RESUMO
Macrophages play critical, but opposite, roles in acute and chronic inflammation and cancer. In response to pathogens or injury, inflammatory macrophages express cytokines that stimulate cytotoxic T cells, whereas macrophages in neoplastic and parasitic diseases express anti-inflammatory cytokines that induce immune suppression and may promote resistance to T cell checkpoint inhibitors. Here we show that macrophage PI 3-kinase γ controls a critical switch between immune stimulation and suppression during inflammation and cancer. PI3Kγ signalling through Akt and mTor inhibits NFκB activation while stimulating C/EBPß activation, thereby inducing a transcriptional program that promotes immune suppression during inflammation and tumour growth. By contrast, selective inactivation of macrophage PI3Kγ stimulates and prolongs NFκB activation and inhibits C/EBPß activation, thus promoting an immunostimulatory transcriptional program that restores CD8+ T cell activation and cytotoxicity. PI3Kγ synergizes with checkpoint inhibitor therapy to promote tumour regression and increased survival in mouse models of cancer. In addition, PI3Kγ-directed, anti-inflammatory gene expression can predict survival probability in cancer patients. Our work thus demonstrates that therapeutic targeting of intracellular signalling pathways that regulate the switch between macrophage polarization states can control immune suppression in cancer and other disorders.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Tolerância Imunológica/imunologia , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Células Cultivadas , Classe Ib de Fosfatidilinositol 3-Quinase/deficiência , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Feminino , Humanos , Inflamação/imunologia , Ativação Linfocitária , Macrófagos/enzimologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neoplasias/imunologia , Neoplasias/patologia , Inibidores de Fosfoinositídeo-3 Quinase , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Serina-Treonina Quinases TOR/metabolismo , Evasão Tumoral/imunologiaRESUMO
The receptor for advanced glycation end products (RAGE) is a multiligand transmembrane receptor implicated in a number of diseases including autoimmune diseases. To further understand the pathogenic mechanism of RAGE in these diseases, we searched for additional ligands. We discovered that C3a bound to RAGE with an EC(50) of 1.9 nM in an ELISA, and the binding was increased both in magnitude (by >2-fold) and in affinity (EC(50) 70 pM) in the presence of human stimulatory unmethylated cytosine-guanine-rich DNA A (hCpGAs). Surface plasmon resonance and fluorescence anisotropy analyses demonstrated that hCpGAs could bind directly to RAGE and C3a and form a ternary complex. In human PBMCs, C3a increased IFN-α production in response to low levels of hCpGAs, and this synergy was blocked by soluble RAGE or by an Ab directed against RAGE. IFN-α production was reduced in response to mouse CpGAs and C3a in RAGE(-/-) mouse bone marrow cells compared wild-type mice. Taken together, these data demonstrate that RAGE is a receptor for C3a and CpGA. Through direct interaction, C3a and CpGA synergize to increase IFN-α production in a RAGE-dependent manner and stimulate an innate immune response. These findings indicate a potential role of RAGE in autoimmune diseases that show accumulation of immunostimulatory DNA and C3a.
Assuntos
Complemento C3a/metabolismo , DNA/metabolismo , Interferon gama/metabolismo , Oligonucleotídeos/metabolismo , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Animais , Complemento C3a/imunologia , DNA/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Interferon gama/imunologia , Camundongos , Camundongos Knockout , Oligonucleotídeos/imunologia , Ligação Proteica , Receptor para Produtos Finais de Glicação Avançada/imunologia , Ressonância de Plasmônio de SuperfícieRESUMO
OBJECTIVES: To investigate the expression and function of triggering receptor expressed on myeloid cells-1 (TREM-1) in the synovium of human RA patients as well as the level of soluble TREM-1 in the plasma of RA patients. METHODS: Twenty-four RA synovial samples were analysed by gene expression oligonucleotide microarrays. Expression levels of TREM-1 mRNA in murine CIA paws were determined by quantitative PCR (qPCR). TREM-1 protein expression was detected by immunohistochemistry in five RA synovial samples and two OA synovial samples. TREM-1-positive cells from five RA synovial tissues were analysed by FACS staining to determine the cell type. Activation of TREM-1 was tested in five RA synovial samples. Soluble TREM-1 was measured in serum from 32 RA patients. RESULTS: The expression of TREM-1 mRNA was found to increase 6.5-fold in RA synovial samples, whereas it was increased 132-fold in CIA paws. Increased numbers of TREM-1-positive cells were seen in RA synovium sections and these cells co-expressed CD14. Using a TREM-1-activating cross-linking antibody in RA synovial cultures, multiple pro-inflammatory cytokines were induced. The average amount of soluble TREM-1 in plasma from RA patients was found to be higher than that in plasma from healthy volunteers. CONCLUSIONS: These findings suggest that the presence of high levels of functionally active TREM-1 in RA synovium may contribute to the development or maintenance of RA, or both. Inhibiting TREM-1 activity may, therefore, have a therapeutic effect on RA. High levels of soluble TREM-1 in the plasma of RA patients compared with healthy volunteers may indicate disease activity.
Assuntos
Artrite Reumatoide/imunologia , Citocinas/biossíntese , Glicoproteínas de Membrana/metabolismo , Receptores Imunológicos/metabolismo , Membrana Sinovial/imunologia , Animais , Artrite Experimental/imunologia , Biomarcadores/metabolismo , Células Cultivadas , Expressão Gênica , Perfilação da Expressão Gênica/métodos , Humanos , Mediadores da Inflamação/metabolismo , Masculino , Glicoproteínas de Membrana/sangue , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos DBA , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , RNA Mensageiro/genética , Receptores Imunológicos/sangue , Receptores Imunológicos/genética , Receptor Gatilho 1 Expresso em Células MieloidesRESUMO
The class I phosphoinoside-3-kinases (PI3Ks) are important enzymes that relay signals from cell surface receptors to downstream mediators driving cellular functions. Elevated PI3K signaling is found in B cell malignancies and lymphocytes of patients with autoimmune disease. The p110δ catalytic isoform of PI3K is a rational target since it is critical for B lymphocyte development, survival, activation, and differentiation. In addition, activating mutations in PIK3CD encoding p110δ cause a human immunodeficiency known as activated PI3K delta syndrome. Currently, idelalisib is the only selective p110δ inhibitor that has been FDA approved to treat certain B cell malignancies. p110δ inhibitors can suppress autoantibody production in mouse models, but limited clinical trials in human autoimmunity have been performed with PI3K inhibitors to date. Thus, there is a need for additional tools to understand the effect of pharmacological inhibition of PI3K isoforms in lymphocytes. In this study, we tested the effects of a potent and selective p110δ inhibitor, IPI-3063, in assays of B cell function. We found that IPI-3063 potently reduced mouse B cell proliferation, survival, and plasmablast differentiation while increasing antibody class switching to IgG1, almost to the same degree as a pan-PI3K inhibitor. Similarly, IPI-3063 potently inhibited human B cell proliferation in vitro. The p110γ isoform has partially overlapping roles with p110δ in B cell development, but little is known about its role in B cell function. We found that the p110γ inhibitor AS-252424 had no significant impact on B cell responses. A novel dual p110δ/γ inhibitor, IPI-443, had comparable effects to p110δ inhibition alone. These findings show that p110δ is the dominant isoform mediating B cell responses and establish that IPI-3063 is a highly potent molecule useful for studying p110δ function in immune cells.
RESUMO
Phosphatidylinositol-3-kinases (PI3K) γ and δ are preferentially enriched in leukocytes, and defects in these signaling pathways have been shown to impair T cell activation. The effects of PI3Kγ and PI3Kδ on alloimmunity remain underexplored. Here, we show that both PI3Kγ -/- and PI3Kδ D910A/D910A mice receiving heart allografts have suppression of alloreactive T effector cells and delayed acute rejection. However, PI3Kδ mutation also dampens regulatory T cells (Treg). After treatment with low dose CTLA4-Ig, PI3Kγ -/- , but not PI3Κδ D910A/D910A , recipients exhibit indefinite prolongation of heart allograft survival. PI3Kδ D910A/D910A Tregs have increased apoptosis and impaired survival. Selective inhibition of PI3Kγ and PI3Kδ (using PI3Kδ and dual PI3Kγδ chemical inhibitors) shows that PI3Kγ inhibition compensates for the negative effect of PI3Kδ inhibition on long-term allograft survival. These data serve as a basis for future PI3K-based immune therapies for transplantation.Phosphatidylinositol-3-kinases (PI3K) γ and δ are key regulators of T cell signaling. Here the author show, using mouse heart allograft transplantation models, that PI3Kγ or PI3Kδ deficiency prolongs graft survival, but selective inhibition of PI3Kγ or PI3Kδ reveals alternative transplant survival outcomes post CTLA4-Ig treatment.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/imunologia , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Transplante de Coração , Ativação Linfocitária/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Transplante de Pele , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante/imunologia , Abatacepte/farmacologia , Animais , Classe I de Fosfatidilinositol 3-Quinases , Classe Ib de Fosfatidilinositol 3-Quinase/genética , Técnicas de Silenciamento de Genes , Sobrevivência de Enxerto/efeitos dos fármacos , Imunossupressores/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Knockout , Modelos Animais , Mutação , Fosfatidilinositol 3-Quinases/genética , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Tolerância ao Transplante/efeitos dos fármacosRESUMO
Optimization of isoquinolinone PI3K inhibitors led to the discovery of a potent inhibitor of PI3K-γ (26 or IPI-549) with >100-fold selectivity over other lipid and protein kinases. IPI-549 demonstrates favorable pharmacokinetic properties and robust inhibition of PI3K-γ mediated neutrophil migration in vivo and is currently in Phase 1 clinical evaluation in subjects with advanced solid tumors.
RESUMO
SOST, a novel bone morphogenetic protein (BMP) antagonist and negative regulator of bone formation, is expressed in osteogenic cells. Null mutations in the SOST gene are associated with the sclerosteosis phenotype typified by high bone mass. We sought to delineate the pathways involved in the regulation of SOST expression in human osteoblastic cells. We evaluated the effects of bone growth factors and hormones on the RNA levels of SOST and the BMP antagonists, noggin and gremlin. Parathyroid hormone (PTH), transforming growth factor-beta1 (TGF-beta1), fibroblast growth factors 1 and 2 (FGF1, FGF2), and insulin-like growth factor-1 (IGF-1) had negligible effects on SOST expression in human osteoblasts. In comparison, BMPs-2, 4, and 6 induced the message levels of SOST in a time- and dose-dependent manner. The levels of noggin and, to a lesser extent, gremlin were also increased by BMPs. BMP's stimulatory effects on SOST were further enhanced by retinoic acid or 1,25-dihydroxyvitamin D3. In contrast, dexamethasone (DEX) blocked the effects of the BMPs on SOST and gremlin, but not on noggin. Retinoic acid and 1,25-dihydroxyvitamin D3 did not affect the BMP-enhanced expression of gremlin or noggin. The steroids did not affect the endogenous levels of the BMP antagonists. These findings show that the levels of SOST are modulated by BMPs and the interactions of the BMPs with steroid hormones in human osteoblasts. These effects differed markedly from that of noggin or gremlin, suggesting that there is an exquisite regulation of the expressions of BMP antagonists in cells of the osteoblast lineage.
Assuntos
Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/fisiologia , Calcitriol/farmacologia , Dexametasona/farmacologia , Regulação da Expressão Gênica/fisiologia , Marcadores Genéticos/genética , Osteoblastos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Bases , Primers do DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
A null mutation in the SOST gene is associated with sclerosteosis, an inherited disorder characterized by a high bone mass phenotype. The protein product of the SOST gene, sclerostin, is a bone morphogenetic protein (BMP) antagonist that decreases osteoblast activity and reduces the differentiation of osteoprogenitors. We sought to delineate the mechanism by which sclerostin modulated osteoblastic function by examining the effects of the protein on differentiating cultures of human mesenchymal stem cells (hMSC). Sclerostin significantly decreased alkaline phosphatase (ALP) activity and the proliferation of hMSC cells. In addition, hMSC cells treated with sclerostin displayed a marked increase in caspase activity. Elevated levels of fragmented histone-associated DNA in these cells were detected by ELISA and by TUNEL staining. Other BMP antagonists including noggin, Chordin, Gremlin, and Twisted gastrulation did not affect caspase activity. The sclerostin-mediated increase in caspase activity was blocked by caspase-1 and caspase-3 inhibitors. Sclerostin-induced changes in ALP activity and the survival of hMSC cells were partially restored by BMP-6, suggesting the involvement of additional growth factors. These findings show that sclerostin selectively controls the apoptosis of bone cells. The ability of sclerostin to interact with important growth factors such as BMPs likely serves as the basis by which it modulates the survival of osteoblasts. By making these growth factors unavailable for cell function, sclerostin promotes the apoptosis of bone cells, providing a novel level of control in the regulation of bone formation.
Assuntos
Apoptose , Proteínas Morfogenéticas Ósseas/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Osteogênese , Proteínas Adaptadoras de Transdução de Sinal , Fosfatase Alcalina/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas Morfogenéticas Ósseas/genética , Inibidores de Caspase , Caspases/metabolismo , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Regulação da Expressão Gênica , Marcadores Genéticos/genética , Humanos , Osteoblastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Osteogênese/genéticaRESUMO
Phosphoinositide-3 kinase (PI3K)-δ and PI3K-γ are preferentially expressed in immune cells, and inhibitors targeting these isoforms are hypothesized to have anti-inflammatory activity by affecting the adaptive and innate immune response. We report on a potent oral PI3K-δ and PI3K-γ inhibitor (IPI-145) and characterize this compound in biochemical, cellular, and in vivo assays. These studies demonstrate that IPI-145 exerts profound effects on adaptive and innate immunity by inhibiting B and T cell proliferation, blocking neutrophil migration, and inhibiting basophil activation. We explored the therapeutic value of combined PI3K-δ and PI3K-γ blockade, and IPI-145 showed potent activity in collagen-induced arthritis, ovalbumin-induced asthma, and systemic lupus erythematosus rodent models. These findings support the hypothesis that inhibition of immune function can be achieved through PI3K-δ and PI3K-γ blockade, potentially leading to significant therapeutic effects in multiple inflammatory, autoimmune, and hematologic diseases.
Assuntos
Artrite/tratamento farmacológico , Asma/tratamento farmacológico , Modelos Animais de Doenças , Isoquinolinas/farmacologia , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/farmacologia , Animais , Artrite/induzido quimicamente , Artrite/imunologia , Asma/induzido quimicamente , Asma/imunologia , Colágeno Tipo II , Relação Dose-Resposta a Droga , Feminino , Humanos , Isoquinolinas/química , Lúpus Eritematoso Sistêmico/imunologia , Estrutura Molecular , Ovalbumina , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Purinas/química , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
A report on the Keystone Symposium 'Innate Immunity: Signaling Mechanisms', Keystone, USA, 24-29 February, 2008.
Assuntos
Imunidade Inata , Transdução de Sinais , Humanos , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/terapia , Receptores Toll-Like/imunologiaRESUMO
High bone mass diseases are caused both by activating mutations in the Wnt pathway and by loss of SOST, a bone morphogenetic protein (BMP) antagonist, leading to the activation of BMP signaling. Given the phenotypic similarity between mutations that activate these signaling pathways, it seems likely that BMPs and Wnts operate in parallel or represent components of the same pathway, modulating osteoblast differentiation. In this study, we show that in C3H10T1/2 cells, Wnt-3A and BMP-6 proteins were inducers of osteoblast differentiation, as measured by alkaline phosphatase (ALP) induction. Surprisingly, sclerostin, noggin, and human BMP receptor 1A (BMPR1A)-FC fusion proteins blocked Wnt-3A-induced ALP as well as BMP-6-induced ALP activity. Dkk-1, a Wnt inhibitor, blocked Wnt-induced ALP activity but not BMP-induced ALP activity. Early Wnt-3A signaling as measured by beta-catenin accumulation was not affected by the BMP antagonists but was blocked by Dkk-1. Wnt-3A induced the appearance of BMP-4 mRNA 12 h prior to that of ALP in C3H10T1/2 cells. We propose that sclerostin and other BMP antagonists do not block Wnt signaling directly. Sclerostin blocks Wnt-induced ALP activity by blocking the activity of BMP proteins produced by Wnt treatment. The expression of BMP proteins in this autocrine loop is essential for Wnt-3A-induced osteoblast differentiation.
Assuntos
Proteínas Morfogenéticas Ósseas/farmacologia , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Fosfatase Alcalina/metabolismo , Animais , Proteína Morfogenética Óssea 6 , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Proteínas do Citoesqueleto/metabolismo , Relação Dose-Resposta a Droga , Marcadores Genéticos , Glicoproteínas , Peptídeos e Proteínas de Sinalização Intercelular , Camundongos , Camundongos Endogâmicos C3H , Modelos Biológicos , Mutação , Osteoblastos/metabolismo , Fenótipo , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Tempo , Transativadores/metabolismo , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3A , beta CateninaRESUMO
Noggin and sclerostin are bone morphogenetic protein (BMP) antagonists that modulate mitogenic activity through sequestering BMPs. Little is known of the interactions among this class of proteins. We show that recombinant sclerostin and noggin bound to each other with high affinity (K(D) = 2.92 nm). This observation has been extended to naturally expressed noggin and sclerostin from the rat osteosarcoma cell line, ROS 17/2.8, supporting a role for the complex in natural systems. The noggin-sclerostin complex was competitive with BMP binding and mutually attenuated the activity of each BMP antagonist. Collectively, the data demonstrate a novel and exquisite paradigm for the regulation of BMP activity through direct neutralization of the BMP and activation by co-localized BMP antagonist expression. The pleiotrophic nature of noggin and sclerostin represents a novel mechanism for the fine-tuning of BMP activity in bone homeostasis.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Ligação Competitiva , Western Blotting , Proteína Morfogenética Óssea 6 , Osso e Ossos/metabolismo , Proteínas de Transporte , Linhagem Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Proteínas de Ligação a DNA/metabolismo , Relação Dose-Resposta Imunológica , Ensaio de Imunoadsorção Enzimática , Marcadores Genéticos , Glicoproteínas , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Camundongos , Camundongos Endogâmicos C3H , Osteossarcoma/metabolismo , Testes de Precipitina , Ligação Proteica , Ratos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Proteínas Smad , Fatores de Tempo , Transativadores/metabolismoRESUMO
There is an unmet medical need for anabolic treatments to restore lost bone. Human genetic bone disorders provide insight into bone regulatory processes. Sclerosteosis is a disease typified by high bone mass due to the loss of SOST expression. Sclerostin, the SOST gene protein product, competed with the type I and type II bone morphogenetic protein (BMP) receptors for binding to BMPs, decreased BMP signaling and suppressed mineralization of osteoblastic cells. SOST expression was detected in cultured osteoblasts and in mineralizing areas of the skeleton, but not in osteoclasts. Strong expression in osteocytes suggested that sclerostin expressed by these central regulatory cells mediates bone homeostasis. Transgenic mice overexpressing SOST exhibited low bone mass and decreased bone strength as the result of a significant reduction in osteoblast activity and subsequently, bone formation. Modulation of this osteocyte-derived negative signal is therapeutically relevant for disorders associated with bone loss.