RESUMO
Cytokines that signal via STAT1 and STAT3 transcription factors instruct decisions affecting tissue homeostasis, antimicrobial host defense, and inflammation-induced tissue injury. To understand the coordination of these activities, we applied RNA sequencing, chromatin immunoprecipitation sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing to identify the transcriptional output of STAT1 and STAT3 in peritoneal tissues from mice during acute resolving inflammation and inflammation primed to drive fibrosis. Bioinformatics focused on the transcriptional signature of the immunomodulatory cytokine IL-6 in both settings and examined how profibrotic IFN-γ-secreting CD4+ T cells altered the interpretation of STAT1 and STAT3 cytokine cues. In resolving inflammation, STAT1 and STAT3 cooperated to drive stromal gene expression affecting antimicrobial immunity and tissue homeostasis. The introduction of IFN-γ-secreting CD4+ T cells altered this transcriptional program and channeled STAT1 and STAT3 to a previously latent IFN-γ activation site motif in Alu-like elements. STAT1 and STAT3 binding to this conserved sequence revealed evidence of reciprocal cross-regulation and gene signatures relevant to pathophysiology. Thus, we propose that effector T cells retune the transcriptional output of IL-6 by shaping a regulatory interplay between STAT1 and STAT3 in inflammation.
Assuntos
Interleucina-6 , Células Th1 , Animais , Camundongos , Citocinas/metabolismo , Inflamação/metabolismo , Interleucina-6/metabolismo , Retroelementos , Fatores de Transcrição STAT/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Th1/metabolismoRESUMO
Oxylipins are potent biological mediators requiring strict control, but how they are removed en masse during infection and inflammation is unknown. Here we show that lipopolysaccharide (LPS) dynamically enhances oxylipin removal via mitochondrial ß-oxidation. Specifically, genetic or pharmacological targeting of carnitine palmitoyl transferase 1 (CPT1), a mitochondrial importer of fatty acids, reveal that many oxylipins are removed by this protein during inflammation in vitro and in vivo. Using stable isotope-tracing lipidomics, we find secretion-reuptake recycling for 12-HETE and its intermediate metabolites. Meanwhile, oxylipin ß-oxidation is uncoupled from oxidative phosphorylation, thus not contributing to energy generation. Testing for genetic control checkpoints, transcriptional interrogation of human neonatal sepsis finds upregulation of many genes involved in mitochondrial removal of long-chain fatty acyls, such as ACSL1,3,4, ACADVL, CPT1B, CPT2 and HADHB. Also, ACSL1/Acsl1 upregulation is consistently observed following the treatment of human/murine macrophages with LPS and IFN-γ. Last, dampening oxylipin levels by ß-oxidation is suggested to impact on their regulation of leukocyte functions. In summary, we propose mitochondrial ß-oxidation as a regulatory metabolic checkpoint for oxylipins during inflammation.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/metabolismo , Metabolismo dos Lipídeos/genética , Mitocôndrias/efeitos dos fármacos , Oxilipinas/metabolismo , Peritonite/genética , Sepse/genética , Acil-CoA Desidrogenase de Cadeia Longa/sangue , Acil-CoA Desidrogenase de Cadeia Longa/genética , Animais , Carnitina O-Palmitoiltransferase/sangue , Carnitina O-Palmitoiltransferase/genética , Coenzima A Ligases/sangue , Coenzima A Ligases/genética , Feminino , Regulação da Expressão Gênica , Humanos , Recém-Nascido , Interferon gama/farmacologia , Lipidômica/métodos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Subunidade beta da Proteína Mitocondrial Trifuncional/sangue , Subunidade beta da Proteína Mitocondrial Trifuncional/genética , Oxirredução , Peritonite/sangue , Peritonite/induzido quimicamente , Peritonite/patologia , Células RAW 264.7 , Sepse/sangue , Sepse/patologiaRESUMO
PGs are important proinflammatory lipid mediators, the significance of which is highlighted by the widespread and efficacious use of nonsteroidal anti-inflammatory drugs in the treatment of inflammation. 4-Octyl itaconate (4-OI), a derivative of the Krebs cycle-derived metabolite itaconate, has recently garnered much interest as an anti-inflammatory agent. In this article, we show that 4-OI limits PG production in murine macrophages stimulated with the TLR1/2 ligand Pam3CSK4. This decrease in PG secretion is due to a robust suppression of cyclooxygenase 2 (COX2) expression by 4-OI, with both mRNA and protein levels decreased. Dimethyl fumarate, a fumarate derivative used in the treatment of multiple sclerosis, with properties similar to itaconate, replicated the phenotype observed with 4-OI. We also demonstrate that the decrease in COX2 expression and inhibition of downstream PG production occurs in an NRF2-independent manner. Our findings provide a new insight into the potential of 4-OI as an anti-inflammatory agent and also identifies a novel anti-inflammatory function of dimethyl fumarate.
Assuntos
Anti-Inflamatórios/farmacologia , Fumarato de Dimetilo/farmacologia , Macrófagos/efeitos dos fármacos , Prostaglandinas/metabolismo , Succinatos/farmacologia , Animais , Ciclo-Oxigenase 2/biossíntese , Humanos , Macrófagos/metabolismo , CamundongosRESUMO
The protozoan parasite Trypanosoma brucei is the causative agent of the neglected tropical disease human African trypanosomiasis, otherwise known as sleeping sickness. Trypanosomes have evolved many immune-evasion mechanisms to facilitate their own survival, as well as prolonging host survival to ensure completion of the parasitic life cycle. A key feature of the bloodstream form of T. brucei is the secretion of aromatic keto acids, which are metabolized from tryptophan. In this study, we describe an immunomodulatory role for one of these keto acids, indole-3-pyruvate (I3P). We demonstrate that I3P inhibits the production of PGs in activated macrophages. We also show that, despite the reduction in downstream PGs, I3P augments the expression of cyclooxygenase (COX2). This increase in COX2 expression is mediated in part via inhibition of PGs relieving a negative-feedback loop on COX2. Activation of the aryl hydrocarbon receptor also participates in this effect. However, the increase in COX2 expression is of little functionality, as we also provide evidence to suggest that I3P targets COX activity. This study therefore details an evasion strategy by which a trypanosome-secreted metabolite potently inhibits macrophage-derived PGs, which might promote host and trypanosome survival.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Indóis/metabolismo , Macrófagos/imunologia , Prostaglandinas/metabolismo , Tripanossomíase Africana/imunologia , Animais , Humanos , Evasão da Resposta Imune/imunologia , Indóis/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Prostaglandinas/imunologia , Trypanosoma brucei brucei/imunologia , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/metabolismoRESUMO
Platelets promote tumor metastasis by inducing promalignant phenotypes in cancer cells and directly contributing to cancer-related thrombotic complications. Platelet-derived extracellular vesicles (EVs) can promote epithelial-mesenchymal transition (EMT) in cancer cells, which confers high-grade malignancy. 12S-hydroxyeicosatetraenoic acid (12-HETE) generated by platelet-type 12-lipoxygenase (12-LOX) is considered a key modulator of cancer metastasis through unknown mechanisms. In platelets, 12-HETE can be esterified into plasma membrane phospholipids (PLs), which drive thrombosis. Using cocultures of human platelets and human colon adenocarcinoma cells (line HT29) and LC-MS/MS, we investigated the impact of platelets on cancer cell biosynthesis of 12S-HETE and its esterification into PLs and whether platelet ability to transfer its molecular cargo might play a role. To this aim, we performed coculture experiments with CFSE[5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester]-loaded platelets. HT29 cells did not generate 12S-HETE or express 12-LOX. However, they acquired the capacity to produce 12S-HETE mainly esterified in plasmalogen phospholipid forms following the uptake of platelet-derived medium-sized EVs (mEVs) expressing 12-LOX. 12-LOX was detected in plasma mEV of patients with adenomas/adenocarcinomas, implying their potential to deliver the protein to cancer cells in vivo. In cancer cells exposed to platelets, endogenous but not exogenous 12S-HETE contributed to changes in EMT gene expression, mitigated by three structurally unrelated 12-LOX inhibitors. In conclusion, we showed that platelets induce the generation of primarily esterified 12-HETE in colon cancer cells following mEV-mediated delivery of 12-LOX. The modification of cancer cell phospholipids by 12-HETE may functionally impact cancer cell biology and represent a novel target for anticancer agent development.
Assuntos
Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/biossíntese , Araquidonato 12-Lipoxigenase/metabolismo , Plaquetas/metabolismo , Neoplasias do Colo/metabolismo , Fosfolipídeos/metabolismo , Adulto , Neoplasias do Colo/patologia , Humanos , Pessoa de Meia-Idade , Células Tumorais Cultivadas , Adulto JovemRESUMO
Atherosclerosis and its complications are responsible for one in three global deaths. Nutraceuticals show promise in the prevention and treatment of atherosclerosis but require an indepth understanding of the mechanisms underlying their actions. A previous study showed that the omega-6 fatty acid, dihomo-γ-linolenic acid (DGLA), attenuated atherosclerosis in the apolipoprotein E deficient mouse model system. However, the mechanisms underlying such protective effects of DGLA are poorly understood and were therefore investigated. We show that DGLA attenuates chemokine-driven monocytic migration together with foam cell formation and the expression of key pro-atherogenic genes induced by three pro-inflammatory cytokines in human macrophages. The effect of DGLA on interferon-γ signaling was mediated via inhibition of signal transducer and activator of transcription-1 phosphorylation on serine 727. In relation to anti-foam cell action, DGLA inhibits modified LDL uptake by both macropinocytosis and receptor-mediated endocytosis, the latter by reduction in expression of two key scavenger receptors (SR-A and CD36), and stimulates cholesterol efflux from foam cells. DGLA also improves macrophage mitochondrial bioenergetic profile by decreasing proton leak. Gamma-linolenic acid and prostaglandin E1, upstream precursor and key metabolite respectively of DGLA, also acted in an anti-atherogenic manner. The actions of DGLA extended to other key atherosclerosis-associated cell types with attenuation of endothelial cell proliferation and migration of smooth muscle cells in response to platelet-derived growth factor. This study provides novel insights into the molecular mechanisms underlying the anti-atherogenic actions of DGLA and supports further assessments on its protective effects on plaque regression in vivo and in human trials.
Assuntos
Ácido 8,11,14-Eicosatrienoico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Citocinas/metabolismo , Células Espumosas/citologia , Células Espumosas/metabolismo , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Monócitos/citologiaRESUMO
Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.
Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Hemorragia/enzimologia , Hemorragia/metabolismo , Fosfolipídeos/metabolismo , Trombina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Coagulação Sanguínea , Fatores de Coagulação Sanguínea/genética , Plaquetas , Ponte Cardiopulmonar/efeitos adversos , Proteínas de Transporte , Cisteína Endopeptidases , Fator IX/genética , Fator VIII/genética , Fator VIIa/metabolismo , Fator X/genética , Hemofilia A , Hemorragia/prevenção & controle , Hemostasia , Humanos , Ácidos Hidroxieicosatetraenoicos , Lipoproteínas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteínas de Neoplasias , Ressonância de Plasmônio de Superfície , Tromboplastina/antagonistas & inibidores , Tromboplastina/metabolismoRESUMO
The inflammatory activation and recruitment of defined myeloid populations is essential for controlling the bridge between innate and adaptive immunity and shaping the immune response to microbial challenge. However, these cells exhibit significant functional heterogeneity and the inflammatory signals that differentially influence their effector characteristics are poorly characterized. In this study, we defined the phenotype of discrete subsets of effective antigen-presenting cells (APCs) in the peritoneal cavity during peritonitis. When the functional properties of these cells were compared to inflammatory monocyte-derived macrophages we noted differential responses to the immune-modulatory cytokine IL-10. In contrast to the suppressive actions of IL-10 on inflammatory macrophages, the recruitment of APCs was relatively refractory and we found no evidence for selective inhibition of APC differentiation. This differential response of myeloid cell subsets to IL-10 may thus have limited impact on development of potentially tissue-damaging adaptive immune responses, while restricting the magnitude of the inflammatory response. These findings may have clinical relevance in the context of peritoneal dialysis patients, where recurrent infections are associated with immune-mediated membrane dysfunction, treatment failure, and increased morbidity.