RESUMO
Inflammation can trigger lasting phenotypes in immune and non-immune cells. Whether and how human infections and associated inflammation can form innate immune memory in hematopoietic stem and progenitor cells (HSPC) has remained unclear. We found that circulating HSPC, enriched from peripheral blood, captured the diversity of bone marrow HSPC, enabling investigation of their epigenomic reprogramming following coronavirus disease 2019 (COVID-19). Alterations in innate immune phenotypes and epigenetic programs of HSPC persisted for months to 1 year following severe COVID-19 and were associated with distinct transcription factor (TF) activities, altered regulation of inflammatory programs, and durable increases in myelopoiesis. HSPC epigenomic alterations were conveyed, through differentiation, to progeny innate immune cells. Early activity of IL-6 contributed to these persistent phenotypes in human COVID-19 and a mouse coronavirus infection model. Epigenetic reprogramming of HSPC may underlie altered immune function following infection and be broadly relevant, especially for millions of COVID-19 survivors.
Assuntos
COVID-19 , Memória Epigenética , Síndrome de COVID-19 Pós-Aguda , Animais , Humanos , Camundongos , Diferenciação Celular , COVID-19/imunologia , Modelos Animais de Doenças , Células-Tronco Hematopoéticas , Inflamação/genética , Imunidade Treinada , Monócitos/imunologia , Síndrome de COVID-19 Pós-Aguda/genética , Síndrome de COVID-19 Pós-Aguda/imunologia , Síndrome de COVID-19 Pós-Aguda/patologiaRESUMO
Cachexia represents a leading cause of morbidity and mortality in various cancers, chronic inflammation and infections. Understanding of the mechanisms that drive cachexia has remained limited, especially for infection-associated cachexia (IAC). In the present paper we describe a model of reversible cachexia in mice with chronic viral infection and identify an essential role for CD8+ T cells in IAC. Cytokines linked to cancer-associated cachexia did not contribute to IAC. Instead, virus-specific CD8+ T cells caused morphologic and molecular changes in the adipose tissue, which led to depletion of lipid stores. These changes occurred at a time point that preceded the peak of the CD8+ T cell response and required T cell-intrinsic type I interferon signaling and antigen-specific priming. Our results link systemic antiviral immune responses to adipose-tissue remodeling and reveal an underappreciated role of CD8+ T cells in IAC.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Caquexia/etiologia , Viroses/complicações , Viroses/imunologia , Tecido Adiposo/diagnóstico por imagem , Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Tecido Adiposo/virologia , Animais , Linfócitos T CD8-Positivos/metabolismo , Caquexia/diagnóstico por imagem , Caquexia/metabolismo , Caquexia/patologia , Doença Crônica , Citocinas/sangue , Citocinas/metabolismo , Feminino , Interferon Tipo I/metabolismo , Metabolismo dos Lipídeos , Lipólise , Ativação Linfocitária/imunologia , Vírus da Coriomeningite Linfocítica , Imageamento por Ressonância Magnética/métodos , Masculino , Camundongos , Transdução de Sinais , Viroses/virologiaRESUMO
Over the past 10 years, the field of immunometabolism made great strides to unveil the crucial role of intracellular metabolism in regulating immune cell function. Emerging insights into how systemic inflammation and metabolism influence each other provide a critical additional dimension on the organismal level. Here, we discuss the concept of systemic immunometabolism and review the current understanding of the communication circuits that underlie the reciprocal impact of systemic inflammation and metabolism across organs in inflammatory and infectious diseases, as well as how these mechanisms apply to homeostasis. We present current challenges of systemic immunometabolic research, and in this context, highlight opportunities and put forward ideas to effectively explore organismal physiological complexity in both health and disease.
Assuntos
Tecido Adiposo/imunologia , Tecido Adiposo/metabolismo , Metabolismo Energético/fisiologia , Sistema Imunitário/metabolismo , Tecido Adiposo/citologia , Homeostase/imunologia , Humanos , Inflamação/metabolismoRESUMO
Infections induce complex host responses linked to antiviral defense, inflammation, and tissue damage and repair. We hypothesized that the liver, as a central metabolic hub, may orchestrate systemic metabolic changes during infection. We infected mice with chronic lymphocytic choriomeningitis virus (LCMV), performed RNA sequencing and proteomics of liver tissue, and integrated these data with serum metabolomics at different infection phases. Widespread reprogramming of liver metabolism occurred early after infection, correlating with type I interferon (IFN-I) responses. Viral infection induced metabolic alterations of the liver that depended on the interferon alpha/beta receptor (IFNAR1). Hepatocyte-intrinsic IFNAR1 repressed the transcription of metabolic genes, including Otc and Ass1, which encode urea cycle enzymes. This led to decreased arginine and increased ornithine concentrations in the circulation, resulting in suppressed virus-specific CD8+ T cell responses and ameliorated liver pathology. These findings establish IFN-I-induced modulation of hepatic metabolism and the urea cycle as an endogenous mechanism of immunoregulation. VIDEO ABSTRACT.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Interferon Tipo I/imunologia , Fígado/metabolismo , Vírus da Coriomeningite Linfocítica/imunologia , Receptor de Interferon alfa e beta/metabolismo , Animais , Arginina/sangue , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Feminino , Hepatócitos/metabolismo , Fígado/imunologia , Fígado/virologia , Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ornitina/sangue , Ornitina Carbamoiltransferase/genética , Transdução de Sinais/imunologia , Ureia/metabolismo , Células VeroRESUMO
Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.
Assuntos
Histona-Lisina N-Metiltransferase/imunologia , NF-kappa B/imunologia , Infecções por Orthomyxoviridae/imunologia , Orthomyxoviridae/imunologia , Pneumonia/imunologia , Superinfecção/imunologia , Animais , Quimiocina CXCL1/imunologia , Suscetibilidade a Doenças , Feminino , Interferon Tipo I/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Infecções por Orthomyxoviridae/enzimologia , Infecções por Orthomyxoviridae/virologia , Pneumonia/enzimologia , Pneumonia/virologia , RNA/química , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Organismos Livres de Patógenos Específicos , Superinfecção/enzimologia , Superinfecção/microbiologiaRESUMO
The mammalian immune system implements a remarkably effective set of mechanisms for fighting pathogens1. Its main components are haematopoietic immune cells, including myeloid cells that control innate immunity, and lymphoid cells that constitute adaptive immunity2. However, immune functions are not unique to haematopoietic cells, and many other cell types display basic mechanisms of pathogen defence3-5. To advance our understanding of immunology outside the haematopoietic system, here we systematically investigate the regulation of immune genes in the three major types of structural cells: epithelium, endothelium and fibroblasts. We characterize these cell types across twelve organs in mice, using cellular phenotyping, transcriptome sequencing, chromatin accessibility profiling and epigenome mapping. This comprehensive dataset revealed complex immune gene activity and regulation in structural cells. The observed patterns were highly organ-specific and seem to modulate the extensive interactions between structural cells and haematopoietic immune cells. Moreover, we identified an epigenetically encoded immune potential in structural cells under tissue homeostasis, which was triggered in response to systemic viral infection. This study highlights the prevalence and organ-specific complexity of immune gene activity in non-haematopoietic structural cells, and it provides a high-resolution, multi-omics atlas of the epigenetic and transcriptional networks that regulate structural cells in the mouse.
Assuntos
Endotélio/imunologia , Células Epiteliais/imunologia , Fibroblastos/imunologia , Regulação da Expressão Gênica/imunologia , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Especificidade de Órgãos/imunologia , Imunidade Adaptativa , Animais , Cromatina/genética , Cromatina/metabolismo , Endotélio/citologia , Epigênese Genética/imunologia , Epigenoma/genética , Células Epiteliais/citologia , Feminino , Fibroblastos/citologia , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Redes Reguladoras de Genes/imunologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Sistema Imunitário/virologia , Imunidade Inata , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Masculino , Camundongos , Especificidade de Órgãos/genética , Transcrição Gênica/imunologia , Transcriptoma/genéticaRESUMO
Vaccination will likely be a key component of strategies to curtail or prevent future sarbecovirus pandemics and to reduce the prevalence of infection and disease by future SARS-CoV-2 variants. A "pan-sarbecovirus" vaccine, that provides maximum possible mitigation of human disease, should elicit neutralizing antibodies with maximum possible breadth. By positioning multiple different receptor binding domain (RBD) antigens in close proximity on a single immunogen, it is postulated that cross-reactive B cell receptors might be selectively engaged. Heteromultimeric vaccines could therefore elicit individual antibodies that neutralize a broad range of viral species. Here, we use model systems to investigate the ability of multimeric sarbecovirus RBD immunogens to expand cross-reactive B cells and elicit broadly reactive antibodies. Homomultimeric RBD immunogens generated higher serum neutralizing antibody titers than the equivalent monomeric immunogens, while heteromultimeric RBD immunogens generated neutralizing antibodies recognizing each RBD component. Moreover, RBD heterodimers elicited a greater fraction of cross-reactive germinal center B cells and cross-reactive RBD binding antibodies than did homodimers. However, when serum antibodies from RBD heterodimer-immunized mice were depleted using one RBD component, neutralization activity against the homologous viral pseudotype was removed, but neutralization activity against pseudotypes corresponding to the other RBD component was unaffected. Overall, simply combining divergent RBDs in a single immunogen generates largely separate sets of individual RBD-specific neutralizing serum antibodies that are mostly incapable of neutralizing viruses that diverge from the immunogen components.
Assuntos
Anticorpos Neutralizantes , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Animais , Camundongos , Humanos , Anticorpos Antivirais , Testes de Neutralização , Vacinação , Glicoproteína da Espícula de Coronavírus/químicaRESUMO
Harnessing the immune system to eradicate tumors requires identification and targeting of tumor antigens, including tumor-specific neoantigens and tumor-associated self-antigens. Tumor-associated antigens are subject to existing immune tolerance, which must be overcome by immunotherapies. Despite many novel immunotherapies reaching clinical trials, inducing self-antigen-specific immune responses remains challenging. Here, we systematically investigate viral-vector-based cancer vaccines encoding a tumor-associated self-antigen (TRP2) for the treatment of established melanomas in preclinical mouse models, alone or in combination with adoptive T cell therapy. We reveal that, unlike foreign antigens, tumor-associated antigens require replication of lymphocytic choriomeningitis virus (LCMV)-based vectors to break tolerance and induce effective antigen-specific CD8+ T cell responses. Immunization with a replicating LCMV vector leads to complete tumor rejection when combined with adoptive TRP2-specific T cell transfer. Importantly, immunization with replicating vectors leads to extended antigen persistence in secondary lymphoid organs, resulting in efficient T cell priming, which renders previously "cold" tumors open to immune infiltration and reprograms the tumor microenvironment to "hot." Our findings have important implications for the design of next-generation immunotherapies targeting solid cancers utilizing viral vectors and adoptive cell transfer.
Assuntos
Vacinas Anticâncer , Neoplasias , Camundongos , Animais , Vírus da Coriomeningite Linfocítica/genética , Linfócitos T CD8-Positivos , Neoplasias/tratamento farmacológico , Antígenos de Neoplasias/genética , Autoantígenos , Microambiente TumoralRESUMO
Listeria monocytogenes (L. monocytogenes) is a food-borne bacterial pathogen. Innate immunity to L. monocytogenes is profoundly affected by type I interferons (IFN-I). Here we investigated host metabolism in L. monocytogenes-infected mice and its potential control by IFN-I. Accordingly, we used animals lacking either the IFN-I receptor (IFNAR) or IRF9, a subunit of ISGF3, the master regulator of IFN-I-induced genes. Transcriptomes and metabolite profiles showed that L. monocytogenes infection induces metabolic rewiring of the liver. This affects various metabolic pathways including fatty acid (FA) metabolism and oxidative phosphorylation and is partially dependent on IFN-I signaling. Livers and macrophages from Ifnar1-/- mice employ increased glutaminolysis in an IRF9-independent manner, possibly to readjust TCA metabolite levels due to reduced FA oxidation. Moreover, FA oxidation inhibition provides protection from L. monocytogenes infection, explaining part of the protection of Irf9-/- and Ifnar1-/- mice. Our findings define a role of IFN-I in metabolic regulation during L. monocytogenes infection. Metabolic differences between Irf9-/- and Ifnar1-/- mice may underlie the different susceptibility of these mice against lethal infection with L. monocytogenes.
Assuntos
Interferon Tipo I/metabolismo , Listeria monocytogenes/metabolismo , Listeriose/metabolismo , Fígado/metabolismo , Animais , Ácidos Graxos/metabolismo , Interferon Tipo I/imunologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Fígado/imunologia , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The liver is a central regulator of metabolic homeostasis and serum metabolite levels. Hepatocytes are the functional units of the liver parenchyma and not only responsible for turnover of biomolecules but also act as central immune signaling platforms. Hepatotropic viruses infect liver tissue, resulting in inflammatory responses, tissue damage and hepatitis. Combining well-established in vitro and in vivo model systems with transcriptomic analyses, we show that type I interferon signaling initiates a robust antiviral immune response in hepatocytes. Strikingly, we also identify IFN-I as both, sufficient and necessary, to induce wide-spread metabolic reprogramming in hepatocytes. IFN-I specifically rewired tryptophan metabolism and induced hepatic tryptophan oxidation to kynurenine via Tdo2, correlating with altered concentrations of serum metabolites upon viral infection. Infected Tdo2-deficient animals displayed elevated serum levels of tryptophan and, unexpectedly, also vast increases in the downstream immune-suppressive metabolite kynurenine. Thus, Tdo2-deficiency did not result in altered serum homeostasis of the tryptophan to kynurenine ratio during infection, which seemed to be independent of hepatocyte-intrinsic compensation via the IDO-axis. These data highlight that inflammation-induced reprogramming of systemic tryptophan metabolism is tightly regulated in viral hepatitis.
Assuntos
Antivirais/metabolismo , Hepatite Viral Animal/imunologia , Hepatócitos/imunologia , Inflamação/imunologia , Cinurenina/metabolismo , Receptor de Interferon alfa e beta/fisiologia , Triptofano/metabolismo , Animais , Feminino , Vírus de Hepatite/isolamento & purificação , Hepatite Viral Animal/metabolismo , Hepatite Viral Animal/virologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Imunidade Inata/imunologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/virologia , Fator Regulador 7 de Interferon/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator de Transcrição STAT1/fisiologia , Triptofano Oxigenase/fisiologiaRESUMO
BACKGROUND & AIMS: 24-Norursodeoxycholic acid (NorUDCA) is a novel therapeutic bile acid used to treat immune-mediated cholestatic liver diseases, such as primary sclerosing cholangitis (PSC), where dysregulated T cells including CD8+ T cells contribute to hepatobiliary immunopathology. We hypothesized that NorUDCA may directly modulate CD8+ T cell function thus contributing to its therapeutic efficacy. METHODS: NorUDCA's immunomodulatory effects were first studied in Mdr2-/- mice, as a cholestatic model of PSC. To differentiate NorUDCA's immunomodulatory effects on CD8+ T cell function from its anticholestatic actions, we also used a non-cholestatic model of hepatic injury induced by an excessive CD8+ T cell immune response upon acute non-cytolytic lymphocytic choriomeningitis virus (LCMV) infection. Studies included molecular and biochemical approaches, flow cytometry and metabolic assays in murine CD8+ T cells in vitro. Mass spectrometry was used to identify potential CD8+ T cell targets modulated by NorUDCA. The signaling effects of NorUDCA observed in murine cells were validated in circulating T cells from patients with PSC. RESULTS: NorUDCA demonstrated immunomodulatory effects by reducing hepatic innate and adaptive immune cells, including CD8+ T cells in the Mdr2-/- model. In the non-cholestatic model of CD8+ T cell-driven immunopathology induced by acute LCMV infection, NorUDCA ameliorated hepatic injury and systemic inflammation. Mechanistically, NorUDCA demonstrated strong immunomodulatory efficacy in CD8+ T cells affecting lymphoblastogenesis, expansion, glycolysis and mTORC1 signaling. Mass spectrometry identified that NorUDCA regulates CD8+ T cells by targeting mTORC1. NorUDCA's impact on mTORC1 signaling was further confirmed in circulating PSC CD8+ T cells. CONCLUSIONS: NorUDCA has a direct modulatory impact on CD8+ T cells and attenuates excessive CD8+ T cell-driven hepatic immunopathology. These findings are relevant for treatment of immune-mediated liver diseases such as PSC. LAY SUMMARY: Elucidating the mechanisms by which 24-norursodeoxycholic acid (NorUDCA) works for the treatment of immune-mediated liver diseases, such as primary sclerosing cholangitis, is of considerable clinical interest. Herein, we uncovered an unrecognized property of NorUDCA in the immunometabolic regulation of CD8+ T cells, which has therapeutic relevance for immune-mediated liver diseases, including PSC.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Inflamação/tratamento farmacológico , Fígado/efeitos dos fármacos , Ácido Ursodesoxicólico/análogos & derivados , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Modelos Animais de Doenças , Inflamação/fisiopatologia , Fígado/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Ácido Ursodesoxicólico/farmacologia , Ácido Ursodesoxicólico/uso terapêuticoRESUMO
OBJECTIVES: External quality assessment (EQA) schemes provide information on individual and general analytical performance of participating laboratories and test systems. The aim of this study was to investigate the use and performance of SARS-CoV-2 virus genome detection systems in Austrian laboratories and their preparedness to face challenges associated with the pandemic. METHODS: Seven samples were selected to evaluate performance and estimate variability of reported results. Notably, a dilution series was included in the panel as a measure of reproducibility and sensitivity. Several performance criteria were evaluated for individual participants as well as in the cohort of all participants. RESULTS: A total of 109 laboratories participated and used 134 platforms, including 67 different combinations of extraction and PCR platforms and corresponding reagents. There were no false positives and 10 (1.2%) false negative results, including nine in the weakly positive sample (Ct â¼35.9, â¼640 copies/mL). Twenty (22%) laboratories reported results of mutation detection. Twenty-five (19%) test systems included amplification of human RNA as evidence of proper sampling. The overall linearity of Ct values from individual test systems for the dilution series was good, but inter-assay variability was high. Both operator-related and systematic failures appear to have caused incorrect results. CONCLUSIONS: Beyond providing certification for participating laboratories, EQA provides the opportunity for participants to evaluate their performance against others so that they may improve operating procedures and test systems. Well-selected EQA samples offer additional inferences to be made about assay sensitivity and reproducibility, which have practical applications.
Assuntos
COVID-19/diagnóstico , Genoma Viral , Garantia da Qualidade dos Cuidados de Saúde , SARS-CoV-2/isolamento & purificação , Áustria/epidemiologia , COVID-19/virologia , Humanos , Laboratórios , Técnicas de Diagnóstico Molecular/métodos , Pandemias , SARS-CoV-2/genética , Sensibilidade e EspecificidadeRESUMO
RNA-dependent RNA polymerases (RdRps) play a key role in the life cycle of RNA viruses and impact their immunobiology. The arenavirus lymphocytic choriomeningitis virus (LCMV) strain Clone 13 provides a benchmark model for studying chronic infection. A major genetic determinant for its ability to persist maps to a single amino acid exchange in the viral L protein, which exhibits RdRp activity, yet its functional consequences remain elusive. To unravel the L protein interactions with the host proteome, we engineered infectious L protein-tagged LCMV virions by reverse genetics. A subsequent mass-spectrometric analysis of L protein pulldowns from infected human cells revealed a comprehensive network of interacting host proteins. The obtained LCMV L protein interactome was bioinformatically integrated with known host protein interactors of RdRps from other RNA viruses, emphasizing interconnected modules of human proteins. Functional characterization of selected interactors highlighted proviral (DDX3X) as well as antiviral (NKRF, TRIM21) host factors. To corroborate these findings, we infected Trim21-/- mice with LCMV and found impaired virus control in chronic infection. These results provide insights into the complex interactions of the arenavirus LCMV and other viral RdRps with the host proteome and contribute to a better molecular understanding of how chronic viruses interact with their host.
Assuntos
RNA Helicases DEAD-box/metabolismo , Vírus da Coriomeningite Linfocítica/enzimologia , Modelos Moleculares , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Repressoras/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Animais , Sistemas CRISPR-Cas , Biologia Computacional , Cruzamentos Genéticos , RNA Helicases DEAD-box/química , Feminino , Células HEK293 , Humanos , Imunoprecipitação , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/veterinária , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Domínios e Motivos de Interação entre Proteínas , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/química , Ribonucleoproteínas/química , Ribonucleoproteínas/genética , Organismos Livres de Patógenos Específicos , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Pathogen encounter results in long-lasting epigenetic imprinting that shapes diseases caused by heterologous pathogens. The breadth of this innate immune memory is of particular interest in the context of respiratory pathogens with increased pandemic potential and wide-ranging impact on global health. Here, we investigated epigenetic imprinting across cell lineages in a disease relevant murine model of SARS-CoV-2 recovery. Past SARS-CoV-2 infection resulted in increased chromatin accessibility of type I interferon (IFN-I) related transcription factors in airway-resident macrophages. Mechanistically, establishment of this innate immune memory required viral pattern recognition and canonical IFN-I signaling and augmented secondary antiviral responses. Past SARS-CoV-2 infection ameliorated disease caused by the heterologous respiratory pathogen influenza A virus. Insights into innate immune memory and how it affects subsequent infections with heterologous pathogens to influence disease pathology could facilitate the development of broadly effective therapeutic strategies.
RESUMO
Cellular metabolism and immune function are closely linked. In this issue of Cell Metabolism, Shen et al. (2021) identify serine metabolism as a central integration hub of cellular metabolism, antiviral immunity, and epigenetic regulation.
Assuntos
Epigênese Genética , Interferons , Antivirais , Serina/metabolismoRESUMO
Cytotoxic T lymphocytes (CTLs) represent key immune effectors of the host response against chronic viruses, due to their cytotoxic response to virus-infected cells. In response to this selection pressure, viruses may accumulate escape mutations that evade CTL-mediated control. To study the emergence of CTL escape mutations, we employed the murine chronic infection model of lymphocytic choriomeningitis virus (LCMV). We developed an amplicon-based next-generation sequencing pipeline to detect low frequency mutations in the viral genome and identified non-synonymous mutations in the immunodominant LCMV CTL epitope, GP33-41, in infected wildtype mice. Infected Rag2-deficient mice lacking CTLs did not contain such viral mutations. By using transgenic mice with T cell receptors specific to GP33-41, we characterized the emergence of viral mutations in this epitope under varying selection pressure. We investigated the two most abundant viral mutations by employing reverse genetically engineered viral mutants encoding the respective mutations. These experiments provided evidence that these mutations prevent activation and expansion of epitope-specific CD8 T cells. Our findings on the mutational dynamics of CTL escape mutations in a widely-studied viral infection model contributes to our understanding of how chronic viruses interact with their host and evade the immune response. This may guide the development of future treatments and vaccines against chronic infections.
Assuntos
Antígenos Virais/metabolismo , Linfócitos T CD8-Positivos/imunologia , Glicoproteínas/metabolismo , Epitopos Imunodominantes/metabolismo , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Fragmentos de Peptídeos/metabolismo , Proteínas Virais/metabolismo , Animais , Antígenos Virais/genética , Células Cultivadas , Modelos Animais de Doenças , Glicoproteínas/genética , Evasão da Resposta Imune , Epitopos Imunodominantes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/genética , Proteínas Virais/genéticaRESUMO
Interrogation of cellular metabolism with high-throughput screening approaches can unravel contextual biology and identify cancer-specific metabolic vulnerabilities. To systematically study the consequences of distinct metabolic perturbations, we assemble a comprehensive metabolic drug library (CeMM Library of Metabolic Drugs; CLIMET) covering 243 compounds. We, next, characterize it phenotypically in a diverse panel of myeloid leukemia cell lines and primary patient cells. Analysis of the drug response profiles reveals that 77 drugs affect cell viability, with the top effective compounds targeting nucleic acid synthesis, oxidative stress, and the PI3K/mTOR pathway. Clustering of individual drug response profiles stratifies the cell lines into five functional groups, which link to specific molecular and metabolic features. Mechanistic characterization of selective responses to the PI3K inhibitor pictilisib, the fatty acid synthase inhibitor GSK2194069, and the SLC16A1 inhibitor AZD3965, bring forth biomarkers of drug response. Phenotypic screening using CLIMET represents a valuable tool to probe cellular metabolism and identify metabolic dependencies at large.
Assuntos
Leucemia Mieloide/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Ácidos Graxos/biossíntese , Genótipo , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/patologia , Transportadores de Ácidos Monocarboxílicos/genética , Fenótipo , Fosfatidilinositol 3-Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Pirimidinonas/metabolismo , Pirimidinonas/farmacologia , Pirrolidinas/metabolismo , Pirrolidinas/farmacologia , Transdução de Sinais , Bibliotecas de Moléculas Pequenas/classificação , Simportadores/genética , Análise de Sistemas , Tiofenos/metabolismo , Tiofenos/farmacologia , Triazóis/metabolismo , Triazóis/farmacologia , Células Tumorais CultivadasRESUMO
OBJECTIVES: We explore the importance of SARS-CoV-2 sentinel surveillance testing in primary care during a regional COVID-19 outbreak in Austria. DESIGN: Prospective cohort study. SETTING: A single sentinel practice serving 22 829 people in the ski-resort of Schladming-Dachstein. PARTICIPANTS: All 73 patients presenting with mild-to-moderate flu-like symptoms between 24 February and 03 April, 2020. INTERVENTION: Nasopharyngeal sampling to detect SARS-CoV-2 using real-time reverse transcriptase-quantitative PCR (RT-qPCR). OUTCOME MEASURES: We compared RT-qPCR at presentation with confirmed antibody status. We split the outbreak in two parts, by halving the period from the first to the last case, to characterise three cohorts of patients with confirmed infection: early acute (RT-qPCR reactive) in the first half; and late acute (reactive) and late convalescent (non-reactive) in the second half. For each cohort, we report the number of cases detected, the accuracy of RT-qPCR, the duration and variety of symptoms, and the number of viral clades present. RESULTS: Twenty-two patients were diagnosed with COVID-19 (eight early acute, seven late acute and seven late convalescent), 44 patients tested SARS-CoV-2 negative and 7 were excluded. The sensitivity of RT-qPCR was 100% among all acute cases, dropping to 68.1% when including convalescent. Test specificity was 100%. Mean duration of symptoms for each group were 2 days (range 1-4) among early acute, 4.4 days (1-7) among late acute and 8 days (2-12) among late convalescent. Confirmed infection was associated with loss of taste. Acute infection was associated with loss of taste, nausea/vomiting, breathlessness, sore throat and myalgia; but not anosmia, fever or cough. Transmission clusters of three viral clades (G, GR and L) were identified. CONCLUSIONS: RT-qPCR testing in primary care can rapidly and accurately detect SARS-CoV-2 among people with flu-like illness in a heterogeneous viral outbreak. Targeted testing in primary care can support national sentinel surveillance of COVID-19.
Assuntos
COVID-19 , SARS-CoV-2 , Áustria , Estudos de Coortes , Humanos , Atenção Primária à Saúde , Estudos Prospectivos , Sensibilidade e EspecificidadeRESUMO
CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.
Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19 , Epitopos de Linfócito T , Antígenos HLA-A/imunologia , Imunidade Celular , Mutação , SARS-CoV-2 , Linfócitos T CD8-Positivos/patologia , COVID-19/genética , COVID-19/imunologia , COVID-19/patologia , Proliferação de Células , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Interferon gama/imunologia , Peptídeos/genética , Peptídeos/imunologia , SARS-CoV-2/genética , SARS-CoV-2/imunologiaRESUMO
Adipose tissue macrophages (ATMs) display tremendous heterogeneity depending on signals in their local microenvironment and contribute to the pathogenesis of obesity. The phosphoinositide 3-kinase (PI3K) signalling pathway, antagonized by the phosphatase and tensin homologue (PTEN), is important for metabolic responses to obesity. We hypothesized that fluctuations in macrophage-intrinsic PI3K activity via PTEN could alter the trajectory of metabolic disease by driving distinct ATM populations. Using mice harbouring macrophage-specific PTEN deletion or bone marrow chimeras carrying additional PTEN copies, we demonstrate that sustained PI3K activity in macrophages preserves metabolic health in obesity by preventing lipotoxicity. Myeloid PI3K signalling promotes a beneficial ATM population characterized by lipid uptake, catabolism and high expression of the scavenger macrophage receptor with collagenous structure (MARCO). Dual MARCO and myeloid PTEN deficiencies prevent the generation of lipid-buffering ATMs, reversing the beneficial actions of elevated myeloid PI3K activity in metabolic disease. Thus, macrophage-intrinsic PI3K signalling boosts metabolic health by driving ATM programmes associated with MARCO-dependent lipid uptake.