RESUMEN
Lymphocyte activation involves a transition from quiescence and associated catabolic metabolism to a metabolic state with noted similarities to cancer cells such as heavy reliance on aerobic glycolysis for energy demands and increased nutrient requirements for biomass accumulation and cell division 1-3 . Following antigen receptor ligation, lymphocytes require spatiotemporally distinct "second signals". These include costimulatory receptor or cytokine signaling, which engage discrete programs that often involve remodeling of organelles and increased nutrient uptake or synthesis to meet changing biochemical demands 4-6 . One such signaling molecule, IL-4, is a highly pleiotropic cytokine that was first identified as a B cell co-mitogen over 30 years ago 7 . However, how IL-4 signaling mechanistically supports B cell proliferation is incompletely understood. Here, using single cell RNA sequencing we find that the cholesterol biosynthetic program is transcriptionally upregulated following IL-4 signaling during the early B cell response to influenza virus infection, and is required for B cell activation in vivo . By limiting lipid availability in vitro , we determine cholesterol to be essential for B cells to expand their endoplasmic reticulum, progress through cell cycle, and proliferate. In sum, we demonstrate that the well-known ability of IL-4 to act as a B cell growth factor is through a previously unknown rewiring of specific lipid anabolic programs, relieving sensitivity of cells to environmental nutrient availability.
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Interleukin-10 (IL-10) is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types1. Loss of IL-10 signalling results in life-threatening inflammatory bowel disease in humans and mice-however, the exact mechanism by which IL-10 signalling subdues inflammation remains unclear2-5. Here we find that increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10 deficiency. Accordingly, genetic deletion of ceramide synthase 2 (encoded by Cers2), the enzyme responsible for VLC ceramide production, limited the exacerbated inflammatory gene expression programme associated with IL-10 deficiency both in vitro and in vivo. The accumulation of saturated VLC ceramides was regulated by a decrease in metabolic flux through the de novo mono-unsaturated fatty acid synthesis pathway. Restoring mono-unsaturated fatty acid availability to cells deficient in IL-10 signalling limited saturated VLC ceramide production and the associated inflammation. Mechanistically, we find that persistent inflammation mediated by VLC ceramides is largely dependent on sustained activity of REL, an immuno-modulatory transcription factor. Together, these data indicate that an IL-10-driven fatty acid desaturation programme rewires VLC ceramide accumulation and aberrant activation of REL. These studies support the idea that fatty acid homeostasis in innate immune cells serves as a key regulatory node to control pathologic inflammation and suggests that 'metabolic correction' of VLC homeostasis could be an important strategy to normalize dysregulated inflammation caused by the absence of IL-10.
Asunto(s)
Inflamación , Interleucina-10 , Esfingolípidos , Animales , Humanos , Ratones , Ceramidas/química , Ceramidas/metabolismo , Ácidos Grasos Insaturados/biosíntesis , Ácidos Grasos Insaturados/metabolismo , Homeostasis , Inmunidad Innata , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-10/deficiencia , Interleucina-10/genética , Interleucina-10/metabolismo , Proteínas Proto-Oncogénicas c-rel , Esfingolípidos/metabolismoRESUMEN
Unchecked chronic inflammation is the underlying cause of many diseases, ranging from inflammatory bowel disease to obesity and neurodegeneration. Given the deleterious nature of unregulated inflammation, it is not surprising that cells have acquired a diverse arsenal of tactics to limit inflammation. IL-10 is a key anti-inflammatory cytokine that can limit immune cell activation and cytokine production in innate immune cell types; however, the exact mechanism by which IL-10 signaling subdues inflammation remains unclear. Here, we find that IL-10 signaling constrains sphingolipid metabolism. Specifically, we find increased saturated very long chain (VLC) ceramides are critical for the heightened inflammatory gene expression that is a hallmark of IL-10-deficient macrophages. Genetic deletion of CerS2, the enzyme responsible for VLC ceramide production, limited exacerbated inflammatory gene expression associated with IL-10 deficiency both in vitro and in vivo , indicating that "metabolic correction" is able to reduce inflammation in the absence of IL-10. Surprisingly, accumulation of saturated VLC ceramides was regulated by flux through the de novo mono-unsaturated fatty acid (MUFA) synthesis pathway, where addition of exogenous MUFAs could limit both saturated VLC ceramide production and inflammatory gene expression in the absence of IL-10 signaling. Together, these studies mechanistically define how IL-10 signaling manipulates fatty acid metabolism as part of its molecular anti-inflammatory strategy and could lead to novel and inexpensive approaches to regulate aberrant inflammation.
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Escherichia coli isolates commonly inhabit the human microbiota, yet the majority of E. coli's small-molecule repertoire remains uncharacterized. We previously employed erythromycin-induced translational stress to facilitate the characterization of autoinducer-3 (AI-3) and structurally related pyrazinones derived from "abortive" tRNA synthetase reactions in pathogenic, commensal, and probiotic E. coli isolates. In this study, we explored the "missing" tryptophan-derived pyrazinone reaction and characterized two other families of metabolites that were similarly upregulated under erythromycin stress. Strikingly, the abortive tryptophanyl-tRNA synthetase reaction leads to a tetracyclic indole alkaloid metabolite (1) rather than a pyrazinone. Furthermore, erythromycin induced two naphthoquinone-functionalized metabolites (MK-hCys, 2; and MK-Cys, 3) and four lumazines (7-10). Using genetic and metabolite analyses coupled with biomimetic synthesis, we provide support that the naphthoquinones are derived from 4-dihydroxy-2-naphthoic acid (DHNA), an intermediate in the menaquinone biosynthetic pathway, and the amino acids homocysteine and cysteine. In contrast, the lumazines are dependent on a flavin intermediate and α-ketoacids from the aminotransferases AspC and TyrB. We show that one of the lumazine members (9), an indole-functionalized analogue, possesses antioxidant properties, modulates the anti-inflammatory fate of isolated TH17 cells, and serves as an aryl-hydrocarbon receptor (AhR) agonist. These three systems described here serve to illustrate that new metabolic branches could be more commonly derived from well-established primary metabolic pathways.
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Escherichia coli , Naftoquinonas , Estrés Fisiológico , Humanos , Eritromicina/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Naftoquinonas/metabolismo , Triptófano/metabolismo , Triptófano-ARNt Ligasa/metabolismo , Biosíntesis de Proteínas/efectos de los fármacosRESUMEN
Neisseria meningitidis is a Gram-negative opportunistic pathogen that is responsible for causing human diseases with high mortality, such as septicemia and meningitis. The molecular mechanisms N.â meningitidis employ to manipulate the immune system, translocate the mucosal and blood-brain barriers, and exert virulence are largely unknown. Human-associated bacteria encode a variety of bioactive small molecules with growing evidence for N-acyl amides as being important signaling molecules. However, only a small fraction of these metabolites has been identified from the human microbiota thus far. Here, we heterologously expressed an N-acyltransferase encoded in the obligate human pathogen N.â meningitidis and identified 30â N-acyl amides with representative members serving as agonists of the G-protein coupled receptor (GPCR) S1PR4. During this process, we also characterized two mammalian N-acyl amides derived from the bovine medium. Both groups of metabolites suppress anti-inflammatory interleukin-10 signaling in human macrophage cell types, but they also suppress the pro-inflammatory interleukin-17A+ population in TH 17-differentiated CD4+ T cells.
Asunto(s)
Neisseria meningitidis , Humanos , Bovinos , Animales , Esfingosina , Amidas/farmacología , Virulencia , Transducción de Señal , MamíferosRESUMEN
Tissue-resident innate lymphoid cells (ILCs) help sustain barrier function and respond to local signals. ILCs are traditionally classified as ILC1, ILC2 or ILC3 on the basis of their expression of specific transcription factors and cytokines1. In the skin, disease-specific production of ILC3-associated cytokines interleukin (IL)-17 and IL-22 in response to IL-23 signalling contributes to dermal inflammation in psoriasis. However, it is not known whether this response is initiated by pre-committed ILCs or by cell-state transitions. Here we show that the induction of psoriasis in mice by IL-23 or imiquimod reconfigures a spectrum of skin ILCs, which converge on a pathogenic ILC3-like state. Tissue-resident ILCs were necessary and sufficient, in the absence of circulatory ILCs, to drive pathology. Single-cell RNA-sequencing (scRNA-seq) profiles of skin ILCs along a time course of psoriatic inflammation formed a dense transcriptional continuum-even at steady state-reflecting fluid ILC states, including a naive or quiescent-like state and an ILC2 effector state. Upon disease induction, the continuum shifted rapidly to span a mixed, ILC3-like subset also expressing cytokines characteristic of ILC2s, which we inferred as arising through multiple trajectories. We confirmed the transition potential of quiescent-like and ILC2 states using in vitro experiments, single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) and in vivo fate mapping. Our results highlight the range and flexibility of skin ILC responses, suggesting that immune activities primed in healthy tissues dynamically adapt to provocations and, left unchecked, drive pathological remodelling.
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Inmunidad Innata/inmunología , Linfocitos/inmunología , Linfocitos/patología , Psoriasis/inmunología , Psoriasis/patología , Piel/inmunología , Piel/patología , Animales , Diferenciación Celular , Linaje de la Célula , Cromatina/genética , Modelos Animales de Enfermedad , Femenino , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interleucina-23/inmunología , Análisis de Clases Latentes , Linfocitos/clasificación , Masculino , Ratones , Psoriasis/genética , ARN Citoplasmático Pequeño/genética , Reproducibilidad de los Resultados , Factores de TiempoRESUMEN
Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.
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Infecciones Bacterianas/inmunología , Toxinas Bacterianas/inmunología , Hidroxicolesteroles/metabolismo , Interferones/aislamiento & purificación , Fagocitos/inmunología , Estreptolisinas/inmunología , Animales , Bacterias/inmunología , Bacterias/metabolismo , Proteínas Bacterianas/administración & dosificación , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades/inmunología , Femenino , Interacciones Microbiota-Huesped/inmunología , Humanos , Microscopía Intravital , Masculino , Ratones , Ratones Transgénicos , Fagocitos/citología , Fagocitos/metabolismo , Cultivo Primario de Células , Esteroide Hidroxilasas/genética , Esteroide Hidroxilasas/metabolismo , Estreptolisinas/administración & dosificación , Estreptolisinas/metabolismoRESUMEN
Macrophages reprogram their lipid metabolism in response to activation signals. However, a systems-level understanding of how different pro-inflammatory stimuli reshape the macrophage lipidome is lacking. Here, we use complementary "shotgun" and isotope tracer mass spectrometry approaches to define the changes in lipid biosynthesis, import, and composition of macrophages induced by various Toll-like receptors (TLRs) and inflammatory cytokines. "Shotgun" lipidomics data revealed that different TLRs and cytokines induce macrophages to acquire distinct lipidomes, indicating their specificity in reshaping lipid composition. Mechanistic studies showed that differential reprogramming of lipid composition is mediated by the opposing effects of MyD88- and TRIF-interferon-signaling pathways. Finally, we applied these insights to show that perturbing reprogramming of lipid composition can enhance inflammation and promote host defense to bacterial challenge. These studies provide a framework for understanding how inflammatory stimuli reprogram lipid composition of macrophages while providing a knowledge platform to exploit differential lipidomics to influence immunity.
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Lipidómica , Macrófagos/metabolismo , Receptores Toll-Like/metabolismo , Animales , Línea Celular , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Transducción de SeñalRESUMEN
An Amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Direct recognition of invading pathogens by innate immune cells is a critical driver of the inflammatory response. However, cells of the innate immune system can also sense their local microenvironment and respond to physiological fluctuations in temperature, pH, oxygen and nutrient availability, which are altered during inflammation. Although cells of the immune system experience force and pressure throughout their life cycle, little is known about how these mechanical processes regulate the immune response. Here we show that cyclical hydrostatic pressure, similar to that experienced by immune cells in the lung, initiates an inflammatory response via the mechanically activated ion channel PIEZO1. Mice lacking PIEZO1 in innate immune cells showed ablated pulmonary inflammation in the context of bacterial infection or fibrotic autoinflammation. Our results reveal an environmental sensory axis that stimulates innate immune cells to mount an inflammatory response, and demonstrate a physiological role for PIEZO1 and mechanosensation in immunity.
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Presión Hidrostática , Inmunidad Innata , Canales Iónicos/metabolismo , Mecanotransducción Celular/inmunología , Animales , Endotelina-1/metabolismo , Femenino , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/microbiología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Pulmón/inmunología , Pulmón/metabolismo , Pulmón/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/inmunología , Transducción de SeñalRESUMEN
The annotation of the mammalian protein-coding genome is incomplete. Arbitrary size restriction of open reading frames (ORFs) and the absolute requirement for a methionine codon as the sole initiator of translation have constrained the identification of potentially important transcripts with non-canonical protein-coding potential1,2. Here, using unbiased transcriptomic approaches in macrophages that respond to bacterial infection, we show that ribosomes associate with a large number of RNAs that were previously annotated as 'non-protein coding'. Although the idea that such non-canonical ORFs can encode functional proteins is controversial3,4, we identify a range of short and non-ATG-initiated ORFs that can generate stable and spatially distinct proteins. Notably, we show that the translation of a new ORF 'hidden' within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. This work expands our interpretation of the protein-coding genome and demonstrates that proteinaceous products generated from non-canonical ORFs are crucial for the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein-coding genes in immunity and disease.
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Inmunidad Mucosa/genética , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas , ARN Largo no Codificante/genética , Animales , Infecciones Bacterianas/genética , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Infecciones Bacterianas/microbiología , Colitis/genética , Colitis/inmunología , Colitis/metabolismo , Inmunidad Mucosa/efectos de los fármacos , Interleucina-12/biosíntesis , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Biosíntesis de Proteínas/efectos de los fármacos , Biosíntesis de Proteínas/genética , ARN Largo no Codificante/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Salmonella typhimurium/inmunología , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
Cellular lipid requirements are achieved through a combination of biosynthesis and import programs. Using isotope tracer analysis, we show that type I interferon (IFN) signaling shifts the balance of these programs by decreasing synthesis and increasing import of cholesterol and long chain fatty acids. Genetically enforcing this metabolic shift in macrophages is sufficient to render mice resistant to viral challenge, demonstrating the importance of reprogramming the balance of these two metabolic pathways in vivo. Unexpectedly, mechanistic studies reveal that limiting flux through the cholesterol biosynthetic pathway spontaneously engages a type I IFN response in a STING-dependent manner. The upregulation of type I IFNs was traced to a decrease in the pool size of synthesized cholesterol and could be inhibited by replenishing cells with free cholesterol. Taken together, these studies delineate a metabolic-inflammatory circuit that links perturbations in cholesterol biosynthesis with activation of innate immunity.
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Colesterol/metabolismo , Inmunidad Innata , Interferón gamma/metabolismo , Transducción de Señal , Animales , Línea Celular Tumoral , Humanos , Interferon beta-1b , Proteínas de la Membrana/metabolismo , Ácido Mevalónico/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismoRESUMEN
Oxysterols are oxidized derivatives of cholesterol that are generated enzymatically or through autoxidation. Initially identified as important lipid signaling molecules in the context of atherosclerosis and inflammation, accumulated evidence indicates that these lipid-signaling molecules can have pleiotropic effects on the fate and function of the immune system. These effects range from the regulation of immune cell survival and proliferation to chemotaxis and antiviral immunity. New studies now indicate that tumor-derived oxysterols can serve to subvert the immune system by recruiting protumorigenic neutrophils into the tumor microenvironment. The consequence of this recruitment is the generation of proangiogenic factors and matrix metalloproteinase proteins that provide a tumor a significant growth and survival advantage. In combination with other recent studies, these data highlight the ongoing cross talk between sterol metabolism and the immune system, and they raise the intriguing possibility that targeting oxysterol pathways could serve as a novel therapeutic approach in the war on cancer.
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Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal , Esteroles/metabolismo , Animales , Homeostasis/inmunología , Humanos , Inmunidad Innata/inmunología , Modelos Biológicos , Neoplasias/inmunología , Transducción de Señal/inmunologíaRESUMEN
The tetracycline antibiotics are widely used in biomedical research as mediators of inducible gene expression systems. Despite many known effects of tetracyclines on mammalian cells-including inhibition of the mitochondrial ribosome-there have been few reports on potential off-target effects at concentrations commonly used in inducible systems. Here, we report that in human cell lines, commonly used concentrations of doxycycline change gene expression patterns and concomitantly shift metabolism towards a more glycolytic phenotype, evidenced by increased lactate secretion and reduced oxygen consumption. We also show that these concentrations are sufficient to slow proliferation. These findings suggest that researchers using doxycycline in inducible expression systems should design appropriate controls to account for potential confounding effects of the drug on cellular metabolism.
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Antibacterianos/farmacología , Doxiciclina/farmacología , Redes y Vías Metabólicas/efectos de los fármacos , Metaboloma/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Humanos , Masculino , Redes y Vías Metabólicas/genética , Metaboloma/genética , Consumo de Oxígeno/efectos de los fármacosRESUMEN
The sterol regulatory element-binding proteins (SREBP) are key transcriptional regulators of lipid metabolism and cellular growth. It has been proposed that SREBP signaling regulates cellular growth through its ability to drive lipid biosynthesis. Unexpectedly, we find that loss of SREBP activity inhibits cancer cell growth and viability by uncoupling fatty acid synthesis from desaturation. Integrated lipid profiling and metabolic flux analysis revealed that cancer cells with attenuated SREBP activity maintain long-chain saturated fatty acid synthesis, while losing fatty acid desaturation capacity. We traced this defect to the uncoupling of fatty acid synthase activity from stearoyl-CoA desaturase 1 (SCD1)-mediated desaturation. This deficiency in desaturation drives an imbalance between the saturated and monounsaturated fatty acid pools resulting in severe lipotoxicity. Importantly, replenishing the monounsaturated fatty acid pool restored growth to SREBP-inhibited cells. These studies highlight the importance of fatty acid desaturation in cancer growth and provide a novel mechanistic explanation for the role of SREBPs in cancer metabolism.
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Regulación Neoplásica de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias/metabolismo , Animales , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Ácido Graso Sintasas/metabolismo , Perfilación de la Expresión Génica , Humanos , Ratones , Ratones Endogámicos NOD , Modelos Estadísticos , Trasplante de Neoplasias , Transducción de Señal , Estearoil-CoA Desaturasa/metabolismo , Esteroles/metabolismoRESUMEN
The human Mediator complex is generally required for expression of protein-coding genes. Here, we show that the GCN5L acetyltransferase stably associates with Mediator together with the TRRAP polypeptide. Yet, contrary to expectations, TRRAP/GCN5L does not associate with the transcriptionally active core Mediator but rather with Mediator that contains the cdk8 subcomplex. Consequently, this derivative 'T/G-Mediator' complex does not directly activate transcription in a reconstituted human transcription system. However, within T/G-Mediator, cdk8 phosphorylates serine-10 on histone H3, which in turn stimulates H3K14 acetylation by GCN5L within the complex. Tandem phosphoacetylation of H3 correlates with transcriptional activation, and ChIP assays demonstrate co-occupancy of T/G-Mediator components at several activated genes in vivo. Moreover, cdk8 knockdown causes substantial reduction of global H3 phosphoacetylation, suggesting that T/G-Mediator is a major regulator of this H3 mark. Cooperative H3 modification provides a mechanistic basis for GCN5L association with cdk8-Mediator and also identifies a biochemical means by which cdk8 can indirectly activate gene expression. Indeed our results suggest that T/G-Mediator directs early events-such as modification of chromatin templates-in transcriptional activation.