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1.
J Immunol ; 198(7): 2772-2784, 2017 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-28258198

RESUMO

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.


Assuntos
Adjuvantes Imunológicos/administração & dosagem , Vacinas Anticâncer/imunologia , Venenos de Cnidários/administração & dosagem , Neoplasias Experimentais/patologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Animais , Venenos de Cnidários/imunologia , Feminino , Citometria de Fluxo , Lipossomos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia
2.
J Immunol ; 194(5): 2319-29, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25637024

RESUMO

Understanding the cellular populations and mechanisms responsible for overcoming immune compartmentalization is valuable for designing vaccination strategies targeting distal mucosae. In this study, we show that the human pathogen Chlamydia trachomatis infects the murine respiratory and genital mucosae and that T cells, but not Abs, elicited through intranasal immunization can protect against a subsequent transcervical challenge. Unlike the genital infection where CD8(+) T cells are primed, yet fail to confer protection, we found that intranasal priming engages both CD4(+) and CD8(+) T cells, allowing for protection against genital infection with C. trachomatis. The protection is largely dependent on IFN-γ secretion by T cells. Moreover, different chemokine receptors are critical for C. trachomatis-specific CD4(+) T cells to home to the lung, rather than the CXCR3- and CCR5-dependent migration observed during genital infection. Overall, this study demonstrates that the cross-mucosa protective immunity against genital C. trachomatis infection following intranasal immunization is not dependent on Ab response but is mediated by not only CD4(+) T cells but also by CD8(+) T cells. This study provides insights for the development of vaccines against mucosal pathogens that threaten reproductive health worldwide.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Chlamydia/prevenção & controle , Imunidade Celular , Interferon gama/metabolismo , Mucosa/imunologia , Administração Intranasal , Animais , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/patologia , Movimento Celular , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/patologia , Chlamydia trachomatis/imunologia , Feminino , Humanos , Imunização , Interferon gama/biossíntese , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mucosa/microbiologia , Mucosa/patologia , Útero/imunologia , Útero/microbiologia , Útero/patologia
3.
PLoS Pathog ; 8(8): e1002809, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22912573

RESUMO

The activation of host cells by interferon gamma (IFNγ) is essential for inhibiting the intracellular replication of most microbial pathogens. Although significant advances have been made in identifying IFNγ-dependent host factors that suppress intracellular bacteria, little is known about how IFNγ enables cells to recognize, or restrict, the growth of pathogens that replicate in the host cytoplasm. The replication of the cytosolic bacterial pathogen Shigella flexneri is significantly inhibited in IFNγ-stimulated cells, however the specific mechanisms that mediate this inhibition have remained elusive. We found that S. flexneri efficiently invades IFNγ-activated mouse embryonic fibroblasts (MEFs) and escapes from the vacuole, suggesting that IFNγ acts by blocking S. flexneri replication in the cytosol. This restriction on cytosolic growth was dependent on interferon regulatory factor 1 (IRF1), an IFNγ-inducible transcription factor capable of inducing IFNγ-mediated cell-autonomous immunity. To identify host factors that restrict S. flexneri growth, we used whole genome microarrays to identify mammalian genes whose expression in S. flexneri-infected cells is controlled by IFNγ and IRF1. Among the genes we identified was the pattern recognition receptor (PRR) retanoic acid-inducible gene I (RIG-I), a cytoplasmic sensor of foreign RNA that had not been previously known to play a role in S. flexneri infection. We found that RIG-I and its downstream signaling adaptor mitochondrial antiviral signaling protein (MAVS)--but not cytosolic Nod-like receptors (NLRs)--are critically important for IFNγ-mediated S. flexneri growth restriction. The recently described RNA polymerase III pathway, which transcribes foreign cytosolic DNA into the RIG-I ligand 5'-triphosphate RNA, appeared to be involved in this restriction. The finding that RIG-I responds to S. flexneri infection during the IFNγ response extends the range of PRRs that are capable of recognizing this bacterium. Additionally, these findings expand our understanding of how IFNγ recognizes, and ultimately restricts, bacterial pathogens within host cells.


Assuntos
Citoplasma/imunologia , RNA Helicases DEAD-box/imunologia , Disenteria Bacilar/imunologia , Imunidade Inata , Interferon gama/imunologia , Shigella flexneri/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Animais , Células Cultivadas , Citoplasma/genética , Citoplasma/microbiologia , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Disenteria Bacilar/genética , Embrião de Mamíferos/imunologia , Embrião de Mamíferos/microbiologia , Fibroblastos/imunologia , Fibroblastos/microbiologia , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/imunologia , Interferon gama/genética , Camundongos , Camundongos Knockout , Transdução de Sinais/genética , Transdução de Sinais/imunologia
4.
Proc Natl Acad Sci U S A ; 107(44): 18997-9001, 2010 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-20944063

RESUMO

Coxiella burnetii and Legionella pneumophila are evolutionarily related pathogens with different intracellular infection strategies. C. burnetii persists within and is transmitted by mammalian hosts, whereas, L. pneumophila is found primarily in the environment associated with protozoan hosts. Although a type IV secretion system encoded by the defect in organelle trafficking (dot) and intracellular multiplication (icm) genes is a virulence determinant that remains highly conserved in both bacteria, the two pathogens encode a different array of effector proteins that are delivered into host cells by the Dot/Icm machinery. This difference suggests that adaptations to evolutionarily distinct hosts may be reflected in the effector protein repertoires displayed by these two pathogens. Here we provide evidence in support of this hypothesis. We show that a unique C. burnetii effector from the ankyrin repeat (Ank) family called AnkG interferes with the mammalian apoptosis pathway. AnkG was found to interact with the host protein gC1qR (p32). Either the addition of AnkG to the repertoire of L. pneumophila effector proteins or the silencing of p32 in mouse dendritic cells resulted in a gain of function that allowed intracellular replication of L. pneumophila in these normally restrictive mammalian host cells by preventing rapid pathogen-induced apoptosis. These data indicate that p32 regulates pathogen-induced apoptosis and that AnkG functions to block this pathway. Thus, emergence of an effector protein that interferes with a proapoptotic signaling pathway directed against intracellular bacteria correlates with adaptation of a pathogen to mammalian hosts.


Assuntos
Apoptose/imunologia , Proteínas de Bactérias/imunologia , Coxiella burnetii/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Receptores de Hialuronatos/imunologia , Legionella pneumophila/fisiologia , Febre Q/imunologia , Motivos de Aminoácidos , Animais , Apoptose/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Coxiella burnetii/patogenicidade , Células Dendríticas/imunologia , Células Dendríticas/microbiologia , Células HEK293 , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Legionella pneumophila/patogenicidade , Camundongos , Proteínas Mitocondriais , Febre Q/genética , Febre Q/metabolismo
5.
PLoS Pathog ; 5(6): e1000478, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19521510

RESUMO

Dendritic cells (DCs) are specialized phagocytes that internalize exogenous antigens and microbes at peripheral sites, and then migrate to lymphatic organs to display foreign peptides to naïve T cells. There are several examples where DCs have been shown to be more efficient at restricting the intracellular replication of pathogens compared to macrophages, a property that could prevent DCs from enhancing pathogen dissemination. To understand DC responses to pathogens, we investigated the mechanisms by which mouse DCs are able to restrict replication of the intracellular pathogen Legionella pneumophila. We show that both DCs and macrophages have the ability to interfere with L. pneumophila replication through a cell death pathway mediated by caspase-1 and Naip5. L. pneumophila that avoided Naip5-dependent responses, however, showed robust replication in macrophages but remained unable to replicate in DCs. Apoptotic cell death mediated by caspase-3 was found to occur much earlier in DCs following infection by L. pneumophila compared to macrophages infected similarly. Eliminating the pro-apoptotic proteins Bax and Bak or overproducing the anti-apoptotic protein Bcl-2 were both found to restore L. pneumophila replication in DCs. Thus, DCs have a microbial response pathway that rapidly activates apoptosis to limit pathogen replication.


Assuntos
Apoptose , Células Dendríticas/microbiologia , Células Dendríticas/fisiologia , Legionella pneumophila/crescimento & desenvolvimento , Doença dos Legionários/imunologia , Animais , Caspase 1/metabolismo , Caspase 3/metabolismo , Fragmentação do DNA , Células Dendríticas/imunologia , Doença dos Legionários/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismo
6.
Mol Immunol ; 131: 144-154, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33422341

RESUMO

Sticholysins (Sts) I and II (StI and StII) are pore-forming proteins (PFPs), purified from the Caribbean Sea anemone Stichodactyla helianthus. StII encapsulated into liposomes induces a robust antigen-specific cytotoxic CD8+ T lymphocytes (CTL) response and in its free form the maturation of bone marrow-derived dendritic cells (BM-DCs). It is probable that the latter is partially supporting in part the immunomodulatory effect on the CTL response induced by StII-containing liposomes. In the present work, we demonstrate that the StII's ability of inducing maturation of BM-DCs is also shared by StI, an isoform of StII. Using heat-denatured Sts we observed a significant reduction in the up-regulation of maturation markers indicating that both PFP's ability to promote maturation of BM-DCs is dependent on their conformational characteristics. StII-mediated DC maturation was abrogated in BM-DCs from toll-like receptor (TLR) 4 and myeloid differentiation primary response gene 88 (MyD88)-knockout mice but not in cells from TLR2-knockout mice. Furthermore, the antigen-specific CTL response induced by StII-containing liposomes was reduced in TLR4-knockout mice. These results indicate that StII, and probably by extension StI, has the ability to induce maturation of DCs through a TLR4/MyD88-dependent pathway, and that this activation contributes to the CTL response generated by StII-containing liposomes.


Assuntos
Venenos de Cnidários/metabolismo , Células Dendríticas/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/fisiologia , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Compostos Orgânicos/metabolismo , Transdução de Sinais/fisiologia
7.
PLoS Pathog ; 4(11): e1000220, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19043549

RESUMO

The immune system must discriminate between pathogenic and nonpathogenic microbes in order to initiate an appropriate response. Toll-like receptors (TLRs) detect microbial components common to both pathogenic and nonpathogenic bacteria, whereas Nod-like receptors (NLRs) sense microbial components introduced into the host cytosol by the specialized secretion systems or pore-forming toxins of bacterial pathogens. The host signaling pathways that respond to bacterial secretion systems remain poorly understood. Infection with the pathogen Legionella pneumophila, which utilizes a type IV secretion system (T4SS), induced an increased proinflammatory cytokine response compared to avirulent bacteria in which the T4SS was inactivated. This enhanced response involved NF-kappaB activation by TLR signaling as well as Nod1 and Nod2 detection of type IV secretion. Furthermore, a TLR- and RIP2-independent pathway leading to p38 and SAPK/JNK MAPK activation was found to play an equally important role in the host response to virulent L. pneumophila. Activation of this MAPK pathway was T4SS-dependent and coordinated with TLR signaling to mount a robust proinflammatory cytokine response to virulent L. pneumophila. These findings define a previously uncharacterized host response to bacterial type IV secretion that activates MAPK signaling and demonstrate that coincident detection of multiple bacterial components enables immune discrimination between virulent and avirulent bacteria.


Assuntos
Citocinas/imunologia , Legionella pneumophila/imunologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Transdução de Sinais/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Ativação Enzimática , Mediadores da Inflamação/imunologia , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , NF-kappa B/metabolismo , Proteína Adaptadora de Sinalização NOD1 , Receptores Toll-Like
8.
Front Immunol ; 9: 2473, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30455685

RESUMO

Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8+ T-cells, a key response for the clearance of intracellular pathogens and tumors. The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation.


Assuntos
Venenos de Cnidários/metabolismo , Células Dendríticas/fisiologia , Macrófagos/fisiologia , Linfócitos T Citotóxicos/imunologia , Vacúolos/metabolismo , Animais , Antígenos/imunologia , Antígenos CD8/metabolismo , Células Cultivadas , Venenos de Cnidários/química , Apresentação Cruzada , Feminino , Leupeptinas/farmacologia , Lipossomos/química , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia
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