RESUMEN
Interleukin receptor associated kinase 4 (IRAK4) plays an important role in innate immune signaling through Toll-like and interleukin-1 receptors and represents an attractive target for the treatment of inflammatory diseases and cancer. We previously reported the development of a potent, selective, and brain-penetrant imidazopyrimidine series of IRAK4 inhibitors. However, lead molecule BIO-7488 (1) suffered from low solubility which led to variable PK, compound accumulation, and poor in vivo tolerability. Herein, we describe the discovery of a series of pyridone analogs with improved solubility which are highly potent, selective and demonstrate desirable PK profiles including good oral bioavailability and excellent brain penetration. BIO-8169 (2) reduced the in vivo production of pro-inflammatory cytokines, was well tolerated in safety studies in rodents and dog at margins well above the predicted efficacious exposure and showed promising results in a mouse model for multiple sclerosis.
Asunto(s)
Encéfalo , Quinasas Asociadas a Receptores de Interleucina-1 , Inhibidores de Proteínas Quinasas , Animales , Perros , Masculino , Ratones , Ratas , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Descubrimiento de Drogas , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Quinasas Asociadas a Receptores de Interleucina-1/antagonistas & inhibidores , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Enfermedades Neuroinflamatorias/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/síntesis química , Pirimidinas/farmacología , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/síntesis química , Pirimidinas/uso terapéutico , Relación Estructura-ActividadRESUMEN
Autoantibodies are a hallmark of numerous neurological disorders, including multiple sclerosis, autoimmune encephalitides and neuromyelitis optica. Whilst well understood in peripheral myeloid cells, the pathophysiological significance of autoantibody-induced Fc receptor signalling in microglia remains unknown, in part due to the lack of a robust in vivo model. Moreover, the application of therapeutic antibodies for neurodegenerative disease also highlights the importance of understanding Fc receptor signalling in microglia. Here, we describe a novel in vivo experimental paradigm that allows for selective engagement of Fc receptors within the CNS by peripherally injecting anti-myelin oligodendrocyte glycoprotein (MOG) monoclonal antibodies into normal wild-type mice. MOG antigen-bound immunoglobulins were detected throughout the CNS and triggered a rapid and tightly regulated proliferative response in both brain and spinal cord microglia. This microglial response was abrogated when anti-MOG antibodies were deprived of Fc receptor effector function or injected into Fcγ receptor knockout mice and was associated with the downregulation of Fc receptors in microglia, but not peripheral myeloid cells, establishing that this response was dependent on central Fc receptor engagement. Downstream of the Fc receptors, BTK was a required signalling node for this response, as microglia proliferation was amplified in BtkE41K knock-in mice expressing a constitutively active form of the enzyme and blunted in mice treated with a CNS-penetrant small molecule inhibitor of BTK. Finally, this response was associated with transient and stringently regulated changes in gene expression predominantly related to cellular proliferation, which markedly differed from transcriptional programs typically associated with Fc receptor engagement in peripheral myeloid cells. Together, these results establish a physiologically-meaningful functional response to Fc receptor and BTK signalling in microglia, while providing a novel in vivo tool to further dissect the roles of microglia-specific Fc receptor and BTK-driven responses to both pathogenic and therapeutic antibodies in CNS homeostasis and disease.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/metabolismo , Autoanticuerpos/inmunología , Encéfalo/patología , Microglía/patología , Glicoproteína Mielina-Oligodendrócito/inmunología , Receptores Fc/metabolismo , Médula Espinal/patología , Animales , Encéfalo/inmunología , Encéfalo/metabolismo , Proliferación Celular/fisiología , Ratones , Microglía/inmunología , Microglía/metabolismo , Médula Espinal/inmunología , Médula Espinal/metabolismoRESUMEN
Objective: Plasmacytoid dendritic cells (pDCs) are a major source of Type-I Interferon (IFN-I), a key driver in cutaneous lupus erythematosus (CLE). Currently evaluated in Phase II clinical trial, 24F4A (BIIB059) is an antibody targeting BDCA2, an inhibitory receptor expressed on pDCs. Given that Hydroxychloroquine (HCQ), a widely-used CLE therapy, and 24F4A are both able to inhibit pDC-derived IFN-I production; this study aimed to determine whether 24F4A would show an additional inhibitory effect on pDC response after ex vivo or in vivo treatment with HCQ. Methods: The effect of 24F4A on pDC-derived IFNα was measured from peripheral blood mononuclear cells (PBMC) either from healthy donors in presence or absence of HCQ or from CLE patients clinically exposed to various levels of HCQ. TLR7, TLR7/8, and TLR9 agonists (ssRNA, R848, and CpG-A) were used for pDC stimulation. Results: PDCs were the only producers of IFNα in response to CpG-A, R848, and ssRNA stimulation in PBMC cultures. CLE patients with higher levels of blood HCQ showed lower ex vivo pDC responses to CpG-A, but not R848 or ssRNA. In contrast, 24F4A reduced the amount of IFNα produced by pDCs from CLE patients in response to all TLR agonists, irrespective of the blood HCQ level. Conclusion: Our findings reveal that clinically-relevant HCQ concentrations partially inhibit the pDC response to TLR9 and weakly affect the response to TLR7/8 stimulation. 24F4A robustly inhibits pDC responses even in the presence of HCQ, highlighting its unique potential to disrupt pDC disease relevant biology, which could provide additional therapeutic benefit for CLE patients.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Células Dendríticas/efectos de los fármacos , Hidroxicloroquina/uso terapéutico , Interferón-alfa/metabolismo , Lectinas Tipo C/metabolismo , Lupus Eritematoso Cutáneo/tratamiento farmacológico , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Adulto , Células Dendríticas/metabolismo , Femenino , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Lupus Eritematoso Cutáneo/metabolismo , Lupus Eritematoso Sistémico/tratamiento farmacológico , Lupus Eritematoso Sistémico/metabolismo , Masculino , Persona de Mediana Edad , Receptores Toll-Like/metabolismoRESUMEN
BACKGROUND: Resected HER2 breast cancer patients treated with adjuvant trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. We previously showed that trastuzumab and chemotherapy induce HER2-specific antibodies which correlate with improved survival in HER2 metastatic breast cancer patients. It remains unclear whether the generation of immunity required trastuzumab and whether endogenous antibody immunity is associated with improved disease-free survival in the adjuvant setting. In this study, we addressed this question by analyzing serum anti-HER2 antibodies from a subset of patients enrolled in the NCCTG trial N9831, which includes an arm (Arm A) in which trastuzumab was not used. Arms B and C received trastuzumab sequentially or concurrently to chemotherapy, respectively. METHODS: Pre-and post-treatment initiation sera were obtained from 50 women enrolled in N9831. Lambda IgG antibodies (to avoid detection of trastuzumab) to HER2 were measured and compared between arms and with disease-free survival. RESULTS: Prior to therapy, across all three arms, N9831 patients had similar mean anti-HER2 IgG levels. Following treatment, the mean levels of antibodies increased in the trastuzumab arms but not the chemotherapy-only arm. The proportion of patients who demonstrated antibodies increased by 4% in Arm A and by 43% in the Arms B and C combined (p = 0.003). Cox modeling demonstrated that larger increases in antibodies were associated with improved disease-free survival in all patients (HR = 0.23; p = 0.04). CONCLUSIONS: These results show that the increased endogenous antibody immunity observed in adjuvant patients treated with combination trastuzumab and chemotherapy is clinically significant, in view of its correlation with improved disease-free survival. The findings may have important implications for predicting treatment outcomes in patients treated with trastuzumab in the adjuvant setting. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00005970 . Registered on July 5, 2000.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Recurrencia Local de Neoplasia/tratamiento farmacológico , Receptor ErbB-2/inmunología , Trastuzumab/administración & dosificación , Adulto , Anciano , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Biomarcadores de Tumor/genética , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Quimioterapia Adyuvante/efectos adversos , Terapia Combinada , Supervivencia sin Enfermedad , Femenino , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Recurrencia , Trastuzumab/efectos adversos , Resultado del TratamientoRESUMEN
Entry of lymphocytes into secondary lymphoid organs (SLOs) involves intravascular arrest and intracellular calcium ion ([Ca(2+)]i) elevation. TCR activation triggers increased [Ca(2+)]i and can arrest T-cell motility in vitro. However, the requirement for [Ca(2+)]i elevation in arresting T cells in vivo has not been tested. Here, we have manipulated the Ca(2+) release-activated Ca(2+) (CRAC) channel pathway required for [Ca(2+)]i elevation in T cells through genetic deletion of stromal interaction molecule (STIM) 1 or by expression of a dominant-negative ORAI1 channel subunit (ORAI1-DN). Interestingly, the absence of CRAC did not interfere with homing of naïve CD4(+) T cells to SLOs and only moderately reduced crawling speeds in vivo. T cells expressing ORAI1-DN lacked TCR activation induced [Ca(2+)]i elevation, yet arrested motility similar to control T cells in vitro. In contrast, antigen-specific ORAI1-DN T cells had a twofold delayed onset of arrest following injection of OVA peptide in vivo. CRAC channel function is not required for homing to SLOs, but enhances spatiotemporal coordination of TCR signaling and motility arrest.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Canales de Calcio/inmunología , Calcio/inmunología , Glicoproteínas de Membrana/inmunología , Animales , Canales de Calcio/genética , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Proteína ORAI1 , Péptidos/inmunología , Péptidos/farmacología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Molécula de Interacción Estromal 1RESUMEN
Lymphocytes mediate cytotoxicity by polarized release of the contents of cytotoxic granules toward their target cells. Here, we have studied the role of the calcium release-activated calcium channel ORAI1 in human lymphocyte cytotoxicity. Natural killer (NK) cells obtained from an ORAI1-deficient patient displayed defective store-operated Ca(2+) entry (SOCE) and severely defective cytotoxic granule exocytosis leading to impaired target cell lysis. Similar findings were obtained using NK cells from a stromal interaction molecule 1-deficient patient. The defect occurred at a late stage of the signaling process, because activation of leukocyte functional antigen (LFA)-1 and cytotoxic granule polarization were not impaired. Moreover, pharmacological inhibition of SOCE interfered with degranulation and target cell lysis by freshly isolated NK cells and CD8(+) effector T cells from healthy donors. In addition to effects on lymphocyte cytotoxicity, synthesis of the chemokine macrophage inflammatory protein-1ß and the cytokines TNF-α and IFN-γ on target cell recognition was impaired in ORAI1-deficient NK cells, as previously described for T cells. By contrast, NK cell cytokine production induced by combinations of IL-12, IL-15, and IL-18 was not impaired by ORAI1 deficiency. Taken together, these results identify a critical role for ORAI1-mediated Ca(2+) influx in granule exocytosis for lymphocyte cytotoxicity as well as for cytokine production induced by target cell recognition.
Asunto(s)
Canales de Calcio/inmunología , Calcio/inmunología , Degranulación de la Célula/inmunología , Citocinas/biosíntesis , Citotoxicidad Inmunológica , Linfocitos T Citotóxicos/inmunología , Quimiocina CCL4/biosíntesis , Humanos , Interferón gamma/biosíntesis , Interleucinas/biosíntesis , Células Asesinas Naturales/patología , Proteína ORAI1 , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
T-cell function is dependent on store-operated Ca(2+) influx that is activated by the stromal interaction molecules (STIM) 1 and 2. We show that mice with T-cell-specific deletion of STIM1 or STIM2 are protected from EAE, a mouse model of multiple sclerosis (MS). While STIM1- and STIM2-deficient T cells could be successfully primed by autoantigen, they failed to produce the proinflammatory cytokines IL-17 and IFN-γ. STIM1-deficient T cells showed reduced expression of IL-23R, required for Th17 cell homeostasis, and had impaired chemokine-dependent T-cell migration caused by a lack of chemokine-induced Ca(2+) influx. Autoantigen-specific STIM1- or STIM2-deficient T cells failed to expand and accumulate in the CNS and lymph nodes following adoptive transfer to passively induce EAE, suggesting that autoantigen-specific restimulation or homeostasis of STIM1- and STIM2-deficient T cells are impaired. Combined deletion of both STIM1 and STIM2, previously shown to impair Treg development and function, completely protected mice from EAE. This indicates that, in the absence of Ca(2+) influx, autoreactive T cells are severely dysfunctional rendering Treg dispensable for the prevention of CNS inflammation. Our findings demonstrate that both STIM1 and STIM2 are critical for T-cell function and autoimmunity in vivo.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Glicoproteínas de Membrana/inmunología , Esclerosis Múltiple/inmunología , Células TH1/inmunología , Células Th17/inmunología , Animales , Calcio/inmunología , Calcio/metabolismo , Canales de Calcio , Movimiento Celular/genética , Movimiento Celular/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Eliminación de Gen , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Esclerosis Múltiple/genética , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2 , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Células TH1/metabolismo , Células TH1/patología , Células Th17/metabolismo , Células Th17/patologíaRESUMEN
ORAI1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, which is responsible for store-operated Ca(2+) entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1(-/-) mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1(KI/KI)) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1(KI/KI) mice die neonatally, but Orai1(KI/KI) fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1(KI/KI) mice display severely impaired store-operated Ca(2+) entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1(KI/KI) mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1(KI/KI) mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity.
Asunto(s)
Autoinmunidad/inmunología , Canales de Calcio/metabolismo , Calcio/metabolismo , Rechazo de Injerto/metabolismo , Linfocitos T/inmunología , Animales , Canales de Calcio/genética , Canales de Calcio/inmunología , Señalización del Calcio/genética , Señalización del Calcio/inmunología , Separación Celular , Citocinas/biosíntesis , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Técnicas de Sustitución del Gen , Rechazo de Injerto/inmunología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Mutación , Proteína ORAI1 , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/metabolismo , Quimera por Trasplante , Trasplante HomólogoRESUMEN
BACKGROUND: Defects in the development or activation of T cells result in immunodeficiency associated with severe infections early in life. T-cell activation requires Ca2+ influx through Ca2+-release activated Ca2+ (CRAC) channels encoded by the gene ORAI1. OBJECTIVE: Investigation of the genetic causes and the clinical phenotype of immunodeficiency in patients with impaired Ca2+ influx and CRAC channel function. METHODS: DNA sequence analysis for mutations in the genes ORAI1, ORAI2, ORAI3, and stromal interaction molecule (STIM) 1 and 2, as well as mRNA and protein expression analysis of ORAI1 in immunodeficient patients. Immunohistochemical analysis of ORAI1 tissue distribution in healthy human donors. RESULTS: We identified mutations in ORAI1 in patients from 2 unrelated families. One patient is homozygous for a frameshift nonsense mutation in ORAI1 (ORAI1-A88SfsX25), and a second patient is compound heterozygous for 2 missense mutations in ORAI1 (ORAI1-A103E/L194P). All 3 mutations abolish ORAI1 expression and impair Ca2+ influx and CRAC channel function. The clinical syndrome associated with ORAI1 deficiency is characterized by immunodeficiency with a defect in the function but not in the development of lymphocytes, congenital myopathy, and anhydrotic ectodermal dysplasia with a defect in dental enamel calcification. In contrast with the limited clinical phenotype, we found ORAI1 protein expression in a wide variety of cell types and organs. CONCLUSION: Ca2+ influx through ORAI1 is crucial for lymphocyte function in vivo. Despite almost ubiquitous ORAI1 expression, the channel has a nonredundant role in only a few cell types judging from the limited clinical phenotype in ORAI1-deficient patients.
Asunto(s)
Canales de Calcio/deficiencia , Displasia Ectodérmica/metabolismo , Síndromes de Inmunodeficiencia/metabolismo , Enfermedades Musculares/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Displasia Ectodérmica/genética , Displasia Ectodérmica/patología , Mutación del Sistema de Lectura , Homocigoto , Humanos , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Enfermedades Musculares/genética , Enfermedades Musculares/patología , Mutación Missense , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Proteína ORAI2 , Molécula de Interacción Estromal 1 , Molécula de Interacción Estromal 2 , TransfecciónRESUMEN
A mutation in ORAI1, the gene encoding the pore-forming subunit of the Ca(2+)-release-activated Ca(2+) (CRAC) channel, abrogates the store-operated entry of Ca(2+) into cells and impairs lymphocyte activation. Stromal interaction molecule 1 (STIM1) in the endoplasmic reticulum activates ORAI1-CRAC channels. We report on three siblings from one kindred with a clinical syndrome of immunodeficiency, hepatosplenomegaly, autoimmune hemolytic anemia, thrombocytopenia, muscular hypotonia, and defective enamel dentition. Two of these patients have a homozygous nonsense mutation in STIM1 that abrogates expression of STIM1 and Ca(2+) influx.
Asunto(s)
Anemia Hemolítica Autoinmune/genética , Codón sin Sentido , Síndromes de Inmunodeficiencia/genética , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Púrpura Trombocitopénica Idiopática/genética , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/metabolismo , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Niño , Resultado Fatal , Femenino , Humanos , Síndromes de Inmunodeficiencia/metabolismo , Lactante , Masculino , Linaje , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Hermanos , Molécula de Interacción Estromal 1 , SíndromeRESUMEN
The intracellular Ca(2+) concentration of many nonexcitable cells is regulated by calcium store release and store-operated calcium entry (SOCE). In platelets, STIM1 was recently identified as the main calcium sensor expressed in the endoplasmic reticulum. To evaluate the role of the SOC channel moiety, Orai1, in platelet SOCE, we generated mice expressing a mutated, inactive form of Orai1 in blood cells only (Orai1(R93W)). Platelets expressing Orai1(R93W) were characterized by markedly reduced SOCE and impaired agonist-induced increases in [Ca(2+)](i). Orai1(R93W) platelets showed reduced integrin activation and impaired degranulation when stimulated with low agonist concentrations under static conditions. This defect, however, did not significantly affect the ability of Orai1(R93W) platelets to aggregate or to adhere to collagen under arterial flow conditions ex vivo. In contrast, these adherent Orai1(R93W) platelets were defective in surface phosphatidylserine exposure, suggesting that Orai1 is crucial for the platelets' procoagulant response rather than for other Ca(2+)-dependent cellular responses.