RESUMO
Interleukin 1ß (IL-1ß) is an important inflammatory mediator of type 2 diabetes. Here we show that oligomers of islet amyloid polypeptide (IAPP), a protein that forms amyloid deposits in the pancreas during type 2 diabetes, triggered the NLRP3 inflammasome and generated mature IL-1ß. One therapy for type 2 diabetes, glyburide, suppressed IAPP-mediated IL-1ß production in vitro. Processing of IL-1ß initiated by IAPP first required priming, a process that involved glucose metabolism and was facilitated by minimally oxidized low-density lipoprotein. Finally, mice transgenic for human IAPP had more IL-1ß in pancreatic islets, which localized together with amyloid and macrophages. Our findings identify previously unknown mechanisms in the pathogenesis of type 2 diabetes and treatment of pathology caused by IAPP.
Assuntos
Amiloide/metabolismo , Proteínas de Transporte/metabolismo , Diabetes Mellitus Tipo 2/imunologia , Interleucina-1beta/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Células Cultivadas , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glibureto/farmacologia , Humanos , Hipoglicemiantes/farmacologia , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Ilhotas Pancreáticas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Ratos , Receptores Citoplasmáticos e Nucleares/antagonistas & inibidores , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Type I interferons (IFN-I) are the principal antiviral molecules of the innate immune system and can be made by most cell types, including central nervous system cells. IFN-I has been implicated in neuroinflammation during neurodegeneration, but its mechanism of induction and its consequences remain unclear. In the current study, we assessed expression of IFN-I in murine prion disease (ME7) and examined the contribution of the IFN-I receptor IFNAR1 to disease progression. The data indicate a robust IFNß response, specifically in microglia, with evidence of IFN-dependent genes in both microglia and astrocytes. This IFN-I response was absent in stimulator of interferon genes (STING-/- ) mice. Microglia showed increased numbers and activated morphology independent of genotype, but transcriptional signatures indicated an IFNAR1-dependent neuroinflammatory phenotype. Isolation of microglia and astrocytes demonstrated disease-associated microglial induction of Tnfα, Tgfb1, and of phagolysosomal system transcripts including those for cathepsins, Cd68, C1qa, C3, and Trem2, which were diminished in IFNAR1 and STING deficient mice. Microglial increases in activated cathepsin D, and CD68 were significantly reduced in IFNAR1-/- mice, particularly in white matter, and increases in COX-1 expression, and prostaglandin synthesis were significantly mitigated. Disease progressed more slowly in IFNAR1-/- mice, with diminished synaptic and neuronal loss and delayed onset of neurological signs and death but without effect on proteinase K-resistant PrP levels. Therefore, STING-dependent IFN-I influences microglial phenotype and influences neurodegenerative progression despite occurring secondary to initial degenerative changes. These data expand our mechanistic understanding of IFN-I induction and its impact on microglial function during chronic neurodegeneration.
Assuntos
Progressão da Doença , Interferon Tipo I/biossíntese , Proteínas de Membrana/deficiência , Microglia/metabolismo , Doenças Neurodegenerativas/metabolismo , Receptor de Interferon alfa e beta/deficiência , Animais , Doença Crônica , Feminino , Interferon Tipo I/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Fenótipo , Receptor de Interferon alfa e beta/genéticaRESUMO
TLR4 interactor with leucine-rich repeats (TRIL) is a brain-enriched accessory protein that is important in TLR3 and TLR4 signaling. In this study, we generated Tril(-/-) mice and examined TLR responses in vitro and in vivo. We found a role for TRIL in both TLR4 and TLR3 signaling in mixed glial cells, consistent with the high level of expression of TRIL in these cells. We also found that TRIL is a modulator of the innate immune response to LPS challenge and Escherichia coli infection in vivo. Tril(-/-) mice produce lower levels of multiple proinflammatory cytokines and chemokines specifically within the brain after E. coli and LPS challenge. Collectively, these data uncover TRIL as a mediator of innate immune responses within the brain, where it enhances neuronal cytokine responses to infection.
Assuntos
Encéfalo/imunologia , Proteínas de Transporte/imunologia , Imunidade Inata/imunologia , Proteínas de Membrana/imunologia , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Células Cultivadas , Quimiocina CCL5/biossíntese , Escherichia coli/imunologia , Infecções por Escherichia coli/imunologia , Peptídeos e Proteínas de Sinalização Intercelular , Interleucina-6/biossíntese , Lipopolissacarídeos , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/imunologia , Poli I-C/farmacologia , Transdução de Sinais/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 7 Toll-Like/imunologia , Receptor 8 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Toll-like receptors (TLRs) and members of their signaling pathway are important in the initiation of the innate immune response to a wide variety of pathogens. The adaptor protein Mal (also known as TIRAP), encoded by TIRAP (MIM 606252), mediates downstream signaling of TLR2 and TLR4 (refs. 4-6). We report a case-control study of 6,106 individuals from the UK, Vietnam and several African countries with invasive pneumococcal disease, bacteremia, malaria and tuberculosis. We genotyped 33 SNPs, including rs8177374, which encodes a leucine substitution at Ser180 of Mal. We found that heterozygous carriage of this variant associated independently with all four infectious diseases in the different study populations. Combining the study groups, we found substantial support for a protective effect of S180L heterozygosity against these infectious diseases (N = 6,106; overall P = 9.6 x 10(-8)). We found that the Mal S180L variant attenuated TLR2 signal transduction.
Assuntos
Bacteriemia/genética , Malária/genética , Proteínas de Membrana Transportadoras/genética , Proteínas da Mielina/genética , Infecções Pneumocócicas/genética , Polimorfismo de Nucleotídeo Único , Proteolipídeos/genética , Tuberculose/genética , África , Estudos de Casos e Controles , Predisposição Genética para Doença , Humanos , Desequilíbrio de Ligação , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras/fisiologia , Modelos Moleculares , Proteínas da Mielina/fisiologia , Proteínas Proteolipídicas Associadas a Linfócitos e Mielina , Polimorfismo de Nucleotídeo Único/fisiologia , Proteolipídeos/fisiologia , Receptores de Interleucina-1/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Reino Unido , VietnãRESUMO
Mutations in HTRA2/Omi/PARK13 have been implicated in Parkinson disease (PD). PARK13 is a neuroprotective serine protease; however, little is known about how PARK13 confers stress protection and which protein targets are directly affected by PARK13. We have reported that Arabidopsis thaliana represents a complementary PD model, and here we demonstrate that AtPARK13, similar to human PARK13 (hPARK13), is a mitochondrial protease. We show that the expression/accumulation of AtPARK13 transcripts are induced by heat stress but not by other stress conditions, including oxidative stress and metals. Our data show that elevated levels of AtPARK13 confer thermotolerance in A. thaliana. Increased temperatures accelerate protein unfolding, and we demonstrate that although AtPARK13 can act on native protein substrates, unfolded proteins represent better AtPARK13 substrates. The results further show that AtPARK13 and hPARK13 can degrade the PD proteins α-synuclein (SNCA) and DJ-1/PARK7 directly, without autophagy involvement, and that misfolded SNCA and DJ-1 represent better substrates than their native counterparts. Comparative proteomic profiling revealed AtPARK13-mediated proteome changes, and we identified four proteins that show altered abundance in response to AtPARK13 overexpression and elevated temperatures. Our study not only suggests that AtPARK13 confers thermotolerance by degrading misfolded protein targets, but it also provides new insight into possible roles of this protease in neurodegeneration.
Assuntos
Adaptação Fisiológica/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Temperatura Alta , Serina Proteases/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Western Blotting , Clonagem Molecular , Perfilação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Dados de Sequência Molecular , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Plantas Geneticamente Modificadas , Proteína Desglicase DJ-1 , Desdobramento de Proteína , Proteólise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Serina Proteases/metabolismo , Especificidade por Substrato , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
The widespread use of herbicides and antibiotics for selection of transgenic plants has not been very successful with regard to commercialization and public acceptance. Hence, alternative selection systems are required. In this study, we describe the use of ipt, the bacterial gene encoding the enzyme isopentenyl transferase from Agrobacterium tumefaciens, as a positive selectable marker for plastid transformation. A comparison between the traditional spectinomycin-based aadA selection system and the ipt selection system demonstrated that selection of transplastomic plants on medium lacking cytokinin was as effective as selection on medium containing spectinomycin. Proof of principle was demonstrated by transformation of the kasIII gene encoding 3-ketoacyl acyl carrier protein synthase III into tobacco plastids. Transplastomic tobacco plants were readily obtained using the ipt selection system, and were phenotypically normal despite over-expression of isopentenyl transferase. Over-expression of KASIII resulted in a significant increase in 16:0 fatty acid levels, and a significant decrease in the levels of 18:0 and 18:1 fatty acids. Our study demonstrates use of a novel positive plastid transformation system that may be used for selection of transplastomic plants without affecting the expression of transgenes within the integrated vector cassette or the resulting activity of the encoded protein. This system has the potential to be applied to monocots, which are typically not amenable to traditional antibiotic-based selection systems, and may be used in combination with a negative selectable marker as part of a two-step selection system to obtain homoplasmic plant lines.
Assuntos
Citocininas/metabolismo , Ácidos Graxos/metabolismo , Nicotiana/metabolismo , Espectinomicina/farmacologia , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Expressão Gênica , Vetores Genéticos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Nicotiana/genética , Transformação Genética , TransgenesRESUMO
The detection of nucleic acids by the innate immune system is an essential host response during viral infection. In recent years, a number of immune sensors capable of recognizing cytosolic DNA have been identified and include the PYHIN family members AIM2, IFI16, and p204 as well as the enzyme, cGAS. Activation of these receptors leads to the induction of antiviral genes including Type-1 interferons and chemokines such as CCL5. We have carried out extensive expression profiling of these DNA sensors and other members of the PYHIN family in highly purified primary astrocytes and microglia and have demonstrated that both cell types express the majority of these proteins at the mRNA level. In microglia, several family members are highly upregulated in response to IFN-ß treatment while both cell types induce robust proinflammatory and antiviral cytokine production (e.g., IL-6, CCL5, IFN-ß) in the presence of immune stimulatory DNA and RNA. The production of IL-6 is partially dependent on the interferon receptor as is IFN-ß itself. Furthermore, we have found that p204 and AIM2 are upregulated in a Type I IFN dependent fashion in vivo, in a murine model of chronic neurodegeneration. Given the propensity of inflammatory responses to cause neuronal damage, increased expression and activation of these receptors, not only during viral infection but also during sterile inflammatory responses, has the potential to exacerbate existing neuroinflammation leading to further damage and impaired neurogenesis.
Assuntos
Astrócitos/metabolismo , Gliose/patologia , Microglia/metabolismo , Doenças Neurodegenerativas/complicações , Proteínas Nucleares/metabolismo , Regulação para Cima/fisiologia , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Gliose/etiologia , Gliose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ácidos Nucleicos/farmacologia , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Fatores de Tempo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Activation of enzymes by low concentrations of denaturants has been reported for a limited number of enzymes including lipocalin-type prostaglandin D synthase (L-PGDS) and adenylate kinase. During unfolding studies on human biliverdin-IXα reductase it was discovered that the enzyme is activated at low concentrations of urea. Under standard assay conditions the native enzyme displays pronounced substrate inhibition with biliverdin as variable substrate; however in the presence of 3M urea, the substrate inhibition is abolished and the enzyme exhibits Michaelian kinetics. When the initial rate kinetics with NADPH as variable substrate are conducted in 3M urea, the Vmax is increased 11-fold to 1.8µmol/min/mg and the apparent Km for biliverdin increases from 1 to 3µM. We report the existence of two kinetically distinct folded intermediates between the native and unfolded forms. When the period of incubation with urea was varied prior to measuring enzyme activity, the apparent Vmax was shown to decay to half that seen at zero time with a half life of 5.8minutes, while the apparent Km for NADPH remains constant at approximately 5µM. With NADH as cofactor the half life of the activated (A) form was 2.9minutes, and this form decays in 3M urea to a less active (LA) form. The apparent Km for NADH increases from 0.33mM to 2mM for the A and LA forms. These kinetically distinct species are reminiscent of the activity-enhanced and inactive forms of L-PGDS observed in the presence of urea and guanidine hydrochloride.
Assuntos
Biliverdina , Oxirredutases/química , Desdobramento de Proteína , Ureia/química , Ativação Enzimática , Humanos , Cinética , Oxirredutases/metabolismoRESUMO
IMPORTANCE: Staphylococcus aureus is one of the leading causes of antimicrobial-resistant infections whose success as a pathogen is facilitated by its massive array of immune evasion tactics, including intracellular survival within critical immune cells such as neutrophils, the immune system's first line of defense. In this study, we describe a novel pathway by which intracellular S. aureus can suppress the antimicrobial capabilities of human neutrophils by using the anti-inflammatory adenosine receptor, adora2a (A2aR). We show that signaling through A2aR suppresses the pentose phosphate pathway, a metabolic pathway used to fuel the antimicrobial NADPH oxidase complex that generates reactive oxygen species (ROS). As such, neutrophils show enhanced ROS production and reduced intracellular S. aureus when treated with an A2aR inhibitor. Taken together, we identify A2aR as a potential therapeutic target for combatting intracellular S. aureus infection.
Assuntos
Anti-Infecciosos , Infecções Estafilocócicas , Humanos , Neutrófilos , Staphylococcus aureus/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Via de Pentose Fosfato , Interações Hospedeiro-Patógeno , Anti-Infecciosos/metabolismo , Receptores Purinérgicos P1/metabolismoRESUMO
Since the recent observation that immune cells undergo metabolic reprogramming upon activation, there has been immense research in this area to not only understand the basis of such changes, but also to exploit metabolic rewiring for therapeutic benefit. In a resting state, macrophages preferentially utilise oxidative phosphorylation to generate energy; however, in the presence of immune cell activators, glycolytic genes are upregulated, and energy is generated through glycolysis. This facilitates the rapid production of biosynthetic intermediates and a pro-inflammatory macrophage phenotype. While this is essential to mount responses to infectious agents, more evidence is accumulating linking dysregulated metabolism to inappropriate immune responses. Given that certain biomaterials are known to promote an inflammatory macrophage phenotype, this prompted us to investigate if biomaterial particulates can impact on macrophage metabolism. Using micron and nano sized hydroxyapatite (HA), we demonstrate for the first time that these biomaterials can indeed drive changes in metabolism, and that this occurs in a size-dependent manner. We show that micronHA, but not nanoHA, particles upregulate surrogate markets of glycolysis including the glucose transporter (GLUT1), hexokinase 2 (HK2), GAPDH, and PKM2. Furthermore, we demonstrate that micronHA alters mitochondrial morphology and promotes a bioenergetic shift to favour glycolysis. Finally, we demonstrate that glycolytic gene expression is dependent on particle uptake and that targeting glycolysis attenuates the pro-inflammatory profile of micronHA-treated macrophages. These results not only further our understanding of biomaterial-based macrophage activation, but also implicate immunometabolism as a new area for consideration in intelligent biomaterial design and therapeutic targeting. STATEMENT OF SIGNIFICANCE: Several recent studies have reported that immune cell activation occurs concurrently with metabolic reprogramming. Furthermore, metabolic reprogramming of innate immune cells plays a prominent role in determining cellular phenotype and function. In this study we demonstrate that hydroxyapatite particle size alters macrophage metabolism, in turn driving their functional phenotype. Specifically, the pro-inflammatory phenotype promoted by micron-sized HA-particles is accompanied by changes in mitochondrial dynamics and a bioenergetic shift favouring glycolysis. This effect is not seen with nano-HA particles and can be attenuated upon inhibition of glycolysis. This study therefore not only identifies immunometabolism as a useful tool for characterising the immune response to biomaterials, but also highlights immunometabolism as a targetable aspect of the host response for therapeutic benefit.
Assuntos
Durapatita , Macrófagos , Durapatita/farmacologia , Tamanho da Partícula , Macrófagos/metabolismo , Materiais Biocompatíveis/farmacologia , Metaboloma , Ativação de MacrófagosRESUMO
Toll-like receptors (TLRs) are a family of proteins that act as the primary sensors of microbial products. Many TLRs require accessory molecules in order to recognize these microbial products and initiate signal transduction cascades. We have identified TRIL (TLR4 interactor with leucine-rich repeats) as a novel modulator of TLR4 signaling showing high expression in the brain. We now show that TRIL also plays a role in TLR3 signaling. TRIL is expressed intracellularly in the astrocytoma cell line U373 and in the monocytic cell line THP1. TRIL co-localizes with the endosomal compartment. These data are consistent with a role for TRIL in TLR3 signaling and endosomal TLR4 signaling. TRIL was induced by the TLR3 ligand poly(I:C). Overexpression of TRIL enhanced cytokine production and interferon-stimulated response element (ISRE) luciferase activity following poly(I:C) stimulation in U373. TRIL interacted with TLR3, and this interaction was enhanced following poly(I:C) stimulation. Transient knockdown of TRIL with siRNA or stable knockdown using shRNA in U373 cells inhibited TLR3 signaling, reducing ISRE luciferase, RANTES, and type I interferon production. Knockdown of TRIL did not affect TLR2 signaling. Most accessory molecules identified to date, such as CD14, gp96, PRAT4a, and Unc93B, all play roles in multiple TLR signaling pathways, and we now show that this is also the case for TRIL.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Linhagem Celular , Endossomos/metabolismo , Células HEK293 , Humanos , Imunidade Inata , Inflamação , Peptídeos e Proteínas de Sinalização Intercelular , Interferon-alfa/metabolismo , Interferon beta/metabolismo , Leucina/química , Ligantes , Proteínas de Membrana , Poli I-C , Elementos de Resposta , Frações Subcelulares/metabolismoRESUMO
The pro-inflammatory cytokines, TNFα, IL-1 and IL-18, amplify cartilage destruction associated with osteoarthritis (OA). Current data suggest that basic calcium phosphate (BCP) crystals are potent drivers of inflammatory mediator and matrix metalloprotease expression in the OA joint. It has previously been demonstrated that synovial macrophages play a role in initiating and driving BCP-induced inflammation. However, the molecular mechanisms by which BCP crystals exert their effects remain unclear. Here we demonstrate that exposure of macrophages to BCP crystals leads to activation of Syk and PI3 kinase. Furthermore, we show that production of pro-inflammatory cytokines and phosphorylation of the downstream kinase, ERK, are suppressed following treatment with Syk and PI3 kinase inhibitors. Finally, we demonstrate that treatment of macrophages with BCP crystals induces the production of the damage-associated molecule, S100A8, in a Syk dependent manner. We therefore identify Syk and PI3 kinase as potential novel targets for the treatment of BCP-related pathologies.
Assuntos
Fosfatos de Cálcio/efeitos adversos , Calgranulina A/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Osteoartrite/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Interleucina-18/metabolismo , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Articulações/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoartrite/enzimologia , Fosforilação , Quinase SykRESUMO
Inflammasome-mediated IL-1beta production is central to the innate immune defects that give rise to certain autoinflammatory diseases and may also be associated with the generation of IL-17-producing CD4(+) T (Th17) cells that mediate autoimmunity. However, the role of the inflammasome in driving adaptive immunity to infection has not been addressed. In this article, we demonstrate that inflammasome-mediated IL-1beta plays a critical role in promoting Ag-specific Th17 cells and in generating protective immunity against Bordetella pertussis infection. Using a murine respiratory challenge model, we demonstrated that the course of B. pertussis infection was significantly exacerbated in IL-1R type I-defective (IL-1RI(-/-)) mice. We found that adenylate cyclase toxin (CyaA), a key virulence factor secreted by B. pertussis, induced robust IL-1beta production by dendritic cells through activation of caspase-1 and the NALP3-containing inflammasome complex. Using mutant toxins, we demonstrate that CyaA-mediated activation of caspase-1 was not dependent on adenylate cyclase enzyme activity but was dependent on the pore-forming capacity of CyaA. In addition, CyaA promoted the induction of Ag-specific Th17 cells in wild-type but not IL-1RI(-/-) mice. Furthermore, the bacterial load was enhanced in IL-17-defective mice. Our findings demonstrate that CyaA, a virulence factor from B. pertussis, promotes innate IL-1beta production via activation of the NALP3 inflammasome and, thereby, polarizes T cell responses toward the Th17 subtype. In addition to its known role in subverting host immunity, our findings suggest that CyaA can promote IL-1beta-mediated Th17 cells, which promote clearance of the bacteria from the respiratory tract.
Assuntos
Toxina Adenilato Ciclase/fisiologia , Bordetella pertussis/imunologia , Linfócitos T CD4-Positivos/imunologia , Mediadores da Inflamação/metabolismo , Interleucina-17/biossíntese , Infecções Respiratórias/prevenção & controle , Toxina Adenilato Ciclase/toxicidade , Animais , Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/microbiologia , Proteínas de Transporte/metabolismo , Caspase 1/metabolismo , Polaridade Celular/imunologia , Células Cultivadas , Epitopos de Linfócito T/imunologia , Inflamação/enzimologia , Inflamação/microbiologia , Inflamação/prevenção & controle , Mediadores da Inflamação/fisiologia , Interleucina-17/deficiência , Interleucina-17/fisiologia , Interleucina-1beta/biossíntese , Interleucina-1beta/fisiologia , Intubação Intratraqueal , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Infecções Respiratórias/enzimologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/patologiaRESUMO
Dysregulated metabolism has long been recognized as a feature of many metabolic disorders. However, recent studies demonstrating that metabolic reprogramming occurs in immune cells have led to a growing interest in the relationship between metabolic rewiring and immune-mediated disease pathogeneses. It is clear now that immune cell subsets engage in different metabolic pathways depending on their activation and/or maturation state. As a result, it may be possible to modulate metabolic reprogramming for clinical benefit. In this review, we provide an overview of immune cell metabolism with focus on endogenous drivers of metabolic reprogramming given their link to a number of immune-mediated disorders.
Assuntos
Metabolismo Energético , Redes e Vias MetabólicasRESUMO
BACKGROUND AND AIMS: Metabolic reprogramming of innate immune cells is emerging as a key player in the progression of a number of chronic diseases, including atherosclerosis, where high rates of glycolysis correlate with plaque instability. This study aimed to investigate if cholesterol crystals, which are key atherosclerosis-associated DAMPs (damage/danger-associated molecular patterns), alter immune cell metabolism and whether this, in turn, impacts on macrophage phenotype and function. METHODS AND RESULTS: Primary human macrophages were treated with cholesterol crystals and expression of M1 (CXCL9, CXCL10) and M2-associated (MRC1, CCL13) macrophage markers, alarmins, and inflammatory cytokines were assessed either by real-time PCR or ELISA. Cholesterol crystal-induced changes in glycolytic markers were determined using real-time PCR and western blotting, while changes in cellular respiration and mitochondrial dynamics were examined via Seahorse analysis, Fluorescence Lifetime Imaging Microscopy (FLIM) and confocal microscopy. Treatment of macrophages with cholesterol crystals upregulated mRNA levels of CXCL9 and CXCL10, while concomitantly downregulating expression of MRC1 and CCL13. Cholesterol crystal--treated macrophages also exhibited a significant shift in metabolism to favour glycolysis, accompanied by the expression of key glycolytic markers GLUT1, Hexokinase 2, HIF1α, GAPDH and PFKFB3. Furthermore, we show that these effects are mediated upstream by the glycolytic enzyme, PKM2, and that direct inhibition of glycolysis or PKM2 nuclear localisation leads to a significant reduction in cholesterol crystal-induced inflammatory readouts. CONCLUSIONS: This study not only provides further insight into how atherosclerosis-associated DAMPs impact on immune cell function, but also highlights metabolic reprogramming as a potential therapeutic target for cholesterol crystal-related inflammation.
Assuntos
Aterosclerose , Ativação de Macrófagos , Aterosclerose/metabolismo , Colesterol/metabolismo , Humanos , Inflamação/metabolismo , Macrófagos/metabolismoRESUMO
In this study, we utilise fluorescence lifetime imaging of NAD(P)H-based cellular autofluorescence as a non-invasive modality to classify two contrasting states of human macrophages by proxy of their governing metabolic state. Macrophages derived from human blood-circulating monocytes were polarised using established protocols and metabolically challenged using small molecules to validate their responding metabolic actions in extracellular acidification and oxygen consumption. Large field-of-view images of individual polarised macrophages were obtained using fluorescence lifetime imaging microscopy (FLIM). These were challenged in real time with small-molecule perturbations of metabolism during imaging. We uncovered FLIM parameters that are pronounced under the action of carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), which strongly stratifies the phenotype of polarised human macrophages; however, this performance is impacted by donor variability when analysing the data at a single-cell level. The stratification and parameters emanating from a full field-of-view and single-cell FLIM approach serve as the basis for machine learning models. Applying a random forests model, we identify three strongly governing FLIM parameters, achieving an area under the receiver operating characteristics curve (ROC-AUC) value of 0.944 and out-of-bag (OBB) error rate of 16.67% when classifying human macrophages in a full field-of-view image. To conclude, 2P-FLIM with the integration of machine learning models is showed to be a powerful technique for analysis of both human macrophage metabolism and polarisation at full FoV and single-cell level.
Assuntos
Macrófagos , NAD , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona , Humanos , Aprendizado de Máquina , Macrófagos/metabolismo , Microscopia de Fluorescência/métodos , NAD/metabolismoRESUMO
The extracellular parasite and causative agent of African sleeping sickness Trypanosoma brucei (T. brucei) has evolved a number of strategies to avoid immune detection in the host. One recently described mechanism involves the conversion of host-derived amino acids to aromatic ketoacids, which are detected at relatively high concentrations in the bloodstream of infected individuals. These ketoacids have been shown to directly suppress inflammatory responses in murine immune cells, as well as acting as potent inducers of the stress response enzyme, heme oxygenase 1 (HO-1), which has proven anti-inflammatory properties. The aim of this study was to investigate the immunomodulatory properties of the T. brucei-derived ketoacids in primary human immune cells and further examine their potential as a therapy for inflammatory diseases. We report that the T. brucei-derived ketoacids, indole pyruvate (IP) and hydroxyphenylpyruvate (HPP), induce HO-1 expression through Nrf2 activation in human dendritic cells (DC). They also limit DC maturation and suppress the production of pro-inflammatory cytokines, which, in turn, leads to a reduced capacity to differentiate adaptive CD4+ T cells. Furthermore, the ketoacids are capable of modulating DC cellular metabolism and suppressing the inflammatory profile of cells isolated from patients with inflammatory bowel disease. This study therefore not only provides further evidence of the immune-evasion mechanisms employed by T. brucei, but also supports further exploration of this new class of HO-1 inducers as potential therapeutics for the treatment of inflammatory conditions.