RESUMO
The ability of recombinant, SARS-CoV-2 Spike (S) protein to modulate the production of two COVID-19 relevant, pro-inflammatory cytokines (IL-6 and IFN-γ) in PBMC cultures of healthy, pre-COVID-19 subjects was investigated. We observed that cytokine production was largely and diversely modulated by the S protein depending on antigen or mitogen stimulation, as well as on the protein source, insect (S-in) or human (S-hu) cells. While both proteins co-stimulated cytokine production by polyclonally CD3-activated T cells, PBMC activation by the mitogenic lectin Concanavalin A (Con A) was up-modulated by S-hu protein and down-modulated by S-in protein. These modulatory effects were likely mediated by the S glycans, as demonstrated by direct Con A-S binding experiments and use of yeast mannan as Con A binder. While being ineffective in modulating memory antigenic T cell responses, the S proteins and mannan were able to induce IL-6 production in unstimulated PBMC cultures and upregulate the expression of the mannose receptor (CD206), a marker of anti-inflammatory M2 macrophage. Our data point to a relevant role of N-glycans, particularly N-mannosidic chains, decorating the S protein in the immunomodulatory effects here reported. These novel biological activities of the S glycan ectodomain may add to the comprehension of COVID-19 pathology and immunity to SARS-CoV-2.
Assuntos
COVID-19 , Interleucina-6 , Lectinas Tipo C , Leucócitos Mononucleares , Receptor de Manose , Lectinas de Ligação a Manose , Receptores de Superfície Celular , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Humanos , Glicoproteína da Espícula de Coronavírus/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Lectinas Tipo C/metabolismo , Receptores de Superfície Celular/metabolismo , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/imunologia , COVID-19/imunologia , COVID-19/virologia , COVID-19/metabolismo , SARS-CoV-2/imunologia , SARS-CoV-2/metabolismo , Lectinas de Ligação a Manose/metabolismo , Interleucina-6/metabolismo , Citocinas/metabolismo , Interferon gama/metabolismo , Células Cultivadas , Polissacarídeos/metabolismo , Voluntários Saudáveis , Linfócitos T/imunologia , Linfócitos T/metabolismo , Ativação Linfocitária , Concanavalina A/metabolismoRESUMO
Cholera toxin (CT) is a potent adjuvant for mucosal vaccination; however, its mechanism of action has not been clarified completely. It is well established that peripheral monocytes differentiate into dendritic cells (DCs) both in vitro and in vivo and that monocytes are the in vivo precursors of mucosal CD103(-) proinflammatory DCs. In this study, we asked whether CT had any effects on the differentiation of monocytes into DCs. We found that CT-treated monocytes, in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4), failed to differentiate into classical DCs (CD14(low) CD1a(high)) and acquired a macrophage-like phenotype (CD14(high) CD1a(low)). Cells differentiated in the presence of CT expressed high levels of major histocompatibility complex class I (MHC-I) and MHC-II and CD80 and CD86 costimulatory molecules and produced larger amounts of IL-1ß, IL-6, and IL-10 but smaller amounts of tumor necrosis factor alpha (TNF-α) and IL-12 than did monocytes differentiated into DCs in the absence of CT. The enzymatic activity of CT was found to be important for the skewing of monocytes toward a macrophage-like phenotype (Ma-DCs) with enhanced antigen-presenting functions. Indeed, treatment of monocytes with scalar doses of forskolin (FSK), an activator of adenylate cyclase, induced them to differentiate in a dose-dependent manner into a population with phenotype and functions similar to those found after CT treatment. Monocytes differentiated in the presence of CT induced the differentiation of naïve T lymphocytes toward a Th2 phenotype. Interestingly, we found that CT interferes with the differentiation of monocytes into DCs in vivo and promotes the induction of activated antigen-presenting cells (APCs) following systemic immunization.
Assuntos
Adjuvantes Imunológicos/farmacologia , Células Apresentadoras de Antígenos/citologia , Diferenciação Celular/efeitos dos fármacos , Toxina da Cólera/farmacologia , Células Dendríticas/citologia , Monócitos/citologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , FenótipoRESUMO
The efficacy of vaccines can be greatly improved by adjuvants that enhance and modify the magnitude and the duration of the immune response. Several approaches to design rational adjuvants are based on the suppression of regulatory T-cell (Treg) function. Here, we evaluated whether removal or addition of Treg at the time of vaccination with tetanus toxoid and the mucosal adjuvant cholera toxin (CT), would affect immune responses. We found that depletion/inactivation of CD4(+)CD25(+) Treg, either by treatment of BALB/c mice with anti-CD25 monoclonal antibodies or by adoptive transfer of CD4(+)CD25(-) T lymphocytes depleted of CD4(+)CD25(+) Treg into nu/nu mice, impaired antibody production after mucosal immunization in the presence of CT. Conversely, transfer of polyclonal, but not Ag-specific, CD4(+)CD25(+)Foxp3(+) Treg to normal BALB/c mice enhanced CT-induced antibody responses. An increased titer of both immunoglobulin IgG1 and IgG2a antibody subclasses was found, however, the ratio between IgG1/IgG2a with or without polyclonal Treg was comparable, suggesting that polyclonal Treg influence the magnitude, but not the quality of the immune response. Recipients of polyclonal Treg that had been immunized with CT had an increased number of Ag-specific CD4(+) T cells with an activated phenotype (CD44(hi)) in the draining lymph nodes. This accumulation of Ag-specific CD4(+) T lymphocytes could favour the germinal centre formation and may promote T-dependent B-cell responses. Overall, our study indicates that Foxp3(+) Treg can not only function as suppressor cells but also as helper T cells, depending on the type of immune response being evaluated and the microenvironment in which the response is generated.
Assuntos
Transferência Adotiva , Centro Germinativo/imunologia , Imunidade Humoral/imunologia , Imunidade nas Mucosas/imunologia , Imunoglobulina G/imunologia , Linfócitos T Reguladores/imunologia , Administração Intranasal , Animais , Diferenciação Celular/imunologia , Toxina da Cólera/imunologia , Fatores de Transcrição Forkhead/metabolismo , Centro Germinativo/citologia , Receptores de Hialuronatos/metabolismo , Imunoglobulina A/biossíntese , Imunoglobulina A/imunologia , Imunoglobulina G/biossíntese , Interferon gama/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Linfócitos T Reguladores/fisiologia , Toxoide Tetânico/imunologia , Células Th2/imunologia , Células Th2/metabolismo , Regulação para Cima/imunologiaRESUMO
CD8+ T cells are an essential component of an effective host immune response to tumors and viral infections. Genetic immunization is particularly suitable for inducing CTL responses, because the encoded proteins enter the MHC class I processing pathway through either transgene expression or cross-presentation. In order to compare the efficiency and persistence of immune response induced by genetic vaccines, BALB/c mice were immunized either twice intramuscularly with DNA plasmid expressing a codon-optimized HIV-1 gp120 Envelope sequence together with murine GM-CSF sequence or with a single immunization using an integrase defective lentiviral vector (IDLV) expressing the same proteins. Results strongly indicated that the schedule based on IDLV vaccine was more efficient in inducing specific immune response, as evaluated three months after the last immunization by IFNgamma ELISPOT in both splenocytes and bone marrow- (BM-) derived cells, chromium release assay in splenocytes, and antibody detection in sera. In addition, IDLV immunization induced high frequency of polyfunctional CD8+ T cells able to simultaneously produce IFNgamma, TNFalpha, and IL2.
RESUMO
The power of cholera toxin (CT) as an effective mucosal adjuvant is well established. Because of the high toxicity of CT, its clinical use is unlikely. Therefore, the need to identify effective and non toxic mucosal adjuvants for human use is important. For this purpose, CT is largely used as a reference molecule for testing the efficacy of new candidate adjuvants in animal models. Here, we evaluated the kinetics and the localization of antigen-specific humoral and cellular immune responses elicited by intranasal immunization with tetanus toxoid antigen in the presence of CT. We show that an antigen-specific cellular immune response localized in the mediastinal lymph nodes can be observed already 1 week after the first immunization. The induction of an appreciable titer of an antibody-specific immune response was assessed after two immunizations. Therefore, we suggest that the efficacy of new candidate mucosal adjuvants can be tested by evaluating the cellular immune response in the mediastinal lymph nodes at early stages of immunization.
Assuntos
Antígenos CD4/imunologia , Epitopos de Linfócito T/imunologia , Imunização/métodos , Linfonodos/imunologia , Mediastino/fisiologia , Linfócitos T/imunologia , Administração Intranasal , Animais , Toxina da Cólera/administração & dosagem , Toxina da Cólera/imunologia , Feminino , Imunidade Celular/efeitos dos fármacos , Imunidade Celular/imunologia , Linfonodos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
B lymphocytes play an important role in the immune response induced by mucosal adjuvants. In this study we investigated the in vitro antigen-presenting cell (APC) properties of human B cells upon treatment with cholera toxin (CT) and Escherichia coli heat-labile enterotoxin (LT) and nontoxic counterparts of these toxins, such as the B subunit of CT (CT-B) and the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Furthermore, forskolin (FSK), a direct activator of adenylate cyclase, and cyclic AMP (cAMP) analogues were used to investigate the role of the increase in intracellular cAMP caused by the A subunit of CT and LT. B lymphocytes were cultured with adjuvants and polyclonal stimuli necessary for activation of B cells in the absence of CD4 T cells. Data indicated that treatment with CT, LT, FSK, or cAMP analogues, but not treatment with CT-B or LTK63, upregulated surface activation markers on B cells, such as CD86 and HLA-DR, and induced inhibition of the proliferation of B cells at early time points, while it increased cell death in long-term cultures. Importantly, B cells treated with CT, LT, or FSK were able to induce pronounced proliferation of both CD4(+) and CD8(+) allogeneic T cells compared with untreated B cells and B cells treated with CT-B and LTK63. Finally, only treatment with toxins or FSK induced antigen-specific T-cell proliferation in Mycobacterium tuberculosis purified protein derivative or tetanus toxoid responder donors. Taken together, these results indicated that the in vitro effects of CT and LT on human B cells are mediated by cAMP.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Toxinas Bacterianas/imunologia , Toxina da Cólera/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Linfócitos B/química , Antígeno B7-2/análise , Toxinas Bacterianas/toxicidade , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proliferação de Células , Células Cultivadas , Toxina da Cólera/toxicidade , Colforsina/farmacologia , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Enterotoxinas/toxicidade , Ativadores de Enzimas/farmacologia , Proteínas de Escherichia coli/toxicidade , Antígenos HLA-DR/análise , Humanos , Fatores Imunológicos/farmacologia , Ativação LinfocitáriaRESUMO
Most microbes invading through mucosal surfaces cause disease and therefore strategies to induce mucosal immune responses are strongly needed. Vitamin A metabolites, such as retinoic acid (RA), play crucial roles in programming T and B cells to home to mucosal compartments, therefore we evaluated the capacity of RA to elicit mucosal immune responses against tuberculosis (TB) after parenteral vaccination. We found that mice immunized through subcutaneous injections with the TB subunit vaccine (CAF01+H56) in presence of RA show enhanced mucosal H56-specific IgA responses and enhanced Ag-specific CD4+ T lymphocytes homing to the lung as compared with control mice. Immunization with CAF01+H56 in presence of RA resulted in lower bacterial loads in the lungs of mice 14 days after challenge with virulent Mycobacterium tuberculosis (Mtb) as compared to mice immunized in the absence of RA or vaccinated with BCG. Higher amounts of IFNγ and IL-17 pro-inflammatory cytokines were found in lung homogenates of mice immunized with CAF01+H56 and RA 24 h after Mtb infection. However, 6 weeks after infection the protection was comparable in vaccinated mice with or without RA even though treatment with RA during immunization is able to better contain the inflammatory response by the host. Furthermore, at later stage of the infection a higher percentage of Mtb specific CD4+PD1+ T lymphocytes were found in the lungs of mice immunized with CAF01+H56 and RA. These data show that an enhanced mucosal immune response is generated during parenteral vaccination in presence of RA. Furthermore, RA treatment contained the bacterial growth at an early stage of the infection and limited the inflammatory response in the lung at later time points.
Assuntos
Imunidade nas Mucosas , Tretinoína/administração & dosagem , Vacinas contra a Tuberculose/administração & dosagem , Tuberculose Pulmonar/prevenção & controle , Vacinas de Subunidades Antigênicas/administração & dosagem , Alérgenos/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Células Produtoras de Anticorpos/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Feminino , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Pulmão/imunologia , Linfócitos/imunologia , Camundongos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/imunologia , Ovalbumina/imunologia , Tuberculose Pulmonar/imunologia , VacinaçãoRESUMO
Cholera toxin (CT) is known to inhibit the proliferation of murine and human T lymphocytes. In this study we have analysed the mechanisms underlying the inhibitory effect of CT on subpopulations of human CD4+ and CD8+ T lymphocytes. We show that CT dramatically prevents the activation of resting T lymphocytes, whereas it has a minor effect on cells that have been previously activated. Analysis of DNA content of the CT-treated T cells showed an arrest in the G(0)/G(1) phase and this correlated with high expression of the cyclin-dependent kinase inhibitor p27(kip). Moreover, we show that CT up-regulates the expression of the inhibitory molecule CTLA-4 in naïve, effector and memory resting CD4+ T cells and in resting CD8+ T lymphocytes. The regulation of CTLA-4 expression by CT is at the transcriptional level. Indeed, in cells treated with CT we observed an increase of two mRNA variants coding for the membrane and the soluble CTLA-4 molecules. In parallel with the up-regulation of the inhibitory CTLA-4, CT down-modulates the costimulatory molecule CD28 on CD4+ and CD8+ resting T cells. The increased expression of CTLA-4 played a role in controlling T cell activation and function as blocking anti-CTLA-4 F(ab')(2) mAbs partially inhibited anti-CD3 mAbs induced proliferation. These findings show that the inhibition of T cell proliferation by CT affects early stages of the T cell activation and involves the modulation of costimulatory molecules CTLA-4 and CD28 on resting T cells.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação/metabolismo , Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Toxina da Cólera/toxicidade , Ativação Linfocitária , Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Antígeno CTLA-4 , Ciclo Celular , Proliferação de Células , Toxina da Cólera/metabolismo , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismoRESUMO
We have previously shown that cholera toxin (CT) and other cAMP-elevating agents induce up-regulation of the inhibitory molecule CTLA-4 on human resting T lymphocytes. In this study, we evaluated the function of these cells. We found that purified human CD4(+) T lymphocytes pretreated with CT were able to inhibit proliferation of autologous PBMC in a dose-dependent manner. It is interesting that this phenomenon was not mediated by inhibitory cytokines such as IL-10, IL-4, or TGF-beta but was in part caused by the release of extracellular cAMP by the CD4(+) T lymphocytes. Purified CD4(+) T cells pretreated with forskolin, a transient cAMP inducer, or with dibutyryl cAMP, an analog of cAMP, did not exert suppressive functions, suggesting that a sustained production of cAMP, such as that induced by CT, was required to identify a novel regulatory function mediated by CD4(+) T cells. Our results show that CD4(+) T lymphocytes can exert regulatory functions through the release of extracellular cAMP and that the cyclic nucleotide acts as a primary messenger, which could play a biological role in the modulation of immune responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , AMP Cíclico/biossíntese , Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Toxina da Cólera/farmacologia , AMP Cíclico/imunologia , AMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Humanos , Interleucina-10/antagonistas & inibidores , Interleucina-4/antagonistas & inibidores , Leucócitos Mononucleares/efeitos dos fármacos , Relação Estrutura-Atividade , Fatores de Tempo , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
Interleukin (IL)-2 plays an important role in the control of the immune responses, and it is released in a variety of tissues in response to inflammatory stimuli. As monocytes and mature dendritic cells (DCs) express CD25, the high-affinity subunit of IL-2 receptor, we examined the effect of exogenous IL-2 on the in vitro generation and maturation of DCs from monocytes. Human monocyte-derived DCs (MDDCs) were generated by culturing monocytes with granulocyte macrophage-colony stimulating factor (GM-CSF) and IL-4 in the presence or absence of IL-2. The cytokine was added at the beginning and after 5 days of culture. Our findings indicate that IL-2 does induce monocytes to differentiate into DCs with the same morphology and phenotype of that obtained in the presence of GM-CSF and IL-4 alone, but with some distinctive functional properties. DCs differentiated in the presence of IL-2 secreted significantly more IL-1beta, TNF-alpha, and IL-12 p70 in response to lipopolysaccharide stimulation and induced allogeneic, naïve T cells to release a significantly higher amount of interferon-gamma if compared with DCs obtained by culturing monocytes with GM-CSF and IL-4. These results indicate unrecognized effects of IL-2 on human MDDCs and suggest that an IL-2-rich environment during differentiation and maturation of DCs can modify their T helper cell-inducing properties.
Assuntos
Citocinas/biossíntese , Interleucina-2/imunologia , Monócitos/imunologia , Células Th1/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Humanos , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/farmacologia , Lipopolissacarídeos/farmacologia , FenótipoRESUMO
Human monocytes can differentiate into dendritic cells (DC) according to the nature of environmental signals. Pathogenic mycobacteria have been demonstrated interfere with this process to generate a not fully competent DC population. This mechanism is not common to non-pathogenic mycobacteria and could be correlated with bacterial virulence. However, it could represents a new mechanism of immune escape by persistent pathogens or a control mechanism of immune system to preserve the life of the organ against persistent infections.
Assuntos
Diferenciação Celular , Células Dendríticas/citologia , Células Dendríticas/imunologia , Mycobacterium/imunologia , Células Dendríticas/microbiologia , Humanos , Modelos ImunológicosRESUMO
We have shown that cholera toxin (CT) and other cyclic AMP (cAMP)-elevating agents induce upregulation of the inhibitory molecule CTLA-4 in human resting CD4+ T lymphocytes, which following the treatment acquired suppressive functions. In this study, we evaluated the effect of cAMP-elevating agents on human CD4+CD25+ T cells, which include the T regulatory cells (Tregs) that play a pivotal role in the maintenance of immunological tolerance. We found that cAMP-elevating agents induce upregulation of CTLA-4 in CD4+CD25- and further enhance its expression in CD4+CD25+ T cells. We observed an increase of two isoforms of mRNA coding for the membrane and the soluble CTLA-4 molecules, suggesting that the regulation of CTLA-4 expression by cAMP is at the transcriptional level. In addition, we found that the increase of cAMP in CD4+CD25+ T cells converts the CD4+CD25+Foxp3- T cells in CD4+CD25+Foxp3+ T cells, whereas the increase of cAMP in CD4+CD25- T cells did not upregulate Foxp3 in the absence of activation stimuli. To investigate the function of these cells, we performed an in vitro suppression assay by culturing CD4+CD25+ T cells untreated or pre-treated with CT with anti-CD3 mAbs-stimulated autologous peripheral blood mononuclear cell. We found that CT enhances the inhibitory function of CD4+CD25+ T cells, CD4+, and CD8+ T cell proliferation and IFNγ production are strongly inhibited by CD4+CD25+ T cells pre-treated with cAMP-elevating agents. Furthermore, we found that CD4+CD25+ T lymphocytes pre-treated with cAMP-elevating agents induce the upregulation of CD80 and CD86 co-stimulatory molecules on immature dendritic cells (DCs) in the absence of antigenic stimulation, however without leading to full DC maturation. These data show that the increase of intracellular cAMP modulates the phenotype and function of human CD4+CD25+ T cells.
RESUMO
Human monocytes can differentiate into dendritic cells (DCs) according to the nature of environmental signals. We tested here whether the infection with the live tuberculosis vaccine bacillus Calmette-Guerin (BCG), which is known to be limited in preventing pulmonary tuberculosis, modulates monocyte and DC differentiation. We found that monocytes infected with BCG differentiate into CD1a- DCs (BCG-DCs) in the presence of granulocyte macrophage-colony stimulating factor and interleukin (IL)-4 and acquired a mature phenotype in the absence of maturation stimuli. In addition, BCG-DCs produced proinflammatory cytokines (tumor necrosis factor alpha, IL-1beta, IL-6) and IL-10 but not IL-12. BCG-DCs were able to stimulate allogeneic T lymphocytes to a similar degree as DCs generated in the absence of infection. However, BCG-DCs induced IL-4 production when cocultured with human cord-blood mononuclear cells. The induction of IL-4 production by DCs generated by BCG-infected monocytes could explain the failure of the BCG vaccine to prevent pulmonary tuberculosis.
Assuntos
Vacina BCG/imunologia , Células Dendríticas/imunologia , Monócitos/microbiologia , Mycobacterium bovis/imunologia , Células Th2/imunologia , Diferenciação Celular , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/citologia , Sangue Fetal , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Lipopolissacarídeos/farmacologiaRESUMO
Salmonella Typhimurium has been shown to be highly effective as antitumor agent. The aim of this study was to investigate the tumor targeting efficacy and the mechanism of action of a specific attenuated mutant strain of Salmonella Typhimurium (STM) devoid of the whole operon coding for the high-affinity zinc transporter ZnuABC, which is required for bacterial growth in environments poor in zinc and for conferring full virulence to different Gram-negative pathogens.We showed that STM is able to penetrate and replicate into tumor cells in in vitro and in vivo models. The subcutaneous administration of STM in mammary adenocarcinoma mouse model led to both reduction of tumor growth and increase in life expectancy of STM treated mice. Moreover, investigating the potential mechanism behind the favorable clinical outcomes, we provide evidence that STM stimulates a potent inflammatory response and a specific immune pattern, recruiting a large number of innate and adaptive immune cells capable to contrast the immunosuppressive environment generated by tumors.
Assuntos
Adenocarcinoma/patologia , Antineoplásicos/farmacologia , Imunoterapia/métodos , Neoplasias Mamárias Experimentais/patologia , Salmonella typhimurium , Transportadores de Cassetes de Ligação de ATP/deficiência , Adenocarcinoma/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Salmonella typhimurium/genéticaRESUMO
In this study, we test the hypothesis that cAMP, acting as an extracellular mediator, affects the physiology and function of human myeloid cells. The cAMP is a second messenger recognized as a universal regulator of several cellular functions in different organisms. Many studies have shown that extracellular cAMP exerts regulatory functions, acting as first mediator in multiple tissues. However, the impact of extracellular cAMP on cells of the immune system has not been fully investigated. We found that human monocytes exposed to extracellular cAMP exhibit higher expression of CD14 and lower amount of MHC class I and class II molecules. When cAMP-treated monocytes are exposed to proinflammatory stimuli, they exhibit an increased production of IL-6 and IL-10 and a lower amount of TNF-α and IL-12 compared with control cells, resembling the features of the alternative-activated macrophages or M2 macrophages. In addition, we show that extracellular cAMP affects monocyte differentiation into DCs, promoting the induction of cells displaying an activated, macrophage-like phenotype with reduced capacity of polarized, naive CD4(+) T cells into IFN-γ-producing lymphocytes compared with control cells. The effects of extracellular cAMP on monocytes are mediated by CD73 ecto-5'-nucleotidase and A2A and A2B adenosine receptors, as selective antagonists could reverse its effects. Of note, the expression of CD73 molecules has been found on the membrane of a small population of CD14(+)CD16(+) monocytes. These findings suggest that an extracellular cAMP-adenosine pathway is active in cells of the immune systems.
Assuntos
AMP Cíclico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , 5'-Nucleotidase/biossíntese , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citocinas/biossíntese , Citocinas/imunologia , Proteínas Ligadas por GPI/biossíntese , Regulação da Expressão Gênica/imunologia , Humanos , Receptores de Lipopolissacarídeos/biossíntese , Receptores de Lipopolissacarídeos/imunologia , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Monócitos/citologia , Monócitos/imunologia , Receptor A2A de Adenosina/imunologia , Receptor A2B de Adenosina/imunologia , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Antigen-specific IFN-gamma producing CD4(+) T cells are the main mediators of protection against Mycobacterium tuberculosis infection both under natural conditions and following vaccination. However these cells are responsible for lung damage and poor vaccine efficacy when not tightly controlled. Discovering new tools to control nonprotective antigen-specific IFN-gamma production without affecting protective IFN-gamma is a challenge in tuberculosis research. METHODS AND FINDINGS: Immunization with DNA encoding Ag85B, a candidate vaccine antigen of Mycobacterium tuberculosis, elicited in mice a low but protective CD4(+) T cell-mediated IFN-gamma response, while in mice primed with DNA and boosted with Ag85B protein a massive increase in IFN-gamma response was associated with loss of protection. Both protective and non-protective Ag85B-immunization generated antigen-specific CD8(+) T cells which suppressed IFN-gamma-secreting CD4(+) T cells. However, ex vivo ligation of 4-1BB, a member of TNF-receptor super-family, reduced the massive, non-protective IFN-gamma responses by CD4(+) T cells in protein-boosted mice without affecting the low protective IFN-gamma-secretion in mice immunized with DNA. This selective inhibition was due to the induction of 4-1BB exclusively on CD8(+) T cells of DNA-primed and protein-boosted mice following Ag85B protein stimulation. The 4-1BB-mediated IFN-gamma inhibition did not require soluble IL-10, TGF-beta, XCL-1 and MIP-1beta. In vivo Ag85B stimulation induced 4-1BB expression on CD8(+) T cells and in vivo 4-1BB ligation reduced the activation, IFN-gamma production and expansion of Ag85B-specific CD4(+) T cells of DNA-primed and protein-boosted mice. CONCLUSION/SIGNIFICANCE: Antigen-specific suppressor CD8(+) T cells are elicited through immunization with the mycobacterial antigen Ag85B. Ligation of 4-1BB receptor further enhanced their suppressive activity on IFN-gamma-secreting CD4(+) T cells. The selective expression of 4-1BB only on CD8(+) T cells in mice developing a massive, non-protective IFN-gamma response opens novel strategies for intervention in tuberculosis pathology and vaccination through T-cell co-stimulatory-based molecular targeting.
Assuntos
Antígenos de Bactérias/imunologia , Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Interferon gama/biossíntese , Mycobacterium tuberculosis/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/fisiologia , Animais , Camundongos , Baço/citologia , Baço/imunologiaRESUMO
Alzheimer's disease (AD) is characterized by abnormal accumulation of amyloid-beta peptide (Abeta) into extracellular fibrillar deposits, paralleled by chronic neuroinflammatory processes. Although Abeta seems to be causative in AD brain damage, the role of the immune system and its mechanisms still remain to be clarified. We have recently shown that normal monocyte-derived dendritic cells (MDDCs), when differentiated in the presence of Abeta1-42, acquire an inflammatory phenotype and a reduced antigen presenting ability. Here we studied MDDCs derived from AD patients in comparison with MDDCs obtained from healthy control subjects (HC). MDDCs from AD patients, at variance with HC-derived cells, were characterized by an augmented cell recovery, a consistent increase in the expression of the pro-inflammatory ICAM-1 molecule, a decrease in the expression of the co-stimulatory CD40 molecule, and an impaired ability to induce T cell proliferation. Furthermore, MDDCs from AD produced higher amounts of IL-6 than HC-derived cells, confirming the more pronounced pro-inflammatory features of these cells in AD patients. Consistent results have been also obtained with monocytes, the MDDC precursors. In fact, while unstimulated monocytes do not appear to be different in AD and HC, after stimulation with lipopolysaccharide, AD monocytes overexpressed ICAM-1 with respect to controls, suggesting that common pathways of monocyte activation and MDDC differentiation are altered in AD. Overall, these findings show that AD-linked dysregulated immune mechanisms exist, which lead to dendritic cell-mediated over-activation of inflammation and impaired antigen presentation, thus supporting the view that immune cell activation could play an important role in AD pathogenesis.
Assuntos
Doença de Alzheimer/patologia , Antígenos CD40/metabolismo , Células Dendríticas/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/metabolismo , Idoso , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Endocitose/efeitos dos fármacos , Feminino , Citometria de Fluxo/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Lipopolissacarídeos , Masculino , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Testes Neuropsicológicos , Fragmentos de Peptídeos/farmacologiaRESUMO
The development of mucosal vaccines for prevention of infectious diseases caused by pathogens entering through the mucosal surfaces is an important and challenging objective. To this purpose, we evaluated the efficacy and durability of immune response induced by sublingual immunization with tetanus toxoid (TT) as an antigen in the presence of mucosal adjuvants, such as E. coli Heat-Labile enterotoxin (LT) or the mutant of LT lacking ADP ribosyltransferase activity (LTK63). Both serum anti-TT IgG and mucosal anti-TT IgA antibodies reached a peak after four immunizations and decreased over time, maintaining detectable titers up to 4 months after the last immunization. Similarly, antigen-specific antibody secreting cells in bone marrow and TT-specific CD4+ and CD8+ T cells in draining lymph nodes and spleen were present up to 4 months from the last immunization. Overall, LT-treated mice showed significantly higher responses compared to LTK63 immunized mice. The efficacy and persistence of the immune response induced by sublingual immunization with different adjuvants strongly suggest that this route represents an appealing and promising alternative to the other mucosal routes of vaccine delivery.
Assuntos
Toxinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Enterotoxinas/imunologia , Proteínas de Escherichia coli/imunologia , Imunidade nas Mucosas , Toxoide Tetânico/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/farmacologia , Administração Sublingual , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Toxinas Bacterianas/administração & dosagem , Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Enterotoxinas/administração & dosagem , Proteínas de Escherichia coli/administração & dosagem , Feminino , Linfonodos/citologia , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologia , Toxoide Tetânico/administração & dosagemRESUMO
Brucellosis is one of the most common bacterial zoonoses worldwide. Infection is usually chronic and sometimes lifelong. Different mechanisms can be postulated as to the basis for the induction of the chronic status of brucellosis, but a comprehensive knowledge is still lacking. Here, we carried out a series of experiments in order to assess if the persistence of Brucella abortus could be ascribed to the effect of a down regulation of the immune response due to activity of regulatory T cells. We demonstrate that CD4+CD25+T regulatory cells are able to limit the effectiveness of CD4+T cells and are able to favor the maintenance and the progression of B. abortus infection.
Assuntos
Brucella abortus/imunologia , Brucelose/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Doença Crônica , Feminino , Humanos , Tolerância Imunológica , Subunidade alfa de Receptor de Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Monocyte differentiation into dendritic cells (DCs) depends on microenvironmental conditions. In this study, the capacity of human monocytes to differentiate into mature DCs and their ability to induce an antiviral immune response was investigated in HIV-infected patients. In healthy subjects, monocytes differentiate into CD1a+ DCs in the presence of granulocyte macrophage colony-stimulating factor and interleukin (IL)-4 and matured in the presence of lipopolysaccharide. Here, we found that in 30% and 45% of HIV-infected white and African subjects, respectively, monocytes gave rise to a homogeneous CD1a* DC population. In the patients who gave rise only to the CD1a* DCs, this population spontaneously produced IL-10 but not IL-12, and induced a T helper 2-like immune response when cultured with human T cells isolated from cord blood mononuclear cells. In patients with monocytes differentiated into CD1a* DCs, a high percentage of HIV-specific CD4 T cells producing IL-4 were seen in the peripheral blood. Furthermore, differentiation of monocytes into DCs with CD1a* phenotype correlated with low CD4 T-cell counts and high viral loads in HIV-infected subjects. These results suggest that the differentiation of monocytes into CD1a* DCs may be a phenotypic marker associated with progression of the disease.