RESUMO
Itaconate is a unique regulatory metabolite that is induced upon Toll-like receptor (TLR) stimulation in myeloid cells. Here, we demonstrate major inflammatory tolerance and cell death phenotypes associated with itaconate production in activated macrophages. We show that endogenous itaconate is a key regulator of the signal 2 of NLR family pyrin domain containing 3 (NLRP3) inflammasome activation after long lipopolysaccharide (LPS) priming, which establishes tolerance to late NLRP3 inflammasome activation. We show that itaconate acts synergistically with inducible nitric oxide synthase (iNOS) and that the ability of various TLR ligands to establish NLRP3 inflammasome tolerance depends on the pattern of co-expression of IRG1 and iNOS. Mechanistically, itaconate accumulation upon prolonged inflammatory stimulation prevents full caspase-1 activation and processing of gasdermin D, which we demonstrate to be post-translationally modified by endogenous itaconate. Altogether, our data demonstrate that metabolic rewiring in inflammatory macrophages establishes tolerance to NLRP3 inflammasome activation that, if uncontrolled, can result in pyroptotic cell death and tissue damage.
Assuntos
Inflamassomos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Succinatos/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Caspase 1/metabolismo , Hidroliases/deficiência , Hidroliases/genética , Hidroliases/metabolismo , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas de Ligação a Fosfato/genética , Proteínas de Ligação a Fosfato/metabolismo , Poli I-C/farmacologia , Piroptose/efeitos dos fármacos , Sepse/induzido quimicamente , Sepse/metabolismo , Sepse/patologia , Transdução de Sinais/efeitos dos fármacos , Receptores Toll-Like/química , Receptores Toll-Like/metabolismoRESUMO
Pyruvate kinase (PK) deficiency is a rare hereditary disorder affecting red cell (RBC) glycolysis, causing changes in metabolism including a deficiency in ATP. This affects red cell homeostasis, promoting premature removal of RBCs from the circulation. In this study we characterized and evaluated the effect of AG-348, an allosteric activator of PK that is currently in clinical trials for treatment of PK deficiency, on RBCs and erythroid precursors from PK-deficient patients. In 15 patients ex vivo treatment with AG-348 resulted in increased enzymatic activity in all patient cells after 24 hours (mean increase 1.8-fold, range 1.2-3.4). ATP levels increased (mean increase 1.5-fold, range 1.0-2.2) similar to control cells (mean increase 1.6-fold, range, 1.4-1.8). Generally, PK thermostability was strongly reduced in PK-deficient RBCs. Ex vivo treatment with AG-348 increased residual activity 1.4 to >10-fold than residual activity of vehicle-treated samples. Protein analyses suggests that a sufficient level of PK protein is required for cells to respond to AG-348 treatment ex-vivo, as treatment effects were minimal in patient cells with very low or undetectable levels of PK-R. In half of the patients, ex vivo treatment with AG-348 was associated with an increase in RBC deformability. These data support the hypothesis that drug intervention with AG-348 effectively upregulates PK enzymatic activity and increases stability in PK-deficient RBCs over a broad range of PKLR genotypes. The concomitant increase in ATP levels suggests that glycolytic pathway activity may be restored. AG-348 treatment may represent an attractive way to correct the underlying pathologies of PK deficiency. (AG-348 is currently in clinical trials for the treatment of PK deficiency. ClinicalTrials.gov: NCT02476916, NCT03853798, NCT03548220, NCT03559699).
Assuntos
Eritrócitos , Piruvato Quinase , Trifosfato de Adenosina , Eritrócitos/metabolismo , Genótipo , Humanos , Piperazinas , Estabilidade Proteica , Piruvato Quinase/genética , QuinolinasRESUMO
Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA-damage response pathways.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Neoplasias Hematológicas/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/antagonistas & inibidores , Pirimidinas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Di-Hidro-Orotato Desidrogenase , Genômica/métodos , Neoplasias Hematológicas/tratamento farmacológico , Neoplasias Hematológicas/etiologia , Neoplasias Hematológicas/patologia , Humanos , Estadiamento de Neoplasias , Proteômica/métodosRESUMO
Following activation, macrophages undergo extensive metabolic rewiring1,2. Production of itaconate through the inducible enzyme IRG1 is a key hallmark of this process3. Itaconate inhibits succinate dehydrogenase4,5, has electrophilic properties6 and is associated with a change in cytokine production4. Here, we compare the metabolic, electrophilic and immunologic profiles of macrophages treated with unmodified itaconate and a panel of commonly used itaconate derivatives to examine its role. Using wild-type and Irg1-/- macrophages, we show that neither dimethyl itaconate, 4-octyl itaconate nor 4-monoethyl itaconate are converted to intracellular itaconate, while exogenous itaconic acid readily enters macrophages. We find that only dimethyl itaconate and 4-octyl itaconate induce a strong electrophilic stress response, in contrast to itaconate and 4-monoethyl itaconate. This correlates with their immunosuppressive phenotype: dimethyl itaconate and 4-octyl itaconate inhibited IκBζ and pro-interleukin (IL)-1ß induction, as well as IL-6, IL-10 and interferon-ß secretion, in an NRF2-independent manner. In contrast, itaconate treatment suppressed IL-1ß secretion but not pro-IL-1ß levels and, surprisingly, strongly enhanced lipopolysaccharide-induced interferon-ß secretion. Consistently, Irg1-/- macrophages produced lower levels of interferon and reduced transcriptional activation of this pathway. Our work establishes itaconate as an immunoregulatory, rather than strictly immunosuppressive, metabolite and highlights the importance of using unmodified itaconate in future studies.
Assuntos
Inflamassomos/efeitos dos fármacos , Interferon Tipo I/farmacologia , Macrófagos/efeitos dos fármacos , Succinatos/química , Succinatos/farmacologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Citocinas/metabolismo , Hidroliases/biossíntese , Hidroliases/genética , Imunidade Celular/efeitos dos fármacos , Interleucina-1beta/antagonistas & inibidores , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/antagonistas & inibidores , Relação Quantitativa Estrutura-AtividadeRESUMO
Epigenetic modifications on DNA and histones regulate gene expression by modulating chromatin accessibility to transcription machinery. Here we identify methionine as a key nutrient affecting epigenetic reprogramming in CD4+ T helper (Th) cells. Using metabolomics, we showed that methionine is rapidly taken up by activated T cells and serves as the major substrate for biosynthesis of the universal methyl donor S-adenosyl-L-methionine (SAM). Methionine was required to maintain intracellular SAM pools in T cells. Methionine restriction reduced histone H3K4 methylation (H3K4me3) at the promoter regions of key genes involved in Th17 cell proliferation and cytokine production. Applied to the mouse model of multiple sclerosis (experimental autoimmune encephalomyelitis), dietary methionine restriction reduced the expansion of pathogenic Th17 cells in vivo, leading to reduced T cell-mediated neuroinflammation and disease onset. Our data identify methionine as a key nutritional factor shaping Th cell proliferation and function in part through regulation of histone methylation.
Assuntos
Encefalomielite Autoimune Experimental , Epigênese Genética/efeitos dos fármacos , Histonas/metabolismo , Metionina , Esclerose Múltipla , Células Th17/metabolismo , Animais , Proliferação de Células , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Células HEK293 , Humanos , Metionina/metabolismo , Metionina/farmacologia , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Células Th17/citologiaRESUMO
Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers.