RESUMEN
While the rapid advancement of immunotherapies has revolutionized cancer treatment, only a small fraction of patients derive clinical benefit. Eradication of large, established tumors appears to depend on engaging and activating both innate and adaptive immune system components to mount a rigorous and comprehensive immune response. Identifying such agents is a high unmet medical need, because they are sparse in the therapeutic landscape of cancer treatment. Here, we report that IL-36 cytokine can engage both innate and adaptive immunity to remodel an immune-suppressive tumor microenvironment (TME) and mediate potent antitumor immune responses via signaling in host hematopoietic cells. Mechanistically, IL-36 signaling modulates neutrophils in a cell-intrinsic manner to greatly enhance not only their ability to directly kill tumor cells but also promote T and NK cell responses. Thus, while poor prognostic outcomes are typically associated with neutrophil enrichment in the TME, our results highlight the pleiotropic effects of IL-36 and its therapeutic potential to modify tumor-infiltrating neutrophils into potent effector cells and engage both the innate and adaptive immune system to achieve durable antitumor responses in solid tumors.
Asunto(s)
Inmunidad Adaptativa , Neutrófilos , Humanos , Citocinas , Terapia de Inmunosupresión , InmunoterapiaRESUMEN
BACKGROUND: Checkpoint inhibitors targeting cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) have demonstrated clinical efficacy in advanced melanoma, but only a subset of patients with inflamed tumors are responsive. Talimogene laherparepvec (T-VEC), a modified herpes simplex virus type 1 (HSV-1) expressing granulocyte-macrophage colony-stimulating factor (GM-CSF), is a first-in-class oncolytic immunotherapy approved for the treatment of melanoma and has been shown to inflame the tumor microenvironment. To evaluate the potential and mechanisms of T-VEC to elicit systemic antitumor immunity and overcome resistance to checkpoint inhibitors in murine tumor models, OncoVEXmGM-CSF was developed similarly to T-VEC, except the human GM-CSF transgene was replaced with murine GM-CSF. Previous work had demonstrated that OncoVEXmGM-CSF generated systemic antitumor immunity dependent on CD8+ T cells in an immune checkpoint-sensitive tumor cell model. METHODS: A novel B16F10 syngeneic tumor model with both HSV-1-permissive subcutaneous tumors and HSV-1-refractory experimental lung metastasis was used to study the local and systemic effects of OncoVEXmGM-CSF treatment alone or in combination with checkpoint inhibitors. RESULTS: Intratumoral injection of OncoVEXmGM-CSF in combination with an anti-CTLA-4 or anti-PD-1 blocking antibody led to increased tumor growth inhibition, a reduction in the number of lung metastases, and prolonged animal survival. OncoVEXmGM-CSF induced both neoantigen-specific and tumor antigen-specific T-cell responses. Furthermore, cured mice from the combination treatment of OncoVEXmGM-CSF and anti-CTLA-4 antibody rejected tumor rechallenges. CONCLUSIONS: These data support the concept that T-VEC and checkpoint inhibition may be an effective combination to treat patients with advanced melanoma.
Asunto(s)
Melanoma , Viroterapia Oncolítica , Humanos , Animales , Ratones , Factor Estimulante de Colonias de Granulocitos y Macrófagos , Linfocitos T CD8-positivos/patología , Antígenos de Neoplasias , Microambiente TumoralRESUMEN
Type I interferon-mediated activation of immune cells can facilitate the generation of productive tumor antigen-specific T cell responses in solid tumors. The cGAS/STING DNA sensing pathway is a critical upstream mediator of type I interferon production and is an important regulator of anti-tumor immunity. Numerous STING pathway agonists are now being tested in clinical trials, but the effectiveness of this approach is not yet clear and a better understanding of the relative importance of this pathway in various tumor settings is needed. We have evaluated syngeneic tumor models with different baseline inflammatory states to determine the contributions of STING activity in both tumor and non-tumor cellular compartments to anti-tumor immune responses. We find that productive anti-tumor immune responses in the poorly immunogenic B16F10 model show a strong dependence on STING expression in non-tumor cells. In the immunogenic MC38 model, constitutive STING activation in tumor cells can partially bypass the requirement for STING-dependent activity from immune cells. Our findings reveal multiple, context-dependent roles for STING activity in the regulation of anti-tumor immunity and the response to immunotherapy. In preclinical models where STING is basally active, checkpoint inhibition is more likely to have a therapeutic effect and removal of STING signaling from either the tumor or the non-tumor compartment has a minimal effect. Removal of STING signaling in both, however, diminishes the efficacy derived from checkpoint therapy. Further work is needed to understand the heterogeneity of STING signaling in patients, both in tumor cells and the tumor microenvironment, and the best means of harnessing this pathway to generate anti-tumor immunity and improve therapeutic outcomes.
Asunto(s)
Interferón Tipo I , Neoplasias , Humanos , ADN , Inmunidad Innata , Inmunoterapia , Transducción de Señal , Microambiente TumoralRESUMEN
Therapeutic approaches are needed to promote T cell-mediated destruction of poorly immunogenic, "cold" tumors typically associated with minimal response to immune checkpoint blockade (ICB) therapy. Bispecific T cell engager (BiTE) molecules induce redirected lysis of cancer cells by polyclonal T cells and have demonstrated promising clinical activity against solid tumors in some patients. However, little is understood about the key factors that govern clinical responses to these therapies. Using an immunocompetent mouse model expressing a humanized CD3ε chain (huCD3e mice) and BiTE molecules directed against mouse CD19, mouse CLDN18.2, or human EPCAM antigens, we investigated the pharmacokinetic and pharmacodynamic parameters and immune correlates associated with BiTE efficacy across multiple syngeneic solid-tumor models. These studies demonstrated that pretreatment tumor-associated T cell density is a critical determinant of response to BiTE therapy, identified CD8+ T cells as important targets and mediators of BiTE activity, and revealed an antagonistic role for CD4+ T cells in BiTE efficacy. We also identified therapeutic combinations, including ICB and 4-1BB agonism, that synergized with BiTE treatment in poorly T cell-infiltrated, immunotherapy-refractory tumors. In these models, BiTE efficacy was dependent on local expansion of tumor-associated CD8+ T cells, rather than their recruitment from circulation. Our findings highlight the relative contributions of baseline T cell infiltration, local T cell proliferation, and peripheral T cell trafficking for BiTE molecule-mediated efficacy, identify combination strategies capable of overcoming resistance to BiTE therapy, and have clinical relevance for the development of BiTE and other T cell engager therapies.
Asunto(s)
Anticuerpos Biespecíficos , Neoplasias , Animales , Anticuerpos Biespecíficos/uso terapéutico , Antígenos CD19 , Complejo CD3 , Linfocitos T CD8-positivos , Claudinas , Humanos , Inmunoterapia , Ratones , Neoplasias/tratamiento farmacológicoRESUMEN
Single-cell RNA sequencing is a powerful tool to examine cellular heterogeneity, novel markers and target genes, and therapeutic mechanisms in human cancers and animal models. Here, we analyzed single-cell RNA sequencing data of T cells obtained from multiple mouse tumor models by PCA-based subclustering coupled with TCR tracking using the STARTRAC algorithm. This approach revealed various differentiated T cell subsets and activation states, and a correspondence of T cell subsets between human and mouse tumors. STARTRAC analyses demonstrated peripheral T cell subsets that were developmentally connected with tumor-infiltrating CD8+ cells, CD4+ Th1 cells, and T reg cells. In addition, large amounts of paired TCRα/ß sequences enabled us to identify a specific enrichment of paired public TCR clones in tumor. Finally, we identified CCR8 as a tumor-associated T reg cell marker that could preferentially deplete tumor-associated T reg cells. We showed that CCR8-depleting antibody treatment provided therapeutic benefit in CT26 tumors and synergized with anti-PD-1 treatment in MC38 and B16F10 tumor models.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Linfocitos Infiltrantes de Tumor/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptor de Muerte Celular Programada 1/inmunología , Células TH1/inmunologíaRESUMEN
Tumor-associated macrophages (TAMs) are abundant in solid tumors where they exhibit immunosuppressive and pro-tumorigenic functions. Inhibition of TAM proliferation and survival through CSF1R blockade has been widely explored as a cancer immunotherapy. To further define mechanisms regulating CSF1R-targeted therapies, we systematically evaluated the effect of anti-CSF1R treatment on tumor growth and tumor microenvironment (TME) inflammation across multiple murine models. Despite substantial macrophage depletion, anti-CSF1R had minimal effects on the anti-tumor immune response in mice bearing established tumors. In contrast, anti-CSF1R treatment concurrent with tumor implantation resulted in more robust tumor growth inhibition and evidence of enhanced anti-tumor immunity. Our findings suggest only minor contributions of CSF1R-dependent TAMs to the inflammatory state of the TME in established tumors, that immune landscape heterogeneity across different tumor models can influence anti-CSF1R activity, and that alternative treatment schedules and/or TAM depletion strategies may be needed to maximize the clinical benefit of this approach.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias/inmunología , Neoplasias/terapia , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Macrófagos Asociados a Tumores/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Inmunoterapia/métodos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Macrófagos Asociados a Tumores/inmunologíaRESUMEN
Inhibitors that block the programmed cell death-1 (PD-1) pathway can potentiate endogenous antitumor immunity and have markedly improved cancer survival rates across a broad range of indications. However, these treatments work for only a minority of patients. The efficacy of anti-PD-1 inhibitors may be extended by cytokines, however, the incorporation of cytokines into therapeutic regimens has significant challenges. In their natural form when administered as recombinant proteins, cytokine treatments are often associated with low response rates. Most cytokines have a short half-life which limits their exposure and efficacy. In addition, cytokines can activate counterregulatory pathways, in the case of immune-potentiating cytokines this can lead to immune suppression and thereby diminish their potential efficacy. Improving the drug-like properties of natural cytokines using protein engineering can yield synthetic cytokines with improved bioavailability and tissue targeting, allowing for enhanced efficacy and reduced off-target effects. Using structure guided engineering we have designed a novel class of antibody-cytokine fusion proteins consisting of a PD-1 targeting antibody fused together with an interleukin-21 (IL-21) cytokine mutein. Our bifunctional fusion proteins can block PD-1/programmed death-ligand 1 (PD-L1) interaction whilst simultaneously delivering IL-21 cytokine to PD-1 expressing T cells. Targeted delivery of IL-21 can improve T cell function in a manner that is superior to anti-PD-1 monotherapy. Fusion of engineered IL-21 variants to anti-PD1 antibodies can improve the drug-like properties of IL-21 cytokine leading to improved cytokine serum half-life allowing for less frequent dosing. In addition, we show that targeted delivery of IL-21 can minimize any potential detrimental effect on local antigen-presenting cells. A highly attenuated IL-21 mutein variant (R9E:R76A) fused to a PD-1 antibody provides protection in a humanized mouse model of cancer that is refractory to anti-PD-1 monotherapy. Collectively, our preclinical data demonstrate that this approach may improve upon and extend the utility of anti-PD-1 therapeutics currently in the clinic.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Linfocitos T CD8-positivos/inmunología , Inmunoterapia , Interleucinas/uso terapéutico , Neoplasias/terapia , Animales , Anticuerpos Monoclonales/inmunología , Antígeno B7-H1/inmunología , Modelos Animales de Enfermedad , Femenino , Humanos , Interleucinas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Neoplasias/inmunología , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéuticoRESUMEN
Bone marrow chimeras represent a key tool employed to understand biological contributions stemming from the hematopoietic versus the stromal compartment. In most institutions, cesium irradiators are used to lethally irradiate recipient animals prior to the injection of donor bone marrow. Cesium irradiators, however, have significant liabilities-including concerns around domestic security. Recently, X-ray irradiators have been implemented as a potential alternative to cesium sources. Only a small number of publications in the literature have attempted to compare these two modalities and, in most cases, the emphasis was on irradiation of human blood productions. We were able to find only a single study that directly compared X-ray and cesium technologies in the generation of murine bone marrow chimeras, a standard laboratory practice. This study focused on chimerism in the blood of recipient animals. In the present study, we begin by comparing cesium and X-ray based sources for irradiation, then transition to using X-ray-based systems for immunology models with an emphasis on immunotherapy of cancer in immunocompetent mouse models-specifically evaluating chimerism in the blood, spleen, and tumor microenvironment. While our data demonstrate that the two platforms are functionally comparable and suggest that X-ray based technology is a suitable alternative to cesium sources. We also highlight a difference in chimerism between the peripheral (blood, spleen) and tumor compartments that is observed using both technologies. While the overall degree of chimerism in the peripheral tissues is very high, the degree of chimerism in the tumor is cell type specific with T and NK cells showing lower chimerism than other cell types.
RESUMEN
Colony-stimulating factor 1 (CSF1) and interleukin 34 (IL34) signal via the CSF1 receptor to regulate macrophage differentiation. Studies in IL34- or CSF1-deficient mice have revealed that IL34 function is limited to the central nervous system and skin during development. However, the roles of IL34 and CSF1 at homeostasis or in the context of inflammatory diseases or cancer in wild-type mice have not been clarified in vivo. By neutralizing CSF1 and/or IL34 in adult mice, we identified that they play important roles in macrophage differentiation, specifically in steady-state microglia, Langerhans cells, and kidney macrophages. In several inflammatory models, neutralization of both CSF1 and IL34 contributed to maximal disease protection. However, in a myeloid cell-rich tumor model, CSF1 but not IL34 was required for tumor-associated macrophage accumulation and immune homeostasis. Analysis of human inflammatory conditions reveals IL34 upregulation that may account for the protection requirement of IL34 blockade. Furthermore, evaluation of IL34 and CSF1 blockade treatment during Listeria infection reveals no substantial safety concerns. Thus, IL34 and CSF1 play non-redundant roles in macrophage differentiation, and therapeutic intervention targeting IL34 and/or CSF1 may provide an effective treatment in macrophage-driven immune-pathologies.
Asunto(s)
Homeostasis/inmunología , Inflamación/inmunología , Interleucinas/inmunología , Factor Estimulante de Colonias de Macrófagos/inmunología , Macrófagos/inmunología , Neoplasias/inmunología , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Modelos Animales de Enfermedad , Homeostasis/genética , Humanos , Inflamación/genética , Inflamación/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Factor Estimulante de Colonias de Macrófagos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos NZB , Ratones Noqueados , Células Mieloides/inmunología , Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismoRESUMEN
IL-17 has been implicated in the pathogenesis of multiple sclerosis (MS). Here, we show that blockade of IL-17A, but not IL-17F, attenuated experimental autoimmune encephalomyelitis (EAE). We further show that IL-17A levels were elevated in the CSF of relapsing-remitting MS (RRMS) patients and that they correlated with the CSF/serum albumin quotient (Qalb), a measure of blood-brain barrier (BBB) dysfunction. We then demonstrated that the combination of IL-17A and IL-6 reduced the expression of tight junction (TJ)-associated genes and disrupted monolayer integrity in the BBB cell line hCMEC/D3. However, unlike IL-17A, IL-6 in the CSF from RRMS patients did not correlate with Qalb. These data highlight the potential importance of targeting IL-17A in preserving BBB integrity in RRMS.
Asunto(s)
Barrera Hematoencefálica , Encefalomielitis Autoinmune Experimental/fisiopatología , Interleucina-17/fisiología , Esclerosis Múltiple Recurrente-Remitente/fisiopatología , Adulto , Anciano , Animales , Células Cultivadas , Encefalomielitis Autoinmune Experimental/terapia , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Femenino , Humanos , Inmunización Pasiva , Interleucina-17/antagonistas & inhibidores , Interleucina-17/líquido cefalorraquídeo , Interleucina-6/farmacología , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/líquido cefalorraquídeo , Receptores de Interleucina-6/fisiología , Proteínas Recombinantes/farmacología , Adulto JovenRESUMEN
Loss of function of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) is associated with a wide spectrum of cancers. We report that tamoxifen-induced BAP1 deletion in adult mice resulted in severe thymic atrophy. BAP1 was critical for T cell development at several stages. In the thymus, BAP1 was required for progression through the pre-T cell receptor checkpoint. Peripheral T cells lacking BAP1 demonstrated a defect in homeostatic and antigen-driven expansion. Deletion of BAP1 resulted in suppression of E2F target genes and defects in cell cycle progression, which was dependent on the catalytic activity of BAP1, but did not require its interaction with host cell factor-1 (HCF-1). Loss of BAP1 led to increased monoubiquitination of histone H2A at Lys119 (H2AK119ub) throughout the T cell lineage, in particular in immature thymocytes, but did not alter trimethylation of histone H3 at Lys27 (H3K27me3). Deletion of BAP1 also abrogated B cell development in the bone marrow. Our findings uncover a nonredundant function for BAP1 in maintaining the lymphoid lineage.
Asunto(s)
Linfocitos T/metabolismo , Timocitos/metabolismo , Timo/metabolismo , Proteínas Supresoras de Tumor/genética , Ubiquitina Tiolesterasa/genética , Animales , Atrofia , Ciclo Celular/genética , Perfilación de la Expresión Génica , Histonas/genética , Histonas/metabolismo , Lisina/genética , Lisina/metabolismo , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Timo/patología , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/metabolismo , UbiquitinaciónRESUMEN
Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.
Asunto(s)
Aminoquinolinas/farmacología , Inflamación/prevención & control , Proteína Adaptadora de Señalización NOD2/antagonistas & inhibidores , Dominios y Motivos de Interacción de Proteínas/efectos de los fármacos , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/metabolismo , Sulfonas/farmacología , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Animales , Células Cultivadas , Humanos , Inflamación/metabolismo , Inflamación/patología , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Fosforilación , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor/antagonistas & inhibidores , Transducción de Señal , Ubiquitina/metabolismo , Ubiquitinación , Proteína Inhibidora de la Apoptosis Ligada a X/antagonistas & inhibidoresRESUMEN
NF-κB-inducing kinase (NIK) mediates non-canonical NF-κB signaling downstream of multiple TNF family members, including BAFF, TWEAK, CD40, and OX40, which are implicated in the pathogenesis of systemic lupus erythematosus (SLE). Here, we show that experimental lupus in NZB/W F1 mice can be treated with a highly selective and potent NIK small molecule inhibitor. Both in vitro as well as in vivo, NIK inhibition recapitulates the pharmacological effects of BAFF blockade, which is clinically efficacious in SLE. Furthermore, NIK inhibition also affects T cell parameters in the spleen and proinflammatory gene expression in the kidney, which may be attributable to inhibition of OX40 and TWEAK signaling, respectively. As a consequence, NIK inhibition results in improved survival, reduced renal pathology, and lower proteinuria scores. Collectively, our data suggest that NIK inhibition is a potential therapeutic approach for SLE.
Asunto(s)
Linfocitos B/efectos de los fármacos , Riñón/efectos de los fármacos , Lupus Eritematoso Sistémico/inmunología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Linfocitos T/efectos de los fármacos , Animales , Linfocitos B/inmunología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocina TWEAK/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Humanos , Técnicas In Vitro , Inflamación/genética , Subunidad p40 de la Interleucina-12/efectos de los fármacos , Subunidad p40 de la Interleucina-12/inmunología , Riñón/inmunología , Riñón/patología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Nefritis Lúpica/inmunología , Nefritis Lúpica/patología , Ratones , Ratones Endogámicos NZB , Terapia Molecular Dirigida , Proteinuria/inmunología , Receptores OX40/metabolismo , Transducción de Señal , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T/inmunología , Quinasa de Factor Nuclear kappa BRESUMEN
Herein we report identification of an imidazopyridine class of potent and selective TYK2 inhibitors, exemplified by prototype 6, through constraint of the rotatable amide bond connecting the pyridine and aryl rings of compound 1. Further optimization led to generation of compound 30 that potently inhibits the TYK2 enzyme and the IL-23 pathway in cells, exhibits selectivity against cellular JAK2 activity, and has good pharmacokinetic properties. In mice, compound 30 demonstrated dose-dependent reduction of IL-17 production in a PK/PD model as well as in an imiquimod-induced psoriasis model. In this efficacy model, the IL-17 decrease was accompanied by a reduction of ear thickness indicating the potential of TYK2 inhibition as a therapeutic approach for psoriasis patients.
Asunto(s)
Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , TYK2 Quinasa/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Humanos , Imidazoles/síntesis química , Imidazoles/química , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Piridinas/síntesis química , Piridinas/química , Relación Estructura-Actividad , TYK2 Quinasa/metabolismoRESUMEN
Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activate the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) in macrophages stimulated through the interleukin-1 receptor (IL-1R), Toll-like receptors (TLRs), or the tumor necrosis factor receptor (TNFR). We describe a conserved and critical role for TPL2 in mediating the effector functions of neutrophils through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Biochemical analyses identified the MAPK kinases MEK3 and MEK6 and the MAPKs p38α and p38δ as downstream effectors of TPL2 in these cells. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with a TPL2-specific inhibitor reduced the production of inflammatory mediators by neutrophils in response to stimulation with the TLR4 agonist lipopolysaccharide (LPS) in vitro, as well as in rodent models of inflammatory disease. Together, these data suggest that TPL2 is a drug target that activates not only MEK1/2-dependent but also MEK3/6-dependent signaling to promote inflammatory responses.
Asunto(s)
Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Activación Neutrófila , Neutrófilos/enzimología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Activación Enzimática , Inflamación/enzimología , Inflamación/genética , MAP Quinasa Quinasa 3/genética , MAP Quinasa Quinasa 3/metabolismo , MAP Quinasa Quinasa 6/genética , MAP Quinasa Quinasa 6/metabolismo , Quinasas Quinasa Quinasa PAM/genética , Ratones , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
Systemic lupus erythematosus (SLE) is often associated with exaggerated B cell activation promoting plasma cell generation, immune-complex deposition in the kidney, renal infiltration of myeloid cells, and glomerular nephritis. Type-I IFNs amplify these autoimmune processes and promote severe disease. Bruton's tyrosine kinase (Btk) inhibitors are considered novel therapies for SLE. We describe the characterization of a highly selective reversible Btk inhibitor, G-744. G-744 is efficacious, and superior to blocking BAFF and Syk, in ameliorating severe lupus nephritis in both spontaneous and IFNα-accelerated lupus in NZB/W_F1 mice in therapeutic regimens. Selective Btk inhibition ablated plasmablast generation, reduced autoantibodies, and - similar to cyclophosphamide - improved renal pathology in IFNα-accelerated lupus. Employing global transcriptional profiling of spleen and kidney coupled with cross-species human modular repertoire analyses, we identify similarities in the inflammatory process between mice and humans, and we demonstrate that G-744 reduced gene expression signatures essential for splenic B cell terminal differentiation, particularly the secretory pathway, as well as renal transcriptional profiles coupled with myeloid cell-mediated pathology and glomerular plus tubulointerstitial disease in human glomerulonephritis patients. These findings reveal the mechanism through which a selective Btk inhibitor blocks murine autoimmune kidney disease, highlighting pathway activity that may translate to human SLE.
Asunto(s)
Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Linfocitos B/inmunología , Nefritis Lúpica/inmunología , Células Mieloides/metabolismo , Células Plasmáticas/patología , Agammaglobulinemia Tirosina Quinasa/metabolismo , Animales , Autoanticuerpos/inmunología , Linfocitos B/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Interferón-alfa/inmunología , Riñón/inmunología , Riñón/patología , Nefritis Lúpica/metabolismo , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos NZB , Células Plasmáticas/efectos de los fármacosRESUMEN
Voltage-gated Kv1.3 and Ca2+-dependent KCa3.1 are the most prevalent K+ channels expressed by human and rat T cells. Despite the preferential upregulation of Kv1.3 over KCa3.1 on autoantigen-experienced effector memory T cells, whether Kv1.3 is required for their induction and function is unclear. Here we show, using Kv1.3-deficient rats, that Kv1.3 is involved in the development of chronically activated antigen-specific T cells. Several immune responses are normal in Kv1.3 knockout (KO) rats, suggesting that KCa3.1 can compensate for the absence of Kv1.3 under these specific settings. However, experiments with Kv1.3 KO rats and Kv1.3 siRNA knockdown or channel-specific inhibition of human T cells show that maximal T-cell responses against autoantigen or repeated tetanus toxoid stimulations require both Kv1.3 and KCa3.1. Finally, our data also suggest that T-cell dependency on Kv1.3 or KCa3.1 might be irreversibly modulated by antigen exposure.
Asunto(s)
Epítopos/inmunología , Memoria Inmunológica , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canal de Potasio Kv1.3/metabolismo , Linfocitos T/metabolismo , Animales , Técnicas de Silenciamiento del Gen , Humanos , Inmunidad/efectos de los fármacos , Memoria Inmunológica/efectos de los fármacos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/deficiencia , Activación de Linfocitos/inmunología , Fenotipo , Bloqueadores de los Canales de Potasio/farmacología , ARN Interferente Pequeño/metabolismo , Ratas , Linfocitos T/efectos de los fármacosRESUMEN
BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is associated with aberrant expression of developmental pathways, including Hedgehog (Hh). As Hh signalling contributes to multiple pro-fibrotic processes, Hh inhibition may represent a therapeutic option for IPF. However, no non-invasive biomarkers are available to monitor lung Hh activity. METHODS: We assessed gene and protein expression in IPF and control lung biopsies, mouse lung, fibroblasts stimulated in vitro with sonic hedgehog (SHh), and plasma in IPF patients versus controls, and cancer patients before and after treatment with vismodegib, a Hh inhibitor. RESULTS: Lung tissue from IPF patients exhibited significantly greater expression of Hh-related genes versus controls. The gene most significantly upregulated in both IPF lung biopsies and fibroblasts stimulated in vitro with SHh was CXCL14, which encodes a soluble secreted chemokine whose expression is inhibited in vitro by the addition of vismodegib. CXCL14 expression was induced by SHh overexpression in mouse lung. Circulating CXCL14 protein levels were significantly higher in plasma from IPF patients than controls. In cancer patients, circulating CXCL14 levels were significantly reduced upon vismodegib treatment. CONCLUSIONS: CXCL14 is a systemic biomarker that could be used to identify IPF patients with increased Hh pathway activity and monitor the pharmacodynamic effects of Hh antagonist therapy in IPF. TRIAL REGISTRATION NUMBER: Post-results, NCT00968981.
Asunto(s)
Quimiocinas CXC/biosíntesis , Proteínas Hedgehog/fisiología , Fibrosis Pulmonar Idiopática/metabolismo , Anciano , Anilidas/farmacología , Animales , Antineoplásicos/farmacología , Biomarcadores/metabolismo , Células Cultivadas , Quimiocinas CXC/sangre , Quimiocinas CXC/efectos de los fármacos , Quimiocinas CXC/genética , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Fibrosis Pulmonar Idiopática/genética , Pulmón/metabolismo , Masculino , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Piridinas/farmacología , Transducción de Señal/genética , Transducción de Señal/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
Dendritic cells (DCs) promote either tolerogenic or immunogenic T cell responses, the latter upon sensing microbes. Using an in vitro system, we analyzed transcriptional determinants that enable mature DCs to direct these opposing T cell outcomes. In the absence of microbial products, the transcription factor interferon regulatory factor 4 (IRF4) promotes regulatory T cell (Treg) generation by enhancing expression of genes required for antigen presentation along with those for T cell tolerance. IRF4-deficient DCs were impaired for Treg generation in vivo. When exposed to microbial stimuli, DCs activated nuclear factor (NF)-κB, which induced expression of a proinflammatory cytokine module that, along with the antigen presentation module, promoted the generation of effector T cells. NF-κB was, however, dispensable for Treg development. Chromatin profiling revealed transcriptional motifs associated with the divergent DC programs. Thus, DCs modulate their ability to prime tolerogenic or immunogenic T cells by expressing a core antigen presentation module that is overlaid by distinctive regulatory modules to promote either tolerance or immunity.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/fisiología , Tolerancia Inmunológica/genética , Tolerancia Inmunológica/inmunología , Transcripción Genética/genética , Animales , Presentación de Antígeno/genética , Presentación de Antígeno/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Citocinas/metabolismo , Células Dendríticas/metabolismo , Tolerancia Inmunológica/fisiología , Factores Reguladores del Interferón/metabolismo , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/fisiología , Transcripción Genética/inmunologíaRESUMEN
We introduce neutron-encoded (NeuCode) amino acid labeling of mice as a strategy for multiplexed proteomic analysis in vivo. Using NeuCode, we characterize an inducible knockout mouse model of Bap1, a tumor suppressor and deubiquitinase whose in vivo roles outside of cancer are not well established. NeuCode proteomics revealed altered metabolic pathways following Bap1 deletion, including profound elevation of cholesterol biosynthetic machinery coincident with reduced expression of gluconeogenic and lipid homeostasis proteins in liver. Bap1 loss increased pancreatitis biomarkers and reduced expression of mitochondrial proteins. These alterations accompany a metabolic remodeling with hypoglycemia, hypercholesterolemia, hepatic lipid loss, and acinar cell degeneration. Liver-specific Bap1 null mice present with fully penetrant perinatal lethality, severe hypoglycemia, and hepatic lipid deficiency. This work reveals Bap1 as a metabolic regulator in liver and pancreas, and it establishes NeuCode as a reliable proteomic method for deciphering in vivo biology.