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1.
J Immunol ; 212(1): 13-23, 2024 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-37991425

RESUMO

4-Octyl itaconate (4-OI) is a derivative of the Krebs cycle-derived metabolite itaconate and displays an array of antimicrobial and anti-inflammatory properties through modifying cysteine residues within protein targets. We have found that 4-OI significantly reduces the production of eosinophil-targeted chemokines in a variety of cell types, including M1 and M2 macrophages, Th2 cells, and A549 respiratory epithelial cells. Notably, the suppression of these chemokines in M1 macrophages was found to be NRF2-dependent. In addition, 4-OI can interfere with IL-5 signaling and directly affect eosinophil differentiation. In a model of eosinophilic airway inflammation in BALB/c mice, 4-OI alleviated airway resistance and reduced eosinophil recruitment to the lungs. Our findings suggest that itaconate derivatives could be promising therapeutic agents for the treatment of eosinophilic asthma.


Assuntos
Eosinófilos , Eosinofilia Pulmonar , Camundongos , Animais , Eosinofilia Pulmonar/tratamento farmacológico , Quimiocinas , Inflamação/tratamento farmacológico
2.
Nat Immunol ; 10(6): 579-86, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19412184

RESUMO

Toll-like receptor 4 (TLR4) signals the induction of transcription factor IRF3-dependent genes from the early endosome via the adaptor TRAM. Here we report a splice variant of TRAM, TAG ('TRAM adaptor with GOLD domain'), which has a Golgi dynamics domain coupled to TRAM's Toll-interleukin 1 receptor domain. After stimulation with lipopolysaccharide, TRAM and TAG localized to late endosomes positive for the GTPase Rab7a. TAG inhibited activation of IRF3 by lipopolysaccharide. Knockdown of TAG with small interfering RNA enhanced induction of the chemokine CCL5 (RANTES), but not of interleukin 8, by lipopolysaccharide in human peripheral blood mononuclear cells. TAG displaced the adaptor TRIF from TRAM. TAG is therefore an example of a specific inhibitor of the adaptor MyD88-independent pathway activated by TLR4. Targeting TAG could be useful in the effort to boost the immunostimulatory effect of TLR4 without causing unwanted inflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Endossomos/metabolismo , Fator Regulador 3 de Interferon/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Quimiocina CCL5/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Lipopolissacarídeos/metabolismo , Camundongos , Dados de Sequência Molecular , Fator 88 de Diferenciação Mieloide/metabolismo , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Especificidade por Substrato , Transfecção , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7
3.
Proc Natl Acad Sci U S A ; 115(36): E8460-E8468, 2018 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-30127006

RESUMO

A variety of innate immune responses and functions are dependent on time of day, and many inflammatory conditions are associated with dysfunctional molecular clocks within immune cells. However, the functional importance of these innate immune clocks has yet to be fully characterized. NRF2 plays a critical role in the innate immune system, limiting inflammation via reactive oxygen species (ROS) suppression and direct repression of the proinflammatory cytokines, IL-1ß and IL-6. Here we reveal that the core molecular clock protein, BMAL1, controls the mRNA expression of Nrf2 via direct E-box binding to its promoter to regulate its activity. Deletion of Bmal1 decreased the response of NRF2 to LPS challenge, resulting in a blunted antioxidant response and reduced synthesis of glutathione. ROS accumulation was increased in Bmal1-/- macrophages, facilitating accumulation of the hypoxic response protein, HIF-1α. Increased ROS and HIF-1α levels, as well as decreased activity of NRF2 in cells lacking BMAL1, resulted in increased production of the proinflammatory cytokine, IL-1ß. The excessive prooxidant and proinflammatory phenotype of Bmal1-/- macrophages was rescued by genetic and pharmacological activation of NRF2, or through addition of antioxidants. Our findings uncover a clear role for the molecular clock in regulating NRF2 in innate immune cells to control the inflammatory response. These findings provide insights into the pathology of inflammatory conditions, in which the molecular clock, oxidative stress, and IL-1ß are known to play a role.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Fatores de Transcrição ARNTL/genética , Animais , Células HEK293 , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/metabolismo , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Macrófagos/patologia , Camundongos , Camundongos Knockout , Fator 2 Relacionado a NF-E2/genética , Espécies Reativas de Oxigênio/metabolismo
4.
Am J Respir Crit Care Med ; 200(11): 1381-1391, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31454256

RESUMO

Rationale: Cystic fibrosis (CF) pulmonary disease is characterized by chronic infection with Pseudomonas aeruginosa and sustained neutrophil-dominant inflammation. The lack of effective antiinflammatory therapies for people with CF (PWCF) represents a significant challenge.Objectives: To identify altered immunometabolism in the CF neutrophil and investigate the feasibility of specific inhibition of the NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome as a CF antiinflammatory strategy in vivo.Methods: Key markers of increased aerobic glycolysis, known as a Warburg effect, including cytosolic PKM2 (pyruvate kinase M2), phosphorylated PKM2, succinate, HIF-1α (hypoxia-inducible factor-1α), lactate, and the IL-1ß precursor pro-IL-1ß, as well as caspase-1 activity and processing of pro-IL-1ß to IL-1ß by the NLRP3 inflammasome, were measured in neutrophils from blood and airway secretions from healthy control subjects (n = 12), PWCF (n = 16), and PWCF after double-lung transplantation (n = 6). The effects of specific inhibition of NLRP3 on airway inflammation and bacterial clearance in a murine CF model were subsequently assessed in vivo.Measurements and Main Results: CF neutrophils display increased aerobic glycolysis in the systemic circulation. This effect is driven by low-level endotoxemia, unaffected by CFTR (cystic fibrosis transmembrane conductance regulator) modulation, and resolves after transplant. The increased pro-IL-1ß produced is processed to its mature active form in the LPS-rich CF lung by the NLRP3 inflammasome via caspase-1. Specific NLRP3 inhibition in vivo with MCC950 inhibited IL-1ß in the lungs of CF mice (P < 0.0001), resulting in significantly reduced airway inflammation and improved Pseudomonas clearance (P < 0.0001).Conclusions: CF neutrophil immunometabolism is altered in response to inflammation. NLRP3 inflammasome inhibition may have an antiinflammatory and anti-infective role in CF.


Assuntos
Anti-Inflamatórios/uso terapêutico , Fibrose Cística/tratamento farmacológico , Furanos/uso terapêutico , Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sulfonamidas/uso terapêutico , Animais , Biomarcadores/análise , Líquido da Lavagem Broncoalveolar/química , Compostos Heterocíclicos de 4 ou mais Anéis , Humanos , Indenos , Interleucina-1beta/análise , Camundongos , Neutrófilos/efeitos dos fármacos , Infecções por Pseudomonas/etiologia , Infecções por Pseudomonas/terapia , Sulfonas
5.
J Immunol ; 198(9): 3558-3564, 2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28298525

RESUMO

PGE2 has been shown to increase the transcription of pro-IL-1ß. However, recently it has been demonstrated that PGE2 can block the maturation of IL-1ß by inhibiting the NLRP3 inflammasome in macrophages. These apparently conflicting results have led us to reexamine the effect of PGE2 on IL-1ß production. We have found that in murine bone marrow-derived macrophages, PGE2 via the cAMP/protein kinase A pathway is potently inducing IL-1ß transcription, as well as boosting the ability of LPS to induce IL-1ß mRNA and pro-IL-1ß while inhibiting the production of TNF-α. This results in an increase in mature IL-1ß production in macrophages treated with ATP. We also examined the effect of endogenously produced PGE2 on IL-1ß production. By blocking PGE2 production with indomethacin, we made a striking finding that endogenous PGE2 is essential for LPS-induced pro-IL-1ß production, suggesting a positive feedback loop. The effect of endogenous PGE2 was mediated by EP2 receptor. In primary human monocytes, where LPS alone is sufficient to induce mature IL-1ß, PGE2 boosted LPS-induced IL-1ß production. PGE2 did not inhibit ATP-induced mature IL-1ß production in monocytes. Because PGE2 mediates the pyrogenic effect of IL-1ß, these effects might be especially relevant for the role of monocytes in the induction of fever. A positive feedback loop from IL-1ß and back to PGE2, which itself is induced by IL-1ß, is likely to be operating. Furthermore, fever might therefore occur in the absence of a septic shock response because of the inhibiting effect of PGE2 on TNF-α production.


Assuntos
Dinoprostona/metabolismo , Febre/imunologia , Interleucina-1beta/metabolismo , Macrófagos/imunologia , Monócitos/imunologia , Trifosfato de Adenosina/farmacologia , Animais , Células Cultivadas , Dinoprostona/antagonistas & inibidores , Retroalimentação Fisiológica , Humanos , Indometacina/farmacologia , Inflamassomos/metabolismo , Interleucina-1beta/genética , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
6.
J Immunol ; 196(6): 2444-9, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26873991

RESUMO

Recent advances in immunometabolism link metabolic changes in stimulated macrophages to production of IL-1ß, a crucial cytokine in the innate immune response to Mycobacterium tuberculosis. To investigate this pathway in the host response to M. tuberculosis, we performed metabolic and functional studies on human alveolar macrophages, human monocyte-derived macrophages, and murine bone marrow-derived macrophages following infection with the bacillus in vitro. M. tuberculosis infection induced a shift from oxidative phosphorylation to aerobic glycolysis in macrophages. Inhibition of this shift resulted in decreased levels of proinflammatory IL-1ß and decreased transcription of PTGS2, increased levels of anti-inflammatory IL-10, and increased intracellular bacillary survival. Blockade or absence of IL-1R negated the impact of aerobic glycolysis on intracellular bacillary survival, demonstrating that infection-induced glycolysis limits M. tuberculosis survival in macrophages through induction of IL-1ß. Drugs that manipulate host metabolism may be exploited as adjuvants for future therapeutic and vaccination strategies.


Assuntos
Imunidade Inata/imunologia , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/microbiologia , Tuberculose Pulmonar/imunologia , Tuberculose Pulmonar/metabolismo , Animais , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Glicólise/imunologia , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Macrófagos Alveolares/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/imunologia , Reação em Cadeia da Polimerase em Tempo Real , Tuberculose Pulmonar/microbiologia
7.
Proc Natl Acad Sci U S A ; 112(23): 7231-6, 2015 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-25995365

RESUMO

The response to an innate immune challenge is conditioned by the time of day, but the molecular basis for this remains unclear. In myeloid cells, there is a temporal regulation to induction by lipopolysaccharide (LPS) of the proinflammatory microRNA miR-155 that correlates inversely with levels of BMAL1. BMAL1 in the myeloid lineage inhibits activation of NF-κB and miR-155 induction and protects mice from LPS-induced sepsis. Bmal1 has two miR-155-binding sites in its 3'-UTR, and, in response to LPS, miR-155 binds to these two target sites, leading to suppression of Bmal1 mRNA and protein in mice and humans. miR-155 deletion perturbs circadian function, gives rise to a shorter circadian day, and ablates the circadian effect on cytokine responses to LPS. Thus, the molecular clock controls miR-155 induction that can repress BMAL1 directly. This leads to an innate immune response that is variably responsive to challenges across the circadian day.


Assuntos
Fatores de Transcrição ARNTL/fisiologia , Ritmo Circadiano , Imunidade Inata , Macrófagos/imunologia , MicroRNAs/fisiologia , Regiões 3' não Traduzidas , Fatores de Transcrição ARNTL/genética , Tecido Adiposo/metabolismo , Animais , Citocinas/biossíntese , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo
8.
Bioessays ; 35(11): 965-73, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24115022

RESUMO

Inflammatory immune cells, when activated, display much the same metabolic profile as a glycolytic tumor cell. This involves a shift in metabolism away from oxidative phosphorylation towards aerobic glycolysis, a phenomenon known as the Warburg effect. The result of this change in macrophages is to rapidly provide ATP and metabolic intermediates for the biosynthesis of immune and inflammatory proteins. In addition, a rise in certain tricarboxylic acid cycle intermediates occurs notably in citrate for lipid biosynthesis, and succinate, which activates the transcription factor Hypoxia-inducible factor. In this review we take a look at the emerging evidence for a role for the Warburg effect in the immune and inflammatory responses. The reprogramming of metabolic pathways in macrophages, dendritic cells, and T cells could have relevance in the pathogenesis of inflammatory and metabolic diseases and might provide novel therapeutic strategies.


Assuntos
Glicólise/fisiologia , Inflamação/patologia , Neoplasias/patologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico/fisiologia , Células Dendríticas/metabolismo , Hexoquinase/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imunidade Inata , Inflamação/imunologia , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/metabolismo , Redes e Vias Metabólicas/fisiologia , Mitocôndrias/metabolismo , Neoplasias/imunologia , Fosforilação Oxidativa , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Ácido Succínico/metabolismo , Hormônios Tireóideos/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Ligação a Hormônio da Tireoide
9.
Cell Res ; 30(4): 300-314, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132672

RESUMO

The growing field of immunometabolism has taught us how metabolic cellular reactions and processes not only provide a means to generate ATP and biosynthetic precursors, but are also a way of controlling immunity and inflammation. Metabolic reprogramming of immune cells is essential for both inflammatory as well as anti-inflammatory responses. Four anti-inflammatory therapies, DMF, Metformin, Methotrexate and Rapamycin all work by affecting metabolism and/or regulating or mimicking endogenous metabolites with anti-inflammatory effects. Evidence is emerging for the targeting of specific metabolic events as a strategy to limit inflammation in different contexts. Here we discuss these recent developments and speculate on the prospect of targeting immunometabolism in the effort to develop novel anti-inflammatory therapeutics. As accumulating evidence for roles of an intricate and elaborate network of metabolic processes, including lipid, amino acid and nucleotide metabolism provides key focal points for developing new therapies, we here turn our attention to glycolysis and the TCA cycle to provide examples of how metabolic intermediates and enzymes can provide potential novel therapeutic targets.


Assuntos
Anti-Inflamatórios/uso terapêutico , Doenças Autoimunes , Imunomodulação , Imunossupressores/uso terapêutico , Inflamação , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Fumarato de Dimetilo/farmacologia , Fumarato de Dimetilo/uso terapêutico , Glicólise/efeitos dos fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Sirolimo/farmacologia , Sirolimo/uso terapêutico
10.
Cell Metab ; 31(2): 391-405.e8, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31761564

RESUMO

Pyruvate kinase (PK) catalyzes the conversion of phosphoenolpyruvate to pyruvate during glycolysis. The PK isoform PKM2 has additional roles in regulation of gene transcription and protein phosphorylation. PKM2 has been shown to control macrophage metabolic remodeling in inflammation, but its role in T cell biology is poorly understood. Here, we report PKM2 upregulation, phosphorylation, and nuclear accumulation in murine and human CD4+ T cells following activation in vitro. Treatment of T cells with TEPP-46, an allosteric activator that induces PKM2 tetramerization and blocks its nuclear translocation, strongly reduces their activation, proliferation, and cytokine production by inhibiting essential signaling pathways and thus preventing the engagement of glycolysis. TEPP-46 limits the development of both T helper 17 (Th17) and Th1 cells in vitro and ameliorates experimental autoimmune encephalomyelitis (EAE) in vivo. Overall, our results suggest that pharmacological targeting of PKM2 may represent a valuable therapeutic approach in T cell-mediated inflammation and autoimmunity.


Assuntos
Proteínas de Transporte/metabolismo , Ativadores de Enzimas/farmacologia , Proteínas de Membrana/metabolismo , Piridazinas/farmacologia , Pirróis/farmacologia , Células Th1 , Hormônios Tireóideos/metabolismo , Animais , Autoimunidade/efeitos dos fármacos , Células Cultivadas , Feminino , Humanos , Inflamação/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Células Th1/citologia , Células Th1/efeitos dos fármacos , Células Th1/imunologia , Proteínas de Ligação a Hormônio da Tireoide
11.
Nat Commun ; 11(1): 1055, 2020 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-32103022

RESUMO

Activated caspase-1 and caspase-11 induce inflammatory cell death in a process termed pyroptosis. Here we show that Prostaglandin E2 (PGE2) inhibits caspase-11-dependent pyroptosis in murine and human macrophages. PGE2 suppreses caspase-11 expression in murine and human macrophages and in the airways of mice with allergic inflammation. Remarkably, caspase-11-deficient mice are strongly resistant to developing experimental allergic airway inflammation, where PGE2 is known to be protective. Expression of caspase-11 is elevated in the lung of wild type mice with allergic airway inflammation. Blocking PGE2 production with indomethacin enhances, whereas the prostaglandin E1 analog misoprostol inhibits lung caspase-11 expression. Finally, alveolar macrophages from asthma patients exhibit increased expression of caspase-4, a human homologue of caspase-11. Our findings identify PGE2 as a negative regulator of caspase-11-driven pyroptosis and implicate caspase-4/11 as a critical contributor to allergic airway inflammation, with implications for pathophysiology of asthma.


Assuntos
Asma/patologia , Caspases Iniciadoras/metabolismo , Dinoprostona/metabolismo , Macrófagos/imunologia , Piroptose/fisiologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Asma/imunologia , Caspases Iniciadoras/genética , Caspases Iniciadoras/imunologia , Células Cultivadas , Sinergismo Farmacológico , Feminino , Humanos , Indometacina/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Misoprostol/farmacologia
12.
Cell Rep ; 29(1): 151-161.e5, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31577945

RESUMO

The NLRP3 inflammasome is a cytosolic complex sensing phagocytosed material and various damage-associated molecular patterns, triggering production of the pro-inflammatory cytokines interleukin-1 beta (IL)-1ß and IL-18 and promoting pyroptosis. Here, we characterize glutathione transferase omega 1-1 (GSTO1-1), a constitutive deglutathionylating enzyme, as a regulator of the NLRP3 inflammasome. Using a small molecule inhibitor of GSTO1-1 termed C1-27, endogenous GSTO1-1 knockdown, and GSTO1-1-/- mice, we report that GSTO1-1 is involved in NLRP3 inflammasome activation. Mechanistically, GSTO1-1 deglutathionylates cysteine 253 in NIMA related kinase 7 (NEK7) to promote NLRP3 activation. We therefore identify GSTO1-1 as an NLRP3 inflammasome regulator, which has potential as a drug target to limit NLRP3-mediated inflammation.


Assuntos
Glutationa Transferase/metabolismo , Inflamassomos/metabolismo , Quinases Relacionadas a NIMA/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Citocinas/metabolismo , Células HEK293 , Humanos , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
13.
Front Immunol ; 9: 270, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29520272

RESUMO

Traditionally cellular respiration or metabolism has been viewed as catabolic and anabolic pathways generating energy and biosynthetic precursors required for growth and general cellular maintenance. However, growing literature provides evidence of a much broader role for metabolic reactions and processes in controlling immunological effector functions. Much of this research into immunometabolism has focused on macrophages, cells that are central in pro- as well as anti-inflammatory responses-responses that in turn are a direct result of metabolic reprogramming. As we learn more about the precise role of metabolic pathways and pathway intermediates in immune function, a novel opportunity to target immunometabolism therapeutically has emerged. Here, we review the current understanding of the regulation of macrophage function through metabolic remodeling.


Assuntos
Metabolismo Energético , Imunomodulação , Inflamação/metabolismo , Macrófagos/metabolismo , Animais , Respiração Celular , Reprogramação Celular , Humanos , Ativação de Macrófagos , Macrófagos/imunologia , Transdução de Sinais
14.
Front Immunol ; 8: 1300, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29081778

RESUMO

Blocking interaction of the immune checkpoint receptor PD-1 with its ligand PD-L1 is associated with good clinical outcomes in a broad variety of malignancies. High levels of PD-L1 promote tumor growth by restraining CD8+ T-cell responses against tumors. Limiting PD-L1 expression and function is therefore critical for allowing the development of antitumor immune responses and effective tumor clearance. Pyruvate kinase isoform M2 (PKM2) is also a key player in regulating cancer as well as immune responses. PKM2 catalyzes the final rate-limiting step of glycolysis. Furthermore, PKM2 as a dimer translocates to the nucleus, where it stimulates hypoxia-inducible factor 1α (Hif-1α) transactivation domain function and recruitment of p300 to the hypoxia response elements (HRE) of Hif-1α target genes. Here, we provide the first evidence of a role for PKM2 in regulating the expression of PD-L1 on macrophages, dendritic cells (DCs), T cells, and tumor cells. LPS-induced expression of PD-L1 in primary macrophages was inhibited by the PKM2 targeting compound TEPP-46. Furthermore, RNA silencing of PKM2 inhibited LPS-induced PD-L1 expression. This regulation occurs through direct binding of PKM2 and Hif-1α to HRE sites on the PD-L1 promoter. Moreover, TEPP-46 inhibited expression of PD-L1 on macrophages, DCs, and T cells as well as tumor cells in a mouse CT26 cancer model. These findings broaden our understanding of how PKM2 may contribute to tumor progression and may explain the upregulation of PD-L1 in the tumor microenvironment.

15.
Front Immunol ; 7: 145, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27148264

RESUMO

Pyruvate kinase (PK) is the enzyme responsible for catalyzing the last step of glycolysis. Of the four PK isoforms expressed in mammalian cells, PKM2 has generated the most interest due to its impact on changes in cellular metabolism observed in cancer as well as in activated immune cells. As our understanding of dysregulated metabolism in cancer develops, and in light of the growing field of immunometabolism, intense efforts are in place to define the mechanism by which PKM2 regulates the metabolic profile of cancer as well as of immune cells. The enzymatic activity of PKM2 is heavily regulated by endogenous allosteric effectors as well as by intracellular signaling pathways, affecting both the enzymatic activity of PKM2 as a PK and the regulation of the recently described non-canonical nuclear functions of PKM2. We here review the current literature on PKM2 and its regulation, and discuss the potential for this protein as a therapeutic target in inflammatory disorders.

16.
Cell Metab ; 21(1): 65-80, 2015 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-25565206

RESUMO

Macrophages activated by the TLR4 agonist LPS undergo dramatic changes in their metabolic activity. We here show that LPS induces expression of the key metabolic regulator Pyruvate Kinase M2 (PKM2). Activation of PKM2 using two well-characterized small molecules, DASA-58 and TEPP-46, inhibited LPS-induced Hif-1α and IL-1ß, as well as the expression of a range of other Hif-1α-dependent genes. Activation of PKM2 attenuated an LPS-induced proinflammatory M1 macrophage phenotype while promoting traits typical of an M2 macrophage. We show that LPS-induced PKM2 enters into a complex with Hif-1α, which can directly bind to the IL-1ß promoter, an event that is inhibited by activation of PKM2. Both compounds inhibited LPS-induced glycolytic reprogramming and succinate production. Finally, activation of PKM2 by TEPP-46 in vivo inhibited LPS and Salmonella typhimurium-induced IL-1ß production, while boosting production of IL-10. PKM2 is therefore a critical determinant of macrophage activation by LPS, promoting the inflammatory response.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Piruvato Quinase/metabolismo , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Ativadores de Enzimas/farmacologia , Expressão Gênica/efeitos dos fármacos , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Interleucina-1beta/genética , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Ligação Proteica , Piruvato Quinase/química , Piruvato Quinase/genética , RNA Mensageiro/metabolismo , Salmonella typhimurium/fisiologia , Receptor 4 Toll-Like/agonistas , Receptor 4 Toll-Like/metabolismo
18.
J Biol Chem ; 283(21): 14277-85, 2008 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-18356163

RESUMO

Phosphorylation of the transcription factor interferon regulatory factor 3 (IRF3) is essential for the induction of promoters which contain the interferon-stimulated response element (ISRE). IRF3 can be activated by Toll-like receptor 3 (TLR3) in response to the double-stranded RNA mimic poly(I-C) and by TLR4 in response to lipopolysaccharide (LPS). Here we have analyzed the effect of the glucocorticoid dexamethasone on this response. Dexamethasone inhibited the induction of the ISRE-dependent gene RANTES (regulated on activation normal T cell expressed and secreted) in both U373-CD14 cells and human peripheral blood mononuclear cells and also an ISRE luciferase construct, activated by either TLR3 or TLR4. It also inhibited increased phosphorylation of IRF3 in its N terminus in response to LPS and in its C terminus on Ser-396 in response to either poly(I-C) or LPS. Several dexamethasone-induced phosphatases were tested for possible involvement in these effects; MKP1 did not appear to be involved, although MKP2 and MKP5 both partially inhibited induction of the ISRE, pointing to their possible involvement in the effect of dexamethasone. Importantly, we found that dexamethasone could inhibit TBK1 kinase activity and TBK1 phosphorylation on Ser-172, both of which are required for IRF3 phosphorylation downstream of TLR3 and TLR4 stimulation. Our study, therefore, demonstrates that TBK1 is a target for dexamethasone, common to both TLR3 and TLR4 signaling.


Assuntos
Glucocorticoides/farmacologia , Fator Regulador 3 de Interferon/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Células Cultivadas , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Dextroanfetamina/farmacologia , Dimerização , Ativação Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter/genética , Humanos , Lipopolissacarídeos/farmacologia , Fosforilação/efeitos dos fármacos , Poli I-C/farmacologia
19.
Proc Natl Acad Sci U S A ; 103(24): 9196-201, 2006 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-16757566

RESUMO

PKCepsilon has been shown to play a key role in the effect of the Gram-negative bacterial product LPS; however, the target for PKCepsilon in LPS signaling is unknown. LPS signaling is mediated by Toll-like receptor 4, which uses four adapter proteins, MyD88, MyD88 adapter-like (Mal), Toll/IL-1R domain-containing adapter inducing IFN-beta (Trif), and Trif-related adapter molecule (TRAM). Here we show that TRAM is transiently phosphorylated by PKCepsilon on serine-16 in an LPS-dependent manner. Activation of IFN regulatory factor 3 and induction of the chemokine RANTES, which are both TRAM-dependent, were attenuated in PKCepsilon-deficient cells. TRAMS16A is inactive when overexpressed and is attenuated in its ability to reconstitute signaling in TRAM-deficient cells. We have therefore uncovered a key process in Toll-like receptor 4 signaling, identifying TRAM as the target for PKCepsilon.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Isoenzimas/metabolismo , Proteína Quinase C-épsilon/metabolismo , Receptores de Interleucina/metabolismo , Transdução de Sinais/fisiologia , Receptor 4 Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Membrana Celular/metabolismo , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Isoenzimas/genética , Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Knockout , Fosforilação , Proteína Quinase C-épsilon/genética , Receptores de Interleucina/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptor 4 Toll-Like/genética
20.
Immunology ; 113(2): 153-62, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15379975

RESUMO

An understanding of lipopolysaccharide (LPS) signal transduction is a key goal in the effort to provide a molecular basis for the lethal effect of LPS during septic shock and point the way to novel therapies. Rapid progress in this field during the last 6 years has resulted in the discovery of not only the receptor for LPS - Toll-like receptor 4 (TLR4) - but also in a better appreciation of the complexity of the signalling pathways activated by LPS. Soon after the discovery of TLR4, the formation of a receptor complex in response to LPS, consisting of dimerized TLR4 and MD-2, was described. Intracellular events following the formation of this receptor complex depend on different sets of adapters. An early response, which is dependent on MyD88 and MyD88-like adapter (Mal), leads to the activation of nuclear factor-kappaB (NF-kappaB). A later response to LPS makes use of TIR-domain-containing adapter-inducing interferon-beta (TRIF) and TRIF-related adapter molecule (TRAM), and leads to the late activation of NF-kappaB and IRF3, and to the induction of cytokines, chemokines, and other transcription factors. As LPS signal transduction is an area of intense research and rapid progress, this review is intended to sum up our present understanding of the events following LPS binding to TLR4, and we also attempt to create a model of the signalling pathways activated by LPS.


Assuntos
Glicoproteínas de Membrana/imunologia , Receptores de Superfície Celular/imunologia , Transdução de Sinais/imunologia , Proteínas de Fase Aguda/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Antígenos de Diferenciação/imunologia , Antígenos de Superfície/imunologia , Proteínas de Transporte/imunologia , Humanos , Receptores de Lipopolissacarídeos/imunologia , Lipopolissacarídeos/imunologia , Antígeno 96 de Linfócito , Modelos Imunológicos , Fator 88 de Diferenciação Mieloide , Receptores Imunológicos/imunologia , Receptor 4 Toll-Like , Receptores Toll-Like
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