RESUMEN
Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.
Asunto(s)
Inflamación/inmunología , Activación de Macrófagos , Macrófagos/inmunología , Mitocondrias/enzimología , Succinato Deshidrogenasa/metabolismo , Ácido Succínico/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Ciclo del Ácido Cítrico , Glucólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/genética , Interleucina-10/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/metabolismo , Malonatos/farmacología , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Proteínas Mitocondriales/metabolismo , Oxidación-Reducción/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Análisis de Secuencia de ARN , Succinato Deshidrogenasa/genética , TranscriptomaRESUMEN
Chronic inflammation is often associated with the development of tissue fibrosis, but how mesenchymal cell responses dictate pathological fibrosis versus resolution and healing remains unclear. Defining stromal heterogeneity and identifying molecular circuits driving extracellular matrix deposition and remodeling stands to illuminate the relationship between inflammation, fibrosis, and healing. We performed single-cell RNA-sequencing of colon-derived stromal cells and identified distinct classes of fibroblasts with gene signatures that are differentially regulated by chronic inflammation, including IL-11-producing inflammatory fibroblasts. We further identify a transcriptional program associated with trans-differentiation of mucosa-associated fibroblasts and define a functional gene signature associated with matrix deposition and remodeling in the inflamed colon. Our analysis supports a critical role for the metalloprotease Adamdec1 at the interface between tissue remodeling and healing during colitis, demonstrating its requirement for colon epithelial integrity. These findings provide mechanistic insight into how inflammation perturbs stromal cell behaviors to drive fibroblastic responses controlling mucosal matrix remodeling and healing.
Asunto(s)
Proteínas ADAM/inmunología , Colitis/inmunología , Matriz Extracelular/metabolismo , Fibroblastos/inmunología , Mucosa Intestinal/inmunología , Células Madre Mesenquimatosas/inmunología , Proteínas ADAM/deficiencia , Proteínas ADAM/genética , Animales , Diferenciación Celular , Colitis/inducido químicamente , Colitis/genética , Colitis/patología , Colon/inmunología , Colon/patología , Matriz Extracelular/inmunología , Fibroblastos/patología , Fibrosis , Regulación de la Expresión Génica , Humanos , Inflamación , Interleucina-11/genética , Interleucina-11/inmunología , Mucosa Intestinal/patología , Masculino , Células Madre Mesenquimatosas/patología , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Dodecil Sulfato de Sodio/administración & dosificación , Transcripción Genética , Transcriptoma , Cicatrización de Heridas/genética , Cicatrización de Heridas/inmunologíaRESUMEN
Genome-wide association studies have identified common genetic variants impacting human diseases; however, there are indications that the functional consequences of genetic polymorphisms can be distinct depending on cell type-specific contexts, which produce divergent phenotypic outcomes. Thus, the functional impact of genetic variation and the underlying mechanisms of disease risk are modified by cell type-specific effects of genotype on pathological phenotypes. In this study, we extend these concepts to interrogate the interdependence of cell type- and stimulation-specific programs influenced by the core autophagy gene Atg16L1 and its T300A coding polymorphism identified by genome-wide association studies as linked with increased risk of Crohn's disease. We applied a stimulation-based perturbational profiling approach to define Atg16L1 T300A phenotypes in dendritic cells and T lymphocytes. Accordingly, we identified stimulus-specific transcriptional signatures revealing T300A-dependent functional phenotypes that mechanistically link inflammatory cytokines, IFN response genes, steroid biosynthesis, and lipid metabolism in dendritic cells and iron homeostasis and lysosomal biogenesis in T lymphocytes. Collectively, these studies highlight the combined effects of Atg16L1 genetic variation and stimulatory context on immune function.
Asunto(s)
Proteínas Relacionadas con la Autofagia/metabolismo , Enfermedad de Crohn/metabolismo , Células Dendríticas/fisiología , Genotipo , Linfocitos T/fisiología , Animales , Proteínas Relacionadas con la Autofagia/genética , Células Cultivadas , Enfermedad de Crohn/genética , Predisposición Genética a la Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Fenotipo , Polimorfismo Genético , Riesgo , Activación TranscripcionalRESUMEN
Identifying immunodominant T cell epitopes remains a significant challenge in the context of infectious disease, autoimmunity, and immuno-oncology. To address the challenge of antigen discovery, we developed a quantitative proteomic approach that enabled unbiased identification of major histocompatibility complex class II (MHCII)-associated peptide epitopes and biochemical features of antigenicity. On the basis of these data, we trained a deep neural network model for genome-scale predictions of immunodominant MHCII-restricted epitopes. We named this model bacteria originated T cell antigen (BOTA) predictor. In validation studies, BOTA accurately predicted novel CD4 T cell epitopes derived from the model pathogen Listeria monocytogenes and the commensal microorganism Muribaculum intestinale. To conclusively define immunodominant T cell epitopes predicted by BOTA, we developed a high-throughput approach to screen DNA-encoded peptide-MHCII libraries for functional recognition by T cell receptors identified from single-cell RNA sequencing. Collectively, these studies provide a framework for defining the immunodominance landscape across a broad range of immune pathologies.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Epítopos Inmunodominantes/genética , Proteómica , Secuencia de Aminoácidos/genética , Presentación de Antígeno/genética , Linfocitos T CD4-Positivos/inmunología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Secuenciación de Nucleótidos de Alto Rendimiento , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Epítopos Inmunodominantes/inmunología , Listeria monocytogenes/genética , Listeria monocytogenes/inmunología , Listeria monocytogenes/patogenicidad , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Análisis de la Célula IndividualRESUMEN
Phagocyte microbiocidal mechanisms and inflammatory cytokine production are temporally coordinated, although their respective interdependencies remain incompletely understood. Here, we identify a nitric-oxide-mediated antioxidant response as a negative feedback regulator of inflammatory cytokine production in phagocytes. In this context, Keap1 functions as a cellular redox sensor that responds to elevated reactive nitrogen intermediates by eliciting an adaptive transcriptional program controlled by Nrf2 and comprised of antioxidant genes, including Prdx5. We demonstrate that engaging the antioxidant response is sufficient to suppress Toll-like receptor (TLR)-induced cytokine production in dendritic cells and that Prdx5 is required for attenuation of inflammatory cytokine production. Collectively, these findings delineate the reciprocal regulation of inflammation and cellular redox systems in myeloid cells.
Asunto(s)
Óxido Nítrico/metabolismo , Peroxirredoxinas/metabolismo , Fagocitos/metabolismo , Animales , Células de la Médula Ósea/metabolismo , Citocinas/biosíntesis , Retroalimentación Fisiológica , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Óxido Nítrico/biosíntesis , Transducción de SeñalRESUMEN
Intestinal reovirus infection can trigger T helper 1 (TH1) immunity to dietary antigen, raising the question of whether other viruses can have a similar impact. Here we show that the acute CW3 strain of murine norovirus, but not the persistent CR6 strain, induces TH1 immunity to dietary antigen. This property of CW3 is dependent on its major capsid protein, a virulence determinant. Transcriptional profiling of mesenteric lymph nodes following infection reveals an immunopathological signature that does not segregate with protective immunity but with loss of oral tolerance, in which interferon regulatory factor 1 is critical. These data show that viral capacity to trigger specific inflammatory pathways at sites where T cell responses to dietary antigens take place interferes with the development of tolerance to an oral antigen. Collectively, these data provide a foundation for the development of therapeutic strategies to prevent TH1-mediated complex immune disorders triggered by viral infections.
Asunto(s)
Infecciones por Caliciviridae/inmunología , Dieta , Norovirus/inmunología , Norovirus/patogenicidad , Ovalbúmina/inmunología , Células TH1/inmunología , Administración Oral , Animales , Infecciones por Caliciviridae/virología , Proteínas de la Cápside/inmunología , Enfermedad Celíaca/inmunología , Modelos Animales de Enfermedad , Femenino , Células HEK293 , Humanos , Inmunidad , Inflamación , Factor 1 Regulador del Interferón/inmunología , Ganglios Linfáticos , Ratones , Ratones Endogámicos C57BL , Ovalbúmina/administración & dosificación , Esparcimiento de VirusRESUMEN
Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.
Asunto(s)
Antígenos/inmunología , Enfermedad Celíaca/inmunología , Enfermedad Celíaca/virología , Glútenes/inmunología , Inflamación/virología , Infecciones por Reoviridae/complicaciones , Infecciones por Reoviridae/inmunología , Células TH1/inmunología , Animales , Dieta/efectos adversos , Modelos Animales de Enfermedad , Ingeniería Genética , Humanos , Tolerancia Inmunológica , Inflamación/inmunología , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/inmunología , Interferón Tipo I/genética , Interferón Tipo I/inmunología , Intestinos/inmunología , Intestinos/patología , Intestinos/virología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor de Interferón alfa y beta/genética , Reoviridae/genéticaRESUMEN
Significant insights into disease pathogenesis have been gleaned from population-level genetic studies; however, many loci associated with complex genetic disease contain numerous genes, and phenotypic associations cannot be assigned unequivocally. In particular, a gene-dense locus on chromosome 11 (61.5-61.65 Mb) has been associated with inflammatory bowel disease, rheumatoid arthritis, and coronary artery disease. Here, we identify TMEM258 within this locus as a central regulator of intestinal inflammation. Strikingly, Tmem258 haploinsufficient mice exhibit severe intestinal inflammation in a model of colitis. At the mechanistic level, we demonstrate that TMEM258 is a required component of the oligosaccharyltransferase complex and is essential for N-linked protein glycosylation. Consequently, homozygous deficiency of Tmem258 in colonic organoids results in unresolved endoplasmic reticulum (ER) stress culminating in apoptosis. Collectively, our results demonstrate that TMEM258 is a central mediator of ER quality control and intestinal homeostasis.