RESUMEN
Mucosal-associated invariant T (MAIT) cells are a subset of unconventional T cells which recognize a limited repertoire of ligands presented by the MHC class-I like molecule MR1. In addition to their key role in host protection against bacterial and viral pathogens, MAIT cells are emerging as potent anti-cancer effectors. With their abundance in human, unrestricted properties, and rapid effector functions MAIT cells are emerging as attractive candidates for immunotherapy. In the current study, we demonstrate that MAIT cells are potent cytotoxic cells, rapidly degranulating and inducing target cell death. Previous work from our group and others has highlighted glucose metabolism as a critical process for MAIT cell cytokine responses at 18 h. However, the metabolic processes supporting rapid MAIT cell cytotoxic responses are currently unknown. Here, we show that glucose metabolism is dispensable for both MAIT cell cytotoxicity and early (<3 h) cytokine production, as is oxidative phosphorylation. We show that MAIT cells have the machinery required to make (GYS-1) and metabolize (PYGB) glycogen and further demonstrate that that MAIT cell cytotoxicity and rapid cytokine responses are dependent on glycogen metabolism. In summary, we show that glycogen-fueled metabolism supports rapid MAIT cell effector functions (cytotoxicity and cytokine production) which may have implications for their use as an immunotherapeutic agent.
Asunto(s)
Glucogenólisis , Células T Invariantes Asociadas a Mucosa , Humanos , Citocinas , Glucógeno , GlucosaRESUMEN
Mutations that result in loss of function of Nod2, an intracellular receptor for bacterial peptidoglycan, are associated with Crohn's disease. Here we found that the E3 ubiquitin ligase Pellino3 was an important mediator in the Nod2 signaling pathway. Pellino3-deficient mice had less induction of cytokines after engagement of Nod2 and had exacerbated disease in various experimental models of colitis. Furthermore, expression of Pellino3 was lower in the colons of patients with Crohn's disease. Pellino3 directly bound to the kinase RIP2 and catalyzed its ubiquitination. Loss of Pellino3 led to attenuation of Nod2-induced ubiquitination of RIP2 and less activation of the transcription factor NF-κB and mitogen-activated protein kinases (MAPKs). Our findings identify RIP2 as a substrate for Pellino3 and Pellino3 as an important mediator in the Nod2 pathway and regulator of intestinal inflammation.
Asunto(s)
Colitis/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Animales , Citrobacter rodentium/inmunología , Colitis/genética , Colitis/inmunología , Colitis/microbiología , Enfermedad de Crohn/genética , Enfermedad de Crohn/inmunología , Enfermedad de Crohn/metabolismo , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Adulto JovenRESUMEN
AIMS/HYPOTHESIS: Mucosal-associated invariant T cells (MAIT cells) are an abundant population of innate T cells. When activated, MAIT cells rapidly produce a range of cytokines, including IFNγ, TNF-α and IL-17. Several studies have implicated MAIT cells in the development of metabolic dysfunction, but the mechanisms through which this occurs are not fully understood. We hypothesised that MAIT cells are associated with insulin resistance in children with obesity, and affect insulin signalling through their production of IL-17. METHODS: In a cross-sectional observational study, we investigated MAIT cell cytokine profiles in a cohort of 30 children with obesity and 30 healthy control participants, of similar age, using flow cytometry. We then used a cell-based model to determine the direct effect of MAIT cells and IL-17 on insulin signalling and glucose uptake. RESULTS: Children with obesity display increased MAIT cell frequencies (2.2% vs 2.8%, p=0.047), and, once activated, these produced elevated levels of both TNF-α (39% vs 28%, p=0.03) and IL-17 (1.25% vs 0.5%, p=0.008). The IL-17-producing MAIT cells were associated with an elevated HOMA-IR (r=0.65, p=0.001). The MAIT cell secretome from adults with obesity resulted in reduced glucose uptake when compared with the secretome from healthy adult control (1.31 vs 0.96, p=0.0002), a defect that could be blocked by neutralising IL-17. Finally, we demonstrated that recombinant IL-17 blocked insulin-mediated glucose uptake via inhibition of phosphorylated Akt and extracellular signal-regulated kinase. CONCLUSIONS/INTERPRETATIONS: Collectively, these studies provide further support for the role of MAIT cells in the development of metabolic dysfunction, and suggest that an IL-17-mediated effect on intracellular insulin signalling is responsible.
Asunto(s)
Resistencia a la Insulina , Células T Invariantes Asociadas a Mucosa , Obesidad Infantil , Adulto , Niño , Estudios Transversales , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Interleucina-17/metabolismo , Activación de Linfocitos , Obesidad Infantil/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Obesity underpins the development of numerous chronic diseases, such as type II diabetes mellitus. It is well established that obesity negatively alters immune cell frequencies and functions. Mucosal-associated invariant T (MAIT) cells are a population of innate T cells, which we have previously reported are dysregulated in obesity, with altered circulating and adipose tissue frequencies and a reduction in their IFN-γ production, which is a critical effector function of MAIT cells in host defense. Hence, there is increased urgency to characterize the key molecular mechanisms that drive MAIT cell effector functions and to identify those which are impaired in the obesity setting. In this study, we found that MAIT cells significantly upregulate their rates of glycolysis upon activation in an mTORC1-dependent manner, and this is essential for MAIT cell IFN-γ production. Furthermore, we show that mTORC1 activation is dependent on amino acid transport via SLC7A5. In obese patients, using RNA sequencing, Seahorse analysis, and a series of in vitro experiments, we demonstrate that MAIT cells isolated from obese adults display defective glycolytic metabolism, mTORC1 signaling, and SLC7A5 aa transport. Collectively, our data detail the intrinsic metabolic pathways controlling MAIT cell cytokine production and highlight mTORC1 as an important metabolic regulator that is impaired in obesity, leading to altered MAIT cell responses.
Asunto(s)
Diabetes Mellitus Tipo 2/inmunología , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Células T Invariantes Asociadas a Mucosa/fisiología , Obesidad/inmunología , Adulto , Células Cultivadas , Femenino , Glucólisis , Humanos , Interferón gamma/metabolismo , Activación de Linfocitos , Masculino , Análisis de Secuencia de ARN , Transducción de SeñalRESUMEN
Mucosal-associated invariant T (MAIT) cells are an abundant population of innate T cells that recognize bacterial ligands and play a key role in host protection against bacterial and viral pathogens. Upon activation, MAIT cells undergo proliferative expansion and increase their production of effector molecules such as cytokines. In this study, we found that both mRNA and protein abundance of the key metabolism regulator and transcription factor MYC was increased in stimulated MAIT cells. Using quantitative mass spectrometry, we identified the activation of two MYC-controlled metabolic pathways, amino acid transport and glycolysis, both of which were necessary for MAIT cell proliferation. Last, we showed that MAIT cells isolated from people with obesity showed decreased MYC mRNA abundance upon activation, which was associated with defective MAIT cell proliferation and functional responses. Collectively, our data uncover the importance of MYC-regulated metabolism for MAIT cell proliferation and provide additional insight into the molecular basis for the functional defects of MAIT cells in obesity.
Asunto(s)
Células T Invariantes Asociadas a Mucosa , Humanos , Células T Invariantes Asociadas a Mucosa/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Obesidad/metabolismo , Glucólisis , Activación de Linfocitos , Proliferación CelularRESUMEN
The NLRP3 inflammasome has an important function in inflammation by promoting the processing of pro-IL-1ß and pro-IL-18 to their mature bioactive forms, and by inducing cell death via pyroptosis. Here we show a critical function of the E3 ubiquitin ligase Pellino2 in facilitating activation of the NLRP3 inflammasome. Pellino2-deficient mice and myeloid cells have impaired activation of NLRP3 in response to toll-like receptor priming, NLRP3 stimuli and bacterial challenge. These functions of Pellino2 in the NLRP3 pathway are dependent on Pellino2 FHA and RING-like domains, with Pellino2 promoting the ubiquitination of NLRP3 during the priming phase of activation. We also identify a negative function of IRAK1 in the NLRP3 inflammasome, and describe a counter-regulatory relationship between IRAK1 and Pellino2. Our findings reveal a Pellino2-mediated regulatory signaling system that controls activation of the NLRP3 inflammasome.
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Inflamasomas/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Proteínas Nucleares/inmunología , Animales , Humanos , Inflamasomas/genética , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Macrófagos/inmunología , Macrófagos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Dominios Proteicos , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Transducción de Señal , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunología , UbiquitinaciónRESUMEN
The immune suppressive and anti-inflammatory capabilities of bone marrow-derived mesenchymal stromal cells (MSCs) represent an innovative new tool in regenerative medicine and immune regulation. The potent immune suppressive ability of MSC over T cells, dendritic cells, and natural killer cells has been extensively characterized, however, the effect of MSC on B cell function has not yet been clarified. In this study, the direct effect of MSC on peripheral blood B cell function is defined and the mechanism utilized by MSC in enhancing B cell survival in vitro identified. Human MSC supported the activation, proliferation, and survival of purified CD19(+) B cells through a cell contact-dependent mechanism. These effects were not mediated through B cell activating factor or notch signaling. However, cell contact between MSC and B cells resulted in increased production of vascular endothelial growth factor (VEGF) by MSC facilitating AKT phosphorylation within the B cell and inhibiting caspase 3-mediated apoptosis. Blocking studies demonstrated that this cell contact-dependent effect was not dependent on signaling through CXCR4-CXCL12 or through the epidermal growth factor receptor (EGFR). These results suggest that direct cell contact between MSC and B cells supports B cell viability and function, suggesting that MSC may not represent a suitable therapy for B cell-mediated disease.
Asunto(s)
Apoptosis/inmunología , Linfocitos B/citología , Caspasa 3/metabolismo , Células Madre Mesenquimatosas/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Antígenos CD19/inmunología , Linfocitos B/inmunología , Células de la Médula Ósea/citología , Proliferación Celular/fisiología , Humanos , Células Asesinas Naturales/inmunología , Trasplante de Células Madre Mesenquimatosas , Linfocitos T/citología , Activación Transcripcional/inmunología , Regulación hacia ArribaRESUMEN
Receptor families of the innate immune response engage in 'cross-talk' to tailor optimal immune responses against invading pathogens. However, these responses are subject to multiple levels of regulation to keep in check aberrant inflammatory signals. Here, we describe a role for the orphan receptor interleukin-17 receptor D (IL-17RD) in negatively regulating Toll-like receptor (TLR)-induced responses. Deficiency of IL-17RD expression in cells leads to enhanced pro-inflammatory signalling and gene expression in response to TLR stimulation, and Il17rd(-/-) mice are more susceptible to TLR-induced septic shock. We demonstrate that the intracellular Sef/IL-17R (SEFIR) domain of IL-17RD targets TIR adaptor proteins to inhibit TLR downstream signalling thus revealing a paradigm involving cross-regulation of members of the IL-17R and TLR families.