RESUMO
OBJECTIVE: In polymyalgia rheumatica (PMR), glucocorticoids (GCs) relieve pain and stiffness, but fatigue may persist. We aimed to explore the effect of disease, GCs and PMR symptoms in the metabolite signatures of peripheral blood from patients with PMR or the related disease, giant cell arteritis (GCA). METHODS: Nuclear magnetic resonance spectroscopy was performed on serum from 40 patients with untreated PMR, 84 with new-onset confirmed GCA, and 53 with suspected GCA who later were clinically confirmed non-GCA, and 39 age-matched controls. Further samples from PMR patients were taken one and six months into glucocorticoid therapy to explore relationship of metabolites to persistent fatigue. 100 metabolites were identified using Chenomx and statistical analysis performed in SIMCA-P to examine the relationship between metabolic profiles and, disease, GC treatment or symptoms. RESULTS: The metabolite signature of patients with PMR and GCA differed from that of age-matched non-inflammatory controls (R2 > 0.7). There was a smaller separation between patients with clinically confirmed GCA and those with suspected GCA who later were clinically confirmed non-GCA (R2 = 0.135). In PMR, metabolite signatures were further altered with glucocorticoid treatment (R2 = 0.42) but did not return to that seen in controls. Metabolites correlated with CRP, pain, stiffness, and fatigue (R2 ≥ 0.39). CRP, pain, and stiffness declined with treatment and were associated with 3-hydroxybutyrate and acetoacetate, but fatigue did not. Metabolites differentiated patients with high and low fatigue both before and after treatment (R2 > 0.9). Low serum glutamine was predictive of high fatigue at both time points (0.79-fold change). CONCLUSION: PMR and GCA alter the metabolite signature. In PMR, this is further altered by glucocorticoid therapy. Treatment-induced metabolite changes were linked to measures of inflammation (CRP, pain and stiffness), but not to fatigue. Furthermore, metabolite signatures distinguished patients with high or low fatigue.
Assuntos
Fadiga , Glucocorticoides , Metaboloma , Metabolômica , Polimialgia Reumática , Humanos , Polimialgia Reumática/tratamento farmacológico , Polimialgia Reumática/metabolismo , Polimialgia Reumática/sangue , Glucocorticoides/uso terapêutico , Fadiga/etiologia , Feminino , Idoso , Masculino , Metabolômica/métodos , Pessoa de Meia-Idade , Arterite de Células Gigantes/tratamento farmacológico , Arterite de Células Gigantes/metabolismo , Arterite de Células Gigantes/sangue , Arterite de Células Gigantes/diagnóstico , Biomarcadores , Idoso de 80 Anos ou mais , Espectroscopia de Ressonância MagnéticaRESUMO
Changes in cellular metabolism have been implicated in mediating the activated fibroblast phenotype in a number of chronic inflammatory disorders, including pulmonary fibrosis, renal disease and rheumatoid arthritis. The aim of this study was therefore to characterise the metabolic profile of synovial joint fluid and synovial fibroblasts under both basal and inflammatory conditions in a cohort of obese and normal-weight hip OA patients. Furthermore, we sought to ascertain whether modulation of a metabolic pathway in OA synovial fibroblasts could alter their inflammatory activity. Synovium and synovial fluid was obtained from hip OA patients, who were either of normal-weight or obese and were undergoing elective joint replacement surgery. The synovial fluid metabolome was determined by 1H NMR spectroscopy. The metabolic profile of isolated synovial fibroblasts in vitro was characterised by lactate secretion, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) using the Seahorse XF Analyser. The effects of a small molecule pharmacological inhibitor and siRNA targeted at glutaminase-1 (GLS1) were assessed to probe the role of glutamine metabolism in OA synovial fibroblast function. Obese OA patient synovial fluid (n = 5) exhibited a different metabotype, compared to normal-weight patient fluid (n = 6), with significantly increased levels of 1, 3-dimethylurate, N-Nitrosodimethylamine, succinate, tyrosine, pyruvate, glucose, glycine and lactate, and enrichment of the glutamine-glutamate metabolic pathway, which correlated with increasing adiposity. In vitro, isolated obese OA fibroblasts exhibited greater basal lactate secretion and aerobic glycolysis, and increased mitochondrial respiration when stimulated with pro-inflammatory cytokine TNFα, compared to fibroblasts from normal-weight patients. Inhibition of GLS1 attenuated the TNFα-induced expression and secretion of IL-6 in OA synovial fibroblasts. These findings suggest that altered cellular metabolism underpins the inflammatory phenotype of OA fibroblasts, and that targeted inhibition of glutamine-glutamate metabolism may provide a route to reducing the pathological effects of joint inflammation in OA patients who are obese.
Assuntos
Osteoartrite do Quadril , Células Cultivadas , Fibroblastos/metabolismo , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Humanos , Ácido Láctico/metabolismo , Obesidade/metabolismo , Osteoartrite do Quadril/patologia , Líquido Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Manipulation of the CD28/CTLA-4 pathway is at the heart of a number of immunomodulatory approaches used in both autoimmunity and cancer. Although it is clear that CTLA-4 is a critical regulator of T cell responses, the immunological contexts in which CTLA-4 controls immune responses are not well defined. In this study, we show that whereas CD80/CD86-dependent activation of resting human T cells caused extensive T cell proliferation and robust CTLA-4 expression, in this context CTLA-4 blocking Abs had no impact on the response. In contrast, in settings where CTLA-4(+) cells were present as "regulators," inhibition of resting T cell responses was dependent on CTLA-4 expression and specifically related to the number of APC. At low numbers of APC or low levels of ligand, CTLA-4-dependent suppression was highly effective whereas at higher APC numbers or high levels of ligand, inhibition was lost. Accordingly, the degree of suppression correlated with the level of CD86 expression remaining on the APC. These data reveal clear rules for the inhibitory function of CTLA-4 on regulatory T cells, which are predicted by its ability to remove ligands from APC.
Assuntos
Anticorpos/farmacologia , Células Dendríticas/imunologia , Modelos Imunológicos , Linfócitos T Reguladores/imunologia , Animais , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Antígenos CD28/genética , Antígenos CD28/imunologia , Células CHO , Antígeno CTLA-4/genética , Antígeno CTLA-4/imunologia , Contagem de Células , Proliferação de Células , Cricetulus , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Endocitose , Regulação da Expressão Gênica , Humanos , Ativação Linfocitária/efeitos dos fármacos , Cultura Primária de Células , Transdução de Sinais , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , TransgenesRESUMO
OBJECTIVES: A genetic variant of the leukocyte phosphatase PTPN22 (R620W) is strongly associated with autoimmune diseases including rheumatoid arthritis (RA). Functional studies on the variant have focussed on lymphocytes, but it is most highly expressed in neutrophils. We have investigated the effects of the variant on neutrophil function in health and in patients with RA. METHODS: Healthy individuals and patients with RA were genotyped for PTPN22 (R620W) and neutrophils isolated from peripheral blood. Neutrophil adhesion and migration across inflamed endothelium were measured. Calcium (Ca(2+)) release and reactive oxygen species (ROS) production in response to fMLP stimulation were also assessed. RESULTS: Expression of R620W enhanced neutrophil migration through cytokine activated endothelium (non-R620W=24%, R620W=45% migrating cells, p<0.001). Following fMLP stimulation, neutrophils that were heterozygous and homozygous for R620W released significantly more Ca(2+) when compared to non-R620W neutrophils, in healthy individuals and patients with RA. fMLP stimulation, after TNF-α priming, provoked more ROS from neutrophils heterozygous for R620W in patients with RA (non-R620W vs R620W=â¼1.75-fold increase) and healthy individuals (non-R620W vs R620W=fourfold increase) and this increase was statistically significant in healthy individuals (p<0.001) but not in patients with RA (p<0.25). CONCLUSIONS: Expression of PTPN22 (R620W) enhanced neutrophil effector functions in health and RA, with migration, Ca(2+) release and production of ROS increased. Neutrophils are found in large numbers in the RA joint, and this hyperactivity of R620W cells may directly contribute to the joint damage, as well as to the initiation and perpetuation of the chronic immune-mediated inflammatory processes driving the disease.
Assuntos
Artrite Reumatoide/genética , Neutrófilos/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 22/genética , Adulto , Artrite Reumatoide/imunologia , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Feminino , Variação Genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Tirosina Fosfatase não Receptora Tipo 22/imunologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/fisiologia , Adulto JovemRESUMO
While the most obvious manifestations of rheumatoid arthritis (RA) involve inflammation and damage in the synovial joints, the systemic effects of the condition are widespread and life-threatening. Of particular interest is the 'lipid paradox' of RA, where patients with a numerically equivocal starting lipid profile have a significantly raised risk of cardiovascular (CV) events and response to therapy results in a 'normalization' of lipid levels and reduction in events. Changes in lipids can be seen before overt disease manifestations which suggest that they are closely linked to the more widespread inflammation-driven metabolic changes associated with tumour necrosis factor (TNF). Cachexia involves a shift in body mass from muscle to fat, which may or may not directly increase the cardiovascular risk. However, since TNF inhibition is associated with reduction in cardiovascular events, it does suggest that these widespread metabolic changes involving lipids are of importance. Analysis of single lipids or metabolites have been used to identify some of the key changes, but more recently, metabolomic and lipidomic approaches have been applied to identify a broad spectrum of small molecule changes and identify potentially altered metabolic pathways. Further work is needed to understand fully the metabolic changes in lipid profiles and identify novel ways of targeting desired profile changes, but work so far does suggest that a better understanding may allow management of patients to downregulate the systemic effects of their disease that puts them at risk of cardiovascular complications.
Assuntos
Artrite Reumatoide/metabolismo , Metabolismo dos Lipídeos/fisiologia , Antirreumáticos/farmacologia , Antirreumáticos/uso terapêutico , Artrite Reumatoide/complicações , Artrite Reumatoide/tratamento farmacológico , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , MetabolômicaRESUMO
BACKGROUND: Metabolomics is a systems approach to the analysis of cellular processes through small-molecule metabolite profiling. Standardisation of sample handling and acquisition approaches has contributed to reproducibility. However, the development of robust methods for the analysis of metabolomic data is a work-in-progress. The tools that do exist are often not well integrated, requiring manual data handling and custom scripting on a case-by-case basis. Furthermore, existing tools often require experience with programming environments such as MATLAB® or R to use, limiting accessibility. Here we present Pathomx, a workflow-based tool for the processing, analysis and visualisation of metabolomic and associated data in an intuitive and extensible environment. RESULTS: The core application provides a workflow editor, IPython kernel and a HumanCyc™-derived database of metabolites, proteins and genes. Toolkits provide reusable tools that may be linked together to create complex workflows. Pathomx is released with a base set of plugins for the import, processing and visualisation of data. The IPython backend provides integration with existing platforms including MATLAB® and R, allowing data to be seamlessly transferred. Pathomx is supplied with a series of demonstration workflows and datasets. To demonstrate the use of the software we here present an analysis of 1D and 2D (1)H NMR metabolomic data from a model system of mammalian cell growth under hypoxic conditions. CONCLUSIONS: Pathomx is a useful addition to the analysis toolbox. The intuitive interface lowers the barrier to entry for non-experts, while scriptable tools and integration with existing tools supports complex analysis. We welcome contributions from the community.
Assuntos
Biologia Computacional/métodos , Bases de Dados Factuais , Macrófagos/metabolismo , Metabolômica/métodos , Software , Fluxo de Trabalho , Células Cultivadas , Humanos , Macrófagos/citologia , Espectroscopia de Ressonância Magnética , Redes e Vias Metabólicas , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: Anti-tumor necrosis factor (anti-TNF) therapies are highly effective in rheumatoid arthritis (RA) and psoriatic arthritis (PsA), but a significant number of patients exhibit only a partial or no therapeutic response. Inflammation alters local and systemic metabolism, and TNF plays a role in this. We undertook this study to determine if the patient's metabolic fingerprint prior to therapy could predict responses to anti-TNF agents. METHODS: Urine was collected from 16 RA patients and 20 PsA patients before and during therapy with infliximab or etanercept. Urine metabolic profiles were assessed using nuclear magnetic resonance spectroscopy. Discriminating metabolites were identified, and the relationship between metabolic profiles and clinical outcomes was assessed. RESULTS: Baseline urine metabolic profiles discriminated between RA patients who did or did not have a good response to anti-TNF therapy according to European League Against Rheumatism criteria, with a sensitivity of 88.9% and a specificity of 85.7%, with several metabolites contributing (in particular histamine, glutamine, xanthurenic acid, and ethanolamine). There was a correlation between baseline metabolic profiles and the magnitude of change in the Disease Activity Score in 28 joints from baseline to 12 months in RA patients (P = 0.04). In both RA and PsA, urinary metabolic profiles changed between baseline and 12 weeks of anti-TNF therapy. Within the responders, urinary metabolite changes distinguished between etanercept and infliximab treatment. CONCLUSION: The clear relationship between urine metabolic profiles of RA patients at baseline and their response to anti-TNF therapy may allow development of novel approaches to the optimization of therapy. Differences in metabolic profiles during treatment with infliximab and etanercept in RA and PsA may reflect distinct mechanisms of action.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Imunoglobulina G/uso terapêutico , Metaboloma , Receptores do Fator de Necrose Tumoral/uso terapêutico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Idoso , Artrite Reumatoide/urina , Inglaterra , Etanercepte , Feminino , Humanos , Infliximab , Espectroscopia de Ressonância Magnética , Masculino , Metabolômica , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
OBJECTIVE: Inflammatory arthritis is associated with systemic manifestations including alterations in metabolism. We used nuclear magnetic resonance (NMR) spectroscopy-based metabolomics to assess metabolic fingerprints in serum from patients with established rheumatoid arthritis (RA) and those with early arthritis. METHODS: Serum samples were collected from newly presenting patients with established RA who were naive for disease-modifying antirheumatic drugs, matched healthy controls, and 2 groups of patients with synovitis of ≤3 months' duration whose outcomes were determined at clinical followup. Serum metabolomic profiles were assessed using 1-dimensional (1) H-NMR spectroscopy. Discriminating metabolites were identified, and the relationships between metabolomic profiles and clinical variables including outcomes were examined. RESULTS: The serum metabolic fingerprint in established RA was clearly distinct from that of healthy controls. In early arthritis, we were able to stratify the patients according to the level of current inflammation, with C-reactive protein correlating with metabolic differences in 2 separate groups (P < 0.001). Lactate and lipids were important discriminators of inflammatory burden in both early arthritis patient groups. The sensitivities and specificities of models to predict the development of either RA or persistent arthritis in patients with early arthritis were low. CONCLUSION: The metabolic fingerprint reflects inflammatory disease activity in patients with synovitis, demonstrating that underlying inflammatory processes drive significant changes in metabolism that can be measured in the peripheral blood. The identification of metabolic alterations may provide insights into disease mechanisms operating in patients with inflammatory arthritis.
Assuntos
Artrite Reumatoide/sangue , Biomarcadores/sangue , Metabolômica/métodos , Sinovite/sangue , Adulto , Idoso , Artrite Reumatoide/diagnóstico , Proteína C-Reativa/análise , Diagnóstico Precoce , Feminino , Humanos , Masculino , Metaboloma , Pessoa de Meia-Idade , Ressonância Magnética Nuclear Biomolecular , Sinovite/diagnósticoRESUMO
Musculoskeletal diseases affect up to 20% of adults worldwide. The gut microbiome has been implicated in inflammatory conditions, but large-scale metagenomic evaluations have not yet traced the routes by which immunity in the gut affects inflammatory arthritis. To characterize the community structure and associated functional processes driving gut microbial involvement in arthritis, the Inflammatory Arthritis Microbiome Consortium investigated 440 stool shotgun metagenomes comprising 221 adults diagnosed with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and 219 healthy controls and individuals with joint pain without an underlying inflammatory cause. Diagnosis explained about 2% of gut taxonomic variability, which is comparable in magnitude to inflammatory bowel disease. We identified several candidate microbes with differential carriage patterns in patients with elevated blood markers for inflammation. Our results confirm and extend previous findings of increased carriage of typically oral and inflammatory taxa and decreased abundance and prevalence of typical gut clades, indicating that distal inflammatory conditions, as well as local conditions, correspond to alterations to the gut microbial composition. We identified several differentially encoded pathways in the gut microbiome of patients with inflammatory arthritis, including changes in vitamin B salvage and biosynthesis and enrichment of iron sequestration. Although several of these changes characteristic of inflammation could have causal roles, we hypothesize that they are mainly positive feedback responses to changes in host physiology and immune homeostasis. By connecting taxonomic alternations to functional alterations, this work expands our understanding of the shifts in the gut ecosystem that occur in response to systemic inflammation during arthritis.
Assuntos
Artrite Reumatoide , Microbioma Gastrointestinal , Microbiota , Humanos , Microbioma Gastrointestinal/genética , Inflamação , Fenótipo , Redes e Vias MetabólicasRESUMO
Neutrophils are the most abundant leukocyte and have a short lifespan, dying by apoptosis approximately five days after leaving the bone marrow. Their apoptosis can be delayed at sites of inflammation to extend their functional lifespan, but inappropriate inhibition of apoptosis contributes to chronic inflammatory disease. Levels of the physiological iron chelator lactoferrin are raised at sites of inflammation and we have shown previously that iron-unsaturated lactoferrin inhibited human neutrophil apoptosis, but the mechanisms involved were not determined. Here we report that the anti-apoptotic effect of lactoferrin is dependent upon its iron saturation status as iron-saturated lactoferrin did not affect neutrophil apoptosis. We also show that the effect of lactoferrin is mediated at an early stage in apoptosis as it inhibited activation of sphingomyelinase, generation of ceramide, activation of caspase 8 and Bax and cleavage of Bid. Lactoferrin did not inhibit apoptosis induced by exogenous ceramide, supporting the proposal that it acts upstream of ceramide generation. We therefore conclude that raised lactoferrin levels are likely to contribute to chronic inflammation by delaying neutrophil apoptosis and that this is achieved by inhibiting proximal apoptotic signaling events.
Assuntos
Apoptose/efeitos dos fármacos , Lactoferrina/farmacologia , Neutrófilos/efeitos dos fármacos , Apoproteínas/farmacologia , Apoptose/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Ceramidas/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Ferro/metabolismo , Neutrófilos/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Esfingomielina Fosfodiesterase/metabolismoRESUMO
While 3,4-methylenedioxymethamphetamine (MDMA/'ecstasy') is cytostatic towards lymphoma cells in vitro, the concentrations required militate against its translation directly to a therapeutic in vivo. The possibility of 'redesigning the designer drug', separating desired anti-lymphoma activity from unwanted psychoactivity and neurotoxicity, was therefore mooted. From an initial analysis of MDMA analogues synthesized with a modified α-substituent, it was found that incorporating a phenyl group increased potency against sensitive, Bcl-2-deplete, Burkitt's lymphoma (BL) cells 10-fold relative to MDMA. From this lead, related analogs were synthesized with the 'best' compounds (containing 1- and 2-naphthyl and para-biphenyl substituents) some 100-fold more potent than MDMA versus the BL target. When assessed against derived lines from a diversity of B-cell tumors MDMA analogues were seen to impact the broad spectrum of malignancy. Expressing a BCL2 transgene in BL cells afforded only scant protection against the analogues and across the malignancies no significant correlation between constitutive Bcl-2 levels and sensitivity to compounds was observed. Bcl-2-deplete cells displayed hallmarks of apoptotic death in response to the analogues while BCL2 overexpressing equivalents died in a caspase-3-independent manner. Despite lymphoma cells expressing monoamine transporters, their pharmacological blockade failed to reverse the anti-lymphoma actions of the analogues studied. Neither did reactive oxygen species account for ensuing cell death. Enhanced cytotoxic performance did however track with predicted lipophilicity amongst the designed compounds. In conclusion, MDMA analogues have been discovered with enhanced cytotoxic efficacy against lymphoma subtypes amongst which high-level Bcl-2--often a barrier to drug performance for this indication--fails to protect.
Assuntos
Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/patologia , Desenho de Fármacos , N-Metil-3,4-Metilenodioxianfetamina/uso terapêutico , Transdução de Sinais , Linfócitos B/patologia , Linfoma de Burkitt/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , N-Metil-3,4-Metilenodioxianfetamina/análogos & derivados , N-Metil-3,4-Metilenodioxianfetamina/química , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We investigated the proposed necrotic mechanism of ingenol mebutate, a natural compound with anti-cancer properties in human keratinocytes, the human squamous cell carcinoma cell line HSC-5, and HeLa cervix carcinoma cells. Topical application of a clinical dose of ingenol mebutate 0.05% (1.15 mM) gel to human reconstituted full-thickness skin equivalents strongly reduced epidermal, but not dermal viability. Ingenol mebutate showed cytotoxic potency between 200-300 M on normal and cancer cells. When keratinocytes were induced to differentiate, they became significantly less sensitive to ingenol mebutate and half-maximal induction of cell death required more than 300 M ingenol mebutate. Cytotoxic concentrations of ingenol mebutate caused rupture of the mitochondrial network within minutes paralleled by cytosolic calcium release in all cells. Subsequently, plasma membrane integrity was lost as seen by propidium uptake into the cells. This was in sharp contrast to lysis of cells with low concentrations of the detergent Triton X-100 that permeabilized the plasma membrane within minutes without affecting organelle morphology. Buffering of intracellular calcium and inhibition of the mitochondrial permeability transition pore reduced the cytotoxic effect of ingenol mebutate in cancer cells, but not in normal keratinocytes. However, these inhibitors could not prevent cell death subsequent to prolonged incubation. Our findings reveal that ingenol mebutate does not mediate cytotoxicity by a simple lytic, necrotic mechanism, but activates distinct processes involving multiple cell organelles in a cell-type and differentiation-dependent manner. These data improve our understanding of ingenol mebutate-target cell interactions and offer new insights relevant to the removal of aberrant cells in human skin.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Diterpenos/farmacologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/patologia , Pele/patologia , Cálcio/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Detergentes/farmacologia , Células HeLa , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/efeitos dos fármacos , Necrose , Octoxinol/farmacologia , Pele/efeitos dos fármacosRESUMO
Dysregulated release of neutrophil azurophilic granules causes increased tissue damage and amplified inflammation during autoimmune disease. Antineutrophil cytoplasmic antibodies (ANCAs) are implicated in the pathogenesis of small vessel vasculitis and promote adhesion and exocytosis in neutrophils. ANCAs activate specific signal transduction pathways in neutrophils that have the potential to be modulated therapeutically to prevent neutrophil activation by ANCAs. We have investigated a role for diacylglycerol kinase (DGK) and its downstream product phosphatidic acid (PA) in ANCA-induced neutrophil exocytosis. Neutrophils incubated with the DGK inhibitor R59022, before treatment with ANCAs, exhibited a reduced capacity to release their azurophilic granules, demonstrated by a component release assay and flow cytometry. PA restored azurophilic granule release in DGK-inhibited neutrophils. Confocal microscopy revealed that R59022 did not inhibit translocation of granules, indicating a role for DGK during the process of granule fusion at the plasma membrane. In investigating possible mechanisms by which PA promotes neutrophil exocytosis, we demonstrated that exocytosis can only be restored in R59022-treated cells through simultaneous modulation of membrane fusion and increasing cytosolic calcium. PA and its associated pathways may represent viable drug targets to reduce tissue injury associated with ANCA-associated vasculitic diseases and other neutrophilic inflammatory disorders.
Assuntos
Anticorpos Anticitoplasma de Neutrófilos/imunologia , Diacilglicerol Quinase/metabolismo , Exocitose/imunologia , Neutrófilos/citologia , Neutrófilos/enzimologia , Ácidos Fosfatídicos/biossíntese , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Cálcio/metabolismo , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Diacilglicerol Quinase/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Imunoglobulina G/imunologia , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Ionomicina/farmacologia , Lisofosfatidilcolinas/metabolismo , Lisofosfatidilcolinas/farmacologia , Fusão de Membrana/efeitos dos fármacos , Neutrófilos/imunologia , Peroxidase/metabolismo , Pirimidinonas/farmacologia , Tetraspanina 30/metabolismo , Tiazóis/farmacologiaRESUMO
Mesenchymal stromal fibroblasts have emerged as key mediators of the inflammatory response and drivers of localised inflammation, in part through their interactions with resident and circulating immune cells at inflammatory sites. As such, they have been implicated in a number of chronic inflammatory conditions as well as in tumour progression through modifying the microenvironment. The connection between metabolic changes and altered phenotype of fibroblasts in inflammatory microenvironments has clear implications for our understanding of how chronic inflammation is regulated and for the development of new anti-inflammatory therapeutics. In this review, we consider the evidence that changes to fibroblast metabolic state underpin chronic inflammation. We examine recent research on fibroblast metabolism in inflammatory microenvironments and consider their involvement in inflammation, providing insight into the role of fibroblasts and metabolism in mediating inflammatory disease progression namely cancer, arthritis and fibrotic disorders including chronic kidney disease, pulmonary fibrosis, heart disease and liver disease.
Assuntos
Fibroblastos/metabolismo , Inflamação/genética , Células-Tronco Mesenquimais/metabolismo , Doenças Metabólicas/genética , Microambiente Celular/genética , Fibroblastos/patologia , Cardiopatias/genética , Cardiopatias/metabolismo , Humanos , Inflamação/metabolismo , Inflamação/patologia , Hepatopatias/genética , Hepatopatias/metabolismo , Células-Tronco Mesenquimais/patologia , Doenças Metabólicas/metabolismo , Doenças Metabólicas/patologia , Fibrose Pulmonar/genética , Fibrose Pulmonar/metabolismo , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismoRESUMO
Background: Systemic inflammation in rheumatoid arthritis (RA) is associated with metabolic changes. We used nuclear magnetic resonance (NMR) spectroscopy-based metabolomics to assess the relationship between an objective measure of systemic inflammation [C-reactive protein (CRP)] and both the serum and urinary metabolome in patients with newly presenting RA. Methods: Serum (n=126) and urine (n=83) samples were collected at initial presentation from disease modifying anti-rheumatic drug naïve RA patients for metabolomic profile assessment using 1-dimensional 1H-NMR spectroscopy. Metabolomics data were analysed using partial least square regression (PLS-R) and orthogonal projections to latent structure discriminant analysis (OPLS-DA) with cross validation. Results: Using PLS-R analysis, a relationship between the level of inflammation, as assessed by CRP, and the serum (p=0.001) and urinary (p<0.001) metabolome was detectable. Likewise, following categorisation of CRP into tertiles, patients in the lowest CRP tertile and the highest CRP tertile were statistically discriminated using OPLS-DA analysis of both serum (p=0.033) and urinary (p<0.001) metabolome. The most highly weighted metabolites for these models included glucose, amino acids, lactate, and citrate. These findings suggest increased glycolysis, perturbation in the citrate cycle, oxidative stress, protein catabolism and increased urea cycle activity are key characteristics of newly presenting RA patients with elevated CRP. Conclusions: This study consolidates our understanding of a previously identified relationship between serum metabolite profile and inflammation and provides novel evidence that there is a relationship between urinary metabolite profile and inflammation as measured by CRP. Identification of these metabolic perturbations provides insights into the pathogenesis of RA and may help in the identification of therapeutic targets.
Assuntos
Artrite Reumatoide/sangue , Artrite Reumatoide/urina , Adulto , Artrite Reumatoide/imunologia , Artrite Reumatoide/metabolismo , Proteína C-Reativa/análise , Feminino , Humanos , Inflamação/sangue , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/urina , Análise dos Mínimos Quadrados , Masculino , Metaboloma , Metabolômica , Pessoa de Meia-Idade , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
Fibroblast-like synoviocytes (FLS) play an important role in maintaining joint homeostasis and orchestrating local inflammatory processes. When activated during injury or inflammation, FLS undergo transiently increased bioenergetic and biosynthetic demand. We aimed to identify metabolic changes which occur early in inflammatory disease pathogenesis which might support sustained cellular activation in persistent inflammation. We took primary human FLS from synovial biopsies of patients with very early rheumatoid arthritis (veRA) or resolving synovitis, and compared them with uninflamed control samples from the synovium of people without arthritis. Metabotypes were compared using NMR spectroscopy-based metabolomics and correlated with serum C-reactive protein levels. We measured glycolysis and oxidative phosphorylation by Seahorse analysis and assessed mitochondrial morphology by immunofluorescence. We demonstrate differences in FLS metabolism measurable after ex vivo culture, suggesting that disease-associated metabolic changes are long-lasting. We term this phenomenon 'metabolic memory'. We identify changes in cell metabolism after acute TNFα stimulation across disease groups. When compared to FLS from patients with early rheumatoid arthritis, FLS from patients with resolving synovitis have significantly elevated mitochondrial respiratory capacity in the resting state, and less fragmented mitochondrial morphology after TNFα treatment. Our findings indicate the potential to restore cell metabotypes by modulating mitochondrial function at sites of inflammation, with implications for treatment of RA and related inflammatory conditions in which fibroblasts play a role.
Assuntos
Artrite Reumatoide/imunologia , Fibroblastos/imunologia , Inflamação/imunologia , Sinoviócitos/imunologia , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Idoso , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Fosforilação Oxidativa , Análise de Regressão , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
Recent advances in emergency medicine and the co-ordinated delivery of trauma care mean more critically-injured patients now reach the hospital alive and survive life-saving operations. Indeed, between 2008 and 2017, the odds of surviving a major traumatic injury in the UK increased by nineteen percent. However, the improved survival rates of severely-injured patients have placed an increased burden on the healthcare system, with major trauma a common cause of intensive care unit (ICU) admissions that last ≥10 days. Improved understanding of the factors influencing patient outcomes is now urgently needed. We investigated the serum metabolomic profile of fifty-five major trauma patients across three post-injury phases: acute (days 0-4), intermediate (days 5-14) and late (days 15-112). Using ICU length of stay (LOS) as a clinical outcome, we aimed to determine whether the serum metabolome measured at days 0-4 post-injury for patients with an extended (≥10 days) ICU LOS differed from that of patients with a short (<10 days) ICU LOS. In addition, we investigated whether combining metabolomic profiles with clinical scoring systems would generate a variable that would identify patients with an extended ICU LOS with a greater degree of accuracy than models built on either variable alone. The number of metabolites unique to and shared across each time segment varied across acute, intermediate and late segments. A one-way ANOVA revealed the most variation in metabolite levels across the different time-points was for the metabolites lactate, glucose, anserine and 3-hydroxybutyrate. A total of eleven features were selected to differentiate between <10 days ICU LOS vs. >10 days ICU LOS. New Injury Severity Score (NISS), testosterone, and the metabolites cadaverine, urea, isoleucine, acetoacetate, dimethyl sulfone, syringate, creatinine, xylitol, and acetone form the integrated biomarker set. Using metabolic enrichment analysis, we found valine, leucine and isoleucine biosynthesis, glutathione metabolism, and glycine, serine and threonine metabolism were the top three pathways differentiating ICU LOS with a p < 0.05. A combined model of NISS and testosterone and all nine selected metabolites achieved an AUROC of 0.824. Differences exist in the serum metabolome of major trauma patients who subsequently experience a short or prolonged ICU LOS in the acute post-injury setting. Combining metabolomic data with anatomical scoring systems allowed us to discriminate between these two groups with a greater degree of accuracy than that of either variable alone.
RESUMO
There is no consensus for diagnosis or treatment of RA muscle loss. We aimed to investigate metabolites in arthritic mice urine as biomarkers of muscle loss. DBA1/J mice comprised collagen-induced arthritis (CIA) and control (CO) groups. Urine samples were collected at 0, 18, 35, 45, 55, and 65 days of disease and subjected to nuclear magnetic resonance spectroscopy. Metabolites were identified using Chenomx and Birmingham Metabolite libraries. The statistical model used principal component analysis, partial least-squares discriminant analysis, and partial least-squares regression analysis. Linear regression and Fisher's exact test via the MetaboAnalyst website were performed (VIP-score). Nearly 100 identified metabolites had CIA vs. CO and disease time-dependent differences (p < 0.05). Twenty-eight metabolites were muscle-associated: carnosine (VIPs 2.8 × 102) and succinyl acetone (VIPs 1.0 × 10) showed high importance in CIA vs. CO models at day 65; CIA pair analysis showed histidine (VIPs 1.2 × 102) days 55 vs. 65, histamine (VIPs 1.1 × 102) days 55 vs. 65, and L-methionine (VIPs 1.1 × 102) days 0 vs. 18. Carnosine was fatigue- (0.039) related, creatine was food intake- (-0.177) and body weight- (-0.039) related, and both metabolites were clinical score- (0.093; 0.050) and paw edema- (0.125; 0.026) related. Therefore, muscle metabolic alterations were detected in arthritic mice urine, enabling further validation in RA patient's urine, targeting prognosis, diagnosis, and monitoring of RA-mediated muscle loss.
RESUMO
We sought to evaluate the diagnostic accuracy of metabolomic biomarker profiles in neurological conditions (idiopathic intracranial hypertension (IIH), multiple sclerosis (MS) and cerebrovascular disease (CVD) compared to controls with either no neurological disease or mixed neurological diseases). Spectra of CSF (n = 87) and serum (n = 72) were acquired using (1)H NMR spectroscopy. Multivariate pattern recognition analysis was used to identify disease-specific metabolite biomarker profiles. The metabolite profiles were then used to predict the diagnosis of a second cohort of patients (n = 25). CSF metabolite profiles were able to predict diagnosis with a sensitivity and specificity of 80% for both IIH and MS. The CVD serum metabolite profile was 75% sensitive and specific. On analysing the second patient cohort, the established metabolite biomarker profiles generated from the first cohort showed moderate ability to segregate patients with IIH and MS (sensitivity:specificity of 63:75% and 67:75%, respectively). These findings suggest that NMR spectroscopic metabolic profiling of CSF and serum can identify differences between IIH, MS, CVD and mixed neurological diseases. Metabolomics may, therefore, have the potential to be developed into a clinically useful diagnostic tool. The identification of disease-unique metabolites may also impart information on disease pathology.