Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 14 de 14
Filtrar
1.
Int Immunol ; 31(6): 385-396, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31051036

RESUMO

Burkholderia cepacia complex (Bcc), which includes B. cenocepacia and B. multivorans, pose a life-threatening risk to patients with cystic fibrosis. Eradication of Bcc is difficult due to the high level of intrinsic resistance to antibiotics, and failure of many innate immune cells to control the infection. Because of the pathogenesis of Bcc infections, we wondered if a novel mechanism of microbial host defense involving direct antibacterial activity by natural killer (NK) cells might play a role in the control of Bcc. We demonstrate that NK cells bound Burkholderia, resulting in Src family kinase activation as measured by protein tyrosine phosphorylation, granule release of effector proteins such as perforin and contact-dependent killing of the bacteria. These studies provide a means by which NK cells could play a role in host defense against Bcc infection.


Assuntos
Infecções por Burkholderia/imunologia , Burkholderia cepacia/fisiologia , Burkholderia/fisiologia , Fibrose Cística/imunologia , Células Matadoras Naturais/imunologia , Adesão Celular , Degranulação Celular , Linhagem Celular , Citotoxicidade Imunológica , Humanos , Imunidade Celular , Perforina/metabolismo , Fosforilação , Transdução de Sinais , Quinases da Família src/metabolismo
2.
J Immunol ; 201(8): 2369-2376, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30201811

RESUMO

Cryptococcus neoformans is a fungal pathogen that causes fatal meningitis and pneumonia. During host defense to Cryptococcus, NK cells directly recognize and kill C. neoformans using cytolytic degranulation analogous to killing of tumor cells. This fungal killing requires independent activation of Src family kinase (SFK) and Rac1-mediated pathways. Recognition of C. neoformans requires the natural cytotoxicity receptor, NKp30; however, it is not known whether NKp30 activates both signal transduction pathways or whether a second receptor is involved in activation of one of the pathways. We used primary human NK cells and a human NK cell line and found that NKp30 activates SFK → PI3K but not Rac1 cytotoxic signaling, which led to a search for the receptor leading to Rac1 activation. We found that NK cells require integrin-linked kinase (ILK) to activate Rac1 for effective fungal killing. This observation led to our identification of ß1 integrin as an essential anticryptococcal receptor. These findings demonstrate that multiple receptors, including ß1 integrins and NKp30 and their proximal signaling pathways, are required for recognition of Cryptococcus, which activates a central cytolytic antimicrobial pathway leading to fungal killing.


Assuntos
Criptococose/imunologia , Cryptococcus neoformans/fisiologia , Integrina beta1/metabolismo , Células Matadoras Naturais/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Adolescente , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Humanos , Imunidade Inata , Masculino , Receptor 3 Desencadeador da Citotoxicidade Natural/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo
3.
J Immunol ; 196(3): 1259-71, 2016 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-26740109

RESUMO

Cryptococcus gattii is an emerging fungal pathogen on the west coast of Canada and the United States that causes a potentially fatal infection in otherwise healthy individuals. In previous investigations of the mechanisms by which C. gattii might subvert cell-mediated immunity, we found that C. gattii failed to induce dendritic cell (DC) maturation, leading to defective T cell responses. However, the virulence factor and the mechanisms of evasion of DC maturation remain unknown. The cryptococcal polysaccharide capsule is a leading candidate because of its antiphagocytic properties. Consequently, we asked if the capsule of C. gattii was involved in evasion of DC maturation. We constructed an acapsular strain of C. gattii through CAP59 gene deletion by homologous integration. Encapsulated C. gattii failed to induce human monocyte-derived DC maturation and T cell proliferation, whereas the acapsular mutant induced both processes. Surprisingly, encapsulation impaired DC maturation independent of its effect on phagocytosis. Indeed, DC maturation required extracellular receptor signaling that was dependent on TNF-α and p38 MAPK, but not ERK activation, and the cryptococcal capsule blocked this extracellular recognition. Although the capsule impaired phagocytosis that led to pH-dependent serine-, threonine-, and cysteine-sensitive protease-dependent Ag processing, it was insufficient to impair T cell responses. In summary, C. gattii affects two independent processes, leading to DC maturation and Ag processing. The polysaccharide capsule masked extracellular detection and reduced phagocytosis that was required for DC maturation and Ag processing, respectively. However, the T cell response was fully restored by inducing DC maturation.


Assuntos
Apresentação de Antígeno/imunologia , Criptococose/imunologia , Cryptococcus gattii/imunologia , Células Dendríticas/imunologia , Cápsulas Fúngicas/imunologia , Evasão da Resposta Imune/imunologia , Western Blotting , Proliferação de Células , Humanos , Ativação Linfocitária/imunologia , Linfócitos T/imunologia
4.
J Biol Chem ; 291(13): 6912-22, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-26867574

RESUMO

The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found thatCryptococcus neoformansindependently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing,Cryptococcusinitiated a novel Rac → PI3K → Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity againstC. neoformans Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to killC. neoformans.


Assuntos
Classe Ia de Fosfatidilinositol 3-Quinase/imunologia , Cryptococcus neoformans/fisiologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Proteínas rac de Ligação ao GTP/imunologia , Proteínas rac1 de Ligação ao GTP/imunologia , Quinases da Família src/imunologia , Linhagem Celular Tumoral , Classe Ia de Fosfatidilinositol 3-Quinase/genética , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/microbiologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Pironas/farmacologia , Quinolinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas rac de Ligação ao GTP/antagonistas & inibidores , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Quinases da Família src/genética , Proteína RAC2 de Ligação ao GTP
5.
PLoS Pathog ; 9(7): e1003439, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23853583

RESUMO

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment.


Assuntos
Degranulação Celular , Microambiente Celular , Cryptococcus gattii/imunologia , Cryptococcus neoformans/imunologia , Citotoxicidade Imunológica , Células Matadoras Naturais/imunologia , Perforina/metabolismo , Adesão Celular , Linhagem Celular , Células Cultivadas , Córtex Cerebral/imunologia , Córtex Cerebral/metabolismo , Córtex Cerebral/microbiologia , Córtex Cerebral/patologia , Criptococose/imunologia , Criptococose/metabolismo , Criptococose/microbiologia , Criptococose/patologia , Cryptococcus gattii/fisiologia , Cryptococcus neoformans/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Células Matadoras Naturais/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Sistema de Sinalização das MAP Quinases , Fosforilação , Processamento de Proteína Pós-Traducional , Regulação para Cima , Replicação Viral , Proteínas ras/genética , Proteínas ras/metabolismo
6.
J Immunol ; 191(1): 249-61, 2013 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-23740956

RESUMO

During adaptive immunity to pathogens, dendritic cells (DCs) capture, kill, process, and present microbial Ags to T cells. Ag presentation is accompanied by DC maturation driven by appropriate costimulatory signals. However, current understanding of the intricate regulation of these processes remains limited. Cryptococcus gattii, an emerging fungal pathogen in the Pacific Northwest of Canada and the United States, fails to stimulate an effective immune response in otherwise healthy hosts leading to morbidity or death. Because immunity to fungal pathogens requires intact cell-mediated immunity initiated by DCs, we asked whether C. gattii causes dysregulation of DC functions. C. gattii was efficiently bound and internalized by human monocyte-derived DCs, trafficked to late phagolysosomes, and killed. Yet, even with this degree of DC activation, the organism evaded pathways leading to DC maturation. Despite the ability to recognize and kill C. gattii, immature DCs failed to mature; there was no increased expression of MHC class II, CD86, CD83, CD80, and CCR7, or decrease of CD11c and CD32, which resulted in suboptimal T cell responses. Remarkably, no increase in TNF-α was observed in the presence of C. gattii. However, addition of recombinant TNF-α or stimulation that led to TNF-α production restored DC maturation and restored T cell responses. Thus, despite early killing, C. gattii evades DC maturation, providing a potential explanation for its ability to infect immunocompetent individuals. We have also established that DCs retain the ability to recognize and kill C. gattii without triggering TNF-α, suggesting independent or divergent activation pathways among essential DC functions.


Assuntos
Imunidade Adaptativa/imunologia , Diferenciação Celular/imunologia , Criptococose/imunologia , Criptococose/patologia , Cryptococcus gattii/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Evasão da Resposta Imune/imunologia , Células Cultivadas , Criptococose/microbiologia , Cryptococcus gattii/crescimento & desenvolvimento , Cryptococcus gattii/patogenicidade , Células Dendríticas/microbiologia , Humanos , Imunofenotipagem , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/fisiologia
7.
Infect Immun ; 81(10): 3912-22, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23918783

RESUMO

Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase-extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse.


Assuntos
Cryptococcus neoformans/fisiologia , Células Matadoras Naturais/metabolismo , Perforina/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Quinases da Família src/metabolismo , Linhagem Celular Tumoral , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/imunologia , Humanos , Microdomínios da Membrana , Perforina/genética , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-fyn/genética , Interferência de RNA , RNA Interferente Pequeno , Tirosina , Quinases da Família src/genética
8.
mBio ; 11(6)2020 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-33234684

RESUMO

Cryptococcus gattii is a major cause of life-threatening mycosis in immunocompetent individuals and responsible for the ongoing epidemic outbreak of cryptococcosis in the Pacific Northwest of North America. This deadly fungus is known to evade important host immune responses, including dendritic cell (DC) maturation and concomitant T cell immunity, via immune evasion mechanisms that remain unclear. Here, we demonstrate that primary human DCs phagocytose C. gattii but the maturation of phagosomes to phagolysosomes was blocked as a result of sustained filamentous actin (F-actin) that entrapped and concealed the phagosomes from recognition. Superresolution structured illumination microscopy (SR-SIM) revealed that the persistent phagosomal F-actin formed a cage-like structure that sterically hindered and functionally blocked the fusion of lysosomes. Blocking lysosome fusion was sufficient to inhibit phagosomal acidification and subsequent intracellular fungal killing by DCs. Retention of phagosomal F-actin by C. gattii also caused DC immunoparalysis. Disrupting the retained F-actin cage with cytochalasin D not only restored DC phagosomal maturation but also promoted DC costimulatory maturation and robust T cell activation and proliferation. Collectively, these results reveal a unique mechanism of DC immune evasion that enhances intracellular fungal pathogenicity and may explain suppressed cell-mediated immunity.IMPORTANCECryptococcus yeast species typically display characteristics of opportunistic pathogens, with the exception of C. gattii, which can cause life-threatening respiratory and disseminated brain infections in otherwise healthy people. The pathogenesis of C. gattii is not well understood, but an important characteristic is that C. gattii is capable of evading host cell-mediated immune defenses initiated by DCs. Here, we report that when virulent C. gattii becomes ingested by a DC, the intracellular compartment containing the fungi is covered by a persistent protein cage structure consisting of F-actin. This F-actin cage acts as a barrier to prevent interaction with other intracellular compartments, and as a result, the DC fails to kill the fungi and activate important cell-mediated immune responses. We propose that this unique immune evasion mechanism permits C. gattii to remain unchallenged within host cells, leading to persistent infection.


Assuntos
Actinas/metabolismo , Cryptococcus gattii/imunologia , Cryptococcus gattii/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Fagossomos/metabolismo , Biomarcadores , Comunicação Celular/imunologia , Criptococose/imunologia , Criptococose/metabolismo , Criptococose/microbiologia , Humanos , Imunofenotipagem , Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/metabolismo , Virulência
9.
Am J Respir Cell Mol Biol ; 39(6): 657-65, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18539953

RESUMO

Patients with cystic fibrosis suffer recurrent pulmonary infections that are characterized by an overactive yet ineffective and destructive inflammatory response that is associated with respiratory infections by Pseudomonas aeruginosa, a pathogen that produces a number of phlogistic molecules. To better understand this process, we used exoenzyme S (ExoS), one of the key P. aeruginosa-secreted exoproducts, which is known to stimulate cells via the Toll-like receptor (TLR) pathway. We found that ExoS induced proinflammatory cytokine production via the NF-kappaB, Erk1/2, and Src kinase pathways. Because Src kinases are concentrated within cholesterol-containing, detergent-resistant membrane microdomains (DRM) (also called lipid rafts) and DRM act as signaling platforms and amplifiers on the surface of cells, we addressed the role of DRM in ExoS signaling. ExoS bound directly to a subset of DRM and induced the phosphorylation of multiple proteins within DRM, including Src kinases. Disruption of DRM by cholesterol extraction prevented NF-kappaB and Erk 1/2 activation and TNF-alpha production in response to ExoS. Activation of monocytic cells by other TLR and Nod-like receptor agonists, such as lipoteichoic acid, lipopolysaccharide, and peptidoglycan, were also dependent on DRM, and disruption prevented TNF-alpha production. Disruption of DRM did not prevent ExoS binding but did release the Src kinase, Lyn, from the DRM fraction into the detergent-soluble fraction, a site in which Src kinases are not active. These studies show that ExoS, a TLR agonist, requires direct binding to DRM for optimal signaling, which suggests that DRM are possible therapeutic targets in cystic fibrosis.


Assuntos
ADP Ribose Transferases/farmacologia , Toxinas Bacterianas/farmacologia , Detergentes/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Linhagem Celular , Humanos , Proteínas I-kappa B/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Microdomínios da Membrana/enzimologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Monócitos/enzimologia , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Ácidos Teicoicos/farmacologia , Receptores Toll-Like/agonistas , Fator de Necrose Tumoral alfa/biossíntese , beta-Ciclodextrinas/farmacologia , Quinases da Família src/metabolismo
10.
Cell Rep ; 24(11): 3017-3032, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30208325

RESUMO

Cryptococcus is the most important cause of fungal meningitis in immunocompromised individuals. Host defense against Cryptococcus involves direct killing by NK cells. That NK cells from HIV-infected patients fail to polarize perforin to the microbial synapse and kill C. neoformans led us to explore the mechanisms used to reposition and polarize the cytolytic granules to the synapse. Using live-cell imaging, we observed microtubule and granule movements in response to Cryptococcus that revealed a kinesin-dependent event. Eg5-kinesin bound to perforin-containing granules and was required for association with the microtubules. Inhibition of Eg5-kinesin abrogated dynein-dependent granule convergence to the MTOC and granule and MTOC polarization to the synapse and suppressed NK cell killing of Cryptococcus. In contrast, Eg5-kinesin was dispensable for tumor killing. This reveals an alternative mechanism of MTOC repositioning and granule polarization, not used in tumor cytotoxicity, in which Eg5-kinesin is required to initiate granule movement, leading to microbial killing.


Assuntos
Cryptococcus/imunologia , Cryptococcus/patogenicidade , Grânulos Citoplasmáticos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Cinesinas/metabolismo , Linhagem Celular , Células Cultivadas , Grânulos Citoplasmáticos/genética , Citotoxicidade Imunológica , Humanos , Cinesinas/genética
11.
J Innate Immun ; 8(6): 617-629, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27560480

RESUMO

NK cells play a central role in innate immunity, acting directly through cell-mediated cytotoxicity and by secreting cytokines. TNFα activation of TNFR2 enhances NK cell cytotoxicity, but its effects on the other essential function of NK cells - cytokine production, for which IFNγ is paramount - are poorly defined. We identify the expression of both TNFα receptors on human peripheral blood NK cells (TNFR2 > TNFR1) and show that TNFα significantly augments IFNγ production from IL-2-/IL-12-treated NK cells in vitro, an effect mimicked by a TNFR2 agonistic antibody. TNFα also enhanced murine NK cell IFNγ production via TNFR2 in vitro. In a mouse model characterized by the hepatic recruitment and activation of NK cells, TNFR2 also regulated NK cell IFNγ production in vivo. Specifically, in this model, after activation of an innate immune response, hepatic numbers of TNFR2-expressing and IFNγ-producing NK cells were both significantly increased; however, the frequency of IFNγ-producing hepatic NK cells was significantly reduced in TNFR2-deficient mice. We delineate an important role for TNFα, acting through TNFR2, in augmenting cytokine-induced NK cell IFNγ production in vivo and in vitro, an effect with significant potential implications for the regulation of innate and adaptive immune responses.


Assuntos
Células Matadoras Naturais/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Anticorpos Bloqueadores/farmacologia , Células Cultivadas , Citotoxicidade Imunológica , Humanos , Imunidade Inata , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-12/imunologia , Interleucina-2/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/imunologia
12.
PLoS One ; 8(6): e66825, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23762498

RESUMO

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.


Assuntos
Neoplasias Encefálicas/prevenção & controle , Glioma/prevenção & controle , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Matadoras Naturais/imunologia , Terapia Viral Oncolítica , Infecções por Poxviridae/prevenção & controle , Infecções Tumorais por Vírus/prevenção & controle , Animais , Western Blotting , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/virologia , Feminino , Glioma/imunologia , Glioma/virologia , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Técnicas Imunoenzimáticas , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Camundongos , Camundongos SCID , Myxoma virus/fisiologia , Infecções por Poxviridae/imunologia , Infecções por Poxviridae/virologia , Células Tumorais Cultivadas , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia
13.
J Leukoc Biol ; 90(1): 189-98, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21498585

RESUMO

Recognition of TLR agonists involves a complex interplay among a variety of serum and cell membrane molecules, including mCD14 and sCD14 that is not fully understood. TLR activation results in downstream signaling that induces inflammatory cytokine production in response to pathogenic molecules, such as ExoS, which is a TLR2 and TLR4 agonist produced by the opportunistic pathogen Pseudomonas aeruginosa. We reasoned that responses to ExoS, a protein, might differ from canonical TLR agonists such as LPS. Stimulating the expression of mCD14 with vitamin D3 enhanced the response to ExoS and LPS. Also, blocking anti-CD14 antibody or removing mCD14 using PLC reduced responses to ExoS and LPS. Furthermore, CD14-deficient cells were unable to bind and respond to ExoS, which was restored by stable transfection of mCD14, indicating that mCD14 was required for the response to ExoS. However, addition of sCD14 to culture enhanced responsiveness to LPS but not ExoS. Moreover, the addition of serum did not alter the response to ExoS but enhanced the response to LPS. Despite differences of adaptor molecule use between ExoS and LPS, lipid antagonists that compete for LPS binding to CD14 also inhibited the response to ExoS. These results highlight a fundamental difference between TLR agonists in their requirements for CD14 and serum components. These results suggest that understanding the dissimilarities and targeting overlapping sites of interaction on CD14 may yield a synergistic, clinical benefit during infections where a variety of TLR agonists are present.


Assuntos
ADP Ribose Transferases/imunologia , Toxinas Bacterianas/imunologia , Membrana Celular/imunologia , Citocinas/biossíntese , Receptores de Lipopolissacarídeos/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/enzimologia , Transdução de Sinais/imunologia , ADP Ribose Transferases/metabolismo , Toxinas Bacterianas/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Citocinas/imunologia , Humanos , Inflamação/imunologia , Inflamação/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/imunologia , Pseudomonas aeruginosa/imunologia
14.
J Immunol ; 173(3): 2031-40, 2004 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-15265938

RESUMO

Some bacterial products possess multiple immunomodulatory effects and thereby complex mechanisms of action. Exogenous administration of an important Pseudomonas aeruginosa virulence factor, exoenzyme S (ExoS) induces potent monocyte activation leading to the production of numerous proinflammatory cytokines and chemokines. However, ExoS is also injected directly into target cells, inducing cell death through its multiple effects on signaling pathways. This study addresses the mechanisms used by ExoS to induce monocyte activation. Exogenous administration resulted in specific internalization of ExoS via an actin-dependent mechanism. However, ExoS-mediated cellular activation was not inhibited if internalization was blocked, suggesting an alternate mechanism of activation. ExoS bound a saturable and specific receptor on the surface of monocytic cells. ExoS, LPS, and peptidoglycan were all able to induce tolerance and cross-tolerance to each other suggesting the involvement of a TLR in ExoS-recognition. ExoS activated monocytic cells via a myeloid differentiation Ag-88 pathway, using both TLR2 and the TLR4/MD-2/CD14 complex for cellular activation. Interestingly, the TLR2 activity was localized to the C-terminal domain of ExoS while the TLR4 activity was localized to the N-terminal domain. This study provides the first example of how different domains of the same molecule activate two TLRs, and also highlights the possible overlapping pathophysiological processes possessed by microbial toxins.


Assuntos
ADP Ribose Transferases/química , Toxinas Bacterianas/química , Glicoproteínas de Membrana/agonistas , Monócitos/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Receptores de Superfície Celular/agonistas , ADP Ribose Transferases/fisiologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Antígenos de Diferenciação/fisiologia , Antígenos de Superfície/fisiologia , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Citocalasina D/farmacologia , Corantes Fluorescentes , Humanos , Receptores de Lipopolissacarídeos/fisiologia , Lipopolissacarídeos/farmacologia , Antígeno 96 de Linfócito , Substâncias Macromoleculares , Monócitos/metabolismo , Fator 88 de Diferenciação Mieloide , Peptidoglicano/farmacologia , Estrutura Terciária de Proteína , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/fisiologia , Proteínas Recombinantes de Fusão/farmacologia , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Receptores Toll-Like , Fator de Necrose Tumoral alfa/biossíntese
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA