RESUMO
The Toll signaling pathway plays an important role in the innate immunity ofDrosophila melanogasterand mammals. The activation and termination of Toll signaling are finely regulated in these animals. Although the primary components of the Toll pathway were identified in shrimp, the functions and regulation of the pathway are seldom studied. We first demonstrated that the Toll signaling pathway plays a central role in host defense againstStaphylococcus aureusby regulating expression of antimicrobial peptides in shrimp. We then found that ß-arrestins negatively regulate Toll signaling in two different ways. ß-Arrestins interact with the C-terminal PEST domain of Cactus through the arrestin-N domain, and Cactus interacts with the RHD domain of Dorsal via the ankyrin repeats domain, forming a heterotrimeric complex of ß-arrestin·Cactus·Dorsal, with Cactus as the bridge. This complex prevents Cactus phosphorylation and degradation, as well as Dorsal translocation into the nucleus, thus inhibiting activation of the Toll signaling pathway. ß-Arrestins also interact with non-phosphorylated ERK (extracellular signal-regulated protein kinase) through the arrestin-C domain to inhibit ERK phosphorylation, which affects Dorsal translocation into the nucleus and phosphorylation of Dorsal at Ser(276)that impairs Dorsal transcriptional activity. Our study suggests that ß-arrestins negatively regulate the Toll signaling pathway by preventing Dorsal translocation and inhibiting Dorsal phosphorylation and transcriptional activity.
Assuntos
Arrestinas/imunologia , Proteínas de Artrópodes/imunologia , Penaeidae/imunologia , Transdução de Sinais/imunologia , Staphylococcus aureus/imunologia , Receptores Toll-Like/imunologia , Transporte Ativo do Núcleo Celular/imunologia , Animais , Núcleo Celular/imunologia , Proteínas de Ligação a DNA/imunologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Fosforilação/imunologia , beta-ArrestinasRESUMO
CCL22 inactivation in vivo occurs by cleavage at the N-terminus; however, it is unclear whether this encompasses the entire site of CCR4 interaction. CCL17 also binds CCR4 and its function requires binding via two discrete binding sites. Using monoclonal antibodies (MAbs), we report that there are two separate sites on CCL22 that are required for CCR4-mediated function. The CCL22-specific antibodies bind with affinities of 632 ± 297 pM (MC2B7) and 308 ± 43 pM (MAB4391) and neither exhibited detectable binding to CCL17. Both antibodies are comparable in their ability to inhibit CCL22-mediated calcium mobilization; however, competition binding studies demonstrate that MC2B7 and MAB4391 bind to distinct epitopes on CCL22. Both antibodies inhibit function through CCR4, which is demonstrated by loss of ß-arrestin recruitment in a reporter cell line. In both assays, blocking either site independently abolished CCL22 function, suggesting that concurrent engagement of both sites with CCR4 is necessary for function. This is the first demonstration that CCL22 has two distinct binding sites that are required for CCR4 function. These antibodies are valuable tools for better understanding the interaction and function of CCL22 and CCR4 and will potentially help further understanding of the differential outcomes of CCL17 and CCL22 interaction with CCR4.
Assuntos
Anticorpos Monoclonais/química , Arrestinas/imunologia , Quimiocina CCL22/imunologia , Epitopos/imunologia , Receptores CCR4/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Arrestinas/genética , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Quimiocina CCL17/genética , Quimiocina CCL17/imunologia , Quimiocina CCL22/genética , Células Dendríticas/citologia , Células Dendríticas/imunologia , Epitopos/química , Epitopos/genética , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Camundongos , Ligação Proteica , Estrutura Terciária de Proteína , Receptores CCR4/genética , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , beta-ArrestinasRESUMO
ß-Arrestins and ß-arrestin2 are important adaptor proteins and signal transduction proteins that are mainly involved in the desensitization and internalization of G-protein-coupled receptors. Fibrosis is characterized by accumulation of excess extracellular matrix (ECM) molecules caused by chronic tissue injury. If highly progressive, the fibrotic process leads to organ malfunction and, eventually, death. The incurable lung fibrosis, renal fibrosis and liver fibrosis are among the most common fibrotic diseases. Recent studies show that ß-arrestins can activate signaling cascades independent of G-protein activation and scaffold many intracellular signaling networks by diverse types of signaling pathways, including the Hedgehog, Wnt, Notch and transforming growth factor-ß pathways, as well as downstream kinases such as MAPK and PI3K. These signaling pathways are involved in the pathological process of fibrosis and fibrotic diseases. This ß-arrestin-mediated regulation not only affects cell growth and apoptosis, but also the deposition of ECM, activation of inflammatory response and development of fibrotic diseases. In this review, we survey the involvement of ß-arrestins in various signaling pathways and highlight different aspects of their regulation of fibrosis. We also discuss the important roles of ß-arrestins in the process of fibrotic diseases by regulating the inflammation and deposit of ECM. It is becoming more evident that targeting ß-arrestins may offer therapeutic potential for the treatment of fibrotic diseases.
Assuntos
Arrestinas/imunologia , Doenças Cardiovasculares/patologia , Inflamação/patologia , Enteropatias/patologia , Hepatopatias/patologia , Pneumopatias/patologia , Transdução de Sinais , Animais , Arrestinas/análise , Doenças Cardiovasculares/imunologia , Sistema Cardiovascular/imunologia , Sistema Cardiovascular/patologia , Matriz Extracelular/imunologia , Matriz Extracelular/patologia , Fibrose , Humanos , Inflamação/imunologia , Enteropatias/imunologia , Intestinos/imunologia , Intestinos/patologia , Fígado/imunologia , Fígado/patologia , Hepatopatias/imunologia , Pulmão/imunologia , Pulmão/patologia , Pneumopatias/imunologia , beta-ArrestinasRESUMO
Arrestins are intracellular scaffolding proteins known to regulate a range of biochemical processes including G protein-coupled receptor (GPCR) desensitization, signal attenuation, receptor turnover and downstream signaling cascades. Their roles in regulation of signaling network have lately been extended to receptors outside of the GPCR family, demonstrating their roles as important scaffolding proteins in various physiological processes including proliferation, differentiation and apoptosis. Recent studies have demonstrated a critical role for arrestins in immunological processes including key functions in inflammatory signaling pathways. In this review, we provide a comprehensive analysis of the different functions of the arrestin family of proteins especially related to immunity and inflammatory diseases.
Assuntos
Arrestinas/imunologia , Inflamação/imunologia , Inflamação/patologia , Transdução de Sinais , Animais , Arrestinas/metabolismo , Humanos , beta-ArrestinasRESUMO
ß-arrestin2 (ß-arr2), identified as a scaffolding protein in G-protein-coupled receptor desensitization, is a negative regulator of inflammation in polymicrobial sepsis. In this study, we wanted to investigate the role of ß-arr2 in intestinal inflammation, a site of persistent microbial stimulation. In the absence of ß-arr2, mice exhibited greater extent of mucosal inflammation determined by cellular infiltration and expression of inflammatory mediators even under homeostatic conditions. Furthermore, ß-arr2-deficient mice were more susceptible to dextran sulfate sodium-induced colitis as demonstrated by greater body weight loss, higher disease activity index, and shortened colon as compared with wild-type mice. We also show that T cells from ß-arr2 knockout mice exhibit altered activation status under both basal and colitic conditions, implicating their involvement in disease induction. Further assessment of the role of ß-arr2 in intrinsic T-cell differentiation confirmed its importance in T-cell polarization. Using the T-cell transfer model of colitis, we demonstrate that T-cell-specific ß-arr2 is important in limiting colitic inflammation; however, it plays a paradoxical role in concurrent systemic wasting disease. Together, our study highlights a critical negative regulatory role of ß-arr2 in intestinal inflammation and demonstrates a distinct role of T-cell-specific ß-arr2 in systemic wasting disease.
Assuntos
Arrestinas/imunologia , Colite/imunologia , Ativação Linfocitária , Linfócitos T/metabolismo , Animais , Arrestinas/deficiência , Arrestinas/genética , Colite/induzido quimicamente , Colite/genética , Sulfato de Dextrana , Mediadores da Inflamação/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Intestinos/imunologia , Intestinos/patologia , Camundongos , Camundongos Knockout , beta-Arrestina 2 , beta-ArrestinasRESUMO
The chemokine receptor CXCR2 is expressed on various immune cells and is essential for neutrophil recruitment and angiogenesis at sites of acute and chronic inflammation caused by tissue injury or infection. CXCR2 and its ligand, CXCL8, are implicated in a number of inflammation-mediated diseases such as chronic obstructive pulmonary disease, cystic fibrosis, and cancer. Though the development of CXCR2-specific small-molecule inhibitors as anti-inflammatory agents has been pursued by pharmaceutical companies within the past decade, there are currently no clinically approved CXCR2 inhibitors. A pharmacophore model based on previously reported CXCR2 antagonists was developed to screen a database of commercially available compounds. Small-molecule compounds identified from the pharmacophore screening were selected for in vitro screening in a cell-based CXCR2-mediated ß-arrestin-2 recruitment assay and further characterized in several cell-based assays and lipopolysaccharide (LPS)-induced lung inflammation studies in mice. CX compounds identified from pharmacophore modeling inhibited cell migration, lung and colon cancer cell proliferation, and colony formation. Mechanistic studies of CX4152 showed that this compound inhibits CXCR2 signaling through downregulation of surface CXCR2. Additionally, CX4152 significantly inhibits CXCL8-mediated neutrophil migration and LPS-induced lung inflammation in mice. Using a CXCR2-inhibitor-based pharmacophore model, we identified a novel set of sulfonamides from a diverse library of small molecules. These compounds inhibit CXCR2/ß-arrestin-2 association, cell migration and proliferation, and acute inflammation in mouse models. CX compounds identified from our pharmacophore models are potential leads for further optimization and development as anti-inflammatory and anticancer agents.
Assuntos
Anti-Inflamatórios/química , Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Receptores de Interleucina-8B/antagonistas & inibidores , Sulfonamidas/química , Sulfonamidas/uso terapêutico , Animais , Anti-Inflamatórios/farmacologia , Arrestinas/imunologia , Células CHO , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cricetulus , Humanos , Inflamação/imunologia , Pulmão/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Receptores de Interleucina-8B/imunologia , Sulfonamidas/farmacologia , beta-Arrestina 2 , beta-ArrestinasRESUMO
Human CMV (HCMV) uses members of the hematopoietic system including neutrophils for dissemination throughout the body. HCMV encodes a viral chemokine, vCXCL-1, that is postulated to attract neutrophils for dissemination within the host. The gene encoding vCXCL-1, UL146, is one of the most variable genes in the HCMV genome. Why HCMV has evolved this hypervariability and how this affects the virus' dissemination and pathogenesis is unknown. Because the vCXCL-1 hypervariability maps to important binding and activation domains, we hypothesized that vCXCL-1s differentially activate neutrophils, which could contribute to HCMV dissemination, pathogenesis, or both. To test whether these viral chemokines affect neutrophil function, we generated vCXCL-1 proteins from 11 different clades from clinical isolates from infants infected congenitally with HCMV. All vCXCL-1s were able to induce calcium flux at a concentration of 100 nM and integrin expression on human peripheral blood neutrophils, despite differences in affinity for the CXCR1 and CXCR2 receptors. In fact, their affinity for CXCR1 or CXCR2 did not correlate directly with chemotaxis, G protein-dependent and independent (ß-arrestin-2) activation, or secondary chemokine (CCL22) expression. Our data suggest that vCXCL-1 polymorphisms affect the binding affinity, receptor usage, and differential peripheral blood neutrophil activation that could contribute to HCMV dissemination and pathogenesis.
Assuntos
Quimiocinas CXC/imunologia , Infecções por Citomegalovirus/imunologia , Citomegalovirus/imunologia , Neutrófilos/imunologia , Receptores de Interleucina-8A/imunologia , Receptores de Interleucina-8B/imunologia , Proteínas Virais/imunologia , Animais , Arrestinas/genética , Arrestinas/imunologia , Cálcio/metabolismo , Quimiocina CCL22/genética , Quimiocina CCL22/imunologia , Quimiocinas CXC/genética , Citomegalovirus/genética , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/patologia , Infecções por Citomegalovirus/virologia , Regulação da Expressão Gênica , Variação Genética , Células HEK293 , Células HL-60 , Interações Hospedeiro-Patógeno , Humanos , Lactente , Neutrófilos/patologia , Neutrófilos/virologia , Cultura Primária de Células , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8B/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Células Sf9 , Transdução de Sinais , Spodoptera , Proteínas Virais/genética , beta-Arrestina 2 , beta-ArrestinasRESUMO
Beta-arrestin-1 (ß-arrestin-1) is an adaptor protein that functions in the termination of G-protein activation and seems to be involved in the mediation of the inflammatory response. Interleukin-1ß (IL-1ß) elicits the expression of inflammatory mediators through a mechanism involving hyaluronan (HA) degradation, thereby contributing to toll-like receptor 4 (TLR-4) and CD44 activation. Stimulation of both receptors induces nuclear factor kappaB (NF-kB) activation that, through transforming-growth-factor-activated-kinase-1 (TAK-1), in turn stimulates the inflammatory mediators of transcription. As ß-arrestin-1 seems to play an inflammatory role in arthritis, we have investigated the involvement of ß-arrestin-1 in a model of IL-1ß-induced inflammatory response in mouse chondrocytes. IL-1ß treatment significantly increases chondrocytes TLR-4, CD44, ß-arrestin-1, TAK-1, and serine/threonine kinase (AKT) mRNA expression and related protein levels. NF-kB is also markedly activated with consequent tumor-necrosis-factor-alpha, interleukin-6, and inducible-nitric-oxide-synthase up-regulation. Treatment of IL-1ß-stimulated chondrocytes with ß-arrestin-1 and/or AKT and/or TAK-1-specific inhibitors significantly reduces all parameters, although the inhibitory effect exerted by TAK-1-mediated pathways is more effective than that of ß-arrestin-1. ß-Arrestin-1-induced NF-kB activation is mediated by the AKT pathway as shown by IL-1ß-stimulated chondrocytes treated with AKT inhibitor. Finally, a specific HA-blocking peptide (Pep-1) has confirmed the inflammatory role of degraded HA as a mediator of the IL-1ß-induced activation of ß-arrestin-1.
Assuntos
Arrestinas/imunologia , Condrócitos/imunologia , Ácido Hialurônico/imunologia , Inflamação/imunologia , Interleucina-1beta/imunologia , Animais , Células Cultivadas , Interleucina-6/imunologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , NF-kappa B/imunologia , Transdução de Sinais , Receptor 4 Toll-Like/imunologia , beta-Arrestina 1 , beta-ArrestinasRESUMO
Over the last decade, functional selectivity (or ligand bias) has evolved from being a peculiar phenomenon to being recognized as an essential feature of synthetic ligands that target Gâ protein-coupled receptors (GPCRs). The CXC chemokine receptorâ 3 (CXCR3) is an outstanding platform to study various aspects of biased signaling, because nature itself uses functional selectivity to manipulate receptor signaling. At the same time, CXCR3 is an attractive therapeutic target in the treatment of autoimmune diseases and cancer. Herein we report the discovery of an 8-azaquinazolinone derivative (N-{1-[3-(4-ethoxyphenyl)-4-oxo-3,4-dihydropyrido[2,3-d]pyrimidin-2-yl]ethyl}-4-(4-fluorobutoxy)-N-[(1-methylpiperidin-4-yl)methyl]butanamide, 1 b) that can inhibit CXC chemokineâ 11 (CXCL11)-dependent Gâ protein activation over ß-arrestin recruitment with 187-fold selectivity. This compound also demonstrates probe-dependent activity, that is, it inhibits CXCL11- over CXCL10-mediated Gâ protein activation with 12-fold selectivity. Together with a previously reported biased negative allosteric modulator from our group, the present study provides additional information on the molecular requirements for allosteric modulation of CXCR3.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Quinazolinonas/química , Quinazolinonas/farmacologia , Receptores CXCR3/imunologia , Arrestinas/imunologia , Compostos Aza/química , Compostos Aza/farmacologia , Quimiocina CXCL11/antagonistas & inibidores , Células HEK293 , Humanos , Ligantes , Transdução de Sinais/efeitos dos fármacos , beta-ArrestinasRESUMO
Inflammasomes are multiprotein complexes that trigger the activation of caspase-1 and the maturation of IL-1ß, which are critical for inflammation and control of pathogen infection. Although the function of inflammasomes in immune response and disease development is well studied, the molecular mechanism by which inflammasomes are activated and assembled remains largely unknown. In this study, we found that ß-arrestin1, a key regulator of the G protein-coupled receptor signaling pathway, was required for nucleotide-binding domain and leucine-rich repeat containing (NLR) family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing protein 4 (NLRC4) inflammasome-mediated IL-1ß production and caspase-1 activation, but it had no effect on absent in melanoma 2 (AIM2) inflammasome activation. Moreover, apoptosis-associated speck-like protein containing a CARD (ASC) pyroptosome, which is ASC aggregation mediating caspase-1 activation, was also impaired in ß-arrestin1-deficient macrophages upon NLRP3 or NLRC4, but not AIM2 inflammasome activation. Mechanistic study revealed that ß-arrestin1 specifically interacted with NLRP3 and NLRC4 and promoted their self-oligomerization. In vivo, in a monosodium urate crystal (MSU)-induced NLRP3-dependent peritonitis model, MSU-induced IL-1ß production and neutrophil flux were significantly reduced in ß-arrestin1 knockout mice. Additionally, ß-arrestin1 deficiency rescued the weight loss of mice upon log-phase Salmonella typhimurium infection, with less IL-1ß production. Taken together, our results indicate that ß-arrestin1 plays a critical role in the assembly and activation of two major canonical inflammasomes, and it may provide a new therapeutic target for inflammatory diseases.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Arrestinas/imunologia , Proteínas de Ligação ao Cálcio/imunologia , Proteínas de Transporte/imunologia , Inflamassomos/imunologia , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Immunoblotting , Imunoprecipitação , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Proteína 3 que Contém Domínio de Pirina da Família NLR , Peritonite/imunologia , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologia , beta-ArrestinasRESUMO
ß-Arrestins (ß-arrs) are regulators and mediators of G protein-coupled receptor signaling, and accumulating evidence suggests that they are functionally involved in inflammation and autoimmune diseases. However, the effect of ß-arrs is unclear in inflammatory bowel disease (IBD), and the role of ß-arr2 is unknown in ulcerative colitis (UC) and Crohn's disease (CD). The aim of this study is to investigate whether ß-arr2 encourages inflammation-induced epithelial apoptosis through endoplasmic reticulum (ER) stress/p53-upregulated modulator of apoptosis (PUMA) in colitis. In the present study, the results showed that ß-arr2 was increased in specimens from patients with UC or CD. Furthermore, a ß-arr2 deficiency significantly repressed intestinal inflammation, ameliorated colitis, and alleviated mucosal apoptosis in mice. In addition, the targeted deletion of ß-arr2 depressed ER stress, inhibited PUMA, and downregulated PUMA-mediated mitochondrial apoptotic signaling in colitis. ß-Arr2, an important modulator of G protein-coupled receptor function, binds eIF2α to activate ER stress signaling. Furthermore, the knockdown of PUMA dramatically prevented ß-arr2-induced apoptosis via alleviating ER stress in vitro. The results suggest that ß-arr2 encourages inflammation-induced epithelial apoptosis through ER stress/PUMA in colitis and that ß-arr2 is a potential therapeutic target for colitis.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Arrestinas/imunologia , Colite Ulcerativa/imunologia , Colite/imunologia , Doença de Crohn/imunologia , Estresse do Retículo Endoplasmático/imunologia , Proteínas Proto-Oncogênicas/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Arrestinas/deficiência , Arrestinas/genética , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/genética , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Doença de Crohn/genética , Doença de Crohn/patologia , Sulfato de Dextrana , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fator de Iniciação 2 em Eucariotos/genética , Fator de Iniciação 2 em Eucariotos/imunologia , Fármacos Gastrointestinais/farmacologia , Regulação da Expressão Gênica , Células HCT116 , Humanos , Infliximab/farmacologia , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/imunologia , Mitocôndrias/patologia , Ligação Proteica , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , beta-Arrestina 2 , beta-ArrestinasRESUMO
Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and ß-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.
Assuntos
Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos/imunologia , Epitopos/imunologia , Receptores de Interleucina-8B/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Arrestinas/imunologia , Arrestinas/metabolismo , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/imunologia , Cálcio/imunologia , Cálcio/metabolismo , Linhagem Celular , Técnicas de Visualização da Superfície Celular/métodos , Quimiocina CXCL1/imunologia , Quimiocina CXCL1/farmacologia , Mapeamento de Epitopos/métodos , Epitopos/metabolismo , Células HEK293 , Humanos , Hibridomas , Imunização , Interleucina-8/imunologia , Interleucina-8/farmacologia , Camundongos Transgênicos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Ligação Proteica/imunologia , Receptores de Interleucina-8B/genética , Receptores de Interleucina-8B/metabolismo , Transdução de Sinais/imunologia , beta-ArrestinasRESUMO
Natural killer (NK) cell activation is well orchestrated by a wide array of NK cell receptor repertoire. T-cell immunoglobulin and ITIM domain (TIGIT) receptor was recently defined as an inhibitory receptor that is expressed on NK cells and T cells. TIGIT receptor/poliovirus receptor (PVR) ligand engagement signaling inhibits cytotoxicity mediated by NK and CD8(+) T cells. However, it is unclear how TIGIT/PVR signaling regulates cytokine secretion in NK cells. Here we show that TIGIT/PVR engagement suppresses interferon-γ (IFN-γ) production of NK cells. TIGIT transgenic NK cells generate less IFN-γ undergoing TIGIT/PVR ligation. Moreover, TIGIT knock-out NK cells produce much more IFN-γ. TIGIT/PVR ligation signaling mediates suppression of IFN-γ production via the NF-κB pathway. We identified a novel adaptor ß-arrestin 2 that associates with phosphorylated TIGIT for further recruitment of SHIP1 (SH2-containing inositol phosphatase 1) through the ITT-like motif. Importantly, SHIP1, but not other phosphatases, impairs the TNF receptor-associated factor 6 (TRAF6) autoubiquitination to abolish NF-κB activation, leading to suppression of IFN-γ production in NK cells.
Assuntos
Arrestinas/metabolismo , Interferon gama/biossíntese , Células Matadoras Naturais/metabolismo , Receptores Imunológicos/metabolismo , Receptores Virais/metabolismo , Transdução de Sinais/fisiologia , Animais , Arrestinas/genética , Arrestinas/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular , Humanos , Capeamento Imunológico/fisiologia , Inositol Polifosfato 5-Fosfatases , Interferon gama/genética , Interferon gama/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Knockout , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Receptores Virais/genética , Receptores Virais/imunologia , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , beta-Arrestina 2 , beta-ArrestinasRESUMO
Gαi-coupled chemoattractant receptors, such as the 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) receptor (OXE-R), are able to switch on Gαißγ protein-dependent and ß-arrestin-related signaling traits. However, which of these signaling pathways are truly important for the chemoattractant functions in leukocytes is not clarified yet. As we recently reported, Gue1654 is a unique Gßγ-biased OXE-R antagonist having no inhibitory activity on Gαi-related signaling, which makes Gue1654 an unprecedented tool for assessing the involvement of G protein subunits in chemoattractant receptor function. ß-arrestin2 recruitment was studied in OXE-R-overexpressing HEK293 cells using bioluminescence resonance energy transfer assays. Activation of leukocytes was assessed by flow cytometric assays and by immunofluorescence microscopy. Leukocyte capture to endothelial cells was addressed under physiological flow conditions. We found that Gue1654 blocks ß-arrestin2 recruitment in HEK293 cells overexpressing OXE-R and ERK1/2 phosphorylation in human eosinophils and neutrophils. Furthermore, Gue1654 was able to prevent several 5-oxo-ETE-triggered functional events in eosinophils and neutrophils, such as activation of CD11b/CD18 integrins, oxidative burst, actin polymerization, and interaction with endothelial cells. In addition, Gue1654 completely prevented 5-oxo-ETE-induced Ca(2+) flux and chemotaxis of human primary monocytes. All of these leukocyte responses to 5-oxo-ETE, except ERK1/2 phosphorylation and oxidative burst, were likewise prevented by pertussis toxin. Therefore, we conclude that chemoattractant receptors require Gαi subunits only as adaptors to transactivate the Gßγ heteromers, which then act responsible for cell activation. Finally, our data characterize Gue1654 as a non-Gαi-biased antagonist of OXE-R that provides a new basis for therapeutic intervention in inflammatory diseases that involve activation of eosinophils, neutrophils, and monocytes.
Assuntos
Benzenoacetamidas/farmacologia , Benzotiazóis/farmacologia , Eosinófilos/imunologia , Subunidades alfa de Proteínas de Ligação ao GTP/imunologia , Monócitos/imunologia , Ativação de Neutrófilo/imunologia , Neutrófilos/imunologia , Receptores Eicosanoides/antagonistas & inibidores , Ácidos Araquidônicos/imunologia , Arrestinas/imunologia , Antígeno CD11b/imunologia , Antígenos CD18/imunologia , Cálcio/imunologia , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Eosinófilos/citologia , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Monócitos/citologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/citologia , Fosforilação/efeitos dos fármacos , Fosforilação/imunologia , Receptores Eicosanoides/imunologia , beta-ArrestinasRESUMO
BACKGROUND: Five different G protein-coupled sphingosine-1-phosphate (S1P) receptors (S1P1-S1P5) regulate a variety of physiologic and pathophysiologic processes, including lymphocyte circulation, multiple sclerosis (MS), and cancer. Although B-lymphocyte circulation plays an important role in these processes and is essential for normal immune responses, little is known about S1P receptors in human B cells. OBJECTIVE: To explore their function and signaling, we studied B-cell lines and primary B cells from control subjects, patients with leukemia, patients with S1P receptor inhibitor-treated MS, and patients with primary immunodeficiencies. METHODS: S1P receptor expression was analyzed by using multicolor immunofluorescence microscopy and quantitative PCR. Transwell assays were used to study cell migration. S1P receptor internalization was visualized by means of time-lapse imaging with fluorescent S1P receptor fusion proteins expressed by using lentiviral gene transfer. B-lymphocyte subsets were characterized by means of flow cytometry and immunofluorescence microscopy. RESULTS: Showing that different B-cell populations express different combinations of S1P receptors, we found that S1P1 promotes migration, whereas S1P4 modulates and S1P2 inhibits S1P1 signals. Expression of CD69 in activated B lymphocytes and B cells from patients with chronic lymphocytic leukemia inhibited S1P-induced migration. Studying B-cell lines, normal B lymphocytes, and B cells from patients with primary immunodeficiencies, we identified Bruton tyrosine kinase, ß-arrestin 2, LPS-responsive beige-like anchor protein, dedicator of cytokinesis 8, and Wiskott-Aldrich syndrome protein as critical signaling components downstream of S1P1. CONCLUSION: Thus S1P receptor signaling regulates human B-cell circulation and might be a factor contributing to the pathology of MS, chronic lymphocytic leukemia, and primary immunodeficiencies.
Assuntos
Subpopulações de Linfócitos B/metabolismo , Imunodeficiência de Variável Comum/metabolismo , Leucemia Linfocítica Crônica de Células B/metabolismo , Esclerose Múltipla/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Tirosina Quinase da Agamaglobulinemia , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Arrestinas/genética , Arrestinas/imunologia , Arrestinas/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Linhagem Celular , Movimento Celular , Imunodeficiência de Variável Comum/genética , Imunodeficiência de Variável Comum/imunologia , Imunodeficiência de Variável Comum/patologia , Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Lisoesfingolipídeo/genética , Receptores de Lisoesfingolipídeo/imunologia , Transdução de Sinais , Imagem com Lapso de Tempo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , beta-Arrestina 2 , beta-ArrestinasRESUMO
Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-ß1-adrenergic (ß1AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at ß1AR. Immunization with the ß1AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at ß1AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through ß-arrestin. In vitro characterization also verified different antibody receptor interactions reflecting the different epitopes on the extracellular surface of ß1AR to which the mAbs bind. The anti-ß1AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with ß1AR agonism.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 1/farmacologia , Anticorpos Monoclonais Murinos/farmacologia , Arrestinas/imunologia , Proteínas Aviárias/imunologia , Receptores Adrenérgicos beta 1/imunologia , Transdução de Sinais/efeitos dos fármacos , Animais , Proteínas Aviárias/agonistas , Feminino , Células HEK293 , Humanos , Camundongos Endogâmicos BALB C , Transdução de Sinais/imunologia , Perus , beta-ArrestinasRESUMO
Multiple sclerosis (MS) is an autoimmune disease characterized by recurrent episodes of demyelination and axonal lesion mediated by CD4+ T cells with a proinflammatory T helper (Th)1 and Th17 phenotypes, macrophages, and soluble inflammatory mediators. The overactive pro-inflammatory Th1 cells and clonal expansion of B cells initiate an inflammatory cascade with several cellular and molecular immune components participating in MS pathogenic mechanisms. In this scenario, autoantibodies and autoantigens have a significant role in immunopathogenesis, diagnosis and therapeutic targets of MS. In this review, we try to introduce the autoantigens and autoantibodies and explain their roles in pathogenesis of MS.
Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Esclerose Múltipla/imunologia , Aquaporina 4/imunologia , Arrestinas/imunologia , Humanos , Esclerose Múltipla/etiologia , Glicoproteína Mielina-Oligodendrócito/imunologia , Fosfopiruvato Hidratase/imunologia , Cadeia B de alfa-Cristalina/imunologia , 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
The chemokine receptor CXCR7, belonging to the membrane-bound G protein-coupled receptor superfamily, is expressed in several tumor types. Inhibition of CXCR7 with either small molecules or small interference (si)RNA has shown promising therapeutic benefits in several tumor models. With the increased interest and effectiveness of biologicals inhibiting membrane-bound receptors we made use of the "Nanobody platform" to target CXCR7. Previously we showed that Nanobodies, i.e. immunoglobulin single variable domains derived from naturally occurring heavy chain-only camelids antibodies, represent new biological tools to efficiently tackle difficult drug targets such as G protein-coupled receptors. In this study we developed and characterized highly selective and potent Nanobodies against CXCR7. Interestingly, the CXCR7-targeting Nanobodies displayed antagonistic properties in contrast with previously reported CXCR7-targeting agents. Several high affinity CXCR7-specific Nanobodies potently inhibited CXCL12-induced ß-arrestin2 recruitment in vitro. A wide variety of tumor biopsies was profiled, showing for the first time high expression of CXCR7 in head and neck cancer. Using a patient-derived CXCR7-expressing head and neck cancer xenograft model in nude mice, tumor growth was inhibited by CXCR7-targeting Nanobody therapy. Mechanistically, CXCR7-targeting Nanobodies did not inhibit cell cycle progression but instead reduced secretion of the angiogenic chemokine CXCL1 from head and neck cancer cells in vitro, thus acting here as inverse agonists, and subsequent angiogenesis in vivo. Hence, with this novel class of CXCR7 inhibitors, we further substantiate the therapeutic relevance of targeting CXCR7 in head and neck cancer.
Assuntos
Neoplasias de Cabeça e Pescoço/imunologia , Receptores CXCR/imunologia , Anticorpos de Domínio Único/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Arrestinas/imunologia , Arrestinas/metabolismo , Ligação Competitiva/imunologia , Camelídeos Americanos/imunologia , Linhagem Celular Tumoral , Quimiocina CXCL12/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/prevenção & controle , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Ensaio Radioligante , Receptores CXCR/genética , Receptores CXCR/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Anticorpos de Domínio Único/farmacologia , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologia , beta-ArrestinasRESUMO
ß-arrestin-2 (ß-arr2) is a scaffolding protein of the arrestin family with a wide variety of cellular functions. Recent studies have demonstrated differential roles for ß-arr2 in inflammation following endotoxemia and cecal ligation and puncture (CLP) models of sepsis. Because CLP-induced inflammation involves response to fecal contents and necrotic cecum in addition to microbial challenge, in this study, we examined the role of ß-arr2 in an exclusively polymicrobial infection (PMI) model. In addition, we examined the role of gene dosage of ß-arr2 in polymicrobial sepsis. Our studies demonstrate that ß-arr2 is a negative regulator of systemic inflammation in response to polymicrobial infection and that one allele is sufficient for this process. Our results further reveal that loss of ß-arr2 leads to increased neutrophil sequestration and overt inflammation specifically in the lungs following polymicrobial infection. Consistent with this, specific NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways were differentially activated in the ß-arr2 knockout (KO) mice lungs compared to the wild type (WT) following PMI. Associated with enhanced inflammation in the KO mice, PMI-induced mortality was also significantly higher in KO mice than in WT mice. To understand the differential role of ß-arr2 in different sepsis models, we used cell culture systems to evaluate inflammatory cytokine production following endotoxin and polymicrobial stimulation. Our results demonstrate cell-type- as well as stimulus-specific roles for ß-arr2 in inflammation. Taken together, our results reveal a negative regulatory role for ß-arr2 in polymicrobial infection-induced inflammation and further demonstrate that one allele of ß-arr2 is sufficient to mediate most of these effects.