RESUMO
Intestinal mesenchymal cells play essential roles in epithelial homeostasis, matrix remodeling, immunity, and inflammation. But the extent of heterogeneity within the colonic mesenchyme in these processes remains unknown. Using unbiased single-cell profiling of over 16,500 colonic mesenchymal cells, we reveal four subsets of fibroblasts expressing divergent transcriptional regulators and functional pathways, in addition to pericytes and myofibroblasts. We identified a niche population located in proximity to epithelial crypts expressing SOX6, F3 (CD142), and WNT genes essential for colonic epithelial stem cell function. In colitis, we observed dysregulation of this niche and emergence of an activated mesenchymal population. This subset expressed TNF superfamily member 14 (TNFSF14), fibroblastic reticular cell-associated genes, IL-33, and Lysyl oxidases. Further, it induced factors that impaired epithelial proliferation and maturation and contributed to oxidative stress and disease severity in vivo. Our work defines how the colonic mesenchyme remodels to fuel inflammation and barrier dysfunction in IBD.
Assuntos
Doenças Inflamatórias Intestinais/fisiopatologia , Mesoderma/fisiologia , Animais , Proliferação de Células , Colite/genética , Colite/fisiopatologia , Colo/fisiologia , Células Epiteliais/metabolismo , Fibroblastos/fisiologia , Heterogeneidade Genética , Homeostase , Humanos , Inflamação , Mucosa Intestinal/imunologia , Mucosa Intestinal/fisiologia , Intestinos/imunologia , Intestinos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos , Pericitos , Células RAW 264.7 , Fatores de Transcrição SOXD/fisiologia , Análise de Célula Única/métodos , Tromboplastina/fisiologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Via de Sinalização Wnt/fisiologiaRESUMO
BACKGROUND: Phosphatidylinositol 3-kinase p110δ isoform (PI3K p110δ) activity is essential for mast cell activation, suggesting that inhibition of PI3K p110δ might be useful in treating allergic diseases. OBJECTIVE: We sought to determine the effect of the PI3K p110δ-selective inhibitor idelalisib on allergic responses. METHODS: This phase 1 randomized, double-blind, placebo-controlled, 2-period crossover study was conducted with the Vienna Challenge Chamber. Grass pollen-induced allergic symptoms were documented during screening. Eligible subjects received idelalisib (100 mg twice daily) or placebo for 7 days, with allergen challenge on day 7. After a 2-week washout period, subjects received the alternate treatment and repeated allergen challenge. Study measures included safety, nasal and nonnasal symptoms, nasal airflow, nasal secretions, basophil activation, and plasma cytokine levels. RESULTS: Forty-one patients with allergic rhinitis received idelalisib/placebo (n = 21) or placebo/idelalisib (n = 20). Idelalisib treatment was well tolerated. Mean total nasal symptom scores were lower during the combined idelalisib treatment periods compared with placebo (treatment difference [idelalisib - placebo], -1.78; 95% CI, -2.53 to -1.03; P < .001). Statistically significant differences were also observed for the combined treatment periods for total symptom scores, nasal airflow, nasal secretion weight, and nasal congestion scores. The percentage of ex vivo-activated basophils (CD63(+)/CCR3(+) cells; after stimulation with grass pollen) was substantially lower for idelalisib-treated compared with placebo-treated subjects. Plasma CCL17 and CCL22 levels were reduced after idelalisib treatment. CONCLUSION: Idelalisib treatment was well tolerated in patients with allergic rhinitis and appears to reduce allergic responses clinically and immunologically after an environmental allergen challenge.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/uso terapêutico , Quinazolinonas/uso terapêutico , Rinite Alérgica/tratamento farmacológico , Adulto , Alérgenos/imunologia , Basófilos/imunologia , Basófilos/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Volume Expiratório Forçado , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Pólen/imunologia , Purinas/farmacologia , Quinazolinonas/farmacologia , Rinite Alérgica/diagnóstico , Rinite Alérgica/metabolismo , Resultado do Tratamento , Adulto JovemRESUMO
Systemic lupus erythematosus (SLE) is a human chronic inflammatory disease generated and maintained throughout life by autoreactive T and B cells. Class I phosphoinositide 3-kinases (PI3K) are heterodimers composed of a regulatory and a catalytic subunit that catalyze phosphoinositide-3,4,5-P3 formation and regulate cell survival, migration, and division. Activity of the PI3Kδ isoform is enhanced in human SLE patient PBLs. In this study, we analyzed the effect of inhibiting PI3Kδ in MRL/lpr mice, a model of human SLE. We found that PI3Kδ inhibition ameliorated lupus progression. Treatment of these mice with a PI3Kδ inhibitor reduced the excessive numbers of CD4(+) effector/memory cells and B cells. In addition, this treatment reduced serum TNF-α levels and the number of macrophages infiltrating the kidney. Expression of inactive PI3Kδ, but not deletion of the other hematopoietic isoform PI3Kγ, reduced the ability of macrophages to cross the basement membrane, a process required to infiltrate the kidney, explaining MRL/lpr mice improvement by pharmacologic inhibition of PI3Kδ. The observations that p110δ inhibitor prolonged mouse life span, reduced disease symptoms, and showed no obvious secondary effects indicates that PI3Kδ is a promising target for SLE.
Assuntos
Adenosina/análogos & derivados , Rim/efeitos dos fármacos , Lúpus Eritematoso Sistêmico/prevenção & controle , Macrófagos/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Quinazolinonas/farmacologia , Adenosina/química , Adenosina/farmacologia , Animais , Anticorpos Antinucleares/sangue , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Citocinas/sangue , Relação Dose-Resposta a Droga , Citometria de Fluxo , Imunoglobulina G/sangue , Rim/metabolismo , Rim/patologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Nefrite Lúpica/prevenção & controle , Contagem de Linfócitos , Macrófagos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos MRL lpr , Microscopia Confocal , Estrutura Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/química , Quinazolinonas/química , Quinoxalinas/farmacologia , Análise de Sobrevida , Tiazolidinedionas/farmacologiaRESUMO
Phosphatidylinositol 3-kinases (PI3K) participate in numerous signaling pathways, and control distinct biological functions. Studies using pan-PI3K inhibitors suggest roles for PI3K in osteoclasts, but little is known about specific PI3K isoforms in these cells. Our objective was to determine effects of isoform-selective PI3K inhibitors on osteoclasts. The following inhibitors were investigated (targets in parentheses): wortmannin and LY294002 (pan-p110), PIK75 (α), GDC0941 (α, δ), TGX221 (ß), AS252424 (γ), and IC87114 (δ). In addition, we characterized a new potent and selective PI3Kδ inhibitor, GS-9820, and explored roles of PI3K isoforms in regulating osteoclast function. Osteoclasts were isolated from long bones of neonatal rats and rabbits. Wortmannin, LY294002, GDC0941, IC87114, and GS-9820 induced a dramatic retraction of osteoclasts within 15-20 min to 65-75% of the initial area. In contrast, there was no significant retraction in response to vehicle, PIK75, TGX221, or AS252424. Moreover, wortmannin and GS-9820, but not PIK75 or TGX221, disrupted actin belts. We examined effects of PI3K inhibitors on osteoclast survival. Whereas PIK75, TGX221, and GS-9820 had no significant effect on basal survival, all blocked RANKL-stimulated survival. When studied on resorbable substrates, osteoclastic resorption was suppressed by wortmannin and inhibitors of PI3Kß and PI3Kδ, but not other isoforms. These data are consistent with a critical role for PI3Kδ in regulating osteoclast cytoskeleton and resorptive activity. In contrast, multiple PI3K isoforms contribute to the control of osteoclast survival. Thus, the PI3Kδ isoform, which is predominantly expressed in cells of hematopoietic origin, is an attractive target for anti-resorptive therapeutics.
Assuntos
Osteoclastos/efeitos dos fármacos , Osteoclastos/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Adenina/análogos & derivados , Adenina/farmacologia , Androstadienos/farmacologia , Animais , Reabsorção Óssea/prevenção & controle , Sobrevivência Celular/efeitos dos fármacos , Cromonas/farmacologia , Citoesqueleto/efeitos dos fármacos , Indazóis/farmacologia , Isoenzimas/antagonistas & inibidores , Morfolinas/farmacologia , Osteoclastos/citologia , Quinazolinas/farmacologia , Ligante RANK/metabolismo , Coelhos , Ratos , Sulfonamidas/farmacologia , WortmaninaRESUMO
In patients with chronic lymphocytic leukemia (CLL), lenalidomide can promote humoral immune responses but also induces a distinct disease-specific toxicity of tumor flare and cytokine release. These CLL-specific events result from increased expression of costimulatory molecules on B cells. Here we demonstrate that lenalidomide activation of CLL cells depends on the phosphatidylinositol 3-kinase p110δ (PI3K-δ) pathway. Inhibition of PI3K-δ signaling by the PI3K-δ-inhibiting drug, CAL-101, or by siRNA knockdown of p110δ, abrogates CLL cell activation, costimulatory molecule expression, and vascular endothelial growth factor and basic fibroblast growth factor gene expression that is induced by lenalidomide. In addition, CAL-101 attenuates lenalidomide-mediated increases in immunoglobulin M production by normal B cells. Collectively, these data demonstrate the importance of PI3K-δ signaling for lenalidomide immune modulation. These findings may guide development of strategies for the treatment of CLL that combine lenalidomide with CAL-101, with other inhibitors of the PI3K-δ pathway, or with other agents that target downstream kinases of this signaling pathway.
Assuntos
Antineoplásicos/imunologia , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Fatores Imunológicos/imunologia , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/enzimologia , Inibidores de Fosfoinositídeo-3 Quinase , Talidomida/análogos & derivados , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Ativação Enzimática/efeitos dos fármacos , Humanos , Fatores Imunológicos/farmacologia , Lenalidomida , Leucemia Linfocítica Crônica de Células B/imunologia , Purinas/farmacologia , Quinazolinonas/farmacologia , RNA Interferente Pequeno/genética , Talidomida/imunologia , Talidomida/farmacologiaRESUMO
Phosphatidylinositol-3-kinase p110δ serves as a central integration point for signaling from cell surface receptors known to promote malignant B-cell proliferation and survival. This provides a rationale for the development of small molecule inhibitors that selectively target p110δ as a treatment approach for patients with B-cell malignancies. We thus identified 5-fluoro-3-phenyl-2-[(S)-1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one (CAL-101), a highly selective and potent p110δ small molecule inhibitor (half-maximal effective concentration [EC(50)] = 8nM). Using tumor cell lines and primary patient samples representing multiple B-cell malignancies, we have demonstrated that constitutive phosphatidylinositol-3-kinase pathway activation is p110δ-dependent. CAL-101 blocked constitutive phosphatidylinositol-3-kinase signaling, resulting in decreased phosphorylation of Akt and other downstream effectors, an increase in poly(ADP-ribose) polymerase and caspase cleavage and an induction of apoptosis. These effects have been observed across a broad range of immature and mature B-cell malignancies, thereby providing a rationale for the ongoing clinical evaluation of CAL-101.
Assuntos
Antineoplásicos/farmacologia , Leucemia de Células B/metabolismo , Linfoma de Células B/metabolismo , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Purinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Western Blotting , Linhagem Celular Tumoral , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 QuinaseRESUMO
Trafficking and recruitment of eosinophils during allergic airway inflammation is mediated by the phosphatidylinositol 3-kinase (PI3K) family of signaling molecules. The role played by the p110δ subunit of PI3K (PI3K p110δ) in regulating eosinophil trafficking and recruitment was investigated using a selective pharmacological inhibitor (IC87114). Treatment with the PI3K p110δ inhibitor significantly reduced murine bone marrow-derived eosinophil (BM-Eos) adhesion to VCAM-1 as well as ICAM-1 and inhibited activation-induced changes in cell morphology associated with reduced Mac-1 expression and aberrant cell surface localization/distribution of Mac-1 and α4. Infused BM-Eos demonstrated significantly decreased rolling and adhesion in inflamed cremaster muscle microvessels of mice treated with IC87114 compared with vehicle-treated mice. Furthermore, inhibition of PI3K p110δ significantly attenuated eotaxin-1-induced BM-Eos migration and prevented eotaxin-1-induced changes in the cytoskeleton and cell morphology. Knockdown of PI3K p110δ with siRNA in BM-Eos resulted in reduced rolling, adhesion, and migration, as well as inhibition of activation-induced changes in cell morphology, validating its role in regulating trafficking and migration. Finally, in a mouse model of cockroach antigen-induced allergic airway inflammation, oral administration of the PI3K p110δ inhibitor significantly inhibited airway eosinophil recruitment, resulting in attenuation of airway hyperresponsiveness in response to methacholine, reduced mucus secretion, and expression of proinflammatory molecules (found in inflammatory zone-1 and intelectin-1). Overall, these findings indicate the important role played by PI3K p110δ in mediating BM-Eos trafficking and migration by regulating adhesion molecule expression and localization/distribution as well as promoting changes in cell morphology that favor recruitment during inflammation.
Assuntos
Asma/imunologia , Eosinofilia/imunologia , Eosinófilos/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Sistema Respiratório/imunologia , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Asma/metabolismo , Células da Medula Óssea , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocina CCL11/metabolismo , Classe I de Fosfatidilinositol 3-Quinases , Eosinofilia/metabolismo , Eosinófilos/metabolismo , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Antígeno de Macrófago 1/biossíntese , Cloreto de Metacolina/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Quinazolinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Sistema Respiratório/metabolismo , Molécula 1 de Adesão de Célula Vascular/imunologia , Molécula 1 de Adesão de Célula Vascular/metabolismoRESUMO
In this study, we demonstrate expression and examined the biologic sequelae of PI3K/p110delta signaling in multiple myeloma (MM). Knockdown of p110delta by small interfering RNA caused significant inhibition of MM cell growth. Similarly, p110delta specific small molecule inhibitor CAL-101 triggered cytotoxicity against LB and INA-6 MM cell lines and patient MM cells, associated with inhibition of Akt phosphorylation. In contrast, CAL-101 did not inhibit survival of normal peripheral blood mononuclear cells. CAL-101 overcame MM cell growth conferred by interleukin-6, insulin-like growth factor-1, and bone marrow stromal cell coculture. Interestingly, inhibition of p110delta potently induced autophagy. The in vivo inhibition of p110delta with IC488743 was evaluated in 2 murine xenograft models of human MM: SCID mice bearing human MM cells subcutaneously and the SCID-hu model, in which human MM cells are injected within a human bone chip implanted subcutaneously in SCID mice. IC488743 significantly inhibited tumor growth and prolonged host survival in both models. Finally, combined CAL-101 with bortezomib induced synergistic cytotoxicity against MM cells. Our studies therefore show that PI3K/p110delta is a novel therapeutic target in MM and provide the basis for clinical evaluation of CAL-101 to improve patient outcome in MM.
Assuntos
Movimento Celular , Mieloma Múltiplo/terapia , Fosfatidilinositol 3-Quinases/metabolismo , Purinas/farmacologia , Quinazolinonas/farmacologia , Animais , Biomarcadores/metabolismo , Western Blotting , Medula Óssea/metabolismo , Adesão Celular , Proliferação de Células , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Imunofluorescência , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , RNA Interferente Pequeno/farmacologia , Células-Tronco/metabolismo , Veias Umbilicais/citologia , Veias Umbilicais/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeted therapy with imatinib in chronic myeloid leukemia (CML) prompted a new treatment paradigm. Unlike CML, chronic lymphocytic leukemia (CLL) lacks an aberrant fusion protein kinase but instead displays increased phosphatidylinositol 3-kinase (PI3K) activity. To date, PI3K inhibitor development has been limited because of the requirement of this pathway for many essential cellular functions. Identification of the hematopoietic-selective isoform PI3K-δ unlocks a new therapeutic potential for B-cell malignancies. Herein, we demonstrate that PI3K has increased enzymatic activity and that PI3K-δ is expressed in CLL cells. A PI3K-δ selective inhibitor CAL-101 promoted apoptosis in primary CLL cells ex vivo in a dose- and time-dependent fashion that was independent of common prognostic markers. CAL-101-mediated cytotoxicity was caspase dependent and was not diminished by coculture on stromal cells. In addition, CAL-101 abrogated protection from spontaneous apoptosis induced by B cell-activating factors CD40L, TNF-α, and fibronectin. In contrast to malignant cells, CAL-101 does not promote apoptosis in normal T cells or natural killer cells, nor does it diminish antibody-dependent cellular cytotoxicity. However, CAL-101 did decrease activated T-cell production of various inflammatory and antiapoptotic cytokines. Collectively, these studies provide rationale for the clinical development of CAL-101 as a first-in-class targeted therapy for CLL and related B-cell lymphoproliferative disorders.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Inibidores de Fosfoinositídeo-3 Quinase , Purinas/farmacologia , Quinazolinonas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos , Apoptose/efeitos dos fármacos , Linfócitos B/enzimologia , Linfócitos B/patologia , Classe I de Fosfatidilinositol 3-Quinases , Inibidores Enzimáticos/farmacologia , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/patologia , Células Tumorais CultivadasRESUMO
Hypoxia-inducible factor-1α (HIF-1α) plays a critical role in immune and inflammatory responses. One of the HIF-1α target genes is vascular endothelial growth factor (VEGF), which is a potent stimulator of inflammation, airway remodeling, and physiologic dysregulation in allergic airway diseases. Using OVA-treated mice and murine tracheal epithelial cells, the signaling networks involved in HIF-1α activation and the role of HIF-1α in the pathogenesis of allergic airway disease were investigated. Transfection of airway epithelial cells with HIF-1α siRNA suppressed VEGF expression. In addition, the increased levels of HIF-1α and VEGF in lung tissues after OVA inhalation were substantially decreased by an HIF-1α inhibitor, 2-methoxyestradiol. Our data also show that the increased numbers of inflammatory cells, increased airway hyperresponsiveness, levels of IL-4, IL-5, IL-13, and vascular permeability in the lungs after OVA inhalation were significantly reduced by 2-methoxyestradiol or a VEGF inhibitor, CBO-P11. Moreover, we found that inhibition of the PI3K p110δ isoform (PI3K-δ) or HIF-1α reduced OVA-induced HIF-1α activation in airway epithelial cells. These findings indicate that HIF-1α inhibition may attenuate antigen-induced airway inflammation and hyperresponsiveness through the modulation of vascular leakage mediated by VEGF, and that PI3K-δ signaling may be involved in the allergen-induced HIF-1α activation.
Assuntos
Remodelação das Vias Aéreas/imunologia , Asma/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/imunologia , 2-Metoxiestradiol , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/citologia , Fatores de Crescimento Endotelial/farmacologia , Células Epiteliais , Estradiol/análogos & derivados , Estradiol/farmacologia , Feminino , Histocitoquímica , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Peptídeos Cíclicos/farmacologia , Fosfatidilinositol 3-Quinases/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Quinazolinas/farmacologia , RNA/química , RNA/genética , RNA Interferente Pequeno/farmacologia , Testes de Função Respiratória , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/imunologia , Organismos Livres de Patógenos Específicos , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The Class IA phosphoinositide 3-kinase delta (PI3Kδ) has been implicated in multiple signaling pathways involved in leukocyte activation and hence is an attractive target in many human autoimmune diseases, including multiple sclerosis (MS). Here, using mice expressing a catalytically inactive form of the PI3Kδ subunit p110δ, we show that signaling through PI3Kδ is required for full and sustained pathology of experimental autoimmune encephalomyelitis (EAE), a Th17-driven model of MS. In p110δ-inactivated mice, T cell activation and function during EAE was markedly reduced and fewer T cells were observed in the central nervous system (CNS). The decrease in T cell activation is unlikely to be due to defects in dendritic cell (DC) function, as p110δ-inactivated DCs migrate and present antigen normally. However, significant increases in the proportion of T cells undergoing apoptosis at early stages of EAE were evident in the absence of PI3Kδ activity. Furthermore, a profound defect in Th17 cellular responses during EAE was apparent in the absence of PI3Kδ activity while Th1 responses were less affected. A highly selective PI3Kδ inhibitor, IC87114, also had greater inhibitory effects on Th17 cell generation in vitro than it did on Th1 cell generation. Thus, PI3Kδ plays an important role in Th17 responses in EAE, suggesting that small molecule inhibitors of PI3Kδ may be useful therapeutics for treatment of MS and other autoimmune diseases.
Assuntos
Apoptose , Encefalomielite Autoimune Experimental/etiologia , Fosfatidilinositol 3-Quinases/fisiologia , Linfócitos T/fisiologia , Células Th17/citologia , Animais , Diferenciação Celular , Classe I de Fosfatidilinositol 3-Quinases , Encefalomielite Autoimune Experimental/imunologia , Feminino , Memória Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Inibidores de Fosfoinositídeo-3 Quinase , Linfócitos T/citologia , Células Th1/citologiaRESUMO
B-1 and marginal zone (MZ) B cells produce natural Abs, make Ab responses to microbial pathogens, and contribute to autoimmunity. Although the delta isoform of the PI3K p110 catalytic subunit is essential for development of these innate-like B cells, its role in the localization, activation, and function of normal B-1 and MZ B cells is not known. Using IC87114, a highly selective inhibitor of p110delta enzymatic activity, we show that p110delta is important for murine B-1 and MZ B cells to respond to BCR clustering, the TLR ligands LPS and CpG DNA, and the chemoattractants CXCL13 and sphingosine 1-phosphate. In these innate-like B cells, p110delta activity mediates BCR-, TLR- and chemoattractant-induced activation of the Akt prosurvival kinase, chemoattractant-induced migration, and TLR-induced proliferation. Moreover, we found that TLR-stimulated Ab responses by B-1 and MZ B cells, as well as the localization of MZ B cells in the spleen, depend on p110delta activity. Finally, we show that the in vivo production of natural Abs requires p110delta and that p110delta inhibitors can reduce in vivo autoantibody responses. Thus, targeting p110delta may be a novel approach for regulating innate-like B cells and for treating Ab-mediated autoimmune diseases.
Assuntos
Autoanticorpos/biossíntese , Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/imunologia , Isoanticorpos/biossíntese , Fosfatidilinositol 3-Quinases/fisiologia , Baço/imunologia , Adenina/administração & dosagem , Adenina/análogos & derivados , Administração Oral , Animais , Anticorpos Antibacterianos/biossíntese , Subpopulações de Linfócitos B/efeitos dos fármacos , Subpopulações de Linfócitos B/microbiologia , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases , Feminino , Técnicas de Introdução de Genes , Homeostase/efeitos dos fármacos , Homeostase/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Peritônio/citologia , Peritônio/imunologia , Peritônio/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/administração & dosagem , Ratos , Ratos Endogâmicos Lew , Baço/citologia , Baço/efeitos dos fármacos , Baço/enzimologiaRESUMO
BACKGROUND: Recent studies indicate that pharmaceutical blockade of phosphoinositide 3-kinase (PI3K) signaling enzymes might be effective in reducing allergic airway inflammation. Signals generated by the p110delta PI3K isoform play critical roles in signaling through antigen and cytokine receptors and were shown to be required for induction of type 2, but not type 1, cytokine responses. OBJECTIVE: We sought to determine the effect of genetic or pharmaceutical inactivation of p110delta PI3K on induction of IgE responses. METHODS: We determined the effect of p110delta inactivation on induction of systemic IgE responses and on the ability of purified B lymphocytes to undergo IgE isotype switch in vitro. IgG and IgE germline transcription, postswitch transcription, protein expression, and secretion were measured, as well as cell division and expression of activation-induced cytidine deaminase, an enzyme required for isotype switch. RESULTS: Paradoxically, inactivation of p110delta PI3K led to markedly increased IgE responses, despite reduced production of other antibody isotypes. This result was seen by using genetic inactivation of p110delta inhibition with IC87114 compound or blockade with the broad-spectrum PI3K inhibitors PIK-90 and PI-103. Significant increases in IgG1/IgE double-positive cells were observed, indicating that inactivation of PI3K leads to uncontrolled sequential switching from IgG1 to IgE. Disruption of p110delta signaling results in increased germline transcription at the epsilon locus and increased activation-induced cytidine deaminase expression, suggesting deregulation at the level of the isotype switch process. CONCLUSION: Blockade of PI3K signaling leads to markedly enhanced B-cell switch to IgE and increased IgE levels in vivo, despite reduced type 2 cytokine production.
Assuntos
Linfócitos B/enzimologia , Inibidores Enzimáticos/farmacologia , Furanos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Imunoglobulina E/biossíntese , Inibidores de Fosfoinositídeo-3 Quinase , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Células Cultivadas , Classe I de Fosfatidilinositol 3-Quinases , Citidina Desaminase/biossíntese , Citidina Desaminase/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Regulação Enzimológica da Expressão Gênica/genética , Switching de Imunoglobulina/efeitos dos fármacos , Switching de Imunoglobulina/genética , Imunoglobulina E/genética , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
P110delta phosphoinositide 3-kinase (PI3K) plays a pivotal role in the recruitment and activation of certain inflammatory cells. Recent findings revealed that the activity of p110delta also contributes to allergen-IgE-induced mast cell activation and vascular permeability. We investigated the role of p110delta in allergic airway inflammation and hyperresponsiveness using IC87114, a selective p110delta inhibitor, in a mouse asthma model. BALB/c mice were sensitized with OVA and, upon OVA aerosol challenge, developed airway eosinophilia, mucus hypersecretion, elevation in cytokine and chemokine levels, up-regulation of ICAM-1 and VCAM-1 expression, and airway hyperresponsiveness. Intratracheal administration of IC87114 significantly (P<0.05) attenuated OVA-induced influx into lungs of total leukocytes, eosinophils, neutrophils, and lymphocytes, as well as levels of IL-4, IL-5, IL-13, and RANTES in a dose-dependent manner. IC87114 also significantly (P<0.05) reduced the serum levels of total IgE and OVA-specific IgE and LTC(4) release into the airspace. Histological studies show that IC87114 inhibited OVA-induced lung tissue eosinophilia, airway mucus production, and inflammation score. In addition, IC87114 significantly (P<0.05) suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. Western blot analyses of whole lung tissue lysates shows that IC87114 markedly attenuated the OVA-induced increase in expression of IL-4, IL-5, IL-13, ICAM-1, VCAM-1, RANTES, and eotaxin. Furthermore, IC87114 treatment markedly attenuated OVA-induced serine phosphorylation of Akt, a downstream effector of PI3K signaling. Taken together, our findings implicate that inhibition of p110delta signaling pathway may have therapeutic potential for the treatment of allergic airway inflammation.
Assuntos
Adenina/análogos & derivados , Asma/terapia , Inibidores de Fosfoinositídeo-3 Quinase , Quinazolinas/uso terapêutico , Adenina/análise , Adenina/farmacologia , Adenina/uso terapêutico , Animais , Asma/patologia , Hiper-Reatividade Brônquica/patologia , Hiper-Reatividade Brônquica/terapia , Testes de Provocação Brônquica , Líquido da Lavagem Broncoalveolar/química , Moléculas de Adesão Celular/biossíntese , Quimiocinas/biossíntese , Quimiotaxia de Leucócito/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Citocinas/biossíntese , Eosinofilia/etiologia , Eosinofilia/prevenção & controle , Feminino , Pulmão/enzimologia , Cloreto de Metacolina , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Muco/metabolismo , Ovalbumina , Fosfatidilinositol 3-Quinases/fisiologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinazolinas/análise , Quinazolinas/farmacologiaRESUMO
There remains a significant need for development of effective small molecules that can inhibit cytokine-mediated inflammation. Phosphoinositide 3 kinase (PI3K) is a direct upstream activator of AKT, and plays a critical role in multiple cell signaling pathways, cell cycle progression, and cell growth, and PI3K inhibitors have been approved or are in clinical development. We examined novel PI3Kdelta inhibitors, which are highly selective for the p110delta isoform of in CD3/CD28 stimulated T-cell cytokine production. In vitro generated CD4+ T effector cells stimulated in the presence of a PI3Kdelta inhibitor demonstrated a dose-dependent suppression of cytokines produced by Th1, Th2, and Th17 cells. This effect was T-cell intrinsic, and we observed similar effects on human PBMCs. Th17 cells expressing a constitutively activated form of AKT were resistant to PI3Kdelta inhibition, suggesting that the inhibitor is acting through AKT signaling pathways. Additionally, PI3Kdelta inhibition decreased IL-17 production in vivo and decreased neutrophil recruitment to the lung in a murine model of acute pulmonary inflammation. These experiments show that targeting PI3Kdelta activity can modulate T-cell cytokine production and reduce inflammation in vivo, suggesting that PI3Kdelta inhibition could have therapeutic potential in treating inflammatory diseases.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Interleucina-17/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Feminino , Interleucina-17/genética , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de SinaisRESUMO
Aberrant signaling of phosphoinositide 3-kinase δ (PI3Kδ) has been implicated in numerous pathologies including hematological malignancies and rheumatoid arthritis. Described in this manuscript are the discovery, optimization, and in vivo evaluation of a novel series of pyridine-containing PI3Kδ inhibitors. This work led to the discovery of 35, a highly selective inhibitor of PI3Kδ which displays an excellent pharmacokinetic profile and is efficacious in a rodent model of rheumatoid arthritis.
RESUMO
Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) is an appealing target for several hematological malignancies and inflammatory diseases. Herein, we describe the discovery and optimization of a series of propeller shaped PI3Kδ inhibitors comprising a novel triaminopyrimidine hinge binder. Combinations of electronic and structural strategies were employed to mitigate aldehyde oxidase mediated metabolism. This medicinal chemistry effort culminated in the identification of 52, a potent and highly selective inhibitor of PI3Kδ that demonstrates efficacy in a rat model of arthritis.
Assuntos
Artrite Experimental/tratamento farmacológico , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Quinazolinonas/farmacologia , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/enzimologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/enzimologia , Células Cultivadas , Colágeno/toxicidade , Cristalografia por Raios X , Modelos Animais de Doenças , Feminino , Hepatócitos/efeitos dos fármacos , Hepatócitos/enzimologia , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacocinética , Pirimidinas/farmacocinética , Quinazolinonas/química , Quinazolinonas/farmacocinética , Ratos , Ratos Endogâmicos Lew , Distribuição TecidualRESUMO
B-cell receptor (BCR) signaling is essential for normal B-cell development, selection, survival, proliferation, and differentiation into antibody-secreting cells. Similarly, this pathway plays a key role in the pathogenesis of multiple B-cell malignancies. Genetic and pharmacological approaches have established an important role for the Spleen tyrosine kinase (Syk), Bruton's tyrosine kinase (Btk), and phosphatidylinositol 3-kinase isoform p110delta (PI3Kδ) in coupling the BCR and other BCRs to B-cell survival, migration, and activation. In the past few years, several small-molecule inhibitory drugs that target PI3Kδ, Btk, and Syk have been developed and shown to have efficacy in clinical trials for the treatment of several types of B-cell malignancies. Emerging preclinical data have also shown a critical role of BCR signaling in the activation and function of self-reactive B cells that contribute to autoimmune diseases. Because BCR signaling plays a major role in both B-cell-mediated autoimmune inflammation and B-cell malignancies, inhibition of this pathway may represent a promising new strategy for treating these diseases. This review summarizes recent achievements in the mechanism of action, pharmacological properties, and clinical activity and toxicity of these BCR signaling inhibitors, with a focus on their emerging role in treating lymphoid malignancies and autoimmune disorders.
Assuntos
Antineoplásicos/uso terapêutico , Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/metabolismo , Leucemia de Células B/tratamento farmacológico , Leucemia de Células B/metabolismo , Linfoma de Células B/tratamento farmacológico , Linfoma de Células B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos B/fisiologia , HumanosRESUMO
During the progression of autoimmune (type 1) diabetes, T cells and macrophages infiltrate the pancreas, disrupt islet function, and destroy insulin-producing beta cells. B-lymphocytes, particularly innate like B-cell populations such as marginal zone B cells and B-1 cells, have been implicated in many autoimmune diseases, and non-obese diabetic (NOD) mice that lack B cells do not develop spontaneous autoimmune diabetes. Hence, inhibitors of B cell signaling pathways could be useful for limiting the autoimmune processes that contribute to type 1 diabetes. Signaling via phosphoinositide 3-kinase (PI3K) regulates many cellular processes. The p110δ isoform of PI3K is expressed primarily in cells of hematopoietic origin and the catalytic activity of p110δ is important for B cell migration, activation, proliferation, and antigen presentation. Because innate-like B cells are particularly sensitive to inhibition of p110δ activity, and p110δ inhibitors also suppress pro-inflammatory functions of other cell types that contribute to autoimmunity, we tested whether a p110δ inhibitor could delay the onset or reduce the incidence of autoimmune diabetes in NOD mice. We found that long-term preventative treatment of pre-diabetic NOD mice with IC87114, a highly selective small molecule inhibitor of p110δ, reduced the infiltration of inflammatory cells into the pancreatic islets and, accordingly, delayed and reduced the loss of glucose homeostasis. Moreover in a therapeutic treatment mode, IC87114 treatment conferred prolonged protection from progression to overt diabetes in a number of animals. These findings suggest that PI3Kδ inhibitors could be useful for managing type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/enzimologia , Células Secretoras de Insulina/imunologia , Inibidores de Fosfoinositídeo-3 Quinase , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autoimunidade/imunologia , Linfócitos B/enzimologia , Linfócitos B/imunologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/imunologia , Progressão da Doença , Feminino , Citometria de Fluxo , Histocitoquímica , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/imunologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Estatísticas não ParamétricasRESUMO
The delta isoform of the p110 catalytic subunit (p110δ) of phosphoinositide 3-kinase is expressed primarily in hematopoietic cells and plays an essential role in B-cell development and function. Studies employing mice lacking a functional p110δ protein, as well as the use of highly-selective chemical inhibitors of p110δ, have revealed that signaling via p110δ-containing PI3K complexes (PI3Kδ) is critical for B-cell survival, migration, and activation, functioning downstream of key receptors on B cells including the B-cell antigen receptor, chemokine receptors, pro-survival receptors such as BAFF-R and the IL-4 receptor, and co-stimulatory receptors such as CD40 and Toll-like receptors (TLRs). Similarly, this PI3K isoform plays a key role in the survival, proliferation, and dissemination of B-cell lymphomas. Herein we summarize studies showing that these processes can be inhibited in vitro and in vivo by small molecule inhibitors of p110δ enzymatic activity, and that these p110δ inhibitors have shown efficacy in clinical trials for the treatment of several types of B-cell malignancies including chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). PI3Kδ also plays a critical role in the activation, proliferation, and tissue homing of self-reactive B cells that contribute to autoimmune diseases, in particular innate-like B-cell populations such as marginal zone (MZ) B cells and B-1 cells that have been strongly linked to autoimmunity. We discuss the potential utility of p110δ inhibitors, either alone or in combination with B-cell depletion, for treating autoimmune diseases such as lupus, rheumatoid arthritis, and type 1 diabetes. Because PI3Kδ plays a major role in both B-cell-mediated autoimmune inflammation and B-cell malignancies, PI3Kδ inhibitors may represent a promising therapeutic approach for treating these diseases.