RESUMO
Secondary bacterial infections contribute to the excess morbidity and mortality of influenza A virus (IAV) infection. Disruption of lung integrity and impaired antibacterial immunity during IAV infection participate in colonization and dissemination of the bacteria out of the lungs. One key feature of IAV infection is the profound alteration of lung myeloid cells, characterized by the recruitment of deleterious inflammatory monocytes. We herein report that IAV infection causes a transient decrease of lung conventional dendritic cells (cDCs) (both cDC1 and cDC2) peaking at day 7 post-infection. While triggering emergency monopoiesis, IAV transiently altered the differentiation of cDCs in the bone marrow, the cDC1-biaised pre-DCs being particularly affected. The impaired cDC differentiation during IAV infection was independent of type I interferons (IFNs), IFN-γ, TNFα and IL-6 and was not due to an intrinsic dysfunction of cDC precursors. The alteration of cDC differentiation was associated with a drop of local and systemic production of Fms-like tyrosine kinase 3 ligand (Flt3-L), a critical cDC differentiation factor. Overexpression of Flt3-L during IAV infection boosted the cDC progenitors' production in the BM, replenished cDCs in the lungs, decreased inflammatory monocytes' infiltration and lowered lung damages. This was associated with partial protection against secondary pneumococcal infection, as reflected by reduced bacterial dissemination and prolonged survival. These findings highlight the impact of distal viral infection on cDC genesis in the BM and suggest that Flt3-L may have potential applications in the control of secondary infections.
Assuntos
Células Dendríticas/imunologia , Vírus da Influenza A/imunologia , Pulmão/imunologia , Proteínas de Membrana/imunologia , Infecções por Orthomyxoviridae/virologia , Infecções Pneumocócicas/imunologia , Superinfecção/imunologia , Animais , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/microbiologia , Células Dendríticas/virologia , Pulmão/microbiologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/complicações , Infecções Pneumocócicas/microbiologia , Infecções Pneumocócicas/virologia , Receptor de Interferon alfa e beta/fisiologia , Streptococcus pneumoniae/imunologiaRESUMO
Immunotherapy aiming at enhancing innate and acquired host immunity is a promising approach for cancer treatment. The invariant NKT (iNKT) cell ligand α-galactosylceramide (α-GalCer) holds great promise in cancer therapy, although several concerns limit its use in clinics, including the uncontrolled response it promotes when delivered in a nonvectorized form. Therefore, development of delivery systems to in vivo target immune cells might be a valuable option to optimize iNKT cell-based antitumor responses. Using dendritic cell (DC)-depleted mice, DC transfer experiments, and in vivo active cell targeting, we show that presentation of α-GalCer by DCs not only triggers optimal primary iNKT cell stimulation, but also maintains secondary iNKT cell activation after challenge. Furthermore, targeted delivery of α-GalCer to CD8α(+) DCs, by means of anti-DEC205 decorated nanoparticles, enhances iNKT cell-based transactivation of NK cells, DCs, and γδ T cells. We report that codelivery of α-GalCer and protein Ag to CD8α(+) DCs triggers optimal Ag-specific Ab and cytotoxic CD8(+) T cell responses. Finally, we show that targeting nanoparticles containing α-GalCer and Ag to CD8α(+) DCs promotes potent antitumor responses, both in prophylactic and in therapeutic settings. Our data may have important implications in tumor immunotherapy and vaccine development.
Assuntos
Antígenos CD8/imunologia , Células Dendríticas/imunologia , Galactosilceramidas/imunologia , Células T Matadoras Naturais/imunologia , Neoplasias Experimentais/imunologia , Animais , Anticorpos/química , Anticorpos/imunologia , Apresentação de Antígeno/imunologia , Antígenos CD/imunologia , Antígenos CD8/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Galactosilceramidas/administração & dosagem , Galactosilceramidas/química , Lectinas Tipo C/imunologia , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Nanopartículas/administração & dosagem , Nanopartículas/química , Células T Matadoras Naturais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Superfície Celular/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Carga Tumoral/imunologiaRESUMO
Interleukin-22 (IL-22) has redundant, protective, or pathogenic functions during autoimmune, inflammatory, and infectious diseases. Here, we addressed the potential role of IL-22 in host defense and pathogenesis during lethal and sublethal respiratory H3N2 influenza A virus (IAV) infection. We show that IL-22, as well as factors associated with its production, are expressed in the lung tissue during the early phases of IAV infection. Our data indicate that retinoic acid receptor-related orphan receptor-γt (RORγt)-positive αß and γδ T cells, as well as innate lymphoid cells, expressed enhanced Il22 transcripts as early as 2 days postinfection. During lethal or sublethal IAV infections, endogenous IL-22 played no role in the control of IAV replication and in the development of the IAV-specific CD8(+) T cell response. During lethal infection, where wild-type (WT) mice succumbed to severe pneumonia, the lack of IL-22 did not accelerate or delay IAV-associated pathogenesis and animal death. In stark contrast, during sublethal IAV infection, IL-22-deficient animals had enhanced lung injuries and showed a lower airway epithelial integrity relative to WT littermates. Of importance, the protective effect of endogenous IL-22 in pulmonary damages was associated with a more controlled secondary bacterial infection. Indeed, after challenge with Streptococcus pneumoniae, IAV-experienced Il22(-/-) animals were more susceptible than WT controls in terms of survival rate and bacterial burden in the lungs. Together, IL-22 plays no major role during lethal influenza but is beneficial during sublethal H3N2 IAV infection, where it limits lung inflammation and subsequent bacterial superinfections.
Assuntos
Infecções Bacterianas/imunologia , Coinfecção/imunologia , Vírus da Influenza A Subtipo H3N2/patogenicidade , Interleucinas/imunologia , Infecções por Orthomyxoviridae/imunologia , Pneumonia/imunologia , Animais , Infecções Bacterianas/microbiologia , Coinfecção/microbiologia , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Pulmão/imunologia , Pulmão/microbiologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Pneumonia/patologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/microbiologia , Streptococcus pneumoniae/patogenicidade , Interleucina 22RESUMO
The humoral and cellular responses against excretory/secretory proteins and soluble extracts of Giardia intestinalis were evaluated in the course of experimental G. intestinalis infection in BALB/c mice. Production of IgG1, IgG2a, IgA, and IgE antibodies against excreted/secreted proteins and soluble extract was detected after infection by G. intestinalis. Specific IgA antibody against E/S proteins and soluble extract form intestinal fluids in infected mice was detected by ELISA. The Western blotting identified proteins of 30, 58, 63, and 83 kDa for IgA and IgG, respectively. High proliferation rate in vitro of spleen cell and secretion of interleukin-4 (IL-4) at 21 days p.i. after stimulation with excreted/secreted proteins and low proliferative response in the presence of soluble extract in infected BALB/c mice was observed. High production of interferon gamma (IFN-γ) and interleukin-5 (IL-5) at the time of decreasing cyst output (14-21 days p.i.) in infected mice was recorded, suggesting the important role of these cytokines in the control of the infection. Interestingly, progressive and gradual increase of the interleukin-10 after stimulation with both preparations was recorded from 7 days until 28 days after infection, indicating the possible regulatory effect of these antigens on the immune response during Giardia infection. Therefore, the infection by Giardia duodenalis stimulates a mixed response Th1 and Th2, mainly stimulated by excretory/secretory antigens. The immunogenicity of these antigens may be a suitable for identification of the proteins related with the effective immune response in the course of infection by G. duodenalsis.
Assuntos
Anticorpos Antiprotozoários/sangue , Antígenos de Protozoários/imunologia , Giardia lamblia/imunologia , Giardíase/imunologia , Imunoglobulina G/sangue , Equilíbrio Th1-Th2 , Animais , Antígenos de Protozoários/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Giardia lamblia/metabolismo , Giardíase/sangue , Giardíase/parasitologia , Interações Hospedeiro-Parasita , Imunoglobulina A/sangue , Imunoglobulina G/classificação , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-4/sangue , Interleucina-5/sangue , Camundongos , Camundongos Endogâmicos BALB C , Baço/imunologia , Baço/parasitologia , Fatores de TempoRESUMO
Invariant natural killer T (iNKT) cells are non-conventional lipid-reactive αß T lymphocytes that play a key role in host responses during viral infections, in particular through the swift production of cytokines. Their beneficial role during experimental influenza A virus (IAV) infection has recently been proposed, although the mechanisms involved remain elusive. Here we show that during in vivo IAV infection, mouse pulmonary iNKT cells produce IFN-γ and IL-22, a Th17-related cytokine critical in mucosal immunity. Although permissive to viral replication, IL-22 production by iNKT cells is not due to IAV infection per se of these cells but is indirectly mediated by IAV-infected dendritic cells (DCs). We show that activation of the viral RNA sensors TLR7 and RIG-I in DCs is important for triggering IL-22 secretion by iNKT cells, whereas the NOD-like receptors NOD2 and NLRP3 are dispensable. Invariant NKT cells respond to IL-1ß and IL-23 provided by infected DCs independently of the CD1d molecule to release IL-22. In vitro, IL-22 protects IAV-infected airway epithelial cells against mortality but has no role on viral replication. Finally, during early IAV infection, IL-22 plays a positive role in the control of lung epithelial damages. Overall, IAV infection of DCs activates iNKT cells, providing a rapid source of IL-22 that might be beneficial to preserve the lung epithelium integrity.
Assuntos
Células Epiteliais/citologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Humana/imunologia , Influenza Humana/fisiopatologia , Interleucinas/imunologia , Pulmão/citologia , Células T Matadoras Naturais/imunologia , Animais , Morte Celular , Células Epiteliais/imunologia , Humanos , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Pulmão/imunologia , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Interleucina 22RESUMO
Influenza A virus (IAV) infection results in a highly contagious respiratory illness leading to substantial morbidity and occasionally death. In this report, we assessed the in vivo physiological contribution of invariant NKT (iNKT) lymphocytes, a subset of lipid-reactive αß T lymphocytes, on the host response and viral pathogenesis using a virulent, mouse-adapted, IAV H3N2 strain. Upon infection with a lethal dose of IAV, iNKT cells become activated in the lungs and bronchoalveolar space to become rapidly anergic to further restimulation. Relative to wild-type animals, C57BL/6 mice deficient in iNKT cells (Jα18(-/-) mice) developed a more severe bronchopneumonia and had an accelerated fatal outcome, a phenomenon reversed by the adoptive transfer of NKT cells prior to infection. The enhanced pathology in Jα18(-/-) animals was not associated with either reduced or delayed viral clearance in the lungs or with a defective local NK cell response. In marked contrast, Jα18(-/-) mice displayed a dramatically reduced IAV-specific CD8(+) T cell response in the lungs and in lung-draining mediastinal lymph nodes. We further show that this defective CD8(+) T cell response correlates with an altered accumulation and maturation of pulmonary CD103(+), but not CD11b(high), dendritic cells in the mediastinal lymph nodes. Taken together, these findings point to a role for iNKT cells in the control of pneumonia as well as in the development of the CD8(+) T cell response during the early stage of acute IAV H3N2 infection.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Inflamação/imunologia , Vírus da Influenza A Subtipo H3N2/imunologia , Pulmão/imunologia , Células T Matadoras Naturais/imunologia , Infecções por Orthomyxoviridae/imunologia , Pneumonia Viral/imunologia , Transferência Adotiva , Animais , Antígenos CD , Broncopneumonia , Antígeno CD11b , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Vírus da Influenza A Subtipo H3N2/patogenicidade , Cadeias alfa de Integrinas , Pulmão/virologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/virologia , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Reação em Cadeia da Polimerase , Carga ViralRESUMO
BACKGROUND: Exogenous activation of pulmonary invariant natural killer T (iNKT) cells, a population of lipid-reactive αß T lymphocytes, with use of mucosal α-galactosylceramide (α-GalCer) administration, is a promising approach to control respiratory bacterial infections. We undertook the present study to characterize mechanisms leading to α-GalCer-mediated protection against lethal infection with Streptococcus pneumoniae serotype 1, a major respiratory pathogen in humans. METHODS AND RESULTS: α-GalCer was administered by the intranasal route before infection with S. pneumoniae. We showed that respiratory dendritic cells (DCs), most likely the CD103(+) subset, play a major role in the activation (IFN-γ and IL-17 release) of pulmonary iNKT cells, whereas alveolar and interstitial macrophages are minor players. After challenge, S. pneumoniae was rapidly (4 hours) eliminated in the alveolar spaces, a phenomenon that depended on respiratory DCs and neutrophils, but not macrophages, and on the early production of both IFN-γ and IL-17. Protection was also associated with the synthesis of various interferon-dependent and IL-17-associated genes as revealed by transcriptomic analysis. CONCLUSIONS: These data imply a new function for pulmonary CD103(+) DCs in mucosal activation of iNKT cells and establish a critical role for both IFN-γ and IL-17 signalling pathways in mediating the innate immune response to S. pneumoniae.
Assuntos
Células Dendríticas/imunologia , Galactosilceramidas/farmacologia , Células T Matadoras Naturais/imunologia , Infecções Pneumocócicas/imunologia , Streptococcus pneumoniae/imunologia , Animais , Antígenos CD/imunologia , Líquido da Lavagem Broncoalveolar/microbiologia , Células Dendríticas/microbiologia , Galactosilceramidas/uso terapêutico , Imunidade Inata/imunologia , Cadeias alfa de Integrinas/imunologia , Interferon gama/imunologia , Interleucina-17/imunologia , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células T Matadoras Naturais/microbiologia , Infecções Pneumocócicas/microbiologia , Transdução de SinaisRESUMO
Dengue virus (DENV), a member of the mosquito-borne flaviviruses, is a serious public health problem in many tropical countries. We assessed the in vivo physiologic contribution of invariant natural killer T (iNKT) cells, a population of nonconventional lipid-reactive αß T lymphocytes, to the host response during experimental DENV infection. We used a mouse-adapted DENV serotype 2 strain that causes a disease that resembles severe dengue in humans. On DENV challenge, splenic and hepatic iNKT cells became activated insofar as CD69 and Fas ligand up-regulation and interferon-γ production. C57BL/6 mice deficient in iNKT cells (Jα18(-/-)) were more resistant to lethal infection than were wild-type animals, and the phenotype was reversed by adoptive transfer of iNKT cells to Jα18(-/-) animals. The absence of iNKT cells in Jα18(-/-) mice was associated with decreased systemic and local inflammatory responses, less liver injury, diminished vascular leak syndrome, and reduced activation of natural killer cells and neutrophils. iNKT cell functions were not necessary for control of primary DENV infection, after either natural endogenous activation or exogenous activation with the canonical iNKT cell agonist α-galactosylceramide. Together, these data reveal a novel and critical role for iNKT cells in the pathogenesis of severe experimental dengue disease.
Assuntos
Vírus da Dengue/imunologia , Dengue/imunologia , Dengue/virologia , Células T Matadoras Naturais/imunologia , Animais , Peso Corporal , Citocinas/biossíntese , Dengue/patologia , Dengue/prevenção & controle , Vírus da Dengue/fisiologia , Feminino , Galactosilceramidas/farmacologia , Mediadores da Inflamação/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ativação de Neutrófilo/imunologia , Análise de Sobrevida , Carga Viral/imunologia , Replicação Viral/fisiologiaRESUMO
Unmethylated CpG oligodeoxynucleotides (ODNs), by activating cells of the innate immune system, such as dendritic cells and NK cells, are potent adjuvants for type 1 immune responses. In the present study, we aimed to investigate the role of invariant NKT (iNKT) cells, a subset of lipid-reactive innate lymphocytes, in CpG ODN-induced innate and acquired type 1 responses. Our data show that, in response to the CpG ODN type B 1826, splenic and hepatic iNKT cells become activated and produce IFN-gamma, but not IL-4, both in vitro and in vivo. This Th1 bias is independent from the Ag-presenting molecule CD1d and strongly requires IL-12, at least in vitro. We also report that iNKT cell activation, in response to CpG ODN type B, results in the transactivation of NK cells. To address the potential role of iNKT cells in type 1 innate immunity induced by CpG ODN, a murine model of malignant melanoma was used. We show that CpG ODN type B protects mice against B16F10-induced lung metastasis in wild-type mice, but in a less efficient manner in iNKT cell-deficient animals. Finally, we report that immunization of wild-type mice with CpG ODN type B plus keyhole limpet hemocyanin biases the immune response toward a Th1 direction, an effect strongly mediated by iNKT cells. We conclude that iNKT cells amplify the innate and acquired response to CpG ODN type B, with potentially important consequences for the regulation of immune responses.
Assuntos
Adjuvantes Imunológicos/farmacologia , DNA/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Animais , DNA/farmacologia , Feminino , Interferon gama/biossíntese , Interferon gama/imunologia , Melanoma Experimental/imunologia , Camundongos , Oligodesoxirribonucleotídeos , Células Th1/imunologiaRESUMO
Splenic marginal zone B (MZB) lymphocytes represent, along with dendritic cells (DC) a first line of defense against blood-borne pathogens. MZB cells express high levels of MHC class II and CD1d molecules but so far their ability to activate and orientate conventional and innate-like T lymphocytes, such as invariant NKT (iNKT) cells, is still elusive. In the present study, we show that murine MZB cells proliferate, mature phenotypically, and secrete cytokines in response to TLR (except TLR3) agonists. When pulsed with OVA peptide (but not whole OVA), MZB cells promote the release of IFN-gamma and IL-4 by Ag-specific CD4(+) T lymphocytes and their stimulation with the TLR9 agonist CpG oligodeoxynucleotide (ODN), a potent MZB cell activator, biases them toward more Th1 inducers. Unlike DC, CpG ODN-stimulated MZB cells fail to stimulate iNKT cells. Although able to activate iNKT hybridomas, MZB cells sensitized with free alpha-galactosylceramide (alpha-GalCer), a CD1d-restricted glycolipid Ag, do not directly activate ex vivo sorted iNKT cells unless DC are added to the culture system. Interestingly, MZB cells amplify the DC-mediated activation of iNKT cells and depletion of MZB cells from total splenocytes strongly reduces iNKT cell activation (cytokine production) in response to alpha-GalCer. Thus, DC and MZB cells provide help to each other to optimize iNKT cell stimulation. Finally, in vivo transfer of alpha-GalCer-loaded MZB cells potently activates iNKT and NK cells. This study confirms and extends the concept that MZB cells are important players in immune responses, a property that might be exploited.
Assuntos
Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Células T Matadoras Naturais/imunologia , Baço/imunologia , Animais , Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/imunologia , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligodesoxirribonucleotídeos/imunologia , Ovalbumina/imunologia , Peptídeos , Baço/citologia , Receptores Toll-Like/imunologiaRESUMO
Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs) and in the induction of immune responses. Few studies have been devoted to address the impact of TLR2 (a fully MyD88-dependent receptor) and TLR3 (a fully TRIF-dependent receptor) co-activation on DC functions, especially in the mouse system. Using canonical agonists, we show that TLR2 acts in concert with TLR3 to induce the synthesis of inflammatory cytokines (TNF-alpha, IL-6), of some IL-12 family members (IL-12p40, IL-12p23, IL-27p28) and of the Notch ligand Delta-4 by mouse DCs. In contrast, TLR2 interferes with the TLR3-induced expression of type I interferon stimulated genes (MIG/CXCL9, IP-10/CXCL10, GARG39) and IL-12p35. We also report that TLR2 cooperates with TLR3 to enhance the DC-mediated production of IFN-gamma by Natural Killer cells and by conventional Ag-specific T lymphocytes. To conclude, our data support the existence of TLR2 and TLR3 synergy and cross-inhibition in DCs that could be important to strengthen immune responses during infection.
Assuntos
Células Dendríticas/metabolismo , Interleucina-12/metabolismo , Receptor Cross-Talk , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Antígenos de Diferenciação , Diferenciação Celular/imunologia , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/imunologia , Epitopos , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Imunidade Celular , Imunofenotipagem , Mediadores da Inflamação/agonistas , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-12/antagonistas & inibidores , Interleucina-12/imunologia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária , Camundongos , Células Mieloides/citologia , Células Mieloides/imunologia , Células Mieloides/metabolismo , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/imunologia , Receptor 3 Toll-Like/agonistas , Receptor 3 Toll-Like/imunologia , Regulação para CimaRESUMO
Toll-like receptors (TLRs) play an important role in the innate recognition of pathogens by dendritic cells (DCs) and in the induction of immune responses. However, relatively little is known about their functions in innate/acquired responses to complex eukaryotic microorganisms, including helminth parasites. That Schistosoma mansoni eggs activate myeloid DCs through TLR2 and TLR3 has been shown by us and others, but the consequences of this combined activation are still unknown. We show that the engagement of both TLR2 and TLR3 by schistosome eggs is important for the production of inflammatory cytokines and interferon-stimulated genes, such as some chemokines, by DCs. Strikingly, DCs sensitized with ovalbumin in the presence of parasite eggs dramatically reduce the release of Th2-type cytokines by ovalbumin-specific T lymphocytes, an effect that fully depends on TLR3. Finally, although TLR2 and TLR3 have no role in host resistance and in egg-induced granuloma formation in S. mansoni-infected mice, they individually and additionally increase the Th1/Th2 balance of the immune response. Thus, TLR2 and TLR3 sensing is required to shape the immune response during murine schistosomiasis, but is dispensable to control infection and pathology.
Assuntos
Células Dendríticas/imunologia , Schistosoma mansoni/patogenicidade , Esquistossomose mansoni/imunologia , Esquistossomose mansoni/patologia , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Contagem de Ovos de Parasitas , Schistosoma mansoni/imunologia , Esquistossomose mansoni/parasitologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
During turnover, the catalytic tyrosine residue (Tyr10) of the sigma class Schistosoma haematobium wild-type glutathione-S-transferase is expected to switch alternately in and out of the reduced glutathione-binding site (G-site). The Tyrout10 conformer forms a pi-cation interaction with the guanidinium group of Arg21. As in other similar glutathione-S-transferases, the catalytic Tyr has a low pKa of 7.2. In order to investigate the catalytic role of Tyr10, and the structural and functional roles of Arg21, we carried out structural studies on two Arg21 mutants (R21L and R21Q) and a Tyr10 mutant, Y10F. Our crystallographic data for the two Arg21 mutants indicate that only the Tyrout10 conformation is populated, thereby excluding a role of Arg21 in the stabilisation of the out conformation. However, Arg21 was confirmed to be catalytically important and essential for the low pKa of Tyr10. Upon comparison with structural data generated for reduced glutathione-bound and inhibitor-bound wild-type enzymes, it was observed that the orientations of Tyr10 and Arg35 are concerted and that, upon ligand binding, minor rearrangements occur within conserved residues in the active site loop. These rearrangements are coupled to quaternary rigid-body movements at the dimer interface and alterations in the localisation and structural order of the C-terminal domain.
Assuntos
Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Glutationa/metabolismo , Mutagênese Sítio-Dirigida , Schistosoma haematobium/enzimologia , Animais , Arginina/metabolismo , Catálise , Clonagem Molecular , Cristalografia por Raios X , Elétrons , Expressão Gênica , Glutationa/análogos & derivados , Glutationa Transferase/química , Glutationa Transferase/isolamento & purificação , Concentração de Íons de Hidrogênio , Cinética , Proteínas Mutantes/isolamento & purificação , Proteínas Mutantes/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Schistosoma haematobium/genética , Tirosina/metabolismoRESUMO
Vaccines designed to abrogate the tolerance of tumor self-antigens and amplify cytotoxic CD8+ T cells (CTLs) have promise for the treatment of cancer. Type I natural killer (NKT) cells have attracted considerable interest in the cancer therapy field. In the current study, we have exploited the unique ability of NKT cells to serve as T-helper cells to license dendritic cells (DCs) for cross priming with the aim to generate efficient CTL antitumor responses. To this end, we designed a nanoparticle-based vaccine to target cross-priming DCs via the Clec9a endocytic pathway. Our results showed for the first time that simultaneous co-delivery of the NKT agonist α-galactosylceramide and tumor self-antigens (Trp2 and gp100) to CD8α+ DCs promotes strong antitumor responses in prophylactic and therapeutic settings (advanced solid tumor model in the mouse). We attributed the vaccine's therapeutic effects to NKT cells (but not to T-helper lymphocytes) and CD8+ T cells. Efficacy was correlated with an elevated ratio between tumor antigen-specific CD8+ T cells and regulatory CD4+ T lymphocytes within the tumor. The nanoparticle-based vaccine actively targeted human CLEC9A-expressing BDCA3+ DCs - the equivalent of murine cross-priming CD8α+ DCs - and induced a strong expansion of effector memory tumor self-antigen (Melan -A)-specific CD8+ T cells from peripheral blood mononuclear cells sourced from healthy donors and melanoma patients. Together, our result shed light on novel therapeutic approaches for controlling tumor development.
RESUMO
Influenza A virus infection can predispose to potentially devastating secondary bacterial infections. Invariant natural killer T (iNKT) cells are unconventional, lipid-reactive T lymphocytes that exert potent immunostimulatory functions. Using a mouse model of postinfluenza invasive secondary pneumococcal infection, we sought to establish whether α-galactosylceramide (α-GalCer [a potent iNKT cell agonist that is currently in clinical development]) could limit bacterial superinfection. Our results highlighted the presence of a critical time window during which α-GalCer treatment can trigger iNKT cell activation and influence resistance to postinfluenza secondary pneumococcal infection. Intranasal treatment with α-GalCer during the acute phase (on day 7) of influenza virus H3N2 and H1N1 infection failed to activate (gamma interferon [IFN-γ] and interleukin-17A [IL-17A]) iNKT cells; this effect was associated with a strongly reduced number of conventional CD103+ dendritic cells in the respiratory tract. In contrast, α-GalCer treatment during the early phase (on day 4) or during the resolution phase (day 14) of influenza was associated with lower pneumococcal outgrowth and dissemination. Less intense viral-bacterial pneumonia and a lower morbidity rate were observed in superinfected mice treated with both α-GalCer (day 14) and the corticosteroid dexamethasone. Our results open the way to alternative (nonantiviral/nonantibiotic) iNKT-cell-based approaches for limiting postinfluenza secondary bacterial infections. IMPORTANCE: Despite the application of vaccination programs and antiviral drugs, influenza A virus (IAV) infection is responsible for widespread morbidity and mortality (500,000 deaths/year). Influenza infections can also result in sporadic pandemics that can be devastating: the 1918 pandemic led to the death of 50 million people. Severe bacterial infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Today's treatments of secondary bacterial (pneumococcal) infections are still not effective enough, and antibiotic resistance is a major issue. Hence, there is an urgent need for novel therapies. In the present study, we set out to evaluate the efficacy of α-galactosylceramide (α-GalCer)-a potent agonist of invariant NKT cells that is currently in clinical development-in a mouse model of postinfluenza, highly invasive pneumococcal pneumonia. Our data indicate that treatment with α-GalCer reduces susceptibility to superinfections and, when combined with the corticosteroid dexamethasone, reduces viral-bacterial pneumonia.
Assuntos
Galactosilceramidas/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Células T Matadoras Naturais/efeitos dos fármacos , Células T Matadoras Naturais/imunologia , Infecções por Orthomyxoviridae/complicações , Infecções Pneumocócicas/imunologia , Infecções Pneumocócicas/prevenção & controle , Administração Intranasal , Animais , Modelos Animais de Doenças , Camundongos , Fatores de Tempo , Resultado do TratamentoRESUMO
Invariant Natural Killer T (iNKT) cells have potent immunostimulatory activities that could be exploited for human therapies. The high-affinity CD1d antigen α-galactosylceramide analogue KRN7000 (KRN) activates a cascade of anti-tumor effector cells and clinical studies have already had some initial success. To improve the efficacy of the treatment, strategies that aim to vectorize KRN would be valuable. In this study, we intended to characterize and compare the effect of KRN encapsulated in poly(lactic-co-glycolic acid) (PLGA)-based nanoparticles (NPs, 90nm) and microparticles instead of macroparticles (MPs, 715nm) on the iNKT cell response. Our data show that whatever the size of the particles, vectorized KRN induced potent primary activation of iNKT cells in vitro and in vivo. We show that endocytosis of PLGA-based particles by dendritic cells is mediated by a clathrin-dependent manner and that this event is important to stimulate iNKT cells. Finally, we report that KRN vectorized in NPs and MPs exhibited different behaviours in vivo in terms of iNKT cell expansion and responsiveness to a recall stimulation. Collectively, our data validate the concept that KRN encapsulated in PLGA-based particles can be used as delivery systems to activate iNKT cells in vitro and in vivo.
Assuntos
Galactosilceramidas/administração & dosagem , Galactosilceramidas/imunologia , Ativação Linfocitária/imunologia , Microesferas , Nanopartículas/química , Células T Matadoras Naturais/imunologia , Animais , Apresentação de Antígeno/imunologia , Antineoplásicos/administração & dosagem , Antineoplásicos/imunologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Clatrina/metabolismo , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Portadores de Fármacos/química , Endocitose/efeitos dos fármacos , Endocitose/imunologia , Galactosilceramidas/farmacologia , Interferon gama/sangue , Interferon gama/metabolismo , Interleucina-2/metabolismo , Interleucina-4/sangue , Interleucina-4/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ácido Láctico/química , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Fígado/citologia , Fígado/imunologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/metabolismo , Tamanho da Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Eletricidade Estática , Propriedades de SuperfícieRESUMO
One important function of conventional dendritic cells (cDC) is their high capacity to capture, process and present Ag to T lymphocytes. Mouse splenic cDC subtypes, including CD8α(+) and CD8α(-) cDC, are not identical in their Ag presenting and T cell priming functions. Surprisingly, few studies have reported functional differences between CD4(-) and CD4(+) CD8α(-) cDC subsets. We show that, when loaded in vitro with OVA peptide or whole protein, and in steady-state conditions, splenic CD4(-) and CD4(+) cDC are equivalent in their capacity to prime and direct CD4(+) and CD8(+) T cell differentiation. In contrast, in response to α-galactosylceramide (α-GalCer), CD4(-) and CD4(+) cDC differentially activate invariant Natural Killer T (iNKT) cells, a population of lipid-reactive non-conventional T lymphocytes. Both cDC subsets equally take up α-GalCer in vitro and in vivo to stimulate the iNKT hybridoma DN32.D3, the activation of which depends solely on TCR triggering. On the other hand, and relative to their CD4(+) counterparts, CD4(-) cDC more efficiently stimulate primary iNKT cells, a phenomenon likely due to differential production of co-factors (including IL-12) by cDC. Our data reveal a novel functional difference between splenic CD4(+) and CD4(-) cDC subsets that may be important in immune responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos CD8/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/citologia , Células Matadoras Naturais/imunologia , Subpopulações de Linfócitos/citologia , Baço/citologia , Animais , Linfócitos T CD4-Positivos/citologia , Células Dendríticas/imunologia , Galactosilceramidas/imunologia , Células Matadoras Naturais/citologia , Subpopulações de Linfócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologiaRESUMO
Invariant NK T (iNKT) cells are a subset of innate/memory lymphocytes that recognize lipid Ags presented by CD1d-expressing APCs such as dendritic cells (DCs). Upon primary stimulation through their TCR, iNKT cells promptly produce large amounts of IFN-gamma and/or IL-4 that play critical roles in the regulation of innate and adaptive immune responses. To date, the role of environmental factors on iNKT cell functions has been poorly investigated. In this study, we addressed the question of whether PGD2, a potent eicosanoid lipid mediator involved in immune responses and inflammation, could be important in DC/iNKT cell cross-talk. We show that PGD2 dramatically reduced the production of IFN-gamma, but not IL-4, by iNKT cells in response to the superagonist alpha-galactosylceramide (alpha-GalCer) both in vitro and in vivo. This effect is mediated by the D prostanoid receptor 1 (DP1) expressed by DCs and iNKT cells and requires protein kinase A activation. We also report that PGD2 and BW245C (a selective DP1 agonist) reduce the protective effects of alpha-GalCer in B16F10-induced melanoma metastasis, an effect that depends on IFN-gamma production by iNKT cells. As a whole, these data reveal novel pathways regulating iNKT cell biologic functions and confirm the immunoregulatory roles of PGD2 on the innate response.
Assuntos
Interferon gama/antagonistas & inibidores , Células Matadoras Naturais/efeitos dos fármacos , Melanoma Experimental/imunologia , Prostaglandina D2/farmacologia , Neoplasias Cutâneas/imunologia , Linfócitos T/efeitos dos fármacos , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Hidantoínas/farmacologia , Imunidade Inata/efeitos dos fármacos , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Melanoma Experimental/secundário , Camundongos , Receptores de Prostaglandina/agonistas , Receptores de Prostaglandina/metabolismo , Neoplasias Cutâneas/patologia , Linfócitos T/imunologiaRESUMO
Invariant natural killer T (iNKT) cells are a subset of innate lymphocytes that recognize lipid antigens in the context of CD1d and mediate potent immune regulatory functions via the rapid production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4). We investigated whether diverse Toll-like receptor (TLR) signals in myeloid dendritic cells (DCs) could differentially stimulate iNKT cells. Together with the lipopolysaccharide-detecting receptor TLR4, activation of the nucleic acid sensors TLR7 and TLR9 in DCs were particularly potent in stimulating iNKT cells to produce IFN-gamma, but not IL-4. iNKT cell activation in response to TLR9 stimulation required combined synthesis of type I interferon and de novo production of charged beta-linked glycosphingolipid(s) by DCs. In addition, DCs stimulated via TLR9 activated both iNKT cells and NK cells in vivo and protected mice against B16F10-induced melanoma metastases. These data underline the role of TLR9 in iNKT cell activation and might have relevance to infectious diseases and cancer.
Assuntos
Glicoesfingolipídeos Acídicos/imunologia , Células Dendríticas/imunologia , Interferon Tipo I/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Receptor Toll-Like 9/imunologia , Glicoesfingolipídeos Acídicos/metabolismo , Animais , Antígenos CD1/imunologia , Antígenos CD1/metabolismo , Antígenos CD1d , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Feminino , Imunoterapia Adotiva , Interferon Tipo I/metabolismo , Interferon gama/imunologia , Interferon gama/metabolismo , Células Matadoras Naturais/metabolismo , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/imunologia , Linfócitos T/metabolismo , Receptor 7 Toll-Like/imunologia , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismoRESUMO
Galectin-3 (Gal-3) is a multifunctional beta-galactoside-binding lectin that senses self-derived and microbial glycoconjugates. Although Gal-3 is important in immune reactions and host defense in some experimental models, the function of Gal-3 during helminthic diseases (e.g., schistosomiasis) is still elusive. We show that, compared to wild-type Schistosoma mansoni-infected mice, infected Gal-3-/- mice have a reduced number of T and B lymphocytes in the spleen, develop reduced liver granulomas at 7 weeks (acute phase) and 14 weeks (chronic phase) postinfection, and mount a biased cellular and humoral Th1 response. In an attempt to understand this latter phenomenon, we studied the role of endogenous Gal-3 in dendritic cells (DCs), the most potent antigen-presenting cells, both in vitro and in vivo. Although Gal-3 deficiency in DCs does not impact their differentiation and maturation processes, it greatly influences the strength (but not the nature) of the adaptive immune response that they trigger, suggesting that Gal-3 deficiency in some other cell types may be important during murine schistosomiasis. As a whole, this study implies that Gal-3 is a modulator of the immune/inflammatory responses during helminthic infection and reveals for the first time that Gal-3 expression in DCs is pivotal to control the magnitude of T-lymphocyte priming.