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Recent developments of novel single-cell analysis techniques have rapidly advanced the fields of immunotoxicology and immunometabolism. Single-cell analyses enable the characterization of immune cells, unraveling heterogeneity, and population dynamics in response to cellular perturbations, including toxicant insults and changes in cellular metabolism. This review provides an overview of current technologies and recent discoveries, illustrating an emerging role of single-cell analyses in the field of immunotoxicology and immunometabolism. Various single-cell techniques, including flow cytometry, mass cytometry, multiplexed imaging, and sequencing, together with their applications to studying immunotoxicology and immunometabolism are discussed. This review emphasizes the potential for single-cell analyses to revolutionize our understanding of immune cell heterogeneity, uncover novel cellular therapeutic targets, and pave the way for novel mechanistic insights.
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T cell receptor (TCR) engagement triggers T cell responses, yet how TCR-mediated activation is regulated at the plasma membrane remains unclear. Here, we report that deleting the membrane scaffolding protein Flotillin-2 (Flot2) increases T cell antigen sensitivity, resulting in enhanced TCR signaling and effector function to weak TCR stimulation. T cell-specific Flot2-deficient mice exhibited reduced tumor growth and enhanced immunity to infection. Flot2-null CD4 + T cells exhibited increased T helper 1 polarization, proliferation, Nur77 induction, and phosphorylation of ZAP70 and LCK upon weak TCR stimulation, indicating a sensitized TCR-triggering threshold. Single cell-RNA sequencing suggested that Flot2 - null CD4 + T cells follow a similar route of activation as wild-type CD4 + T cells but exhibit higher occupancy of a discrete activation state under weak TCR stimulation. Given prior reports that TCR clustering influences sensitivity of T cells to stimuli, we evaluated TCR distribution with super-resolution microscopy. Flot2 ablation increased the number of surface TCR nanoclusters on naïve CD4 + T cells. Collectively, we posit that Flot2 modulates T cell functionality to weak TCR stimulation, at least in part, by regulating surface TCR clustering. Our findings have implications for improving T cell reactivity in diseases with poor antigenicity, such as cancer and chronic infections.
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Lipid accumulation in macrophages (Mφs) is a hallmark of atherosclerosis, yet how lipid accumulation affects inflammatory responses through rewiring of Mφ metabolism is poorly understood. We modeled lipid accumulation in cultured wild-type mouse thioglycolate-elicited peritoneal Mφs and bone marrow-derived Mφs with conditional (Lyz2-Cre) or complete genetic deficiency of Vhl, Hif1a, Nos2, and Nfe2l2. Transfection studies employed RAW264.7 cells. Mφs were cultured for 24 h with oxidized low-density lipoprotein (oxLDL) or cholesterol and then were stimulated with LPS. Transcriptomics revealed that oxLDL accumulation in Mφs downregulated inflammatory, hypoxia, and cholesterol metabolism pathways, whereas the antioxidant pathway, fatty acid oxidation, and ABC family proteins were upregulated. Metabolomics and extracellular metabolic flux assays showed that oxLDL accumulation suppressed LPS-induced glycolysis. Intracellular lipid accumulation in Mφs impaired LPS-induced inflammation by reducing both hypoxia-inducible factor 1-α (HIF-1α) stability and transactivation capacity; thus, the phenotype was not rescued in Vhl-/- Mφs. Intracellular lipid accumulation in Mφs also enhanced LPS-induced NF erythroid 2-related factor 2 (Nrf2)-mediated antioxidative defense that destabilizes HIF-1α, and Nrf2-deficient Mφs resisted the inhibitory effects of lipid accumulation on glycolysis and inflammatory gene expression. Furthermore, oxLDL shifted NADPH consumption from HIF-1α- to Nrf2-regulated apoenzymes. Thus, we postulate that repurposing NADPH consumption from HIF-1α to Nrf2 transcriptional pathways is critical in modulating inflammatory responses in Mφs with accumulated intracellular lipid. The relevance of our in vitro models was established by comparative transcriptomic analyses, which revealed that Mφs cultured with oxLDL and stimulated with LPS shared similar inflammatory and metabolic profiles with foamy Mφs derived from the atherosclerotic mouse and human aorta.
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Aterosclerosis , Hipercolesterolemia , Humanos , Ratones , Animales , Factor 2 Relacionado con NF-E2/metabolismo , Lipopolisacáridos/metabolismo , NADP/metabolismo , Macrófagos/metabolismo , Lipoproteínas LDL/metabolismo , Glucólisis , Aterosclerosis/metabolismo , Colesterol/metabolismo , Antioxidantes/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismoRESUMEN
Immunity-related GTPase family M (IRGM), located on human chromosome 5q33.1, encodes a protein that promotes autophagy and suppresses the innate immune response. The minor allele of rs13361189 (-4299T>C), a single nucleotide polymorphism in the IRGM promoter, has been associated with several diseases, including Crohn's disease and tuberculosis. Although patterns of linkage disequilibrium and minor allele frequency for this polymorphism differ dramatically between subjects of European and African descent, studies of rs13361189 have predominantly been conducted in Europeans and the mechanism of association is poorly understood. We recruited a cohort of 68 individuals (30 White, 34 African American, 4 other race) with varying rs13361189 genotypes and assessed a panel of immune response measures including whole blood cytokine induction following ex vivo stimulation with Toll-like Receptor ligands. Minor allele carriers were found to have increased serum immunoglobulin M, C-reactive protein, and circulating CD8+ T cells. No differences in whole blood cytokines were observed between minor allele carriers and non-carriers in the overall study population; however, minor allele status was associated with increased induction of a subset of cytokines among African American subjects, and decreased induction among White subjects. These findings underline the importance of broad racial inclusion in genetic studies of immunity.
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Citocinas , Predisposición Genética a la Enfermedad , Humanos , Alelos , Citocinas/genética , Linfocitos T CD8-positivos , Estudios de Casos y Controles , Proteínas de Unión al GTP/genética , Polimorfismo de Nucleótido SimpleRESUMEN
In vitro models to study simultaneous development of different human immune cells and hematopoietic lineages are lacking. We identified and characterized, using single-cell methods, an in vitro stromal cell-free culture system of human hematopoietic stem and progenitor cell (HSPC) differentiation that allows concurrent development of multiple immune cell lineages. The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor influencing many biological processes in diverse cell types. Using this in vitro model, we found that AHR activation by the highly specific AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, drives differentiation of human umbilical cord blood-derived CD34+ HSPCs toward monocytes and granulocytes with a significant decrease in lymphoid and megakaryocyte lineage specification that may lead to reduced immune competence. To our knowledge, we also discovered for the first time, using single-cell modalities, that AHR activation decreased the expression of BCL11A and IRF8 in progenitor cells, which are critical genes involved in hematopoietic lineage specification processes at both transcriptomic and protein levels. Our in vitro model of hematopoiesis, coupled with single-cell tools, therefore allows for a better understanding of the role played by AHR in modulating hematopoietic differentiation.
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Células Madre Hematopoyéticas , Receptores de Hidrocarburo de Aril , Humanos , Células Madre Hematopoyéticas/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Ligandos , Hematopoyesis , Diferenciación CelularRESUMEN
Introduction: Asthma is a chronic disease of the airways that impairs normal breathing. The etiology of asthma is complex and involves multiple factors, including the environment and genetics, especially the distinct genetic architecture associated with ancestry. Compared to early-onset asthma, little is known about genetic predisposition to late-onset asthma. We investigated the race/ethnicity-specific relationship among genetic variants within the major histocompatibility complex (MHC) region and late-onset asthma in a North Carolina-based multiracial cohort of adults. Methods: We stratified all analyses by self-reported race (i.e., White and Black) and adjusted all regression models for age, sex, and ancestry. We conducted association tests within the MHC region and performed fine-mapping analyses conditioned on the race/ethnicity-specific lead variant using whole-genome sequencing (WGS) data. We applied computational methods to infer human leukocyte antigen (HLA) alleles and residues at amino acid positions. We replicated findings in the UK Biobank. Results: The lead signals, rs9265901 on the 5' end of HLA-B, rs55888430 on HLA-DOB, and rs117953947 on HCG17, were significantly associated with late-onset asthma in all, White, and Black participants, respectively (OR = 1.73, 95%CI: 1.31 to 2.14, p = 3.62 × 10-5; OR = 3.05, 95%CI: 1.86 to 4.98, p = 8.85 × 10-6; OR = 19.5, 95%CI: 4.37 to 87.2, p = 9.97 × 10-5, respectively). For the HLA analysis, HLA-B*40:02 and HLA-DRB1*04:05, HLA-B*40:02, HLA-C*04:01, and HLA-DRB1*04:05, and HLA-DRB1*03:01 and HLA-DQB1 were significantly associated with late-onset asthma in all, White, and Black participants. Conclusion: Multiple genetic variants within the MHC region were significantly associated with late-onset asthma, and the associations were significantly different by race/ethnicity group.
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Single-cell RNA sequencing (scRNA-seq) of BAL cells has provided insights into coronavirus disease (COVID-19). However, reports have been limited by small patient cohorts. We performed a meta-analysis of BAL scRNA-seq data from healthy control subjects (n = 13) and patients with COVID-19 (n = 20), sourced from six independent studies (167,280 high-quality cells in total). Consistent with the source reports, increases in infiltrating leukocyte subtypes were noted, several with type I IFN signatures and unique gene expression signatures associated with transcellular chemokine signaling. Noting dramatic reductions of inferred NKX2-1 and NR4A1 activity in alveolar epithelial type II (AT-II) cells, we modeled pseudotemporal AT-II-to-AT-I progression. This revealed changes in inferred AT-II cell metabolic activity, increased transitional cells, and a previously undescribed AT-I state. This cell state was conspicuously marked by the induction of genes of the epidermal differentiation complex, including the cornified envelope protein SPRR3 (small proline-rich protein 3), upregulation of multiple KRT (keratin) genes, inferred mitochondrial dysfunction, and cell death signatures including apoptosis and ferroptosis. Immunohistochemistry of lungs from patients with COVID-19 confirmed upregulation and colocalization of KRT13 and SPRR3 in the distal airspaces. Forced overexpression of SPRR3 in human alveolar epithelial cells ex vivo did not activate caspase-3 or upregulate KRT13, suggesting that SPRR3 marks an AT-I cornification program in COVID-19 but is not sufficient for phenotypic changes.
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Células Epiteliales Alveolares , COVID-19 , Humanos , COVID-19/genética , COVID-19/metabolismo , Pulmón , Células Epiteliales/metabolismo , Análisis de Secuencia de ARNRESUMEN
Mitochondrial abnormalities have been noted in lupus, but the causes and consequences remain obscure. Autophagy-related genes ATG5, ATG7 and IRGM have been previously implicated in autoimmune disease. We reasoned that failure to clear defective mitochondria via mitophagy might be a foundational driver in autoimmunity by licensing mitochondrial DNA-dependent induction of type I interferon. Here, we show that mice lacking the GTPase IRGM1 (IRGM homolog) exhibited a type I interferonopathy with autoimmune features. Irgm1 deletion impaired the execution of mitophagy with cell-specific consequences. In fibroblasts, mitochondrial DNA soiling of the cytosol induced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-dependent type I interferon, whereas in macrophages, lysosomal Toll-like receptor 7 was activated. In vivo, Irgm1-/- tissues exhibited mosaic dependency upon nucleic acid receptors. Whereas salivary and lacrimal gland autoimmune pathology was abolished and lung pathology was attenuated by cGAS and STING deletion, pancreatic pathology remained unchanged. These findings reveal fundamental connections between mitochondrial quality control and tissue-selective autoimmune disease.
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Enfermedades Autoinmunes/metabolismo , Autoinmunidad , Fibroblastos/metabolismo , Proteínas de Unión al GTP/metabolismo , Mitocondrias/metabolismo , Mitofagia , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Células Cultivadas , Fibroblastos/inmunología , Fibroblastos/patología , Proteínas de Unión al GTP/deficiencia , Proteínas de Unión al GTP/genética , Regulación de la Expresión Génica , Macrófagos/inmunología , Macrófagos/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Mitocondrias/genética , Mitocondrias/inmunología , Mitocondrias/patología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Receptor Toll-Like 7/genética , Receptor Toll-Like 7/metabolismoRESUMEN
Histone crotonylation is a non-acetyl histone lysine modification that is as widespread as acetylation. However, physiological functions associated with histone crotonylation remain almost completely unknown. Here we report that histone crotonylation is crucial for endoderm differentiation. We demonstrate that key crotonyl-coenzyme A (CoA)-producing enzymes are specifically induced in endodermal cells during differentiation of human embryonic stem cells (hESCs) in vitro and in mouse embryos, where they function to increase histone crotonylation and enhance endodermal gene expression. Chemical enhancement of histone crotonylation promotes endoderm differentiation of hESCs, whereas deletion of crotonyl-CoA-producing enzymes reduces histone crotonylation and impairs meso/endoderm differentiation in vitro and in vivo. Our study uncovers a histone crotonylation-mediated mechanism that promotes endodermal commitment of pluripotent stem cells, which may have important implications for therapeutic strategies against a number of human diseases.
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Histonas , Células Madre Embrionarias Humanas , Acetilación , Animales , Diferenciación Celular , Histonas/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Lisina/metabolismo , Ratones , Procesamiento Proteico-PostraduccionalRESUMEN
A defining feature of adaptive immunity is the development of long-lived memory T cells to curtail infection. Recent studies have identified a unique stem-like T-cell subset amongst exhausted CD8-positive T cells in chronic infection1-3, but it remains unclear whether CD4-positive T-cell subsets with similar features exist in chronic inflammatory conditions. Amongst helper T cells, TH17 cells have prominent roles in autoimmunity and tissue inflammation and are characterized by inherent plasticity4-7, although how such plasticity is regulated is poorly understood. Here we demonstrate that TH17 cells in a mouse model of autoimmune disease are functionally and metabolically heterogeneous; they contain a subset with stemness-associated features but lower anabolic metabolism, and a reciprocal subset with higher metabolic activity that supports transdifferentiation into TH1-like cells. These two TH17-cell subsets are defined by selective expression of the transcription factors TCF-1 and T-bet, and by discrete levels of CD27 expression. We also identify signalling via the kinase complex mTORC1 as a central regulator of TH17-cell fate decisions by coordinating metabolic and transcriptional programmes. TH17 cells with disrupted mTORC1 signalling or anabolic metabolism fail to induce autoimmune neuroinflammation or to develop into TH1-like cells, but instead upregulate TCF-1 expression and acquire stemness-associated features. Single-cell RNA sequencing and experimental validation reveal heterogeneity in fate-mapped TH17 cells, and a developmental arrest in the TH1 transdifferentiation trajectory upon loss of mTORC1 activity or metabolic perturbation. Our results establish that the dichotomy of stemness and effector function underlies the heterogeneous TH17 responses and autoimmune pathogenesis, and point to previously unappreciated metabolic control of plasticity in helper T cells.
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Transdiferenciación Celular , Células Madre/citología , Células Madre/metabolismo , Células Th17/citología , Células Th17/metabolismo , Animales , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/metabolismo , Enfermedades Autoinmunes/patología , Modelos Animales de Enfermedad , Femenino , Memoria Inmunológica/inmunología , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Proteína Reguladora Asociada a mTOR/deficiencia , Proteína Reguladora Asociada a mTOR/genética , Análisis de Secuencia de ARN , Transducción de Señal , Análisis de la Célula Individual , Células Madre/inmunología , Factor 1 de Transcripción de Linfocitos T/biosíntesis , Factor 1 de Transcripción de Linfocitos T/metabolismo , Proteínas de Dominio T Box/biosíntesis , Proteínas de Dominio T Box/metabolismo , Células Th17/inmunología , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
An emerging emphasis on mechanism-focused and human-relevant alternatives to animal use in toxicology underlies the toxicology testing in the twenty-first-century initiative. Herein we describe in vitro high-throughput screening programs seeking to address this goal, as well as strategies established to integrate assay results to build weight of evidence in support of hazard assessment. Furthermore, we discuss unique challenges facing the application of such alternatives for assessing immunotoxicity given the complexity of immune responses. Addressing these challenges will require the development of novel in vitro assays that evaluate well-characterized biochemical processes involved in immune response to help inform on putative adverse outcomes in vivo.
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Sistema Inmunológico/metabolismo , Pruebas de Toxicidad/historia , Pruebas de Toxicidad/métodos , Animales , Ensayos Analíticos de Alto Rendimiento , Historia del Siglo XXI , Humanos , InmunizaciónRESUMEN
Dendritic cells orchestrate the crosstalk between innate and adaptive immunity. CD8α+ dendritic cells present antigens to CD8+ T cells and elicit cytotoxic T cell responses to viruses, bacteria and tumours 1 . Although lineage-specific transcriptional regulators of CD8α+ dendritic cell development have been identified 2 , the molecular pathways that selectively orchestrate CD8α+ dendritic cell function remain elusive. Moreover, metabolic reprogramming is important for dendritic cell development and activation3,4, but metabolic dependence and regulation of dendritic cell subsets are largely uncharacterized. Here we use a data-driven systems biology algorithm (NetBID) to identify a role of the Hippo pathway kinases Mst1 and Mst2 (Mst1/2) in selectively programming CD8α+ dendritic cell function and metabolism. Our NetBID analysis reveals a marked enrichment of the activities of Hippo pathway kinases in CD8α+ dendritic cells relative to CD8α- dendritic cells. Dendritic cell-specific deletion of Mst1/2-but not Lats1 and Lats2 (Lats1/2) or Yap and Taz (Yap/Taz), which mediate canonical Hippo signalling-disrupts homeostasis and function of CD8+ T cells and anti-tumour immunity. Mst1/2-deficient CD8α+ dendritic cells are impaired in presentation of extracellular proteins and cognate peptides to prime CD8+ T cells, while CD8α- dendritic cells that lack Mst1/2 have largely normal function. Mechanistically, compared to CD8α- dendritic cells, CD8α+ dendritic cells exhibit much stronger oxidative metabolism and critically depend on Mst1/2 signalling to maintain bioenergetic activities and mitochondrial dynamics for their functional capacities. Further, selective expression of IL-12 by CD8α+ dendritic cells depends on Mst1/2 and the crosstalk with non-canonical NF-κB signalling. Our findings identify Mst1/2 as selective drivers of CD8α+ dendritic cell function by integrating metabolic activity and cytokine signalling, and highlight that the interplay between immune signalling and metabolic reprogramming underlies the unique functions of dendritic cell subsets.
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Antígenos CD8/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Algoritmos , Animales , Antígenos CD8/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/citología , Vía de Señalización Hippo , Homeostasis , Interleucina-12/inmunología , Interleucina-12/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Serina-Treonina Quinasa 3 , Proteínas Supresoras de TumorRESUMEN
Myelopoiesis is necessary for the generation of mature myeloid cells during homeostatic turnover and immunological insults; however, the metabolic requirements for this process remain poorly defined. Here, we demonstrate that myelopoiesis, including monocyte and macrophage differentiation, requires mechanistic target of rapamycin complex 1 (mTORC1) signaling and anabolic metabolism. Loss of mTORC1 impaired myelopoiesis under steady state and dampened innate immune responses against Listeria monocytogenes infection. Stimulation of hematopoietic progenitors with macrophage colony-stimulating factor (M-CSF) resulted in mTORC1-dependent anabolic metabolism, which in turn promoted expression of M-CSF receptor and transcription factors PU.1 and IRF8, thereby constituting a feed-forward loop for myelopoiesis. Mechanistically, mTORC1 engaged glucose metabolism and initiated a transcriptional program involving Myc activation and sterol biosynthesis after M-CSF stimulation. Perturbation of glucose metabolism or disruption of Myc function or sterol biosynthesis impaired myeloid differentiation. Integrative metabolomic and genomic profiling further identified one-carbon metabolism as a central node in mTORC1-dependent myelopoiesis. Therefore, the interplay between mTORC1 signaling and metabolic reprogramming underlies M-CSF-induced myelopoiesis.
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Factor Estimulante de Colonias de Macrófagos/fisiología , Complejos Multiproteicos/fisiología , Mielopoyesis/fisiología , Serina-Treonina Quinasas TOR/fisiología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Proteínas Portadoras/fisiología , Técnicas de Sustitución del Gen , Glucosa/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Metabolómica , Ratones , Ratones Endogámicos C57BL , Proteína Asociada al mTOR Insensible a la Rapamicina , Proteína Reguladora Asociada a mTOR , Transducción de Señal/fisiologíaRESUMEN
Immunotoxicology assessments have historically focused on the effects that xenobiotics exhibit directly on immune cells. These studies are invaluable as they identify immune cell targets and help characterize mechanisms and/or adverse outcome pathways of xenobiotics within the immune system. However, leukocytes can receive environmental cues by cell-cell contact or via released mediators from cells of organs outside of the immune system. These organs include, but are not limited to, the mucosal areas such as the lung and the gut, the liver, and the central nervous system. Homeostatic perturbation in these organs induced directly by toxicants can initiate and alter the outcome of local and systemic immunity. This review will highlight some of the identified nonimmune influences on immune homeostasis and provide summaries of how immunotoxic mechanisms of selected xenobiotics involve nonimmune cells or mediators. Thus, this review will identify data gaps and provide possible alternative mechanisms by which xenobiotics alter immune function that could be considered during immunotoxicology safety assessment.
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Comunicación Celular/efectos de los fármacos , Sistema Inmunológico/efectos de los fármacos , Toxicología/métodos , Xenobióticos/toxicidad , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Sistema Nervioso Central/efectos de los fármacos , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Humanos , Sistema Inmunológico/inmunología , Sistema Inmunológico/metabolismo , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Medición de Riesgo , Transducción de Señal/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Células del Estroma/inmunología , Células del Estroma/metabolismo , Timo/efectos de los fármacos , Timo/inmunología , Timo/metabolismoRESUMEN
mTOR signaling links bioenergetic and biosynthetic metabolism to immune responses. mTOR is activated by diverse upstream stimuli, including immune signals, growth factors, and nutrients. Recent studies highlight crucial roles of mTOR signaling in immune functions mediated by conventional T cells and Tregs In this review, we discuss the regulation of mTOR signaling in T cells and the functional impacts of mTOR and metabolic pathways on T cell-mediated immune responses, with a particular focus on the differentiation and function of Tregs.
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Memory CD8(+) T cells are an essential component of protective immunity. Signaling via mechanistic target of rapamycin (mTOR) has been implicated in the regulation of the differentiation of effector and memory T cells. However, little is understood about the mechanisms that control mTOR activity, or the effector pathways regulated by mTOR. We describe here that tuberous sclerosis 1 (Tsc1), a regulator of mTOR signaling, plays a crucial role in promoting the differentiation and function of memory CD8(+) T cells in response to Listeria monocytogenes infection. Mice with specific deletion of Tsc1 in antigen-experienced CD8(+) T cells evoked normal effector responses, but were markedly impaired in the generation of memory T cells and their recall responses to antigen reexposure in a cell-intrinsic manner. Tsc1 deficiency suppressed the generation of memory-precursor effector cells while promoting short-lived effector cell differentiation. Transcriptome analysis indicated that Tsc1 coordinated gene expression programs underlying immune function, transcriptional regulation, and cell metabolism. Furthermore, Tsc1 deletion led to excessive mTORC1 activity and dysregulated glycolytic and oxidative metabolism in response to IL-15 stimulation. These findings establish a Tsc1-mediated checkpoint in linking immune signaling and cell metabolism to orchestrate memory CD8(+) T-cell development and function.
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Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/metabolismo , Diferenciación Celular/inmunología , Memoria Inmunológica/genética , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Antígenos/inmunología , Diferenciación Celular/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Endogámicos C57BL , Complejos Multiproteicos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteína 1 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/deficienciaRESUMEN
Dendritic cells (DCs) are a heterogeneous cell population of great importance in the immune system. The emergence of new genetic technology utilizing the CD11c promoter and Cre recombinase has facilitated the dissection of functional significance and molecular regulation of DCs in immune responses and homeostasis in vivo. For the first time, this strategy allows observation of the effects of DC-specific gene deletion on immune system function in an intact organism. In this review, we present the latest findings from studies using the Cre recombinase system for cell type-specific deletion of key molecules that mediate DC homeostasis and function. Our focus is on the molecular pathways that orchestrate DC life span, migration, antigen presentation, pattern recognition, and cytokine production and signaling.
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Células Dendríticas/metabolismo , Presentación de Antígeno , Apoptosis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/inmunología , Eliminación de Gen , Integrasas/genética , Integrasas/metabolismo , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Naive T cells respond to antigen stimulation by exiting from quiescence and initiating clonal expansion and functional differentiation, but the control mechanism is elusive. Here we describe that Raptor-mTORC1-dependent metabolic reprogramming is a central determinant of this transitional process. Loss of Raptor abrogated T cell priming and T helper 2 (Th2) cell differentiation, although Raptor function is less important for continuous proliferation of actively cycling cells. mTORC1 coordinated multiple metabolic programs in T cells including glycolysis, lipid synthesis, and oxidative phosphorylation to mediate antigen-triggered exit from quiescence. mTORC1 further linked glucose metabolism to the initiation of Th2 cell differentiation by orchestrating cytokine receptor expression and cytokine responsiveness. Activation of Raptor-mTORC1 integrated T cell receptor and CD28 costimulatory signals in antigen-stimulated T cells. Our studies identify a Raptor-mTORC1-dependent pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates lymphocyte activation and fate decisions in adaptive immunity.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Diferenciación Celular , Activación de Linfocitos/fisiología , Linfocitos T/citología , Linfocitos T/inmunología , Serina-Treonina Quinasas TOR/metabolismo , Células Th2/citología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Ciclo Celular , Proliferación Celular , Células Cultivadas , Citocinas/metabolismo , Eliminación de Gen , Glucosa/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Reguladora Asociada a mTOR , Transducción de Señal , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Δ(9)-tetrahydrocannabinol (Δ(9)-THC) has potent immune modulatory properties and can impair pathogen-induced immune defenses, which in part have been attributed to ligation of the cannabinoid receptors 1 (CB(1)) and 2 (CB(2)). Most recently, dendritic cells (DC) were identified for their potential to enhance influenza-induced immunopathology in mice lacking CB(1) and CB(2) (CB(1) (-/-)CB(2) (-/-)). This study focused on the modulation of the inflammatory immune response to influenza by Δ(9)-THC and the role of CB(1) and/or CB(2) as receptor targets for Δ(9)-THC. C57Bl/6 (wild type) and CB(1) (-/-)CB(2) (-/-) mice were administered Δ(9)-THC (75 mg/kg) surrounding the intranasal instillation of A/PR/8/34 influenza virus. Three days post infection (dpi), Δ(9)-THC broadly decreased expression levels of mRNA induced by the innate immune response to influenza, suppressed the percentage of interferon-gamma (IFN-γ)-producing CD4(+) and interleukin-17-producing NK1.1(+) cells, and reduced the influx of antigen-presenting cells (APC), including inflammatory myeloid cells and monocytes/macrophages, into the lung in a CB(1)- and/or CB(2)-dependent manner. Δ(9)-THC had little effect on the expression of CD86, major histocompatibility complex I (MHC I), and MHC II by APC isolated from the lung. In vitro studies demonstrated that lipopolysaccharide (LPS)-induced maturation was suppressed by Δ(9)-THC in bone marrow-derived DC (bmDC). Furthermore, antigen-specific IFN-γ production by CD8(+) T cells after coculture was reduced by Δ(9)-THC treatment of bmDC in a CB(1)- and/or CB(2)-dependent manner. Collectively, these studies suggest that Δ(9)-THC potently suppresses myeloid cell immune function, in a manner involving CB(1) and/or CB(2), thereby impairing immune responses to influenza infection.