RESUMO
It is clear that obesity increases the risk of many types of cancer, including breast cancer. However, the underlying molecular mechanisms by which obesity is linked to cancer risk remain to be defined. Herein, we report that circulating adipose fatty acid binding protein (A-FABP) promotes obesity-associated breast cancer development. Using clinical samples, we demonstrated that circulating A-FABP levels were significantly increased in obese patients with breast cancer in comparison with those without breast cancer. Circulating A-FABP released by adipose tissue directly targeted mammary tumor cells, enhancing tumor stemness and aggressiveness through activation of the IL-6/STAT3/ALDH1 pathway. Importantly, genetic deletion of A-FABP successfully reduced tumor ALHD1 activation and obesity-associated mammary tumor growth and development in different mouse models. Collectively, these data suggest circulating A-FABP as a new link between obesity and breast cancer risk, thereby revealing A-FABP as a potential new therapeutic target for treatment of obesity-associated cancers.
Assuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/etiologia , Proteínas de Ligação a Ácido Graxo/sangue , Obesidade/complicações , Família Aldeído Desidrogenase 1 , Animais , Biomarcadores Tumorais/sangue , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Modelos Animais de Doenças , Progressão da Doença , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Humanos , Interleucina-6/metabolismo , Isoenzimas/metabolismo , Camundongos Endogâmicos C57BL , Invasividade Neoplásica/patologia , Obesidade/sangue , Obesidade/metabolismo , Obesidade/patologia , Retinal Desidrogenase/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Obesity is associated with elevated levels of free fatty acids (FAs) and proinflammatory CD11c+ macrophages. However, whether and how free FAs contribute to CD11c+ macrophage differentiation and proinflammatory functions remain unclear. Here we report that dietary saturated FAs, but not unsaturated FAs, promoted the differentiation and function of CD11c+ macrophages. Specifically, we demonstrated that stearic acid (SA) significantly induced CD11c expression in monocytes through activation of the nuclear retinoid acid receptor. More importantly, cytosolic expression of epidermal FA binding protein (E-FABP) in monocytes/macrophages was shown to be critical to the mediation of the SA-induced effect. Depletion of E-FABP not only inhibited SA-induced CD11c upregulation in macrophages in vitro but also abrogated high-saturated-fat diet-induced skin lesions in obese mouse models in vivo. Altogether, our data demonstrate a novel mechanism by which saturated FAs promote obesity-associated inflammation through inducing E-FABP/retinoid acid receptor-mediated differentiation of CD11c+ macrophages.
Assuntos
Antígeno CD11c/metabolismo , Proteínas de Ligação a Ácido Graxo/metabolismo , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Ácidos Esteáricos/farmacologia , Animais , Ácidos Graxos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Obesidade/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Skin lipids (e.g., fatty acids) are essential for normal skin functions. Epidermal FABP (E-FABP) is the predominant FABP expressed in skin epidermis. However, the role of E-FABP in skin homeostasis and pathology remains largely unknown. Herein, we utilized the 7,12-dimethylbenz(a)anthracene and 12-O-tetradecanolyphorbol-13-acetate-induced skin tumorigenesis model to assess the role of E-FABP in chemical-induced skin tumorigenesis. Compared to their wild-type littermates, mice deficient in E-FABP, but not adipose FABP, developed more skin tumors with higher incidence. 12-O-tetradecanolyphorbol-13-acetate functioning as a tumor promoter induced E-FABP expression and initiated extensive flaring inflammation in skin. Interestingly, 12-O-tetradecanolyphorbol-13-acetate -induced production of IFN-ß and IFN-λ in the skin tissue was dependent on E-FABP expression. Further protein and gene expression arrays demonstrated that E-FABP was critical in enhancing IFN-induced p53 responses and in suppressing SOX2 expression in keratinocytes. Thus, E-FABP expression in skin suppresses chemical-induced skin tumorigenesis through regulation of IFN/p53/SOX2 pathway. Collectively, our data suggest an unknown function of E-FABP in prevention of skin tumor development, and offer E-FABP as a therapeutic target for improving skin innate immunity in chemical-induced skin tumor prevention.
Assuntos
Proteínas de Ligação a Ácido Graxo/genética , Regulação Neoplásica da Expressão Gênica , Interferon beta/genética , Queratinócitos/metabolismo , Proteínas de Neoplasias/genética , Fatores de Transcrição SOXB1/genética , Neoplasias Cutâneas/genética , Proteína Supressora de Tumor p53/genética , Animais , Carcinogênese , Epiderme/metabolismo , Epiderme/patologia , Proteínas de Ligação a Ácido Graxo/biossíntese , Interferon beta/metabolismo , Queratinócitos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Neoplasias/biossíntese , Neoplasias Experimentais , RNA Neoplásico/genética , Fatores de Transcrição SOXB1/metabolismo , Neoplasias Cutâneas/metabolismo , Acetato de Tetradecanoilforbol/toxicidade , Proteína Supressora de Tumor p53/metabolismoRESUMO
Tumor-associated macrophages (TAM) play a critical role in cancer development and progression. However, the heterogeneity of TAM presents a major challenge to identify clinically relevant markers for protumor TAM. Here, we report that expression of adipocyte/macrophage fatty acid-binding protein (A-FABP) in TAM promotes breast cancer progression. Although upregulation of A-FABP was inversely associated with breast cancer survival, deficiency of A-FABP significantly reduced mammary tumor growth and metastasis. Furthermore, the protumor effect of A-FABP was mediated by TAM, in particular, in a subset of TAM with a CD11b+F4/80+MHCII-Ly6C- phenotype. A-FABP expression in TAM facilitated protumor IL6/STAT3 signaling through regulation of the NFκB/miR-29b pathway. Collectively, our results suggest A-FABP as a new functional marker for protumor TAM.Significance: These findings identify A-FABP as a functional marker for protumor macrophages, thus offering a new target for tumor immunotherapy. Cancer Res; 78(9); 2343-55. ©2018 AACR.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proteínas de Ligação a Ácido Graxo/genética , Regulação Neoplásica da Expressão Gênica , Macrófagos/metabolismo , Animais , Biomarcadores Tumorais , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Macrófagos/patologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Metástase NeoplásicaRESUMO
The intestinal immune system is continuously exposed to massive amounts of nanoparticles derived from food. Whether nanoparticles from plants we eat daily have a role in maintaining intestinal immune homeostasis is poorly defined. Here, we present evidence supporting our hypothesis that edible nanoparticles regulate intestinal immune homeostasis by targeting dendritic cells (DCs). Using three mouse colitis models, our data show that orally given nanoparticles isolated from broccoli extracts protect mice against colitis. Broccoli-derived nanoparticle (BDN)-mediated activation of adenosine monophosphate-activated protein kinase (AMPK) in DCs plays a role in not only prevention of DC activation but also induction of tolerant DCs. Adoptively transferring DCs pre-pulsed with total BDN lipids, but not sulforaphane (SFN)-depleted BDN lipids, prevented DSS-induced colitis in C57BL/6 (B6) mice, supporting the role of BDN SFN in the induction of DC tolerance. Adoptively transferring AMPK+/+, but not AMPK-/-, DCs pre-pulsed with SFN prevented DSS-induced colitis in B6 mice, further supporting the DC AMPK role in SFN-mediated prevention of DSS-induced colitis. This finding could open new preventive or therapeutic avenues to address intestinal-related inflammatory diseases via activating AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/genética , Anti-Inflamatórios/farmacologia , Brassica/química , Colite Ulcerativa/prevenção & controle , Células Dendríticas/efeitos dos fármacos , Nanopartículas/química , Proteínas Quinases Ativadas por AMP/metabolismo , Administração Oral , Transferência Adotiva , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/imunologia , Colite Ulcerativa/patologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/transplante , Modelos Animais de Doenças , Ativação Enzimática/efeitos dos fármacos , Expressão Gênica , Humanos , Tolerância Imunológica , Isotiocianatos/química , Lipídeos/isolamento & purificação , Lipídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Nanopartículas/administração & dosagem , Extratos Vegetais/química , Dodecilsulfato de Sódio , SulfóxidosRESUMO
Macrophages play a critical role in obesity-associated chronic inflammation and disorders. However, the molecular mechanisms underlying the response of macrophages to elevated fatty acids (FAs) and their contribution to metabolic inflammation in obesity remain to be fully elucidated. In this article, we report a new mechanism by which dietary FAs, in particular, saturated FAs (sFAs), are able to directly trigger macrophage cell death. We demonstrated that excess sFAs, but not unsaturated FAs, induced the production of cytotoxic ceramides (Cers) in macrophage cell lines. Most importantly, expression of adipose FA binding protein (A-FABP) in macrophages facilitated metabolism of excess sFAs for Cer synthesis. Inhibition or deficiency of A-FABP in macrophage cell lines decreased sFA-induced Cer production, thereby resulting in reduced cell death. Furthermore, we validated the role of A-FABP in promoting sFA-induced macrophage cell death with primary bone marrow-derived macrophages and high-fat diet-induced obese mice. Altogether, our data reveal that excess dietary sFAs may serve as direct triggers in induction of Cer production and macrophage cell death through elevated expression of A-FABP, thus establishing A-FABP as a new molecular sensor in triggering macrophage-associated sterile inflammation in obesity.
Assuntos
Ceramidas/biossíntese , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/efeitos adversos , Macrófagos/patologia , Animais , Western Blotting , Morte Celular , Dieta Hiperlipídica , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Obesidade/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
As a master metabolic sensor, AMP-activated protein kinase (AMPK) is involved in different fundamental cellular processes. Regulation of AMPK activity either by agonists (e.g., AICAR) or by antagonists (e.g., Compound C) has been widely employed to study the physiological functions of AMPK. However, mounting evidence indicates AMPK-independent effects for these chemicals and how they regulate immune cell functions remains largely unknown. Herein, using T cells from AMPK conditional knockout mice and their wild type littermates, we demonstrate that AICAR and Compound C can, indeed, activate or inhibit AMPK activity in T cells, respectively. Specifically, AICAR inhibits, but Compound C promotes, Ca2+-induced T cell death in an AMPK-dependent manner. In contrast, our data also demonstrate that AICAR and Compound C inhibit T cell activation and cytokine production in an AMPK-independent manner. Moreover, we find that the AMPK-independent activity of AICAR and Compound Cis mediated via the mTOR signaling pathway in activated T cells. Our results not only reveal the critical role of AMPK in regulating T cell survival and function, but also demonstrate AMPK-dependent and independent rolesof AICAR/Compound C in regulating T cell responses, thus suggesting a context-dependent effect of these "AMPK regulators".
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Ativadores de Enzimas/farmacologia , Fatores Imunológicos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ribonucleosídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Genótipo , Camundongos Knockout , Fenótipo , Linfócitos T/enzimologia , Linfócitos T/imunologia , Linfócitos T/patologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease in which dysregulated immune cells attack myelin in the central nervous system (CNS), leading to irreversible neuronal degeneration. Our previous studies have demonstrated that epidermal fatty acid binding protein (E-FABP), widely expressed in immune cells, in particular in dendritic cells (DCs) and T lymphocytes, fuels the overactive immune responses in the mouse model of experimental autoimmune encephalomyelitis (EAE). METHODS: In the present study, we conducted an intensive computational docking analysis to identify novel E-FABP inhibitors for regulation of immune cell functions and for treatment of EAE. RESULTS: We demonstrate that compound [2-(4-acetylphenoxy)-9,10-dimethoxy-6,7-dihydropyrimido[6,1-a]isoquinolin-4-one; designated as EI-03] bound to the lipid binding pocket of E-FABP and enhanced the expression of peroxisome proliferator-activating receptor (PPAR) γ. Further in vitro experiments showed that EI-03 regulated DC functions by inhibition of TNFα production while promoting IL-10 secretion. Moreover, EI-03 treatment counterregulated T cell balance by decreasing effector T cell differentiation (e.g. Th17, Th1) while increasing regulatory T cell development. Most importantly, mice treated with this newly identified compound exhibited reduced clinical symptoms of EAE in mouse models. CONCLUSIONS: Taken together, we have identified a new compound which displays a potential therapeutic benefit for treatment of MS by targeting E-FABP.
Assuntos
Encefalomielite Autoimune Experimental/terapia , Proteínas de Ligação a Ácido Graxo/antagonistas & inibidores , Terapia de Alvo Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Animais , Diferenciação Celular , Citocinas/biossíntese , Células Dendríticas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/metabolismo , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismoRESUMO
A number of studies have linked AMPK, a major metabolic sensor coordinating of multiple cellular functions, to tumor development and progression. However, the exact role of AMPK in tumor development is still controversial. Here we report that activation of AMPK promotes survival and anti-tumor function of T cells, in particular CD8+ T cells, resulting in superior tumor suppression in vivo. While AMPK expression is dispensable for T cell development, genetic deletion of AMPK promotes T cell death during in vitro activation and in vivo tumor development. Moreover, we demonstrate that protein phosphatases are the key mediators of AMPK-dependent effects on T cell death, and inhibition of phosphatase activity by okadaic acid successfully restores T cell survival and function. Altogether, our data suggest a novel mechanism by which AMPK regulates protein phosphatase activity in control of survival and function of CD8+ T cells, thereby enhancing their role in tumor immunosurveillance.
Assuntos
Proteínas Quinases Ativadas por AMP/deficiência , Linfócitos T CD8-Positivos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Linfócitos T CD8-Positivos/enzimologia , Linfócitos T CD8-Positivos/imunologia , Morte Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , HumanosRESUMO
Chronic inflammation in obese adipose tissue is linked to endoplasmic reticulum (ER) stress and systemic insulin resistance. Targeted deletion of the murine fatty acid binding protein (FABP4/aP2) uncouples obesity from inflammation although the mechanism underlying this finding has remained enigmatic. Here, we show that inhibition or deletion of FABP4/aP2 in macrophages results in increased intracellular free fatty acids (FFAs) and elevated expression of uncoupling protein 2 (UCP2) without concomitant increases in UCP1 or UCP3. Silencing of UCP2 mRNA in FABP4/aP2-deficient macrophages negated the protective effect of FABP loss and increased ER stress in response to palmitate or lipopolysaccharide (LPS). Pharmacologic inhibition of FABP4/aP2 with the FABP inhibitor HTS01037 also upregulated UCP2 and reduced expression of BiP, CHOP, and XBP-1s. Expression of native FABP4/aP2 (but not the non-fatty acid binding mutant R126Q) into FABP4/aP2 null cells reduced UCP2 expression, suggesting that the FABP-FFA equilibrium controls UCP2 expression. FABP4/aP2-deficient macrophages are resistant to LPS-induced mitochondrial dysfunction and exhibit decreased mitochondrial protein carbonylation and UCP2-dependent reduction in intracellular reactive oxygen species. These data demonstrate that FABP4/aP2 directly regulates intracellular FFA levels and indirectly controls macrophage inflammation and ER stress by regulating the expression of UCP2.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Inflamação/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Metabolismo dos Lipídeos/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Tecido Adiposo/metabolismo , Animais , Linhagem Celular , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos não Esterificados/genética , Inflamação/genética , Lipopolissacarídeos/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/metabolismo , Obesidade/genética , Obesidade/metabolismo , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2RESUMO
AMP-activated protein kinase (AMPK) is a conserved serine/threonine kinase with a critical function in the regulation of metabolic pathways in eukaryotic cells. Recently, AMPK has been shown to play an additional role as a regulator of inflammatory activity in leukocytes. Treatment of macrophages with chemical AMPK activators, or forced expression of a constitutively active form of AMPK, results in polarization to an anti-inflammatory phenotype. In addition, we reported previously that stimulation of macrophages with anti-inflammatory cytokines such as IL-10, IL-4, and TGF-ß results in rapid activation of AMPK, suggesting that AMPK contributes to the suppressive function of these cytokines. In this study, we investigated the role of AMPK in IL-10-induced gene expression and anti-inflammatory function. IL-10-stimulated wild-type macrophages displayed rapid activation of PI3K and its downstream targets Akt and mammalian target of rapamycin complex (mTORC1), an effect that was not seen in macrophages generated from AMPKα1-deficient mice. AMPK activation was not impacted by treatment with either the PI3K inhibitor LY294002 or the JAK inhibitor CP-690550, suggesting that IL-10-mediated activation of AMPK is independent of PI3K and JAK activity. IL-10 induced phosphorylation of both Tyr(705) and Ser(727) residues of STAT3 in an AMPKα1-dependent manner, and these phosphorylation events were blocked by inhibition of Ca(2+)/calmodulin-dependent protein kinase kinase ß, an upstream activator of AMPK, and by the mTORC1 inhibitor rapamycin, respectively. The impaired STAT3 phosphorylation in response to IL-10 observed in AMPKα1-deficient macrophages was accompanied by reduced suppressor of cytokine signaling 3 expression and an inadequacy of IL-10 to suppress LPS-induced proinflammatory cytokine production. Overall, our data demonstrate that AMPKα1 is required for IL-10 activation of the PI3K/Akt/mTORC1 and STAT3-mediated anti-inflammatory pathways regulating macrophage functional polarization.
Assuntos
Proteínas Quinases Ativadas por AMP/imunologia , Interleucina-10/imunologia , Macrófagos/imunologia , Transdução de Sinais/imunologia , Proteínas Quinases Ativadas por AMP/genética , Animais , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-10/genética , Interleucina-4/genética , Interleucina-4/imunologia , Lipopolissacarídeos/toxicidade , Macrófagos/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologiaRESUMO
Description of macrophage activation is currently contentious and confusing. Like the biblical Tower of Babel, macrophage activation encompasses a panoply of descriptors used in different ways. The lack of consensus on how to define macrophage activation in experiments in vitro and in vivo impedes progress in multiple ways, including the fact that many researchers still consider there to be only two types of activated macrophages, often termed M1 and M2. Here, we describe a set of standards encompassing three principles-the source of macrophages, definition of the activators, and a consensus collection of markers to describe macrophage activation-with the goal of unifying experimental standards for diverse experimental scenarios. Collectively, we propose a common framework for macrophage-activation nomenclature.
Assuntos
Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Terminologia como Assunto , Animais , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Guias como Assunto , Humanos , Fator Estimulador de Colônias de Macrófagos/imunologia , Camundongos , PesquisaRESUMO
Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C4 (LTC4) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC4 and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC4 and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC4 precursor leukotriene A4 (LTA4) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC4 synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC4 secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC4 and contribute to the insulin resistant state.
Assuntos
Tecido Adiposo/imunologia , Proteínas de Ligação a Ácido Graxo/imunologia , Ácidos Graxos/imunologia , Leucotrieno C4/imunologia , Macrófagos/imunologia , Obesidade/imunologia , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/análise , Ácido 12-Hidroxi-5,8,10,14-Eicosatetraenoico/imunologia , Tecido Adiposo/patologia , Animais , Linhagem Celular , Células Cultivadas , Ácidos Graxos/análise , Feminino , Ácidos Hidroxieicosatetraenoicos/análise , Ácidos Hidroxieicosatetraenoicos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/patologiaRESUMO
AMPK is a serine/threonine kinase that regulates energy homeostasis and metabolic stress in eukaryotes. Previous work from our laboratory, as well as by others, has provided evidence that AMPKα1 acts as a negative regulator of TLR-induced inflammatory function. Herein, we demonstrate that AMPKα1-deficient macrophages and DCs exhibit heightened inflammatory function and an enhanced capacity for antigen presentation favoring the promotion of Th1 and Th17 responses. Macrophages and DCs generated from AMPKα1-deficient mice produced higher levels of proinflammatory cytokines and decreased production of the anti-inflammatory cytokine IL-10 in response to TLR and CD40 stimulation as compared with WT cells. In assays of antigen presentation, AMPKα1 deficiency in the myeloid APC and T cell populations contributed to enhanced IL-17 and IFN-γ production. Focusing on the CD154-CD40 interaction, we found that CD40 stimulation resulted in increased phosphorylation of ERK1/2, p38, and NF-κB p65 and decreased activation of the anti-inflammatory Akt -GSK3ß-CREB pathway in DCs deficient for AMPKα1. Our data demonstrate that AMPKα1 serves to attenuate LPS and CD40-mediated proinflammatory activity of myeloid APCs and that AMPKα1 activity in both APC and T cells contributes to T cell functional polarization during antigen presentation.
Assuntos
Proteínas Quinases Ativadas por AMP/imunologia , Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Antígenos CD40/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Células Mieloides/imunologia , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/enzimologia , Antígenos CD40/genética , Antígenos CD40/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-17/metabolismo , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Células Mieloides/citologia , Células Mieloides/enzimologia , Células Th1/citologia , Células Th1/enzimologia , Células Th1/imunologia , Células Th17/citologia , Células Th17/enzimologia , Células Th17/imunologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Fator de Transcrição RelA/metabolismoRESUMO
The role of JAK-3 in TLR-mediated innate immune responses is poorly understood, although the suppressive function of JAK3 inhibition in adaptive immune response has been well studied. In this study, we found that JAK3 inhibition enhanced TLR-mediated immune responses by differentially regulating pro- and anti- inflammatory cytokine production in innate immune cells. Specifically, JAK3 inhibition by pharmacological inhibitors or specific small interfering RNA or JAK3 gene knockout resulted in an increase in TLR-mediated production of proinflammatory cytokines while concurrently decreasing the production of IL-10. Inhibition of JAK3 suppressed phosphorylation of PI3K downstream effectors including Akt, mammalian target of rapamycin complex 1, glycogen synthase kinase 3ß (GSK3ß), and CREB. Constitutive activation of Akt or inhibition of GSK3ß abrogated the capability of JAK3 inhibition to enhance proinflammatory cytokines and suppress IL-10 production. In contrast, inhibition of PI3K enhanced this regulatory ability of JAK3 in LPS-stimulated monocytes. At the transcriptional level, JAK3 knockout lead to the increased phosphorylation of STATs that could be attenuated by neutralization of de novo inflammatory cytokines. JAK3 inhibition exhibited a GSK3 activity-dependent ability to enhance phosphorylation levels and DNA binding of NF-κB p65. Moreover, JAK3 inhibition correlated with an increased CD4(+) T cell response. Additionally, higher neutrophil infiltration, IL-17 expression, and intestinal epithelium erosion were observed in JAK3 knockout mice. These findings demonstrate the negative regulatory function of JAK3 and elucidate the signaling pathway by which JAK3 differentially regulates TLR-mediated inflammatory cytokine production in innate immune cells.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Inflamação/imunologia , Janus Quinase 3/metabolismo , Receptores Toll-Like/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Humanos , Imunidade Inata/imunologia , Inflamação/genética , Interleucina-10/biossíntese , Interleucina-17/biossíntese , Mucosa Intestinal/imunologia , Intestinos/imunologia , Janus Quinase 3/genética , Lipopolissacarídeos , Ativação Linfocitária/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Neutrófilos/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/metabolismo , Fator de Transcrição RelA/metabolismoRESUMO
The therapeutic effects of probiotic treatment in alcoholic liver disease (ALD) have been studied in both patients and experimental animal models. Although the precise mechanisms of the pathogenesis of ALD are not fully understood, gut-derived endotoxin has been postulated to play a crucial role in hepatic inflammation. Previous studies have demonstrated that probiotic therapy reduces circulating endotoxin derived from intestinal gram-negative bacteria in ALD. In this study, we investigated the effects of probiotics on hepatic tumor necrosis factor-α (TNFα) production and inflammation in response to chronic alcohol ingestion. Mice were fed Lieber DeCarli liquid diet containing 5% alcohol for 8weeks, and Lactobacillus rhamnosus GG (LGG) was supplemented in the last 2 weeks. Eight-week alcohol feeding caused a significant increase in hepatic inflammation as shown by histological assessment and hepatic tissue myeloperoxidase activity assay. Two weeks of LGG supplementation reduced hepatic inflammation and liver injury and markedly reduced TNFα expression. Alcohol feeding increased hepatic mRNA expression of Toll-like receptors (TLRs) and CYP2E1 and decreased nuclear factor erythroid 2-related factor 2 expression. LGG supplementation attenuated these changes. Using human peripheral blood monocytes-derived macrophages, we also demonstrated that incubation with ethanol primes both lipopolysaccharide- and flagellin-induced TNFα production, and LGG culture supernatant reduced this induction in a dose-dependent manner. In addition, LGG treatment also significantly decreased alcohol-induced phosphorylation of p38 MAP kinase. In conclusion, probiotic LGG treatment reduced alcohol-induced hepatic inflammation by attenuation of TNFα production via inhibition of TLR4- and TLR5-mediated endotoxin activation.
Assuntos
Inflamação/terapia , Lacticaseibacillus rhamnosus , Hepatopatias Alcoólicas/terapia , Probióticos/uso terapêutico , Fator de Necrose Tumoral alfa/metabolismo , Animais , Citocromo P-450 CYP2E1/genética , Citocromo P-450 CYP2E1/metabolismo , Endotoxinas/efeitos adversos , Endotoxinas/metabolismo , Etanol/metabolismo , Flagelina/efeitos adversos , Flagelina/metabolismo , Bactérias Gram-Negativas/metabolismo , Humanos , Inflamação/patologia , Mucosa Intestinal/metabolismo , Intestinos/microbiologia , Leucócitos Mononucleares/metabolismo , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/metabolismo , Fígado/patologia , Hepatopatias Alcoólicas/patologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Probióticos/administração & dosagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptor 5 Toll-Like/genética , Receptor 5 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C4 (LTC4) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC4 and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC4 and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC4 precursor leukotriene A4 (LTA4) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC4 synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC4 secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC4 and contribute to the insulin resistant state.
RESUMO
Activation of the PI3K pathway plays a pivotal role in regulating the inflammatory response. The loss of mTORC2 has been shown to abrogate the activation of Akt, a critical downstream component of PI3K signaling. However, the biological importance of mTORC2 in innate immunity is currently unknown. Here we demonstrate that rictor, a key component of mTORC2, plays a critical role in controlling the innate inflammatory response via its ability to regulate FoxO1. Upon LPS stimulation, both rictor-deficient mouse embryonic fibroblasts (MEFs) and rictor knockdown dendritic cells exhibited a hyperinflammatory phenotype. The hyperinflammatory phenotype was due to a defective Akt signaling axis, because both rictor-deficient MEFs and rictor knockdown dendritic cells exhibited attenuated Akt phosphorylation and kinase activity. Analysis of downstream Akt targets revealed that phosphorylation of FoxO1 was impaired in rictor-deficient cells, resulting in elevated nuclear FoxO1 levels and diminished nuclear export of FoxO1 upon LPS stimulation. Knockdown of FoxO1 attenuated the hyperinflammatory phenotype exhibited by rictor-deficient MEFs. Moreover, FoxO1 deletion in dendritic cells attenuated the capacity of LPS to induce inflammatory cytokine expression. These findings identify a novel signaling pathway by which mTORC2 regulates the TLR-mediated inflammatory response through its ability to regulate FoxO1.
Assuntos
Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/imunologia , Imunidade Inata/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Transativadores/imunologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/imunologia , Embrião de Mamíferos/metabolismo , Fibroblastos/citologia , Fibroblastos/imunologia , Fibroblastos/metabolismo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Inflamação/induzido quimicamente , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Transgênicos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/imunologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Companheira de mTOR Insensível à Rapamicina , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de TranscriçãoRESUMO
The generation of oxidized phospholipids in lipoproteins has been linked to vascular inflammation in atherosclerotic lesions. Products of phospholipid oxidation increase endothelial activation; however, their effects on macrophages are poorly understood, and it is unclear whether these effects are regulated by the biochemical pathways that metabolize oxidized phospholipids. We found that incubation of 1-palmitoyl-2-(5'-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC) with THP-1-derived macrophages upregulated the expression of cytokine genes, including granulocyte/macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein 1 (MCP-1), interleukin (IL)-1ß, IL-6, and IL-8. In these cells, reagent POVPC was either hydrolyzed to lyso-phosphatidylcholine (lyso-PC) or reduced to 1-palmitoyl-2-(5-hydroxy-valeroyl)-sn-glycero-3-phosphocholine (PHVPC). Treatment with the phospholipase A(2) (PLA(2)) inhibitor, pefabloc, decreased POVPC hydrolysis and increased PHVPC accumulation. Pefabloc also increased the induction of cytokine genes in POVPC-treated cells. In contrast, PHVPC accumulation and cytokine production were decreased upon treatment with the aldose reductase (AR) inhibitor, tolrestat. In comparison with POVPC, lyso-PC led to 2- to 3-fold greater and PHVPC 10- to 100-fold greater induction of cytokine genes. POVPC-induced cytokine gene induction was prevented in bone-marrow derived macrophages from AR-null mice. These results indicate that although hydrolysis is the major pathway of metabolism, reduction further increases the proinflammatory responses to POVPC. Thus, vascular inflammation in atherosclerotic lesions is likely to be regulated by metabolism of phospholipid aldehydes in macrophages.
Assuntos
Inflamação/metabolismo , Éteres Fosfolipídicos/metabolismo , Éteres Fosfolipídicos/farmacologia , Aldeído Redutase/metabolismo , Animais , Linhagem Celular , Citocinas/genética , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Oxirredução , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Alcohol abuse has long-term deleterious effects on the immune system, and results in a depletion and loss of function of CD4(+) T lymphocytes, which regulate both innate and adaptive immunity. T-lymphocyte activation via T-cell receptor (TCR) involves the lipid raft colocalization and aggregation of proteins into the immunological signalosome, which triggers a signaling cascade resulting in the production of interleukin-2 (IL-2). IL-2 regulates the proliferation and clonal expansion of activated T cells and is essential for an effective immune response. The present work examines the mechanisms underlying ethanol-induced dysfunction of CD4(+) T lymphocytes based on the hypothesis that ethanol downregulates lipid raft-mediated TCR signal transduction and resultant IL-2 production. METHODS: Primary or cultured human T lymphocytes were exposed to ethanol for 24 hours prior to stimulation with anti-CD3/anti-CD28 antibodies or phytohemagglutinin. Effects of ethanol exposure on TCR-signaling (including activation of Lck, ZAP70, LAT, and PLCγ1) and IL-2 gene expression were examined. RESULTS: Exposure of both primary and cultured human CD4(+) T lymphocytes to physiologically relevant concentrations of ethanol leads to down-regulation of IL-2 mRNA and protein via inhibition of DNA-binding activity of NFAT, the essential transcription factor for IL-2. Ethanol decreases tyrosine phosphorylation and activation of upstream signaling proteins PLCγ1, LAT, ZAP70, and Lck. These effects are prevented by inhibition of metabolism of ethanol. Sucrose density gradient fractionation and confocal microscopy revealed that ethanol inhibited essential upstream lipid raft-mediated TCR-dependent signaling events, namely colocalization of Lck, ZAP70, LAT, and PLCγ1 with plasma membrane lipid rafts. CONCLUSIONS: Overall, our data demonstrate that ethanol inhibits lipid raft-mediated TCR-signaling in CD4(+) T lymphocytes, resulting in suppression of IL-2 production. These findings may represent a novel mechanism underlying alcohol abuse-associated immune suppression and may be particularly relevant in diseases such as HIV/AIDS and hepatitis C virus infection where alcohol abuse is a known comorbidity.