrated that IL-23-induced GM-CSF mediates the pathogenicity of CD4(+) T cells in EAM. The neutralization of GM-CSF abrogated cardiac inflammation. However, sustained IL-23 signaling is required to maintain IL-17A production in CD4(+) T cells. Despite inducing inflammation in Il23a(-/-) recipients comparable to wild-type (WT), autoreactive CD4(+) T cells downregulated IL-17A production without persistent IL-23 signaling. This divergence on the controls of GM-CSF-dependent pathogenicity on one side and IL-17A production on the other side may contribute to the discrepant efficacies of anti-IL-23 therapy in different autoimmune diseases.

Collaborative Interferon-γ and Interleukin-17 Signaling Protects the Oral Mucosa from Staphylococcus aureus.

Infections with Staphylococcus aureus are a continuing and growing problem in community and hospital settings. Preclinical animal modeling of S. aureus relies on experimental infection, which carries some limitations. We describe here a novel, spontaneous model of oral staphylococcal infection in double knockout mice, deficient in the receptors for IL-17 (IL-17RA) and interferon (IFN)-γ (IFNγRI), beginning at 6 to 8 weeks of age. IFNγRI(-/-)IL17RA(-/-) (GRAKO) mice developed progressive oral abscesses. Cytometric methods revealed extensive neutrophilic infiltration of oral tissues in GRAKO mice; further investigation evidenced that IL-17 predominated neutrophil defects in these mice. To investigate the contribution of IFN-γ signaling to this native host defense to S. aureus, we observed perturbations of monocyte recruitment and macrophage differentiation in the oral tissues of GRAKO mice, and CXCL9/chemokine ligand receptor (CXCR)3-driven recruitment of T-cell oral tissues and draining lymph nodes. To address the former finding, we depleted macrophages and monocytes in vivo from IL17RA(-/-) mice using liposomes loaded with clodronate. This treatment elicited oral abscesses, recapitulating the phenotype of GRAKO mice. From these findings, we propose novel collaborative functions of IL-17 and IFN-γ, acting through neutrophils and macrophages, respectively, in native mucocutaneous host defenses to S. aureus.

Regulation of autoimmune myocarditis by host responses to the microbiome.

The extensive, diverse communities that constitute the microbiome are increasingly appreciated as important regulators of human health and disease through inflammatory, immune, and metabolic pathways. We sought to elucidate pathways by which microbiota contribute to inflammatory, autoimmune cardiac disease. We employed an animal model of experimental autoimmune myocarditis (EAM), which results in inflammatory and autoimmune pathophysiology and subsequent maladaptive cardiac remodeling and heart failure. Antibiotic dysbiosis protected mice from EAM and fibrotic cardiac dysfunction. Additionally, mice derived from different sources with different microbiome colonization profiles demonstrated variable susceptibility to disease. Unexpectedly, it did not track with segmented filamentous bacteria (SFB)-driven Th17 programming of CD4+ T cells in the steady-state gut. Instead, we found disease susceptibility to track with presence of type 3 innate lymphoid cells (ILC3s). Ablating ILCs by antibody depletion or genetic tools in adoptive transfer variants of the EAM model demonstrated that ILCs and microbiome profiles contributed to the induction of CCL20/CCR6-mediated inflammatory chemotaxis to the diseased heart. From these data, we conclude that sensing of the microbiome by ILCs is an important checkpoint in the development of inflammatory cardiac disease processes through their ability to elicit cardiotropic chemotaxis.

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.

The Cardiac Microenvironment Instructs Divergent Monocyte Fates and Functions in Myocarditis.

Two types of monocytes, Ly6Chi and Ly6Clo, infiltrate the heart in murine experimental autoimmune myocarditis (EAM). We discovered a role for cardiac fibroblasts in facilitating monocyte-to-macrophage differentiation of both Ly6Chi and Ly6Clo cells, allowing these macrophages to perform divergent functions in myocarditis progression. During the acute phase of EAM, IL-17A is highly abundant. It signals through cardiac fibroblasts to attenuate efferocytosis of Ly6Chi monocyte-derived macrophages (MDMs) and simultaneously prevents Ly6Clo monocyte-to-macrophage differentiation. We demonstrated an inverse clinical correlation between heart IL-17A levels and efferocytic receptor expressions in humans with heart failure (HF). In the absence of IL-17A signaling, Ly6Chi MDMs act as robust phagocytes and are less pro-inflammatory, whereas Ly6Clo monocytes resume their differentiation into MHCII+ macrophages. We propose that MHCII+Ly6Clo MDMs are associated with the reduction of cardiac fibrosis and prevention of the myocarditis sequalae.

Eosinophils in Autoimmune Diseases.

Eosinophils are multifunctional granulocytes that contribute to initiation and modulation of inflammation. Their role in asthma and parasitic infections has long been recognized. Growing evidence now reveals a role for eosinophils in autoimmune diseases. In this review, we summarize the function of eosinophils in inflammatory bowel diseases, neuromyelitis optica, bullous pemphigoid, autoimmune myocarditis, primary biliary cirrhosis, eosinophilic granulomatosis with polyangiitis, and other autoimmune diseases. Clinical studies, eosinophil-targeted therapies, and experimental models have contributed to our understanding of the regulation and function of eosinophils in these diseases. By examining the role of eosinophils in autoimmune diseases of different organs, we can identify common pathogenic mechanisms. These include degranulation of cytotoxic granule proteins, induction of antibody-dependent cell-mediated cytotoxicity, release of proteases degrading extracellular matrix, immune modulation through cytokines, antigen presentation, and prothrombotic functions. The association of eosinophilic diseases with autoimmune diseases is also examined, showing a possible increase in autoimmune diseases in patients with eosinophilic esophagitis, hypereosinophilic syndrome, and non-allergic asthma. Finally, we summarize key future research needs.

Eosinophil-derived IL-4 drives progression of myocarditis to inflammatory dilated cardiomyopathy.

Inflammatory dilated cardiomyopathy (DCMi) is a major cause of heart failure in children and young adults. DCMi develops in up to 30% of myocarditis patients, but the mechanisms involved in disease progression are poorly understood. Patients with eosinophilia frequently develop cardiomyopathies. In this study, we used the experimental autoimmune myocarditis (EAM) model to determine the role of eosinophils in myocarditis and DCMi. Eosinophils were dispensable for myocarditis induction but were required for progression to DCMi. Eosinophil-deficient ΔdblGATA1 mice, in contrast to WT mice, showed no signs of heart failure by echocardiography. Induction of EAM in hypereosinophilic IL-5Tg mice resulted in eosinophilic myocarditis with severe ventricular and atrial inflammation, which progressed to severe DCMi. This was not a direct effect of IL-5, as IL-5TgΔdblGATA1 mice were protected from DCMi, whereas IL-5-/- mice exhibited DCMi comparable with WT mice. Eosinophils drove progression to DCMi through their production of IL-4. Our experiments showed eosinophils were the major IL-4-expressing cell type in the heart during EAM, IL-4-/- mice were protected from DCMi like ΔdblGATA1 mice, and eosinophil-specific IL-4 deletion resulted in improved heart function. In conclusion, eosinophils drive progression of myocarditis to DCMi, cause severe DCMi when present in large numbers, and mediate this process through IL-4.