RESUMO
The complement system is an ancient and critical part of innate immunity. Recent studies have highlighted novel roles of complement beyond lysis of invading pathogens with implications in regulating the innate immune response, as well as contributing to metabolic reprogramming of T-cells, synoviocytes as well as cells in the CNS. These findings hint that complement can be an immunometabolic regulator, but whether this is also the case for the terminal step of the complement pathway, the membrane attack complex (MAC) is not clear. In this study we focused on determining whether MAC is an immunometabolic regulator of the innate immune response in human monocyte-derived macrophages. Here, we uncover previously uncharacterized metabolic changes and mitochondrial dysfunction occurring downstream of MAC deposition. These alterations in glycolytic flux and mitochondrial morphology and function mediate NLRP3 inflammasome activation, pro-inflammatory cytokine release and gasdermin D formation. Together, these data elucidate a novel signalling cascade, with metabolic alterations at its center, in MAC-stimulated human macrophages that drives an inflammatory consequence in an immunologically relevant cell type.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Humanos , Inflamassomos/metabolismo , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismoRESUMO
MicroRNAs are critical regulators of gene expression controlling cellular processes including inflammation. We explored their role in the pathogenesis of inflammatory bowel disease (IBD) and identified reduced expression of miR-374a-5p in IBD monocytes that correlated with a module of up-regulated genes related to the inflammatory response. Key proinflammatory module genes, including for example TNFα, IL1A, IL6, and OSM, were inversely correlated with miR-374a-5p and were validated in vitro. In colonic biopsies, miR-374a-5p was again reduced in expression and inversely correlated with the same inflammatory module, and its levels predicted subsequent response to anti-TNF therapy. Increased miR-374a-5p expression was shown to control macrophage-driven inflammation by suppressing proinflammatory mediators and to reduce the capacity of monocytes to migrate and activate T cells. Our findings suggest that miR-374a-5p reduction is a central driver of inflammation in IBD, and its therapeutic supplementation could reduce monocyte-driven inflammation in IBD or other immune-mediated diseases.
Assuntos
Colite , Doenças Inflamatórias Intestinais , MicroRNAs , Humanos , Doenças Inflamatórias Intestinais/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Monócitos/metabolismo , Inibidores do Fator de Necrose TumoralRESUMO
Human rhinovirus (HRV), like coronavirus (HCoV), are positive-strand RNA viruses that cause both upper and lower respiratory tract illness, with their replication facilitated by concentrating RNA-synthesizing machinery in intracellular compartments made of modified host membranes, referred to as replication organelles (ROs). Here we report a non-canonical, essential function for stimulator of interferon genes (STING) during HRV infections. While the canonical function of STING is to detect cytosolic DNA and activate inflammatory responses, HRV infection triggers the release of STIM1-bound STING in the ER by lowering Ca2+, thereby allowing STING to interact with phosphatidylinositol 4-phosphate (PI4P) and traffic to ROs to facilitates viral replication and transmission via autophagy. Our results thus hint a critical function of STING in HRV viral replication and transmission, with possible implications for other RO-mediated RNA viruses.
Assuntos
Enterovirus , Vírus de RNA , Humanos , Organelas , Rhinovirus , Replicação Viral/fisiologiaAssuntos
Alergia e Imunologia , Pesquisa Biomédica , Sistema Imunitário/imunologia , Mediadores da Inflamação/imunologia , Inflamação/imunologia , Animais , Anti-Inflamatórios/uso terapêutico , Difusão de Inovações , Descoberta de Drogas , Humanos , Sistema Imunitário/efeitos dos fármacos , Sistema Imunitário/metabolismo , Sistema Imunitário/patologia , Imunoterapia , Inflamação/diagnóstico , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Terapia de Alvo Molecular , Transdução de SinaisRESUMO
Macrophages are important drivers of pathogenesis and progression to AIDS in HIV infection. The virus in the later phases of the infection is often predominantly macrophage-tropic and this tropism contributes to a chronic inflammatory and immune activation state that is observed in HIV patients. Pattern recognition receptors of the innate immune system are the key molecules that recognise HIV and mount the inflammatory responses in macrophages. The innate immune response against HIV-1 is potent and elicits caspase-1-dependent pro-inflammatory cytokine production of IL-1ß and IL-18. Although, NLRP3 has been reported as an inflammasome sensor dictating this response little is known about the pattern recognition receptors that trigger the "priming" signal for inflammasome activation, the NLRs involved or the HIV components that trigger the response. Using a combination of siRNA knockdowns in monocyte derived macrophages (MDMs) of different TLRs and NLRs as well as chemical inhibition, it was demonstrated that HIV Vpu could trigger inflammasome activation via TLR4/NLRP3 leading to IL-1ß/IL-18 secretion. The priming signal is triggered via TLR4, whereas the activation signal is triggered by direct effects on Kv1.3 channels, causing K+ efflux. In contrast, HIV gp41 could trigger IL-18 production via NAIP/NLRC4, independently of priming, as a one-step inflammasome activation. NAIP binds directly to the cytoplasmic tail of HIV envelope protein gp41 and represents the first non-bacterial ligand for the NAIP/NLRC4 inflammasome. These divergent pathways represent novel targets to resolve specific inflammatory pathologies associated with HIV-1 infection in macrophages.
Assuntos
Infecções por HIV/virologia , Inflamassomos/imunologia , Interleucina-18/metabolismo , Interleucina-1beta/metabolismo , Macrófagos/virologia , Fragmentos de Peptídeos/metabolismo , Comunicação Celular/genética , Comunicação Celular/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Infecções por HIV/metabolismo , Humanos , Imunidade Inata/genética , Imunidade Inata/imunologia , Inflamassomos/metabolismo , Macrófagos/imunologia , Proteína Inibidora de Apoptose Neuronal/genética , Transdução de Sinais/imunologiaRESUMO
It has been reported that a GM-CSFâCCL17 pathway, originally identified in vitro in macrophage lineage populations, is implicated in the control of inflammatory pain, as well as arthritic pain and disease. We explore, in this study and in various inflammation models, the cellular CCL17 expression and its GM-CSF dependence as well as the function of CCL17 in inflammation and pain. This study used models allowing the convenient cell isolation from Ccl17E/+ reporter mice; it also exploited both CCL17-dependent and unique CCL17-driven inflammatory pain and arthritis models, the latter permitting a radiation chimera approach to help identify the CCL17 responding cell type(s) and the mediators downstream of CCL17 in the control of inflammation and pain. We present evidence that 1) in the particular inflammation models studied, CCL17 expression is predominantly in macrophage lineage populations and is GM-CSF dependent, 2) for its action in arthritic pain and disease development, CCL17 acts on CCR4+ non-bone marrow-derived cells, and 3) for inflammatory pain development in which a GM-CSFâCCL17 pathway appears critical, nerve growth factor, CGRP, and substance P all appear to be required.
Assuntos
Artrite Experimental/imunologia , Quimiocina CCL17/metabolismo , Dor/imunologia , Peritonite/imunologia , Pneumonia/imunologia , Animais , Artrite Experimental/complicações , Artrite Experimental/patologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Quimiocina CCL17/genética , Genes Reporter/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Fator de Crescimento Neural/metabolismo , Dor/diagnóstico , Dor/patologia , Medição da Dor , Peritonite/complicações , Peritonite/patologia , Pneumonia/complicações , Pneumonia/patologia , Transdução de Sinais/imunologia , Substância P/metabolismoRESUMO
Memory T cells possess functional differences from naïve T cells that powerfully contribute to the efficiency of secondary immune responses. These abilities are imprinted during the primary response, linked to the acquisition of novel patterns of gene expression. Underlying this are alterations at the chromatin level (epigenetic modifications) that regulate constitutive and inducible gene transcription. T cell epigenetic memory can persist long-term, contributing to long-lasting immunity after infection or vaccination. However, acquired epigenetic states can also hinder effective tumor immunity or contribute to autoimmunity. The growing understanding of epigenetic gene regulation as it relates to both the stability and malleability of T cell memory may offer the potential to selectively modify T cell memory in disease by targeting epigenetic mechanisms.
Assuntos
Epigênese Genética , Memória Imunológica , Linfócitos T , Cromatina/imunologia , Epigênese Genética/imunologia , Regulação da Expressão Gênica , Humanos , Memória Imunológica/genética , Linfócitos T/imunologiaRESUMO
Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Mediadores da Inflamação/farmacologia , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , Citocinas/metabolismo , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Lisina/metabolismo , Malonil Coenzima A/metabolismo , Camundongos Endogâmicos C57BL , Mutagênese , Polirribossomos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.
Assuntos
Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/agonistas , Fator 2 Relacionado a NF-E2/metabolismo , Succinatos/metabolismo , Alquilação , Animais , Carboxiliases , Bovinos , Cisteína/química , Cisteína/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Retroalimentação Fisiológica , Feminino , Células HEK293 , Humanos , Hidroliases/biossíntese , Interferon beta/imunologia , Interferon beta/farmacologia , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Proteínas/metabolismo , Ratos , Ratos Wistar , Succinatos/químicaRESUMO
Different immune activation states require distinct metabolic features and activities in immune cells. For instance, inhibition of fatty acid synthase (FASN), which catalyzes the synthesis of long-chain fatty acids, prevents the proinflammatory response in macrophages; however, the precise role of this enzyme in this response remains poorly defined. Consistent with previous studies, we found here that FASN is essential for lipopolysaccharide-induced, Toll-like receptor (TLR)-mediated macrophage activation. Interestingly, only agents that block FASN upstream of acetoacetyl-CoA synthesis, including the well-characterized FASN inhibitor C75, inhibited TLR4 signaling, while those acting downstream had no effect. We found that acetoacetyl-CoA could overcome C75's inhibitory effect, whereas other FASN metabolites, including palmitate, did not prevent C75-mediated inhibition. This suggested an unexpected role for acetoacetyl-CoA in inflammation that is independent of its role in palmitate synthesis. Our evidence further suggested that acetoacetyl-CoA arising from FASN activity promotes cholesterol production, indicating a surprising link between fatty acid synthesis and cholesterol synthesis. We further demonstrate that this process is required for TLR4 to enter lipid rafts and facilitate TLR4 signaling. In conclusion, we have uncovered an unexpected link between FASN and cholesterol synthesis that appears to be required for TLR signal transduction and proinflammatory macrophage activation.
Assuntos
Colesterol/biossíntese , Ácido Graxo Sintase Tipo I/metabolismo , Ativação de Macrófagos , Macrófagos/enzimologia , Transdução de Sinais , Acil Coenzima A/metabolismo , Animais , Inflamação/enzimologia , Camundongos , Ácido Palmítico/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Poor solubility and cationic amphiphilic drug-likeness were liabilities identified for a lead series of S1P3-sparing, S1P1 agonists originally developed from a high-throughput screening campaign. This work describes the subsequent optimization of these leads by balancing potency, selectivity, solubility and overall molecular charge. Focused SAR studies revealed favorable structural modifications that, when combined, produced compounds with overall balanced profiles. The low brain exposure observed in rat suggests that these compounds would be best suited for the potential treatment of peripheral autoimmune disorders.
Assuntos
Oxidiazóis/farmacologia , Receptores de Lisoesfingolipídeo/agonistas , Tiadiazóis/farmacologia , Animais , Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Células Hep G2 , Humanos , Ligação de Hidrogênio , Cinética , Oxidiazóis/sangue , Oxidiazóis/síntese química , Ratos , Solubilidade , Relação Estrutura-Atividade , Tiadiazóis/sangue , Tiadiazóis/síntese químicaRESUMO
The role of microglia and monocyte-derived macrophages in experimental autoimmune encephalomyelitis pathogenesis has been controversial. To gain insight into their respective roles, we developed a method for differentiating between microglia and monocyte-derived macrophages in the CNS by flow cytometry utilizing anti-CD44 antibodies. We used this system to monitor changes in cell number, activation status, and gene expression by RNA sequencing over the course of disease. This in vivo characterization and RNA-Seq dataset improves our understanding of macrophage biology in the brain under inflammatory conditions and may lead to strategies to identify therapies for neuroinflammatory diseases.
Assuntos
Sequência de Bases/fisiologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Macrófagos Peritoneais/metabolismo , Microglia/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Sequência de Bases/genética , Proliferação de Células , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Feminino , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/toxicidade , Transdução de Sinais/imunologia , Fatores de TempoRESUMO
The discovery of a new series of selective S1P1 agonists is described. This series of piperazinyl-oxadiazole derivatives was rapidly optimized starting from high-throughput screening hit 1 to afford potent and selective lead compound 10d. Further SAR studies showed that 10d was converted to the active phosphate metabolite 29 in vivo. Oral administration of compound 10d to rats was shown to induce lymphopenia at 3 mg/kg.
Assuntos
Oxidiazóis/farmacologia , Piperazinas/farmacologia , Receptores de Lisoesfingolipídeo/agonistas , Administração Oral , Animais , Relação Dose-Resposta a Droga , Feminino , Linfopenia/induzido quimicamente , Linfopenia/patologia , Estrutura Molecular , Oxidiazóis/administração & dosagem , Oxidiazóis/química , Piperazinas/administração & dosagem , Piperazinas/química , Ratos , Ratos Endogâmicos Lew , Receptores de Esfingosina-1-Fosfato , Relação Estrutura-AtividadeRESUMO
Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1-/- animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freund's adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1-/- mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.
Assuntos
Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Dor/imunologia , Receptores CCR1/imunologia , Ácido Acético , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Movimento Celular/genética , Movimento Celular/imunologia , Citometria de Fluxo , Adjuvante de Freund , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/imunologia , Leucócitos/imunologia , Leucócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , Infiltração de Neutrófilos/genética , Neutrófilos/metabolismo , Dor/induzido quimicamente , Dor/genética , Medição da Dor/métodos , Peritonite/genética , Peritonite/imunologia , Peritonite/metabolismo , Receptores CCR1/antagonistas & inibidores , Receptores CCR1/genéticaRESUMO
GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Receptores Acoplados a Proteínas G/agonistas , Animais , Células CHO , Análise por Conglomerados , Cricetulus , AMP Cíclico , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/tratamento farmacológico , Feminino , Perfilação da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Células Mieloides/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Bile acids (BAs) and BA receptors, including G protein-coupled bile acid receptor 1 (GPBAR1), represent novel targets for the treatment of metabolic and inflammatory disorders. However, BAs elicit myriad effects on cardiovascular function, although this has not been specifically ascribed to GPBAR1. This study was designed to test whether stimulation of GPBAR1 elicits effects on cardiovascular function that are mechanism based that can be identified in acute ex vivo and in vivo cardiovascular models, to delineate whether effects were due to pathways known to be modulated by BAs, and to establish whether a therapeutic window between in vivo cardiovascular liabilities and on-target efficacy could be defined. The results demonstrated that the infusion of three structurally diverse and selective GPBAR1 agonists produced marked reductions in vascular tone and blood pressure in dog, but not in rat, as well as reflex tachycardia and a positive inotropic response, effects that manifested in an enhanced cardiac output. Changes in cardiovascular function were unrelated to modulation of the levothyroxine/thyroxine axis and were nitric oxide independent. A direct effect on vascular tone was confirmed in dog isolated vascular rings, whereby concentration-dependent decreases in tension that were tightly correlated with reductions in vascular tone observed in vivo and were blocked by iberiotoxin. Compound concentrations in which cardiovascular effects occurred, both ex vivo and in vivo, could not be separated from those necessary for modulation of GPBAR1-mediated efficacy, resulting in project termination. These results are the first to clearly demonstrate direct and potent peripheral arterial vasodilation due to GPBAR1 stimulation in vivo through activation of large conductance Ca(2+) activated potassium channel K(Ca)1.1.
Assuntos
Artérias/efeitos dos fármacos , Receptores Acoplados a Proteínas G/agonistas , Vasodilatação/efeitos dos fármacos , Animais , Artérias/fisiologia , Fator Natriurético Atrial/sangue , Células CHO , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/patologia , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Citocinas/sangue , Dinitrofluorbenzeno/análogos & derivados , Cães , Endotelina-1/sangue , Humanos , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Óxido Nítrico/biossíntese , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Tiroxina/sangue , Triazóis/farmacologiaRESUMO
Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs, and S1P receptor modulators suppress lymphocyte circulation. However, the role of S1P receptors on monocytes is less clear. To elucidate this, we systematically evaluated monocytes in rats and mice, both in naive and inflammatory conditions, with S1P receptor modulators FTY720 and BAF312. We demonstrate that S1P receptor modulators reduce circulating monocytes in a similar time course as lymphocytes. Furthermore, total monocyte numbers were increased in the spleen and bone marrow, suggesting that S1P receptor modulation restricts egress from hematopoietic organs. Monocytes treated ex vivo with FTY720 had reduced CD40 expression and TNF-α production, suggesting a direct effect on monocyte activation. Similar reductions in protein expression and cytokine production were also found in vivo. Suppression of experimental autoimmune encephalomyelitis in mice and rats by FTY720 correlated with reduced numbers of lymphocytes and monocytes. These effects on monocytes were independent of S1P3, as treatment with BAF312, a S1P1,4,5 modulator, led to similar results. These data reveal a novel role for S1P receptors on monocytes and offer additional insights on the mechanism of action of S1P receptor modulators in disease.
Assuntos
Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Propilenoglicóis/farmacologia , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Animais , Medula Óssea/efeitos dos fármacos , Medula Óssea/metabolismo , Movimento Celular/imunologia , Citocinas/biossíntese , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Cloridrato de Fingolimode , Células Matadoras Naturais/metabolismo , Contagem de Leucócitos , Camundongos , Monócitos/imunologia , Neutrófilos/metabolismo , Ratos , Esfingosina/farmacologia , Baço/efeitos dos fármacos , Baço/metabolismoRESUMO
Sphingosine-1-phospate (S1P) and S1P receptor agonists elicit mechanism-based effects on cardiovascular function in vivo. Indeed, FTY720 (non-selective S1P(X) receptor agonist) produces modest hypertension in patients (2-3 mmHg in 1-yr trial) as well as acute bradycardia independent of changes in blood pressure. However, the precise receptor subtypes responsible is controversial, likely dependent upon the cardiovascular response in question (e.g. bradycardia, hypertension), and perhaps even species-dependent since functional differences in rodent, rabbit, and human have been suggested. Thus, we characterized the S1P receptor subtype specificity for each compound in vitro and, in vivo, the cardiovascular effects of FTY720 and the more selective S1P1,5 agonist, BAF312, were tested during acute i.v. infusion in anesthetized rats and after oral administration for 10 days in telemetry-instrumented conscious rats. Acute i.v. infusion of FTY720 (0.1, 0.3, 1.0 mg/kg/20 min) or BAF312 (0.5, 1.5, 5.0 mg/kg/20 min) elicited acute bradycardia in anesthetized rats demonstrating an S1P1 mediated mechanism-of-action. However, while FTY720 (0.5, 1.5, 5.0 mg/kg/d) elicited dose-dependent hypertension after multiple days of oral administration in rat at clinically relevant plasma concentrations (24-hr mean blood pressureâ=â8.4, 12.8, 16.2 mmHg above baseline vs. 3 mmHg in vehicle controls), BAF312 (0.3, 3.0, 30.0 mg/kg/d) had no significant effect on blood pressure at any dose tested suggesting that hypertension produced by FTY720 is mediated S1P3 receptors. In summary, in vitro selectivity results in combination with studies performed in anesthetized and conscious rats administered two clinically tested S1P agonists, FTY720 or BAF312, suggest that S1P1 receptors mediate bradycardia while hypertension is mediated by S1P3 receptor activation.