RESUMO
Deubiquitinases (DUBs) are a new class of drug targets, although the physiological function of only few DUBs has been characterized. Here we identified the DUB USP15 as a crucial negative regulator of T cell activation. USP15 stabilized the E3 ubiquitin ligase MDM2, which in turn negatively regulated T cell activation by targeting the degradation of the transcription factor NFATc2. USP15 deficiency promoted T cell activation in vitro and enhanced T cell responses to bacterial infection and tumor challenge in vivo. USP15 also stabilized MDM2 in cancer cells and regulated p53 function and cancer-cell survival. Our results suggest that inhibition of USP15 may both induce tumor cell apoptosis and boost antitumor T cell responses.
Assuntos
Fatores de Transcrição NFATC/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/imunologia , Células Th1/imunologia , Proteases Específicas de Ubiquitina/imunologia , Transferência Adotiva , Animais , Apoptose/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células HCT116 , Humanos , Leupeptinas/farmacologia , Listeria monocytogenes/imunologia , Listeriose/imunologia , Ativação Linfocitária/imunologia , Melanoma Experimental/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-mdm2/genética , Evasão Tumoral , Proteína Supressora de Tumor p53/imunologia , Proteases Específicas de Ubiquitina/genética , Ubiquitinação/genética , Ubiquitinação/imunologiaRESUMO
Psoralen and UVA (PUVA) has immunosuppressive and proapoptotic effects, which are thought to be responsible alone or in combination for its therapeutic efficacy. However, the molecular mechanism by which PUVA mediates its effects is not well understood. Activation of the serotonin (5-hydroxytryptamine, 5-HT) pathway has been suggested to be involved in the modulation of T-cell responses and found to mediate UVB-induced immune suppression. In particular, the activation of the 5-HT2A receptor has been proposed as one mechanism responsible for UV-induced immune suppression. We therefore hypothesized that 5-HT may play a role in PUVA-induced effects. The model of systemic suppression of delayed-type hypersensitivity (DTH) to Candida albicans was used to study immune function after exposure of C3H and KIT(W) (-Sh/W-Sh) mice to a minimal inflammatory dose of topical PUVA. The intra-peritoneal injection of the 5-HT2 receptor antagonist ketanserin or cyproheptadine or an anti-5-HT antibody immediately before PUVA exposure entirely abrogated suppression of DTH but had no significant effect on inflammation, as measured by swelling and cellular infiltration of the skin, and apoptosis as determined by the number of sunburn cells in C3H mice. Importantly, the systemic injection of 5-HT recapitulated PUVA immune suppression of DTH but did not induce inflammation or apoptosis in the skin. KIT(W) (-Sh/W-Sh) mice (exhibiting myelopoietic abnormalities, including lack of 5-HT-containing mast cells) were resistant to PUVA-induced suppression of DTH but not local skin swelling. Thus, this points towards a crucial role of 5-HT signalling in PUVA-induced immune suppression but not inflammation or apoptosis in situ in the skin.
Assuntos
Hipersensibilidade Tardia/metabolismo , Terapia PUVA , Serotonina/metabolismo , Animais , Apoptose , Feminino , Hipersensibilidade Tardia/tratamento farmacológico , Inflamação/metabolismo , Mastócitos/fisiologia , Camundongos Endogâmicos C3HRESUMO
Heterozygous mutations in the transcriptional regulator GATA-2 associate with multilineage immunodeficiency, myelodysplastic syndrome (MDS), and acute myeloid leukemia (AML). The majority of these mutations localize in the zinc finger (ZnF) domains, which mediate GATA-2 DNA binding. Deregulated hematopoiesis with GATA-2 mutation frequently develops in adulthood, yet GATA-2 function in the bone marrow remains unresolved. To investigate this, we conditionally deleted the GATA-2 C-terminal ZnF (C-ZnF) coding sequences in adult mice. Upon Gata2 C-ZnF deletion, we observed rapid peripheral cytopenia, bone marrow failure, and decreased c-Kit expression on hematopoietic progenitors. Transplant studies indicated GATA-2 has a cell-autonomous role in bone marrow hematopoiesis. Moreover, myeloid lineage populations were particularly sensitive to Gata2 hemizygosity, while molecular assays indicated GATA-2 regulates c-Kit expression in multilineage progenitor cells. Enforced c-Kit expression in Gata2 C-ZnF-deficient hematopoietic progenitors enhanced myeloid colony activity, suggesting GATA-2 sustains myelopoiesis via a cell intrinsic role involving maintenance of c-Kit expression. Our results provide insight into mechanisms regulating hematopoiesis in bone marrow and may contribute to a better understanding of immunodeficiency and bone marrow failure associated with GATA-2 mutation.
Assuntos
Anemia Aplástica/genética , Doenças da Medula Óssea/genética , Medula Óssea/patologia , Fator de Transcrição GATA2/genética , Hemoglobinúria Paroxística/genética , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Proto-Oncogênicas c-kit/deficiência , Deleção de Sequência , Dedos de Zinco/genética , Anemia Aplástica/diagnóstico , Anemia Aplástica/metabolismo , Anemia Aplástica/mortalidade , Animais , Biomarcadores , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Doenças da Medula Óssea/diagnóstico , Doenças da Medula Óssea/metabolismo , Doenças da Medula Óssea/mortalidade , Transtornos da Insuficiência da Medula Óssea , Osso e Ossos/patologia , Imunoprecipitação da Cromatina , Descalcificação Patológica/genética , Modelos Animais de Doenças , Fator de Transcrição GATA2/química , Fator de Transcrição GATA2/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Frequência do Gene , Genes Reporter , Genótipo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinúria Paroxística/diagnóstico , Hemoglobinúria Paroxística/metabolismo , Hemoglobinúria Paroxística/mortalidade , Sequenciamento de Nucleotídeos em Larga Escala , Imunofenotipagem , Camundongos , Camundongos Knockout , Prognóstico , Células da Side PopulationRESUMO
Dermal mast cells protect the skin from inflammatory effects of ultraviolet (UV) radiation and are required for UV-induced immune suppression. We sought to determine a potential mechanistic role of mast cells in reducing the sensitivity to UV radiation (i.e. phototolerance induction) through photohardening. We administered single UV exposures as well as a chronic UV irradiation regime to mast cell-deficient Kit(W-Sh/W-Sh) mice and their controls. The chronic irradiation protocol was similar to that given for prophylaxis in certain photodermatoses in humans. Compared to controls, UV-exposed Kit(W-Sh/W-Sh) mice were more susceptible to epidermal hyperplasia and dermal oedema which was linked to blood vessel dilation. Unexpectedly, Kit(W-Sh/W-Sh) mice exhibited an excessive scratching behaviour following broadband UVB plus UVA or solar simulated UV irradiation at doses far below their minimal skin-swelling dose. Protection from this UV-induced scratching phenotype was dependent on mast cells, as engraftment of bone marrow-derived cultured mast cells abated it entirely. Kit(W-Sh/W-Sh) mice were entirely resistant to phototolerance induction by photohardening treatment. Compared to controls, these mice also showed reduced numbers of regulatory T cells and neutrophils in the skin 24 h after UV irradiation. While it is well known that mast cell-deficient mice are resistant to UV-induced immune suppression, we have discovered that they are prone to develop photo-itch and are more susceptible to UV-induced epidermal hyperplasia and skin oedema.
Assuntos
Mastócitos/imunologia , Mastócitos/efeitos da radiação , Pele/imunologia , Pele/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Animais , Edema/etiologia , Edema/imunologia , Hiperplasia , Tolerância Imunológica/efeitos da radiação , Mastócitos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neutrófilos/imunologia , Neutrófilos/efeitos da radiação , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/imunologia , Prurido/etiologia , Prurido/imunologia , Prurido/fisiopatologia , Pele/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos da radiação , Vasodilatação/imunologia , Vasodilatação/efeitos da radiaçãoRESUMO
The most prevalent cancer diagnosed in the world is sunlight-induced skin cancer. In addition to being a complete carcinogen, UV radiation, the causative agent of skin cancer, induces immune suppression. Because UV-induced immune suppression is a well-recognized risk factor for skin cancer induction, it is crucial to understand the mechanisms underlying UV-induced immune suppression. Mast cells, which have recently emerged as immune regulatory cells, are particularly important in UV-induced immune suppression. UV exposure does not induce immune suppression in mast cell-deficient mice. We report that UV irradiation blocks germinal center (GC) formation, Ab secretion, and T follicular helper (Tfh) cell function, in part by altering the expression of transcription factors BCL-6 and BLIMP-1. No suppression of GC formation, Tfh cell IL-21 expression, or Ab secretion was observed in UV-irradiated mast cell-deficient (Kit(W-sh/W-sh)) mice. When mast cell-deficient mice were reconstituted with wild type mast cells, immune suppression was restored. Reconstituting the mast cell-deficient mice with bone marrow-derived mast cells from IL-10-deficient mice failed to restore the ability of UV radiation to suppress GC formation. Our findings demonstrate a function for mast cells, suppression of Tfh cell production, GC formation, and Ab production in vivo.
Assuntos
Diferenciação Celular/imunologia , Centro Germinativo/citologia , Centro Germinativo/imunologia , Inibidores do Crescimento/fisiologia , Interleucina-10/fisiologia , Ativação Linfocitária/imunologia , Mastócitos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Formação de Anticorpos/efeitos da radiação , Transplante de Medula Óssea/imunologia , Diferenciação Celular/efeitos da radiação , Centro Germinativo/efeitos da radiação , Interleucina-10/deficiência , Interleucina-10/genética , Interleucinas/antagonistas & inibidores , Interleucinas/biossíntese , Interleucinas/efeitos da radiação , Ativação Linfocitária/genética , Mastócitos/metabolismo , Mastócitos/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/biossíntese , Proteínas Proto-Oncogênicas c-bcl-6/efeitos da radiação , Proteínas Proto-Oncogênicas c-kit/genética , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/efeitos da radiação , Raios UltravioletaRESUMO
BACKGROUND: Inhalational anthrax caused by Bacillus anthracis is associated with high mortality primarily due to toxin-mediated injury. Raxibacumab is a human IgG1lambda monoclonal antibody directed against protective antigen, a component of the anthrax toxin. METHODS: We evaluated the efficacy of raxibacumab as a prophylactic agent and after disease onset in a total of four randomized, placebo-controlled studies conducted in rabbits and monkeys. Animals were exposed to an aerosolized target exposure of B. anthracis spores that was approximately 100 times (in the prophylactic studies) and 200 times (in the therapeutic-intervention studies) the median lethal dose. In the therapeutic-intervention studies, animals were monitored for the onset of symptoms. Animals with detectable protective antigen in serum, a significant increase in temperature, or both received a single intravenous bolus of placebo or raxibacumab at a dose of either 20 mg per kilogram of body weight or 40 mg per kilogram. The primary end point was survival at day 14 (in rabbits) or at day 28 (in monkeys). Safety studies were conducted with intravenous raxibacumab (40 mg per kilogram) in 333 healthy human volunteers. RESULTS: In both rabbits and monkeys, the time to detection of protective antigen correlated with the time to bacteremia (r=0.9, P<0.001). In the therapeutic-intervention studies, the survival rate was significantly higher among rabbits that received raxibacumab at a dose of 40 mg per kilogram (44% [8 of 18]) than among rabbits that received placebo (0% [0 of 18]; P=0.003). Raxibacumab treatment also significantly increased survival in monkeys (64% [9 of 14], vs. 0% [0 of 12] with placebo; P<0.001). In human subjects, intravenous raxibacumab at a dose of 40 mg per kilogram had a half-life of 20 to 22 days and provided a maximum concentration of the drug in excess of levels that are protective in animals. Concentrations of raxibacumab provide a surrogate end point that should be predictive of clinical benefit. CONCLUSIONS: A single dose of raxibacumab improved survival in rabbits and monkeys with symptomatic inhalational anthrax. (ClinicalTrials.gov number, NCT00639678.)
Assuntos
Antraz/tratamento farmacológico , Anti-Infecciosos/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Bacillus anthracis , Infecções Respiratórias/tratamento farmacológico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antraz/imunologia , Anti-Infecciosos/efeitos adversos , Anticorpos Antibacterianos/sangue , Anticorpos Monoclonais/efeitos adversos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais Humanizados , Bacteriemia , Ciprofloxacina/efeitos adversos , Ciprofloxacina/uso terapêutico , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Feminino , Humanos , Imunoglobulina G , Macaca fascicularis , Masculino , Pessoa de Meia-Idade , Coelhos , Distribuição Aleatória , Análise de Sobrevida , Adulto JovemRESUMO
Ultraviolet exposure alters the morphology and function of epidermal Langerhans cells (LCs), which play a role in UV-induced immune suppression. It is generally believed that UV exposure triggers the migration of immature LCs from the skin to the draining lymph nodes (LNs), where they induce tolerance. However, because most of the previous studies employed in vitro UV-irradiated LCs, the data generated may not adequately reflect what is happening in vivo. In this study, we isolated migrating LCs from the LNs of UV-irradiated mice and studied their function. We found prolonged LC survival in the LNs of UV-irradiated mice. LCs were necessary for UV-induced immune suppression because no immune suppression was observed in LC-deficient mice. Transferring LCs from UV-irradiated mice into normal recipient animals transferred immune suppression and induced tolerance. We found that LCs colocalized with LN NKT cells. No immune suppression was observed when LCs were transferred from UV-irradiated mice into NKT cell-deficient mice. NKT cells isolated from the LNs of UV-irradiated mice secreted significantly more IL-4 than NKT cells isolated from nonirradiated controls. Injecting the wild-type mice with anti-IL-4 blocked the induction of immune suppression. Our findings indicate that UV exposure activates the migration of mature LC to the skin draining LNs, where they induce immune regulation in vivo by activating NKT cells.
Assuntos
Tolerância Imunológica/imunologia , Células de Langerhans/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Animais , Quimiotaxia de Leucócito/imunologia , Quimiotaxia de Leucócito/efeitos da radiação , Imunofluorescência , Tolerância Imunológica/efeitos da radiação , Células de Langerhans/efeitos da radiação , Ativação Linfocitária/efeitos da radiação , Camundongos , Células T Matadoras Naturais/efeitos da radiação , Raios Ultravioleta/efeitos adversosRESUMO
BACKGROUND: The incidence of non-Hodgkin's lymphoma has increased over recent years. The exact etiology of lymphoma remains unknown. Ultraviolet light exposure has been associated with the development of internal lymphoid malignancies and some reports suggest that it may play a role in the development of lymphoma in humans. Here we describe the characterization and progression of lymphoma in p53 heterozygous mice exposed to UVB irradiation. METHODS: UVB-irradiated p53+/- mice developed enlargement of the spleen. Isolated spleen cells were transplanted into Rag deficient hosts. The UV-induced tumor cells were analyzed by flow cytometry. The tumor cells were tagged with GFP to study their metastatic potential. SKY and karyotypic analysis were carried out for the detection of chromosomal abnormalities. Functional assays included in vitro class switch recombination assay, immunoglobulin rearrangement assay, as well as cytokine profiling. RESULTS: UVB-exposed mice showed enlargement of the spleen and lymph nodes. Cells transplanted into Rag deficient mice developed aggressive tumors that infiltrated the lymph nodes, the spleen and the bone marrow. The tumor cells did not grow in immune competent syngeneic C57Bl/6 mice yet showed a modest growth in UV-irradiated B6 mice. Phenotypic analysis of these tumor cells revealed these cells are positive for B cell markers CD19+, CD5+, B220+, IgM+ and negative for T cell, NK or dendritic cell markers. The UV-induced tumor cells underwent robust in vitro immunoglobulin class switch recombination in response to lipopolysaccharide. Cytogenetic analysis revealed a t(14;19) translocation and trisomy of chromosome 6. These tumor cells secret IL-10, which can promote tumor growth and cause systemic immunosuppression. CONCLUSION: UV-irradiated p53+/- mice developed lymphoid tumors that corresponded to a mature B cell lymphoma. Our results suggest that an indirect mechanism is involved in the development of internal tumors after chronic exposure to UV light. The induction of B cell lymphoma in UV-irradiated p53 heterozygous mice may provide a useful model for lymphoma development in humans.
Assuntos
Genes p53 , Haploinsuficiência , Linfoma de Células B/etiologia , Linfoma de Células B/genética , Raios Ultravioleta , Animais , Aberrações Cromossômicas , Citocinas/biossíntese , Interleucina-10/biossíntese , Interleucina-10/metabolismo , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Ultraviolet (UV) radiation plays a critical role in the induction of nonmelanoma skin cancer. UV radiation is also immune suppressive, and the immune suppression induced by UV irradiation has been identified as a major risk factor for skin cancer induction. Previously, we showed that UV exposure activates a cytokine cascade involving prostaglandin (PG)E(2), interleukin (IL)-4, and IL-10 that induces immune suppression. However, the earliest molecular events that occur immediately after UV exposure, especially those upstream of PGE2, are not well defined. UV-irradiated keratinocytes secrete the inflammatory phospholipid mediator, platelet-activating factor (PAF). Because PAF upregulates the production of immunomodulatory compounds, including PGE2, we tested the hypothesis that UV-induced PAF activates cytokine production and initiates UV-induced immune suppression. Both UV and PAF activated cyclooxygenase (COX)-2 and IL-10 reporter gene construct transcription. PAF mimicked the effects of UV in vivo and suppressed delayed-type hypersensitivity (DTH). Furthermore, immune suppression was blocked when UV-irradiated mice were injected with PAF receptor antagonists. In addition to the well-known role of PAF as a proinflammatory lipid mediator, we propose that the PAF receptor senses cellular damage through the recognition of PAF and/or PAF-like molecules, such as oxidized phosphatidylcholine, which activates cytokine transcription and induces systemic immune suppression.
Assuntos
Terapia de Imunossupressão , Queratinócitos/imunologia , Fator de Ativação de Plaquetas/imunologia , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Animais , Ciclo-Oxigenase 2 , Ativação Enzimática/efeitos da radiação , Isoenzimas/imunologia , Isoenzimas/metabolismo , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C3H , Fator de Ativação de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/imunologia , Glicoproteínas da Membrana de Plaquetas/metabolismo , Prostaglandina-Endoperóxido Sintases/imunologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Receptores de Interleucina/biossíntese , Receptores de Interleucina/imunologia , Receptores de Interleucina-10 , Transdução de Sinais/imunologia , Transcrição Gênica/efeitos da radiação , Raios UltravioletaRESUMO
The relationship between exposure to UV radiation and development of skin cancer has been well established. Several studies have shown that UVB induces unique mutations (C-->T and CC-->TT transitions) in the p53 tumor suppressor gene that are not commonly induced by other carcinogens. Our studies have demonstrated that UV-induced mouse skin cancers contain p53 mutations at a high frequency and that these mutations can be detected in UV-irradiated mouse skin well before the appearance of skin tumors. This observation suggested that it might be possible to use p53 mutations as a biologic endpoint for testing the efficacy of sunscreens in photoprotection studies. Indeed, application of SPF 15 sunscreens to mouse skin before each UVB irradiation resulted in reduction in the number of p53 mutations. Because p53 mutations represent an early essential step in photocarcinogenesis, these results imply that inhibition of this event may protect against skin cancer development. This hypothesis was confirmed by our finding that sunscreens used in p53 mutation inhibition experiments also protected mice against UVB-induced skin cancer.
Assuntos
Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/prevenção & controle , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Animais , Humanos , Queratinócitos/metabolismo , Queratinócitos/efeitos da radiação , Mutação/genética , Neoplasias Cutâneas/genética , Protetores Solares/farmacologiaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. It has an excessive desmoplastic stroma that can limit the intratumoral delivery of chemotherapy drugs, and protect tumor cells against radiotherapy. Therefore, both stromal and tumor compartments need to be addressed in order to effectively treat PDAC. We hereby co-deliver a sonic hedgehog inhibitor, cyclopamine (CPA), and a cytotoxic chemotherapy drug paclitaxel (PTX) with a polymeric micelle formulation (M-CPA/PTX). CPA can deplete the stroma-producing cancer-associated fibroblasts (CAFs), while PTX can inhibit tumor proliferation. Here we show that in clinically relevant PDAC models, M-CPA effectively modulates stroma by increasing microvessel density, alleviating hypoxia, reducing matrix stiffness while maintaining the tumor-restraining function of extracellular matrix. M-CPA/PTX also significantly extends animal survival by suppressing tumor growth and lowering the percentages of poorly to moderately differentiated tumor phenotypes. Our study suggests that using multifunctional nanoparticles to simultaneously target stromal and tumor compartments is a promising strategy for PDAC therapy.
Assuntos
Neoplasias Pancreáticas/tratamento farmacológico , Animais , Proliferação de Células/efeitos dos fármacos , Feminino , Camundongos , Camundongos Nus , Micelas , Paclitaxel/química , Paclitaxel/uso terapêutico , Polímeros/química , Transdução de Sinais/efeitos dos fármacos , Neoplasias PancreáticasRESUMO
Tuberculosis is one of the leading causes of human morbidity and mortality. Mycobacterium tuberculosis (Mtb) employs different strategies to evade and counterattack immune responses persisting for years. Mast cells are crucial during innate immune responses and help clear infections via inflammation or by direct antibacterial activity through extracellular traps (MCETs). Whether Mtb induce MCETs production is unknown. In this study, we report that viable Mtb did not induce DNA release by mast cells, but heat-killed Mtb (HK-Mtb) did. DNA released by mast cells after stimulation with HK-Mtb was complexed with histone and tryptase. MCETs induced with PMA and HK-Mtb were unable to kill live Mtb bacilli. Mast cells stimulated with HK-Mtb induced hydrogen peroxide production, whereas cells stimulated with viable Mtb did not. Moreover, MCETs induction by HK-Mtb was dependent of NADPH oxidase activity, because its blockade resulted in a diminished DNA release by mast cells. Interestingly, catalase-deficient Mtb induced a significant production of hydrogen peroxide and DNA release by mast cells, indicating that catalase produced by Mtb prevents MCETs release by degrading hydrogen peroxide. Our findings show a new strategy employed by Mtb to overcome the immune response through inhibiting MCETs formation, which could be relevant during early stages of infection.
Assuntos
Proteínas de Bactérias/imunologia , Catalase/imunologia , Armadilhas Extracelulares/imunologia , Imunidade Inata , Mastócitos/imunologia , Mycobacterium tuberculosis/imunologia , Animais , Proteínas de Bactérias/metabolismo , Catalase/metabolismo , Linhagem Celular , Armadilhas Extracelulares/metabolismo , Humanos , Mastócitos/enzimologia , Camundongos , Mycobacterium tuberculosis/enzimologia , Triptases/imunologia , Triptases/metabolismo , Tuberculose/enzimologia , Tuberculose/imunologia , Tuberculose/patologiaRESUMO
Although treatment with immune checkpoint inhibitors provides promising benefit for patients with cancer, optimal use is encumbered by high resistance rates and requires a thorough understanding of resistance mechanisms. We observed that tumors treated with PD-1/PD-L1 blocking antibodies develop resistance through the upregulation of CD38, which is induced by all-trans retinoic acid and IFNß in the tumor microenvironment. In vitro and in vivo studies demonstrate that CD38 inhibits CD8+ T-cell function via adenosine receptor signaling and that CD38 or adenosine receptor blockade are effective strategies to overcome the resistance. Large data sets of human tumors reveal expression of CD38 in a subset of tumors with high levels of basal or treatment-induced T-cell infiltration, where immune checkpoint therapies are thought to be most effective. These findings provide a novel mechanism of acquired resistance to immune checkpoint therapy and an opportunity to expand their efficacy in cancer treatment.Significance: CD38 is a major mechanism of acquired resistance to PD-1/PD-L1 blockade, causing CD8+ T-cell suppression. Coinhibition of CD38 and PD-L1 improves antitumor immune response. Biomarker assessment in patient cohorts suggests that a combination strategy is applicable to a large percentage of patients in whom PD-1/PD-L1 blockade is currently indicated. Cancer Discov; 8(9); 1156-75. ©2018 AACR.See related commentary by Mittal et al., p. 1066This article is highlighted in the In This Issue feature, p. 1047.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Antineoplásicos Imunológicos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Glicoproteínas de Membrana/metabolismo , ADP-Ribosil Ciclase 1/antagonistas & inibidores , Animais , Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon gama/metabolismo , Neoplasias Pulmonares/imunologia , Melanoma/genética , Melanoma/imunologia , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptores Purinérgicos P1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tretinoína/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dermal exposure to military (JP-8) and/or commercial (Jet-A) jet fuel suppresses cell-mediated immune reactions. Immune regulatory cytokines and biological modifiers, including platelet activating factor (PAF), prostaglandin E(2), and interleukin-10, have been implicated in the pathway of events leading to immune suppression. It is estimated that approximately 260 different hydrocarbons are found in jet fuel, and the exact identity of the active immunotoxic agent(s) is unknown. The recent availability of synthetic jet fuel (S-8), which is refined from natural gas, and is devoid of aromatic hydrocarbons, made it feasible to design experiments to address this problem. Here we tested the hypothesis that the aromatic hydrocarbons present in jet fuel are responsible for immune suppression. We report that applying S-8 to the skin of mice does not upregulate the expression of epidermal cyclooxygenase-2 (COX-2) nor does it induce immune suppression. Adding back a cocktail of seven of the most prevalent aromatic hydrocarbons found in jet fuel (benzene, toluene, ethylbenzene, xylene, 1,2,4-trimethlybenzene, cyclohexylbenzene, and dimethylnaphthalene) to S-8 upregulated epidermal COX-2 expression and suppressed a delayed-type hypersensitivity (DTH) reaction. Injecting PAF receptor antagonists, or a selective cycloozygenase-2 inhibitor into mice treated with S-8 supplemented with the aromatic cocktail, blocked suppression of DTH, similar to data previously reported using JP-8. These findings identify the aromatic hydrocarbons found in jet fuel as the agents responsible for suppressing DTH, in part by the upregulation of COX-2, and the production of immune regulatory factors and cytokines.
Assuntos
Ciclo-Oxigenase 2/metabolismo , Hidrocarbonetos/toxicidade , Hipersensibilidade Tardia/tratamento farmacológico , Querosene/toxicidade , Pele/efeitos dos fármacos , Animais , Candida albicans/imunologia , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Edema/induzido quimicamente , Edema/tratamento farmacológico , Edema/imunologia , Feminino , Pé , Hidrocarbonetos Aromáticos/toxicidade , Hipersensibilidade Tardia/induzido quimicamente , Hipersensibilidade Tardia/imunologia , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Camundongos , Camundongos Endogâmicos C3H , Pele/metabolismo , Absorção Cutânea/imunologia , Organismos Livres de Patógenos Específicos , Regulação para CimaRESUMO
MEKK2 is a member of the mitogen-activated protein kinase (MAPK) kinase kinase gene family involved in regulating multiple MAPK signaling pathways. To elucidate the in vivo function of MEKK2, we generated mice carrying a targeted mutation in the Mekk2 locus. Mekk2(-/-) mice are viable and fertile. Major subsets of thymic and spleen T cells in Mekk2-deficient mice were indistinguishable from those in wild-type mice. B-cell development appeared to proceed similarly in the bone marrow of Mekk2-deficient and wild-type mice. However, Mekk2(-/-) T-cell proliferation was augmented in response to anti-CD3 monoclonal antibody (MAb) stimulation, and these T cells produced more interleukin 2 and gamma interferon than did the wild-type T cells, suggesting that MEKK2 may be involved in controlling the strength of T-cell receptor (TCR) signaling. Consistently, Mekk2(-/-) thymocytes were more susceptible than wild-type thymocytes to anti-CD3 MAb-induced cell death. Furthermore, TCR-mediated c-Jun N-terminal kinase activation was not blocked but moderately enhanced in Mekk2(-/-) T cells. Neither extracellular signal-regulated kinase nor p38 MAPK activation was affected in Mekk2(-/-) T cells. In conclusion, we found that MEKK2 may be required for controlling the strength of TCR/CD3 signaling.
Assuntos
MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/fisiologia , Animais , Anticorpos/administração & dosagem , Apoptose/fisiologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/fisiologia , Linfócitos B/efeitos da radiação , Células Cultivadas , Dexametasona/farmacologia , Ativação Enzimática , Citometria de Fluxo , Marcação de Genes , Glucocorticoides/farmacologia , Interferon gama/metabolismo , Interleucina-2/metabolismo , MAP Quinase Quinase Quinase 2 , MAP Quinase Quinase Quinases/imunologia , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Receptores de Antígenos de Linfócitos T/imunologia , Recombinação Genética , Transdução de Sinais/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/fisiologia , Linfócitos T/efeitos da radiação , Timo/citologia , Raios UltravioletaRESUMO
Exposing experimental animals or human volunteers to UVA II (320-340 nm) radiation after immunization suppresses immunologic memory and the elicitation of delayed-in-time hypersensitivity reactions. Previous studies indicated that the mechanisms underlying UVA-induced immune suppression are similar to those described for UVB-induced immune suppression, i.e. transferred by T regulatory cells, overcome by repairing DNA damage, neutralizing interleukin (IL)-10 activity, or injecting recombinant IL-12. Here we continued our examination of the mechanisms involved in UVA II-induced suppression. Antibodies to cis-urocanic acid blocked UVA-induced immune suppression. Treating UVA-irradiated mice with histamine receptor antagonists, calcitonin gene-related peptide (CGRP) receptor antagonists or platelet activating factor receptor antagonists blocked immune suppression in UVA-irradiated mice. In light of the fact that cis-urocanic acid and CGRP target mast cells, which can then release platelet activating factor and histamine, we measured UVA-induced immune suppression in mast cell-deficient mice. No immune suppression was noted in UVA-irradiated mast cell-deficient mice. These findings indicate that exposure to UVA II activates many of the same immune regulatory factors activated by UVB to induce immune suppression. Moreover, they indicate that mast cells play a critical role in UVA-induced suppression of secondary immune reactions.
Assuntos
Mastócitos/efeitos da radiação , Raios Ultravioleta , Animais , Peptídeo Relacionado com Gene de Calcitonina/fisiologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidoresRESUMO
Mast cells are part of the immune system and characteristically contain histamine- and heparin-rich basophilic granules. While these cells are usually associated with allergy and anaphylaxis, they also promote wound healing and angiogenesis and confer protection against pathogens. The presence of these cells is sometimes indicative of a poor prognosis, especially in skin cancer, pancreatic cancer, and lymphoma. Toluidine blue staining of acid-fast granules is an established method for the identification and quantification of mast cells. Generating detailed information on the location of mast cells within tissues is problematic using this technique and often requires serial sections from adjacent tissue to be separately stained with hematoxylin and eosin (H&E). Staining serial sections is not always possible, particularly if the sample is very small or rare. In such cases, a method of simultaneously identifying and localizing mast cells in a tissue would be advantageous. Toluidine blue and H&E are not commonly combined because H&E includes repetitive washes in water, which may affect the efficacy of the aqueous-soluble toluidine blue. We have developed and tested a novel staining technique that integrates toluidine blue between hematoxylin and eosin in one simple procedure. This protocol works on both frozen and formalin-fixed, paraffin-embedded tissue and readily allows for the identification of purple-stained mast cells against a clean H&E background. This facilitates a more accurate localization and proper counting of mast cells in normal and affected tissue.
Assuntos
Histocitoquímica , Mastócitos/metabolismo , Mastócitos/patologia , Cloreto de Tolônio , Animais , Amarelo de Eosina-(YS) , Hematoxilina , Histocitoquímica/métodos , Camundongos , Camundongos KnockoutRESUMO
Ultraviolet (UV)-irradiated keratinocytes secrete the lipid mediator of inflammation, platelet-activating factor (PAF). PAF plays an essential role in UV-induced immune suppression and skin cancer induction. Dermal mast cell migration from the skin to the draining lymph nodes plays a prominent role in activating systemic immune suppression. UV-induced PAF activates mast cell migration by up-regulating mast cell CXCR4 surface expression. Recent findings indicate that PAF up-regulates CXCR4 expression via histone acetylation. UV-induced PAF also activates cell cycle arrest and disrupts DNA repair, in part by increasing p21 expression. Do epigenetic alterations play a role in p21 up-regulation? Here we show that PAF increases Acetyl-CREB-binding protein (CBP/p300) histone acetyltransferase expression in a time and dose-dependent fashion. Partial deletion of the HAT domain in the CBP gene, blocked these effects. Chromatin immunoprecipitation assays indicated that PAF-treatment activated the acetylation of the p21 promoter. PAF-treatment had no effect on other acetylating enzymes (GCN5L2, PCAF) indicating it is not a global activator of histone acetylation. This study provides further evidence that PAF activates epigenetic mechanisms to affect important cellular processes, and we suggest this bioactive lipid can serve as a link between the environment and the epigenome.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Código das Histonas , Fator de Ativação de Plaquetas/farmacologia , Acetilação , Linhagem Celular , Inibidor de Quinase Dependente de Ciclina p21/genética , Humanos , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores CXCR4/genética , Receptores CXCR4/metabolismoRESUMO
Mast cells play an essential role in different immunological phenomena including allergy and infectious diseases. Several bacteria induce mast cell activation leading to degranulation and the production of several cytokines and chemokines. However, mast cells also have different microbicidal activities such as phagocytosis and the release of DNA with embedded granular proteins known as Mast Cell Extracellular Traps (MCETs). Although previous reports indicate that extracellular bacteria are able to induce MCETs little is known if intracellular bacteria can induce these structures. In this work, we evaluated MCETs induction by the intracellular bacteria Listeria monocytogenes. We found that mast cells released DNA after stimulation with L. monocytogenes, and this DNA was complexed to histone and tryptase. Before extracellular DNA release, L. monocytogenes induced modifications to the mast cell nuclear envelope and DNA was detected outside the nucleus. L. monocytogenes stimulated mast cells to produce significant amounts of reactive oxygen species (ROS) and blocking NADPH oxidase diminished DNA release by mast cells. Finally, MCETs showed antimicrobial activity against L. monocytogenes that was partially blocked when ß-hexosaminidase activity was inhibited. These results show that L. monocytogenes induces mast cells to produce microbicidal MCETs, suggesting a role for mast cells in containing infection beyond the induction of inflammation.
Assuntos
Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Listeria monocytogenes/imunologia , Mastócitos/imunologia , Mastócitos/metabolismo , Linhagem Celular , DNA/metabolismo , Histonas/metabolismo , Humanos , Listeriose , Mastócitos/ultraestrutura , Membrana Nuclear/ultraestrutura , Fagocitose/imunologia , Espécies Reativas de Oxigênio/metabolismo , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
Vascular endothelial cells (EC) participate in the process of bone formation through the production of factors regulating osteoclast differentiation and function. In this study, we report the selective expression in primary human microvascular EC of Osteostat/TNF superfamily 18, a ligand of the TNF superfamily. Osteostat protein is detectable in human microvascular EC and is highly up-regulated by IFN-alpha and IFN-beta. Moreover, an anti-Osteostat antibody strongly binds to the vascular endothelium in human tissues, demonstrating that the protein is present in the EC layers surrounding blood vessels. Functional in vitro assays were used to define Osteostat involvement in osteoclastogenesis. Both recombinant and membrane-bound Osteostat inhibit differentiation of osteoclasts from monocytic precursor cells. Osteostat suppresses the early stage of osteoclastogenesis via inhibition of macrophage colony-stimulating factor-induced receptor activator of NF-kappaB (RANK) expression in the osteoclast precursor cells. This effect appears to be specific for the differentiation pathway of the osteoclast lineage, because Osteostat does not inhibit lipopolysaccharide-induced RANK expression in monocytes and dendritic cells, or activation-induced RANK expression in T cells. These findings demonstrate that Osteostat is a novel regulator of osteoclast generation and substantiate the major role played by the endothelium in bone physiology.