RESUMO
Signal transduction by the Toll-like receptors (TLRs) is a key component of innate immunity against many pathogens and also underlies a large burden of human diseases. Therefore, the mechanisms and regulation of signaling from the TLRs are of considerable interest. Here we seek to determine the molecular mechanism by which TLR2 and TLR4, members of the Toll-like receptor family, are activated by bacterial LPS, hyperoxia, and zymosan respectively. Our central hypothesis is that the oxidation state of cysteine thiols on the ectodomain of TLR2 and TLR4 are critical for pathogen-initiated intracellular signaling as well in hyperoxia. Cysteine thiols of TLR4 and its co-receptor MD2 have been shown to aid binding between the two molecules and also bacterial LPS binding to the receptor complex. We extend these findings by demonstrating the oxidation of free thiols on the ectodomain of hTLR4, after exposure to LPS or hyperoxia suggesting that the cysteines on the ectodomain of TLR4 could form intra- or intermolecular disulfide bonds. We also demonstrated blockade of intracellular signaling from TLR4 and TLR2 by thiol-modifying compounds which suggest a novel therapeutic intervention for sepsis, hyperoxia-induced cell injury and yeast infection. In these experiments CHO-3E10, HEK293 cells expressing hTLR2 or hTLR4 and mouse peritoneal macrophages cells were pretreated with cell impermeable maleimides to alkylate thiols on the extracellular domain of TLRs, cells were then exposed to LPS, hyperoxia or zymosan. In all of these models, we detected decreased intracellular signaling from TLR2 or TLR4. Furthermore, incubation with phenyl arsine oxide - which forms stable complexes with vicinal cysteine residues - prevented LPS induced HEK293/hTLR4 intracellular signaling which was reversed by DMPS. Sequence analysis of different TLRs revealed Leucine-Rich Repeat C-terminal (LRRCT) domain that contains 4 conserved cysteines. Further work is required to pinpoint the role of each cysteine in receptor dimerization, pathogen binding, hyperoxia modulation, and intracellular signaling.
Assuntos
Cisteína/metabolismo , Transdução de Sinais/fisiologia , Compostos de Sulfidrila/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Células CHO , Linhagem Celular , Cricetulus , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Oxirredução/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Cystic fibrosis (CF) is an inherited disorder caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that promotes persistent lung infection and inflammation and progressive loss of lung function. Patients with CF have increased lung lymphoid follicles (LFs) and B cell-activating factor of tumor necrosis factor family (BAFF) that regulates B cell survival and maturation. A direct role for CFTR in B cell activation and disease pathogenesis in CF remains unclear. METHODS: The number of LFs, BAFF+, TLR4+ and proliferation marker Ki67+ B cells in lung explants or resections from subjects with CF and normal controls was quantified by immunostaining. The role of CFTR in B cell activation and LF development was then examined in two independent cohorts of uninfected CFTR-deficient mice (Cftr -/-) and wild type controls. The number of lung LFs, B cells and BAFF+, CXCR4+, immunoglobulin G+ B cells was examined by immunostaining. Lung and splenocyte B cell activation marker and major histocompatibility complex class II (MHC class II) expression was quantified by flow cytometry. Inflammatory cytokine levels were measured in supernatants from isolated B cells from Cftr -/- and wild type mice stimulated in vitro with Pseudomonas aeruginosa lipopolysaccharide (LPS). RESULTS: There was a significant increase in well-formed LFs in subjects with CF compared to normal controls. Increased B cell activation and proliferation was observed in lung LFs from CF subjects as was quantified by a significant increase in B cell BAFF, TLR4 and Ki67 expression. Uninfected Cftr -/- mice had increased lung LFs and BAFF+ and CXCR4+ B cells compared to wild type controls. Lung B cells isolated from uninfected Cftr -/- mice demonstrated increased MHC class II expression. In vitro, isolated B cells from Cftr -/- mice produced increased IL-6 when stimulated with LPS compared to wild type controls. CONCLUSIONS: These data support a direct role for CFTR in B cell activation, proliferation and inflammatory cytokine production that promotes lung LF follicle development in cystic fibrosis.
Assuntos
Linfócitos B/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/metabolismo , Estruturas Linfoides Terciárias/metabolismo , Adolescente , Animais , Fibrose Cística/patologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Abdominal aortic aneurysm (AAA) remains the second most frequent vascular disease with high mortality but has no approved medical therapy. We investigated the direct role of apelin (APLN) in AAA and identified a unique approach to enhance APLN action as a therapeutic intervention for this disease. Loss of APLN potentiated angiotensin II (Ang II)-induced AAA formation, aortic rupture, and reduced survival. Formation of AAA was driven by increased smooth muscle cell (SMC) apoptosis and oxidative stress in Apln-/y aorta and in APLN-deficient cultured murine and human aortic SMCs. Ang II-induced myogenic response and hypertension were greater in Apln-/y mice, however, an equivalent hypertension induced by phenylephrine, an α-adrenergic agonist, did not cause AAA or rupture in Apln-/y mice. We further identified Ang converting enzyme 2 (ACE2), the major negative regulator of the renin-Ang system (RAS), as an important target of APLN action in the vasculature. Using a combination of genetic, pharmacological, and modeling approaches, we identified neutral endopeptidase (NEP) that is up-regulated in human AAA tissue as a major enzyme that metabolizes and inactivates APLN-17 peptide. We designed and synthesized a potent APLN-17 analog, APLN-NMeLeu9-A2, that is resistant to NEP cleavage. This stable APLN analog ameliorated Ang II-mediated adverse aortic remodeling and AAA formation in an established model of AAA, high-fat diet (HFD) in Ldlr-/- mice. Our findings define a critical role of APLN in AAA formation through induction of ACE2 and protection of vascular SMCs, whereas stable APLN analogs provide an effective therapy for vascular diseases.
Assuntos
Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/patologia , Apelina/metabolismo , Neprilisina/metabolismo , Idoso , Idoso de 80 Anos ou mais , Angiotensina II/administração & dosagem , Enzima de Conversão de Angiotensina 2 , Animais , Aorta Abdominal/citologia , Aneurisma da Aorta Abdominal/tratamento farmacológico , Aneurisma da Aorta Abdominal/etiologia , Apelina/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Fármacos Cardiovasculares/química , Fármacos Cardiovasculares/farmacologia , Fármacos Cardiovasculares/uso terapêutico , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos Transgênicos , Pessoa de Meia-Idade , Miócitos de Músculo Liso , Neprilisina/genética , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Peptidil Dipeptidase A/metabolismo , Fenilefrina/administração & dosagem , Cultura Primária de Células , Proteólise/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/genéticaRESUMO
BACKGROUND: Loss of bronchoprotection (LOBP) with a regularly used long-acting ß2-adrenergic receptor agonist (LABA) is well documented. LOBP has been attributed to ß2-adrenergic receptor (B2AR) downregulation, a process requiring farnesylation, which is inhibited by alendronate. OBJECTIVE: We sought to determine whether alendronate can reduce LABA-associated LOBP in inhaled corticosteroid (ICS)-treated patients. METHODS: We conducted a randomized, double-blind, placebo-controlled, parallel-design, proof-of-concept trial. Seventy-eight participants with persistent asthma receiving 250 µg of fluticasone twice daily for 2 weeks were randomized to receive alendronate or placebo while initiating salmeterol for 8 weeks. Salmeterol-protected methacholine challenges (SPMChs) and PBMC B2AR numbers (radioligand binding assay) and signaling (cyclic AMP ELISA) were assessed before randomization and after 8 weeks of ICS plus LABA treatment. LOBP was defined as a more than 1 doubling dose reduction in SPMCh PC20 value. RESULTS: The mean doubling dose reduction in SPMCh PC20 value was 0.50 and 0.27 with alendronate and placebo, respectively (P = .62). Thirty-eight percent of participants receiving alendronate and 33% receiving placebo had LOBP (P = .81). The after/before ICS plus LABA treatment ratio of B2AR number was 1.0 for alendronate (P = .86) and 0.8 for placebo (P = .15; P = .31 for difference between treatments). The B2AR signaling ratio was 0.89 for alendronate (P = .43) and 1.02 for placebo (P = .84; P = .44 for difference). Changes in lung function and B2AR number and signaling were similar between those who did and did not experience LOBP. CONCLUSION: This study did not find evidence that alendronate reduces LABA-associated LOBP, which relates to the occurrence of LOBP in only one third of participants. LOBP appears to be less common than presumed in concomitant ICS plus LABA-treated asthmatic patients. B2AR downregulation measured in PBMCs does not appear to reflect LOBP.
Assuntos
Corticosteroides/administração & dosagem , Alendronato/administração & dosagem , Asma , Fluticasona/administração & dosagem , Receptores Adrenérgicos beta 2/metabolismo , Xinafoato de Salmeterol/administração & dosagem , Administração por Inalação , Adulto , Asma/tratamento farmacológico , Asma/patologia , Asma/fisiopatologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Estudo de Prova de ConceitoRESUMO
BACKGROUND: Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Insight into the transcriptomic landscape of early cardiac regeneration from model organisms will shed light on evolutionarily conserved pathways in successful cardiac regeneration. METHODS: Here we describe a cross-species transcriptomic screen in 3 model organisms for cardiac regeneration: axolotl, neonatal mice, and zebrafish. Apical resection to remove ≈10% to 20% of ventricular mass was carried out in these model organisms. RNA-sequencing analysis was performed on the hearts harvested at 3 time points: 12, 24, and 48 hours after resection. Sham surgery was used as internal control. RESULTS: Genes associated with inflammatory processes were found to be upregulated in a conserved manner. Complement receptors (activated by complement components, part of the innate immune system) were found to be highly upregulated in all 3 species. This approach revealed induction of gene expression for complement 5a receptor 1 in the regenerating hearts of zebrafish, axolotls, and mice. Inhibition of complement 5a receptor 1 significantly attenuated the cardiomyocyte proliferative response to heart injury in all 3 species. Furthermore, after left ventricular apical resection, the cardiomyocyte proliferative response was diminished in mice with genetic deletion of complement 5a receptor 1. CONCLUSIONS: These data reveal that activation of complement 5a receptor 1 mediates an evolutionarily conserved response that promotes cardiomyocyte proliferation after cardiac injury and identify complement pathway activation as a common pathway of successful heart regeneration.
Assuntos
Evolução Molecular , Coração/fisiologia , Receptor da Anafilatoxina C5a/metabolismo , Regeneração/fisiologia , Ambystoma mexicanum , Animais , Animais Recém-Nascidos , Proliferação de Células , Perfilação da Expressão Gênica , Ontologia Genética , Camundongos , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Peptídeos Cíclicos/farmacologia , RNA/química , RNA/isolamento & purificação , RNA/metabolismo , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/genética , Análise de Sequência de RNA , Troponina T/análise , Peixe-ZebraRESUMO
Cystic fibrosis (CF) remains the most lethal genetic disease in the Caucasian population. However, there is great variability in clinical phenotypes and survival times, even among patients harboring the same genotype. We identified five patients with CF and a homozygous F508del mutation in the CFTR gene who were in their fifth or sixth decade of life and had shown minimal changes in lung function over a longitudinal period of more than 20 years. Because of the rarity of this long-term nonprogressive phenotype, we hypothesized these individuals may carry rare genetic variants in modifier genes that ameliorate disease severity. Individuals at the extremes of survival time and lung-function trajectory underwent whole-exome sequencing, and the sequencing data were filtered to include rare missense, stopgain, indel, and splicing variants present with a mean allele frequency of <0.2% in general population databases. Epithelial sodium channel (ENaC) mutants were generated via site-directed mutagenesis and expressed for Xenopus oocyte assays. Four of the five individuals carried extremely rare or never reported variants in the SCNN1D and SCNN1B genes of the ENaC. Separately, an independently enriched rare variant in SCNN1D was identified in the Exome Variant Server database associated with a milder pulmonary disease phenotype. Functional analysis using Xenopus oocytes revealed that two of the three variants in δ-ENaC encoded by SCNN1D exhibited hypomorphic channel activity. Our data suggest a potential role for δ-ENaC in controlling sodium reabsorption in the airways, and advance the plausibility of ENaC as a therapeutic target in CF.
Assuntos
Sequência de Aminoácidos , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Canais Epiteliais de Sódio/metabolismo , Deleção de Sequência , Animais , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Canais Epiteliais de Sódio/genética , Feminino , Humanos , Masculino , Xenopus , Xenopus laevisRESUMO
Substance P neuropeptide and its receptor, neurokinin-1 receptor (NK1R), are reported to present on the ocular surface. In this study, mice lacking functional NK1R exhibited an excessive desquamation of apical corneal epithelial cells in association with an increased epithelial cell proliferation and increased epithelial cell density, but decreased epithelial cell size. The lack of NK1R also resulted in decreased density of corneal nerves, corneal epithelial dendritic cells (DCs), and a reduced volume of basal tears. Interestingly, massive accumulation of CD11c+CD11b+ conventional DCs was noted in the bulbar conjunctiva and near the limbal area of corneas from NK1R-/- mice. After ocular HSV-1 infection, the number of conventional DCs and neutrophils infiltrating the infected corneas was significantly higher in NK1R-/- than C57BL/6J mice. This was associated with an increased viral load in infected corneas of NK1R-/- mice. As a result, the number of IFN-γ-secreting virus-specific CD4 T cells in the draining lymph nodes of NK1R-/- mice was much higher than in infected C57BL/6J mice. An increased number of CD4 T cells and mature neutrophils (CD11b+Ly6ghigh) in the inflamed corneas of NK1R-/- mice was associated with an early development of severe herpes stromal keratitis. Collectively, our results show that the altered corneal biology of uninfected NK1R-/- mice along with an enhanced immunological response after ocular HSV-1 infection causes an early development of herpes stromal keratitis in NK1R-/- mice.
Assuntos
Córnea/imunologia , Córnea/patologia , Herpesvirus Humano 1/imunologia , Ceratite Herpética/virologia , Receptores da Neurocinina-1/fisiologia , Animais , Linfócitos T CD4-Positivos/imunologia , Túnica Conjuntiva/imunologia , Túnica Conjuntiva/patologia , Túnica Conjuntiva/virologia , Córnea/virologia , Células Dendríticas/imunologia , Herpesvirus Humano 1/fisiologia , Homeostase , Interferon gama/imunologia , Ceratite Herpética/imunologia , Ceratite Herpética/fisiopatologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Receptores da Neurocinina-1/deficiência , Receptores da Neurocinina-1/imunologia , Carga ViralRESUMO
LPS-induced lung injury in the mouse is one of the most robust experimental models used for studies of acute lung injury (ALI) and acute respiratory distress syndrome in humans. Prior clinical and experimental studies support an important role for complement activation, particularly production of C5a, in the pathophysiology of human ALI/acute respiratory distress syndrome. In the mouse model, however, the precise role of C5a and its receptors is unclear. C5L2, an enigmatic second receptor for C5a, has been characterized, and results have generated substantial debate regarding its in vivo function. Our previous work with human neutrophils revealed a unique role for C5L2 in negatively modulating C5a-C5a receptor (C5aR)-mediated cellular activation, in which antibody-mediated blockade of C5L2 resulted in augmented C5a-C5aR responses. Here, we demonstrate that C5L2-/- mice (BALB/c background) administered intranasal LPS exhibit significantly more airway edema and hemorrhage than do wild-type animals. Bronchoalveolar lavage fluid and lung homogenates have significantly more neutrophils and myeloperoxidase activity, as well as proinflammatory cytokines and chemokines. When a blocking antibody against the C5aR was administered before LPS administration, the increased neutrophilic infiltration and cytokine levels were reversed. Thus, our data show not only that C5a contributes significantly to LPS-induced ALI in the mouse, but also that C5L2 plays an important antiinflammatory role in this model through actions resulting at least in part from negative modulation of C5a receptor activation.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Receptores de Quimiocinas/metabolismo , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/fisiopatologia , Administração Intranasal , Animais , Líquido da Lavagem Broncoalveolar , Quimiocinas/genética , Quimiocinas/metabolismo , Edema/complicações , Edema/patologia , Edema/fisiopatologia , Regulação da Expressão Gênica , Hemorragia/complicações , Hemorragia/patologia , Hemorragia/fisiopatologia , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/administração & dosagem , Pulmão/patologia , Camundongos Endogâmicos BALB C , Neutrófilos/metabolismo , Peroxidase/metabolismo , Receptor da Anafilatoxina C5a , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/deficiência , Mecânica Respiratória , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Staphylococcus aureus is well adapted to the human host. Evasion of the host phagocyte response is critical for successful infection. The staphylococcal bicomponent pore-forming toxins Panton-Valentine leukocidin LukSF-PV (PVL) and γ-hemolysin CB (HlgCB) target human phagocytes through interaction with the complement receptors C5aR1 and C5aR2. Currently, the apparent redundancy of both toxins cannot be adequately addressed in experimental models of infection because mice are resistant to PVL and HlgCB. The molecular basis for species specificity of the two toxins in animal models is not completely understood. We show that PVL and HlgCB feature distinct activity toward neutrophils of different mammalian species, where activity of PVL is found to be restricted to fewer species than that of HlgCB. Overexpression of various mammalian C5a receptors in HEK cells confirms that cytotoxicity toward neutrophils is driven by species-specific interactions of the toxins with C5aR1. By taking advantage of the species-specific engagement of the toxins with their receptors, we demonstrate that PVL and HlgCB differentially interact with human C5aR1 and C5aR2. In addition, binding studies illustrate that different parts of the receptor are involved in the initial binding of the toxin and the subsequent formation of lytic pores. These findings allow a better understanding of the molecular mechanism of pore formation. Finally, we show that the toxicity of PVL, but not of HlgCB, is neutralized by various C5aR1 antagonists. This study offers directions for the development of improved preclinical models for infection, as well as for the design of drugs antagonizing leukocidin toxicity.
Assuntos
Proteínas de Bactérias/imunologia , Toxinas Bacterianas/imunologia , Exotoxinas/imunologia , Proteínas Hemolisinas/imunologia , Leucocidinas/imunologia , Receptor da Anafilatoxina C5a/imunologia , Receptores de Quimiocinas/imunologia , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Células HEK293 , Humanos , Evasão da Resposta Imune/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Neutrófilos/imunologia , Fagócitos/imunologia , Ligação Proteica , Estrutura Terciária de Proteína , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptores de Quimiocinas/antagonistas & inibidores , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/patogenicidadeRESUMO
Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO-induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO-induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.
Assuntos
Vasculite Associada a Anticorpo Anticitoplasma de Neutrófilos/prevenção & controle , Autoantígenos/imunologia , Glomerulonefrite/prevenção & controle , Peroxidase/imunologia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Administração Oral , Animais , Complemento C6/imunologia , Via Alternativa do Complemento , Relação Dose-Resposta a Droga , Técnicas de Introdução de Genes , Glomerulonefrite/complicações , Glomerulonefrite/imunologia , Hematúria/etiologia , Hematúria/prevenção & controle , Humanos , Imunização Passiva , Leucócitos , Erros Inatos do Metabolismo/complicações , Erros Inatos do Metabolismo/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidase/deficiência , Proteinúria/etiologia , Proteinúria/prevenção & controle , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/fisiologia , Proteínas Recombinantes de Fusão , Urina/citologiaRESUMO
Chemokines display considerable promiscuity with multiple ligands and receptors shared in common, a phenomenon that is thought to underlie their biochemical "redundancy." Their receptors are part of a larger seven-transmembrane receptor superfamily, commonly referred to as G protein-coupled receptors, which have been demonstrated to be able to signal with different efficacies to their multiple downstream signaling pathways, a phenomenon referred to as biased agonism. Biased agonism has been primarily reported as a phenomenon of synthetic ligands, and the biologic prevalence and importance of such signaling are unclear. Here, to assess the presence of biased agonism that may underlie differential signaling by chemokines targeting the same receptor, we performed a detailed pharmacologic analysis of a set of chemokine receptors with multiple endogenous ligands using assays for G protein signaling, ß-arrestin recruitment, and receptor internalization. We found that chemokines targeting the same receptor can display marked differences in their efficacies for G protein- or ß-arrestin-mediated signaling or receptor internalization. This ligand bias correlates with changes in leukocyte migration, consistent with different mechanisms underlying the signaling downstream of these receptors induced by their ligands. These findings demonstrate that biased agonism is a common and likely evolutionarily conserved biological mechanism for generating qualitatively distinct patterns of signaling via the same receptor in response to different endogenous ligands.
Assuntos
Receptores de Quimiocinas/agonistas , Receptores de Quimiocinas/metabolismo , Arrestinas/metabolismo , Quimiocinas/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Cinética , Ligantes , Modelos Biológicos , Transdução de Sinais , beta-ArrestinasRESUMO
The complement anaphylatoxin C5a is a critical mediator of allergic contact dermatitis, bridging essential aspects of innate and adaptive immunity. This anaphylatoxin functions by interacting with two 7-transmembrane segment receptors, the C5aR and C5L2. The C5aR is a classical G protein coupled receptor, whereas C5L2 is deficient in coupling to G proteins because of variations in the sequence. Our previous work in human neutrophils revealed a unique role for C5L2 in negatively modulating anaphylatoxin receptor mediated cellular activation through interactions with ß-arrestin. When C5L2 is deficient, C5aR-mediated ß-arrestin signaling is greatly enhanced. The work described in this study was undertaken first to determine the effect of C5L2 deficiency in a murine model of contact sensitivity, and second to determine whether the resultant exacerbation of inflammatory parameters reflects a negative modulatory function of C5L2 on the C5aR. First, we find dramatic increases in inflammation in C5L2(-/-) animals compared with wild type mice. Second, these increases are completely reversed following administration of mAb against the C5aR. Thus, in allergic contact sensitivity, as in isolated human neutrophils, C5L2 functions to suppress C5a-C5aR-mediated responses, further underscoring its role as a negative regulator of anaphylatoxin activity.
Assuntos
Complemento C5a/imunologia , Dermatite Alérgica de Contato/imunologia , Inflamação/imunologia , Receptor da Anafilatoxina C5a/imunologia , Receptores de Quimiocinas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Arrestinas/imunologia , Arrestinas/metabolismo , Feminino , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptores de Quimiocinas/deficiência , Receptores de Quimiocinas/genética , Transdução de Sinais , beta-ArrestinasRESUMO
OBJECTIVE: To investigate the impact of whole body C5a receptor (C5aR) deficiency on energy metabolism and fat storage. DESIGN: Male wildtype (WT) and C5aR knockout (C5aRKO) mice were fed a low fat (CHOW) or a high fat high sucrose diet-induced obesity (DIO) diet for 14 weeks. Body weight and food intake were measured weekly. Indirect calorimetry, dietary fatload clearance, insulin and glucose tolerance tests were also evaluated. Liver, muscle and adipose tissue mRNA gene expression were measured by RT-PCR. RESULTS: At week one and 12, C5aRKO mice on DIO had increased oxygen consumption. After 12 weeks, although food intake was comparable, C5aRKO mice had lower body weight (-7% CHOW, -12% DIO) as well as smaller gonadal (-38% CHOW, -36% DIO) and inguinal (-29% CHOW, -30% DIO) fat pads than their WT counterparts. Conversely, in WT mice, C5aR was upregulated in DIO vs CHOW diets in gonadal adipose tissue, muscle and liver, while C5L2 mRNA expression was lower in C5aRKO on both diet. Furthermore, blood analysis showed lower plasma triglyceride and non-esterified fatty acid levels in both C5aRKO groups, with faster postprandial triglyceride clearance after a fatload. Additionally, C5aRKO mice showed lower CD36 expression in gonadal and muscle on both diets, while DGAT1 expression was higher in gonadal (CHOW) and liver (CHOW and DIO) and PPARγ was increased in muscle and liver. CONCLUSION: These observations point towards a role (either direct or indirect) for C5aR in energy expenditure and fat storage, suggesting a dual role for C5aR in metabolism as well as in immunity.
Assuntos
Tecido Adiposo/metabolismo , Metabolismo Energético , Receptor da Anafilatoxina C5a/deficiência , Animais , Dieta Hiperlipídica/efeitos adversos , Ingestão de Alimentos , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Homeostase , Insulina/metabolismo , Resistência à Insulina , Fígado/citologia , Fígado/metabolismo , Masculino , Camundongos , Atividade Motora , Músculos/citologia , Músculos/metabolismo , Obesidade/etiologia , Obesidade/metabolismo , Oxigênio/metabolismo , Receptor da Anafilatoxina C5a/genética , Receptores de Quimiocinas/genéticaRESUMO
According to the current paradigm, lymphocyte homing to the small intestine requires the expression of two tissue-specific homing receptors, the integrin α4ß7 and the CCL25 receptor CCR9. In this study, we investigated the organ distribution and the homing molecule expression of IgA Ab-secreting cells (ASCs) induced by intrarectal immunization with a particulate Ag, in comparison with other mucosal immunization routes. Intrarectal immunization induces gut-homing IgA ASCs that localize not only in the colon but also in the small intestine, although they are not responsive to CCL25, unlike IgA ASCs induced by oral immunization. The mucosal epithelial chemokine CCL28, known to attract all IgA ASCs, does not compensate for the lack of CCL25 responsiveness, because the number of Ag-specific cells is not decreased in the gut of CCR10-deficient mice immunized by the intrarectal route. However, Ag-specific IgA ASCs induced by intrarectal immunization express the integrin α4ß7, and their number is considerably decreased in the gut of ß7-deficient mice immunized by the intrarectal route, indicating that α4ß7 enables these cells to migrate into the small intestine, even without CCL25 responsiveness. In contrast, IgA ASCs induced by intranasal immunization express low α4ß7 levels and are usually excluded from the gut. Paradoxically, after intranasal immunization, Ag-specific IgA ASCs are significantly increased in the small intestine of ß7-deficient mice, demonstrating that lymphocyte homing is a competitive process and that integrin α4ß7 determines not only the intestinal tropism of IgA ASCs elicited in GALTs but also the intestinal exclusion of lymphocytes primed in other inductive sites.
Assuntos
Células Produtoras de Anticorpos/imunologia , Imunoglobulina A/imunologia , Intestino Delgado/imunologia , Administração Retal , Animais , Células Produtoras de Anticorpos/metabolismo , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Quimiocinas CC/imunologia , Quimiocinas CC/metabolismo , Colo/imunologia , Colo/metabolismo , Feminino , Imunização/métodos , Imunoglobulina A/metabolismo , Integrinas/imunologia , Integrinas/metabolismo , Intestino Delgado/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mucoproteínas , Mucosa/imunologia , Mucosa/metabolismo , Receptores CCR10/imunologia , Receptores CCR10/metabolismoRESUMO
Tachykinins are a large group of neuropeptides with both central and peripheral activity. Despite the increasing number of studies reporting a growth supportive effect of tachykinin peptides in various in vitro stem cell systems, it remains unclear whether these findings are applicable in vivo. To determine how neurokinin-1 receptor (NK-1R) deficient hematopoietic stem cells would behave in a normal in vivo environment, we tested their reconstitution efficiency using competitive bone marrow repopulation assays. We show here that bone marrow taken from NK-1R deficient mice (Tacr1(-/-)) showed lineage specific B and T cell engraftment deficits compared to wild-type competitor bone marrow cells, providing evidence for an involvement of NK-1R signalling in adult hematopoiesis. Tachykinin knockout mice lacking the peptides SP and/or HK-1 (Tac1 (-/-), Tac4 (-/-) and Tac1 (-/-)/Tac4 (-/-) mice) repopulated a lethally irradiated wild-type host with similar efficiency as competing wild-type bone marrow. The difference between peptide and receptor deficient mice indicates a paracrine and/or endocrine mechanism of action rather than autocrine signalling, as tachykinin peptides are supplied by the host environment.
Assuntos
Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Receptores da Neurocinina-1/metabolismo , Transdução de Sinais , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Linhagem da Célula , Feminino , Técnicas de Inativação de Genes , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Precursores de Proteínas/deficiência , Receptores da Neurocinina-1/deficiência , Receptores da Neurocinina-1/genética , Substância P/deficiência , Linfócitos T/citologia , Linfócitos T/metabolismo , Taquicininas/deficiênciaRESUMO
BACKGROUND: Mast cells express receptors for complement anaphylatoxins C3a and C5a (ie, C3a receptor [C3aR] and C5a receptor [C5aR]), and C3a and C5a are generated during various IgE-dependent immediate hypersensitivity reactions in vivo. However, it is not clear to what extent mast cell expression of C3aR or C5aR influences C3a- or C5a-induced cutaneous responses or IgE-dependent mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo. OBJECTIVE: We sought to assess whether mouse skin mast cell expression of C3aR or C5aR influences (1) the cells' responsiveness to intradermal injections of C3a or C5a or (2) the extent of IgE-dependent mast cell degranulation and PCA in vivo. METHODS: We measured the magnitude of cutaneous responses to intradermal injections of C3a or C5a and the extent of IgE-dependent mast cell degranulation and PCA responses in mice containing mast cells that did or did not express C3aR or C5aR. RESULTS: The majority of the skin swelling induced by means of intradermal injection of C3a or C5a required that mast cells at the site expressed C3aR or C5aR, respectively, and the extent of IgE-dependent degranulation of skin mast cells and IgE-dependent PCA was significantly reduced when mast cells lacked either C3aR or C5aR. IgE-dependent PCA responses associated with local increases in C3a levels occurred in antibody-deficient mice but not in mice deficient in FcÉRIγ. CONCLUSION: Expression of C3aR and C5aR by skin mast cells contributes importantly to the ability of C3a and C5a to induce skin swelling and can enhance mast cell degranulation and inflammation during IgE-dependent PCA in vivo.
Assuntos
Imunoglobulina E/metabolismo , Inflamação/imunologia , Mastócitos/imunologia , Receptor da Anafilatoxina C5a/biossíntese , Receptores de Complemento/biossíntese , Pele/imunologia , Anafilatoxinas/genética , Anafilatoxinas/imunologia , Animais , Células Cultivadas , Complemento C3a/genética , Complemento C3a/imunologia , Complemento C3a/metabolismo , Complemento C5a/genética , Complemento C5a/imunologia , Complemento C5a/metabolismo , Feminino , Imunoglobulina E/genética , Inflamação/genética , Inflamação/metabolismo , Inflamação/patologia , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/imunologia , Receptores de Complemento/genética , Receptores de Complemento/imunologia , Receptores de Complemento/metabolismo , Pele/metabolismo , Pele/patologiaRESUMO
Respiratory syncytial virus (RSV) infection is associated with serious lung disease in infants and immunocompromised individuals and is linked to development of asthma. In mice, acute RSV infection causes airway hyperresponsiveness (AHR), inflammation, and mucus hypersecretion. Infected cells induce complement activation, producing the anaphylatoxin C3a. In this paper, we show RSV-infected wild-type mice produce Th17 cytokines, a response not previously associated with viral infections. Mice deficient in the C3aR fail to develop AHR following acute RSV infection, and production of Th17 cytokines was significantly attenuated. Tachykinin production also has been implicated in RSV pathophysiology, and tachykinin receptor-null mice were similarly protected from developing AHR. These animals were also deficient in production of Th17 cytokines. Tachykinin release was absent in mice deficient in C3aR, whereas C3a levels were unchanged in tachykinin receptor-null animals. Thus, our data reveal a crucial sequence following acute RSV infection where initial C3a production causes tachykinin release, followed by activation of the IL-17A pathway. Deficiency of either receptor affords protection from AHR, identifying two potential therapeutic targets.
Assuntos
Hiper-Reatividade Brônquica/imunologia , Complemento C3a/imunologia , Interleucina-17/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Taquicininas/imunologia , Animais , Hiper-Reatividade Brônquica/metabolismo , Hiper-Reatividade Brônquica/virologia , Separação Celular , Complemento C3a/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Expressão Gênica , Perfilação da Expressão Gênica , Interleucina-17/metabolismo , Camundongos , Camundongos Knockout , Infecções por Vírus Respiratório Sincicial/metabolismo , Vírus Sinciciais Respiratórios/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taquicininas/metabolismoRESUMO
Heterotopic ossification (HO) is a disabling condition associated with neurologic injury, inflammation, and overactive bone morphogenetic protein (BMP) signaling. The inductive factors involved in lesion formation are unknown. We found that the expression of the neuro-inflammatory factor Substance P (SP) is dramatically increased in early lesional tissue in patients who have either fibrodysplasia ossificans progressiva (FOP) or acquired HO, and in three independent mouse models of HO. In Nse-BMP4, a mouse model of HO, robust HO forms in response to tissue injury; however, null mutations of the preprotachykinin (PPT) gene encoding SP prevent HO. Importantly, ablation of SP(+) sensory neurons, treatment with an antagonist of SP receptor NK1r, deletion of NK1r gene, or genetic down-regulation of NK1r-expressing mast cells also profoundly inhibit injury-induced HO. These observations establish a potent neuro-inflammatory induction and amplification circuit for BMP-dependent HO lesion formation, and identify novel molecular targets for prevention of HO.