RESUMEN
The TLR5 agonist flagellin is a potent adjuvant and is currently being developed for use in vaccines. The mechanisms that drive flagellin's activity are influenced by its administration route. Previous studies showed that lung structural cells (especially epithelial cells lining the conducting airways) are pivotal for the efficacy of intranasally administered flagellin-containing vaccines. In this study, we looked at how the airway epithelial cells (AECs) regulate the flagellin-dependent stimulation of Ag-specific CD4+ T cells and the Ab response in mice. Our results demonstrate that after sensing flagellin, AECs trigger the release of GM-CSF in a TLR5-dependent fashion and the doubling of the number of activated type 2 conventional dendritic cells (cDC2s) in draining lymph nodes. Furthermore, the neutralization of GM-CSF reduced cDC2s activation. This resulted in lower of Ag-specific CD4+ T cell count and Ab titers in mice. Our data indicate that during pulmonary immunization, the GM-CSF released by AECs orchestrates the cross-talk between cDC2s and CD4+ T cells and thus drives flagellin's adjuvant effect.
Asunto(s)
Células Epiteliales/metabolismo , Flagelina/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Mucosa Respiratoria/inmunología , Vacunas/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Administración Intranasal , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Comunicación Celular/inmunología , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Epiteliales/inmunología , Femenino , Flagelina/administración & dosificación , Inmunidad Mucosa , Inmunogenicidad Vacunal , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Noqueados , Modelos Animales , Cultivo Primario de Células , Mucosa Respiratoria/citología , Mucosa Respiratoria/metabolismo , Receptor Toll-Like 5/agonistas , Receptor Toll-Like 5/genética , Vacunas/administración & dosificaciónRESUMEN
The 3-O-desacyl-4'-monophosphoryl lipid A (MPL) activates immunity through Toll-like receptor 4 (TLR4) signaling. The Adjuvant System AS01 contains MPL and is used in the candidate malaria vaccine and the licensed zoster vaccine. Recent studies reported that AS01 adjuvant activity depends on a transient inflammation at the site of vaccination, but the role of stromal or structural cells in the adjuvant effect is unknown. We investigated this question in mouse models by assessing the role of TLR4 on hematopoietic versus resident structural cells during immunization with AS01-adjuvanted vaccines. We first established that TLR4-deficient animals had a reduced immune response to an AS01-adjuvanted vaccine. Using bone marrow chimera, we consistently found that Tlr4 expression in radio-sensitive cells, i.e., hematopoietic cells, was required for an optimal adjuvant effect on antibody and T-cell responses. At day 1 after injection, the pro-inflammatory reaction at the site of injection was strongly dependent on TLR4 signaling in hematopoietic cells. Similarly, activation of dendritic cells in muscle-draining lymph nodes was strictly associated with the radio-sensitive cells expressing Tlr4. Altogether, these data suggest that MPL-mediated TLR4-signaling in hematopoietic cells is critical in the mode of action of AS01.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Células Madre Hematopoyéticas/inmunología , Lípido A/análogos & derivados , Saponinas/farmacología , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/inmunología , Animales , Combinación de Medicamentos , Femenino , Células Madre Hematopoyéticas/citología , Humanos , Lípido A/farmacología , Masculino , Ratones , Ratones Noqueados , Transducción de Señal/genética , Transducción de Señal/inmunología , Receptor Toll-Like 4/genética , Vacunas/farmacologíaRESUMEN
Signaling through Toll-like receptors (TLRs), the main receptors in innate immunity, is essential for the defense of mucosal surfaces. It was previously shown that systemic TLR5 stimulation by bacterial flagellin induces an immediate, transient interleukin-22 (IL-22)-dependent antimicrobial response to bacterial or viral infections of the mucosa. This process was dependent on the activation of type 3 innate lymphoid cells (ILCs). The objective of the present study was to analyze the effects of flagellin treatment in a murine model of oral infection with Yersinia pseudotuberculosis (an invasive, Gram-negative, enteropathogenic bacterium that targets the small intestine). We found that systemic administration of flagellin significantly increased the survival rate after intestinal infection (but not systemic infection) by Y. pseudotuberculosis This protection was associated with a low bacterial count in the gut and the spleen. In contrast, no protection was afforded by administration of the TLR4 agonist lipopolysaccharide, suggesting the presence of a flagellin-specific effect. Lastly, we found that TLR5- and MyD88-mediated signaling was required for the protective effects of flagellin, whereas neither lymphoid cells nor IL-22 was involved.
Asunto(s)
Flagelina/inmunología , Interleucinas/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Infecciones por Yersinia pseudotuberculosis/inmunología , Infecciones por Yersinia pseudotuberculosis/metabolismo , Yersinia pseudotuberculosis/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Flagelina/administración & dosificación , Interleucinas/genética , Mucosa Intestinal/microbiología , Lipopolisacáridos/inmunología , Ratones , Ratones Noqueados , Proteínas Recombinantes de Fusión , Transducción de Señal , Receptores Toll-Like/metabolismo , Infecciones por Yersinia pseudotuberculosis/microbiología , Infecciones por Yersinia pseudotuberculosis/mortalidad , Interleucina-22RESUMEN
Bacterial superantigens (SAgs) are immunostimulatory toxins that induce acute diseases mainly through the massive release of inflammatory cytokines. Yersinia pseudotuberculosis is the only Gram-negative bacterium known to produce a SAg (Y. pseudotuberculosis-derived mitogen [YPM]). This SAg binds major histocompatibility complex class II molecules on antigen-presenting cells and T cell receptors (TcR) bearing the variable region Vß3, Vß9, Vß13.1, or Vß13.2 (in humans) and Vß7 or Vß8 (in mice). We have previously shown that YPM exacerbates the virulence of Y. pseudotuberculosis in mice. With a view to understanding the mechanism of YPM's toxicity, we compared the immune response in BALB/c mice infected with a YPM-producing Y. pseudotuberculosis or the corresponding isogenic, SAg-deficient mutant. Five days after infection, we observed strong CD4(+) Vß7(+) T cell expansion and marked interleukin-4 (IL-4) production in mice inoculated with SAg-producing Y. pseudotuberculosis. These phenomena were correlated with the activation of ypm gene transcription in liver and spleen. A transcriptomic analysis revealed that the presence of YPM also increased expression of granzyme and perforin genes in the host's liver and spleen. This expression was attributed to a CD4(+) T cell subset, rather than to natural killer T (NKT) cells that display a TcR with a Vß region that is potentially recognized by YPM. Increased production of cytotoxic molecules was correlated with hepatotoxicity, as demonstrated by an increase in plasma alanine aminotransferase activity. Our results demonstrate that YPM activates a potentially hepatotoxic CD4(+) T cell population.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Granzimas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Superantígenos/inmunología , Yersinia pseudotuberculosis/inmunología , Animales , Perfilación de la Expresión Génica , Hígado/inmunología , Hígado/patología , Ratones Endogámicos BALB C , Bazo/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/microbiologíaRESUMEN
Mucosal sites are continuously exposed to pathogenic microorganisms and are therefore equipped to control respiratory infections. Type 3 innate lymphoid cells (ILC3) are key players in antimicrobial defense in intestinal mucosa, through interleukin 17 and interleukin 22 (IL-22) production. The present study aimed at analyzing the distribution and function of ILC3 in the respiratory tract. We first observed that lung mucosa harbors a discrete population of ILC3 expressing CD127, CD90, CCR6, and the transcriptional factor RORγt. In addition, lung ILC3 were identified as a major source of IL-22 in response to interleukin 23 stimulation. During Streptococcus pneumoniae infection, ILC3 rapidly accumulated in the lung tissue to produce IL-22. In response to S. pneumoniae, dendritic cells and MyD88, an important adaptor of innate immunity, play critical functions in IL-22 production by ILC3. Finally, administration of the Toll-like receptor 5 agonist flagellin during S. pneumoniae challenge exacerbated IL-22 production by ILC3, a process that protects against lethal infection. In conclusion, boosting lung ILC3 might represent an interesting strategy to fight respiratory bacterial infections.
Asunto(s)
Interleucinas/metabolismo , Pulmón/metabolismo , Linfocitos/clasificación , Linfocitos/fisiología , Neumonía Neumocócica/inmunología , Neumonía Neumocócica/metabolismo , Animales , Femenino , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucinas/genética , Activación de Linfocitos , Ratones , Ratones Noqueados , Streptococcus pneumoniae , Interleucina-22RESUMEN
In adaptive immunity, Th17 lymphocytes produce the IL-17 and IL-22 cytokines that stimulate mucosal antimicrobial defenses and tissue repair. In this study, we observed that the TLR5 agonist flagellin induced swift and transient transcription of genes encoding IL-17 and IL-22 in lymphoid, gut, and lung tissues. This innate response also temporarily enhanced the expression of genes associated with the antimicrobial Th17 signature. The source of the Th17-related cytokines was identified as novel populations of CD3(neg)CD127(+) immune cells among which CD4-expressing cells resembling lymphoid tissue inducer cells. We also demonstrated that dendritic cells are essential for expression of Th17-related cytokines and so for stimulation of innate cells. These data define that TLR-induced activation of CD3(neg)CD127(+) cells and production of Th17-related cytokines may be crucial for the early defenses against pathogen invasion of host tissues.
Asunto(s)
Interleucina-17/inmunología , Interleucinas/inmunología , Membrana Mucosa/inmunología , Transducción de Señal/inmunología , Bazo/inmunología , Receptor Toll-Like 5/inmunología , Animales , Complejo CD3/genética , Complejo CD3/inmunología , Complejo CD3/metabolismo , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Flagelina/farmacología , Citometría de Flujo , Expresión Génica/efectos de los fármacos , Expresión Génica/inmunología , Íleon/efectos de los fármacos , Íleon/inmunología , Íleon/metabolismo , Interleucina-17/genética , Interleucina-17/metabolismo , Subunidad alfa del Receptor de Interleucina-7/genética , Subunidad alfa del Receptor de Interleucina-7/inmunología , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Tejido Linfoide/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones SCID , Ratones Transgénicos , Membrana Mucosa/citología , Membrana Mucosa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Bazo/citología , Bazo/metabolismo , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/metabolismo , Interleucina-22RESUMEN
Canonical and alternative NF-kappaB pathways depend on distinct NF-kappaB members and regulate expression of different gene subset in inflammatory and steady state conditions, respectively. In intestinal epithelial cells, both pathways control the transcription of the gene coding the CCL20 chemokine. Lymphotoxin beta receptor (LTbetaR) mediates long lasting CCL20 expression whereas Toll-like receptor 5 (TLR5) signals promote inducible and transient activation. Here, we investigated whether the regulation of ccl20 expression involves different promoter sites and NF-kappaB molecules in response to TLR5 and LTbetaR stimulation. In epithelial cells, both stimulation required the same promoter regions, especially the NF-kappaB binding site but involved different NF-kappaB isoforms: p65/p50 and p52/RelB, for TLR5 and LTbetaR-dependent activation, respectively. The dynamic of activation and interaction with CCL20-specific NF-kappaB site correlated with gene transcription. Similar Ccl20 expression and NF-kappaB activation was found in the small intestine of mice stimulated with TLR5 and LTbetaR agonists. In summary, different NF-kappaB pathways modulate CCL20 transcription by operating on the same NF-kappaB binding site in the same cell type.
Asunto(s)
Quimiocina CCL20/genética , Receptor beta de Linfotoxina/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 5/metabolismo , Transporte Activo de Núcleo Celular , Animales , Secuencia de Bases , Sitios de Unión , Células CACO-2 , Línea Celular , Núcleo Celular/metabolismo , Quimiocina CCL20/biosíntesis , Quimiocina CCL20/metabolismo , Flagelina/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Receptor beta de Linfotoxina/agonistas , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Transducción de SeñalRESUMEN
TLRs trigger immunity by detecting microbe-associated molecular patterns (MAMPs). Flagellin is a unique MAMP because it harbors 1) an antigenic hypervariable region and 2) a conserved domain involved in TLR5-dependent systemic and mucosal proinflammatory and adjuvant activities. In this study, the contribution of the flagellin domains in TLR5 activation was investigated. We showed that TLR5 signaling can be neutralized in vivo by flagellin-specific Abs, which target the conserved domain. However, deletions of flagellin's hypervariable region abrogated the protein's intrinsic ability to trigger the production of neutralizing Abs. The fact that MAMP-specific Abs block TLR-mediated responses shows that this type of neutralization is a novel mechanism for down-regulating innate immunity. The stimulation of mucosal innate immunity and adjuvancy to foreign Ag was not altered by the hypervariable domain deletions. In contrast, this domain is essential to trigger systemic innate immunity, suggesting that there are distinct mechanisms for TLR5 activation in systemic and mucosal compartments. In summary, specific MAMP determinants control the production of neutralizing Abs and the compartmentalization of innate responses.
Asunto(s)
Regiones Determinantes de Complementariedad/inmunología , Flagelina/inmunología , Flagelina/metabolismo , Receptor Toll-Like 5/inmunología , Receptor Toll-Like 5/metabolismo , Animales , Células CACO-2 , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/farmacología , Femenino , Flagelina/genética , Eliminación de Gen , Humanos , Inmunidad , Inmunidad Innata/inmunología , Ratones , Modelos Moleculares , Estructura Cuaternaria de Proteína , Salmonella enterica/genética , Salmonella enterica/inmunología , Salmonella enterica/metabolismoRESUMEN
Bacterial infections of the respiratory tract constitute a major cause of death worldwide. Given the constant rise in bacterial resistance to antibiotics, treatment failure is increasingly frequent. In this context, innovative therapeutic strategies are urgently needed. Stimulation of innate immune cells in the respiratory tract [via activation of Toll-like receptors (TLRs)] is an attractive approach for rapidly activating the body's immune defenses against a broad spectrum of microorganisms. Previous studies of the TLR5 agonist flagellin in animal models showed that standalone TLR stimulation does not result in the effective treatment of pneumococcal respiratory infection but does significantly improve the therapeutic outcome of concomitant antibiotic treatment. Here, we investigated the antibacterial interaction between antibiotic and intranasal flagellin in a mouse model of pneumococcal respiratory infection. Using various doses of orally administered amoxicillin or systemically administered cotrimoxazole, we found that the intranasal instillation of flagellin (a dose that promotes maximal lung pro-inflammatory responses) induces synergistic rather than additive antibacterial effects against antibiotic-susceptible pneumococcus. We next set up a model of infection with pneumococcus that is resistant to multiple antibiotics in the context of influenza superinfection. Remarkably, the combination of amoxicillin and flagellin effectively treated superinfection with the amoxicillin-resistant pneumococcus since the bacterial clearance was increased by more than 100-fold compared to standalone treatments. Our results also showed that, in response to flagellin, the lung tissue generated an innate immune response even though it had been damaged by the influenza virus and pneumococcal infections. In conclusion, we demonstrated that the selective boosting of lung innate immunity is a conceptually advantageous approach for improving the effectiveness of antibiotic treatment and fighting antibiotic-resistant bacteria.
Asunto(s)
Antibacterianos/farmacología , Pulmón/metabolismo , Infecciones Neumocócicas/tratamiento farmacológico , Neumonía Neumocócica/tratamiento farmacológico , Streptococcus pneumoniae/efectos de los fármacos , Receptor Toll-Like 5/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Inmunidad Innata/efectos de los fármacos , Pulmón/microbiología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Infecciones Neumocócicas/metabolismo , Infecciones Neumocócicas/microbiología , Neumonía Neumocócica/metabolismo , Neumonía Neumocócica/microbiologíaRESUMEN
Bacterial flagellin activates the innate immune system and ultimately the adaptive immune system through a Toll-like receptor 5 (TLR5)-dependent signaling mechanism. Given that TLR5 is widely distributed in epithelia, flagellin is currently being developed as a mucosal adjuvant. Flagellin FliC from Salmonella enterica has four domains: the conserved D0 and D1 domains and the hypervariable D2 and D3 domains. The deletion of D3 and partial deletion of D2 in the recombinant FliCΔ174-400 strongly impairs flagellin's intrinsic antigenicity but does not affect the TLR5-dependent immunostimulation activity, i.e., the capacity to promote innate responses and adaptive responses to co-administered antigens. Here, we describe the development of novel recombinant flagellins with various deletions encompassing all of D2 and D3, and part of D1. Most of the recombinant molecules conserved an α-helical secondary structure that was as resistant to heat denaturation as the native protein. Whereas the recombinant flagellins' ability to trigger TLR5 varied markedly in vitro, most gave equivalent in vivo TLR5-dependent innate immune responses following intranasal administration of 2⯵g of flagellin to mice. Concordantly, the recombinant flagellins were also valuable respiratory adjuvants for eliciting antibody responses to the foreign antigen ovalbumin, although their intrinsic antigenicity was decreased compared to the native flagellin and not increased compared to FliCΔ174-400. Our results show that the additional deletions of D2 and the distal part of D1 of FliCΔ174-400 does not impact on antigenicity and does not significantly modify the immunostimulatory adjuvant activity. Altogether, this study generated a novel set of recombinant flagellin that constitutes a portfolio of TLR5-dependent candidate adjuvants for vaccination.
Asunto(s)
Adyuvantes Inmunológicos/genética , Flagelina/genética , Flagelina/inmunología , Proteínas Recombinantes/inmunología , Animales , Inmunidad Innata , Inmunidad Mucosa , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Salmonella enterica/genética , Salmonella enterica/inmunología , Eliminación de Secuencia , Transducción de Señal , Receptor Toll-Like 5/inmunologíaRESUMEN
Influenza infections remain a burden on health care systems despite vaccination programs and marketed antiviral drugs. Immunomodulation through activation of innate sensors could represent innovative approaches to fight the flu. This study evaluated the ability of flagellin, agonist of Toll-like receptor 5 (TLR5), to control the replication of influenza A virus (IAV) in mice. First, we showed that systemic or intranasal administration of flagellin activated transcription of anti-viral genes in lung tissue. Prophylactic and therapeutic flagellin administration resulted in decreased levels of viral RNA and infectious virus in the lungs of H3N2 IAV-infected mice. The effect of the flagellin on viral replication was also observed in Ifnar-/- and Il22-/- IAV-infected mice, suggesting a mechanism independent of type I interferon and interleukin 22 signaling. In addition, a combination therapy associating the neuraminidase inhibitor oseltamivir and flagellin was more effective than standalone treatments in reducing pulmonary viral replication. Thus, this study highlights the therapeutic potential of the flagellin to control the replication of the influenza virus.
Asunto(s)
Antivirales/administración & dosificación , Flagelina/administración & dosificación , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Oseltamivir/administración & dosificación , Receptor Toll-Like 5/agonistas , Replicación Viral/efectos de los fármacos , Animales , Virus de la Influenza A/efectos de los fármacos , Interferón Tipo I/deficiencia , Interleucinas/deficiencia , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Interleucina-22RESUMEN
Mycobacterium tuberculosis, the etiologic agent of tuberculosis, adheres to, invades and multiplies in both professional phagocytes and epithelial cells. Adherence to epithelial cells is predominantly mediated by the 28-kDa heparin-binding haemagglutinin adhesin (HBHA), which is also required for the extrapulmonary dissemination of the bacilli. To study the cellular mechanisms that might result in HBHA-mediated extrapulmonary dissemination, we used a transwell model of cellular barrier and fluorescence microscopy and found that HBHA induces a reorganization of the actin filament network in confluent endothelial cells, but does not affect the tight junctions that link them. When coupled to colloidal gold particles, HBHA-mediated a rapid attachment of the particles to the membrane of human laryngeal epithelial cells (non polarized HEp-2 cells) and human type II pneumocytes (polarized A-549 pneumocytes). After attachment, the particles were internalized in membrane-bound vacuoles that migrated across the polarized pneumocytes to reach the basal side. Attachment of the HBHA-coated particles was not observed when the epithelial cells were pretreated with heparinase III, a lyase that specifically cleaves the heparan sulfate chains borne by the proteoglycans. Furthermore, no binding was observed when the gold particles were coated with HBHA lacking its C-terminal heparin-binding domain. These observations indicate that HBHA induces receptor-mediated endocytosis through the recognition of heparan sulfate-containing proteoglycans by the heparin-binding domain of the adhesin. In addition, the transcellular migration of the endocytic vacuoles containing HBHA-coated particles suggests that HBHA induces epithelial transcytosis, which may represent a macrophage-independent extrapulmonary dissemination mechanism leading to systemic infection by M. tuberculosis.
Asunto(s)
Endocitosis/efectos de los fármacos , Lectinas/farmacología , Mycobacterium tuberculosis , Proteoglicanos/metabolismo , Uniones Estrechas/efectos de los fármacos , Actinas/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Bovinos , Línea Celular , Citoesqueleto/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/ultraestructura , Humanos , Neuroglía/metabolismo , Ratas , Receptores de Superficie Celular/metabolismo , Uniones Estrechas/fisiología , Vesículas Transportadoras/metabolismoRESUMEN
Lactate is an essential component of carbon metabolism in mammals. Recently, lactate was shown to signal through the G protein coupled receptor 81 (GPR81) and to thus modulate inflammatory processes. This study demonstrates that lactate inhibits pro-inflammatory signaling in a GPR81-independent fashion. While lipopolysaccharide (LPS) triggered expression of IL-6 and IL-12 p40, and CD40 in bone marrow-derived macrophages, lactate was able to abrogate these responses in a dose dependent manner in Gpr81-/- cells as well as in wild type cells. Macrophage activation was impaired when glycolysis was blocked by chemical inhibitors. Remarkably, lactate was found to inhibit LPS-induced glycolysis in wild type as well as in Gpr81-/- cells. In conclusion, our study suggests that lactate can induce GPR81-independent metabolic changes that modulate macrophage pro-inflammatory activation.
Asunto(s)
Inflamación/metabolismo , Ácido Láctico/farmacología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Receptores Acoplados a Proteínas G/metabolismo , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Reprogramación Celular/efectos de los fármacos , Espacio Extracelular/metabolismo , Femenino , Glucólisis/efectos de los fármacos , Inflamación/patología , Lipopolisacáridos , Macrófagos Peritoneales/efectos de los fármacos , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Pseudotuberculosis is an infection caused by the bacterial enteropathogen Yersinia pseudotuberculosis and is considered to be a significant problem in veterinary medicine. We previously found that intranasal administration of a recombinant Lactococcus lactis strain that secretes the low-calcium response V (LcrV) antigen from Y. pseudotuberculosis (Ll-LcrV) confers protection against a lethal Y. pseudotuberculosis infection. Here, we aimed at characterizing the immunological basis of this LcrV-elicited protective response and at determining the duration of vaccine-induced immunity. METHODS: Splenocytes from BALB/c mice intranasally immunized with Ll-LcrV or Ll as control were immunostained then analyzed by flow cytometry. Protection against a lethal intravenous injection of Y. pseudotuberculosis was also determined (i) in immunized BALB/c mice depleted or not of CD4+, CD8+ or CD25+ cells and (ii) in naïve BALB/c mice receiving serum from immunized mice by counting the number of bacteria in liver and spleen. Lastly, survival rate of immunized BALB/c mice following a lethal intravenous injection of Y. pseudotuberculosis was followed up to 9-months. RESULTS: We found that T and B lymphocytes but not non-conventional lymphoid cells were affected by Ll-LcrV immunization. We also observed that depletion of CD4+ and CD25+ but not CD8+ cells in immunized mice eradicated protection against a lethal systemic Y. pseudotuberculosis infection, suggesting that activated CD4+ T lymphocytes are required for vaccine-induced protection. Adoptive transfer of LcrV-specific antibodies from Ll-LcrV-immunized animals significantly reduced the bacterial counts in the liver compared to non-vaccinated mice. Lastly, the protective immunity conferred by Ll-LcrV decreased slightly over time; nevertheless almost 60% of the mice survived a lethal bacterial challenge at 9months post-vaccination. CONCLUSION: Mucosal vaccination of mice with Ll-LcrV induced cell- and antibody-mediated protective immunity against Y. pseudotuberculosis infection in the mouse and the protection is long-lasting.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/inmunología , Inmunidad Activa/inmunología , Lactococcus lactis/inmunología , Proteínas Citotóxicas Formadoras de Poros/inmunología , Infecciones por Yersinia pseudotuberculosis/prevención & control , Yersinia pseudotuberculosis/inmunología , Administración Intranasal , Animales , Antígenos Bacterianos/genética , Carga Bacteriana , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Femenino , Humanos , Inyecciones Intravenosas , Subunidad alfa del Receptor de Interleucina-2/inmunología , Lactococcus lactis/genética , Ratones , Ratones Endogámicos BALB C , Proteínas Citotóxicas Formadoras de Poros/genética , Cultivo Primario de Células , Bazo/inmunología , Bazo/microbiología , Estadísticas no Paramétricas , Factores de Tiempo , Vacunación , Vacunas Sintéticas/inmunología , Yersinia pseudotuberculosis/genéticaRESUMEN
Lactate has long been considered as a metabolic by-product of cells. Recently, this view has been changed by the observation that lactate can act as a signaling molecule and regulates critical functions of the immune system. We previously identified lactate as the component responsible for the modulation of innate immune epithelial response of fermented milk supernatants in vitro. We have also shown that lactate downregulates proinflammatory responses of macrophages and dendritic cells. So far, in vivo effects of lactate on intestinal inflammation have not been reported. We evaluated the effect of intrarectal administration of lactate in a murine model of colitis induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS). The increase in lactate concentration in colon promoted protective effects against TNBS-induced colitis preventing histopathological damage, as well as bacterial translocation and rise of IL-6 levels in serum. Using intestinal epithelial reporter cells, we found that flagellin treatment induced reporter gene expression, which was abrogated by lactate treatment as well as by glycolysis inhibitors. Furthermore, lactate treatment modulated glucose uptake, indicating that high levels of extracellular lactate can impair metabolic reprograming induced by proinflammatory activation. These results suggest that lactate could be a potential beneficial microbiota metabolite and may constitute an overlooked effector with modulatory properties.
RESUMEN
The Toll-like receptor 5 (TLR5) agonist flagellin is an effective adjuvant for vaccination. Recently, we demonstrated that the adaptive responses stimulated by intranasal administration of flagellin and antigen were linked to TLR5 signaling in the lung epithelium. The present study sought to identify the antigen presenting cells involved in this adjuvant activity. We first found that the lung dendritic cells captured antigen very efficiently in a process independent of TLR5. However, TLR5-mediated signaling specifically enhanced the maturation of lung dendritic cells. Afterward, the number of antigen-bound and activated conventional dendritic cells (both CD11b(+) and CD103(+)) increased in the mediastinal lymph nodes in contrast to monocyte-derived dendritic cells. These data suggested that flagellin-activated lung conventional dendritic cells migrate to the draining lymph nodes. The lymph node dendritic cells, in particular CD11b(+) cells, were essential for induction of CD4 T-cell response. Lastly, neutrophils and monocytes were recruited into the lungs by flagellin administration but did not contribute to the adjuvant activity. The functional activation of conventional dendritic cells was independent of direct TLR5 signaling, thereby supporting the contribution of maturation signals produced by flagellin-stimulated airway epithelium. In conclusion, our results demonstrated that indirect TLR5-dependent stimulation of airway conventional dendritic cells is essential to flagellin's mucosal adjuvant activity.
Asunto(s)
Adyuvantes Inmunológicos/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Flagelina/metabolismo , Mucosa Respiratoria/inmunología , Sistema Respiratorio/inmunología , Receptor Toll-Like 5/agonistas , Animales , Inmunidad Innata , Inmunidad Mucosa , Ratones Endogámicos BALB C , Ratones Endogámicos C3HRESUMEN
The use of short chain fatty acids to modulate gastrointestinal inflammatory conditions such as ulcerative colitis has produced encouraging results either in animal models or also in clinical trials. Identifying the key cellular and molecular targets of this activity will contribute to establish the appropriate combinations/targeting strategies to maximize the efficacy of anti-inflammatory interventions. In the present work, we evaluated in vitro the interaction of lactate, acetate, propionate and butyrate on cells relevant for innate immune response of the gastrointestinal tract. All molecules tested regulate the production of proinflammatory cytokines by TLR-4 and TLR-5 activated intestinal epithelial cells in a dose response manner. Furthermore SCFAs and lactate modulate cytokine secretion of TLR-activated bone marrow derived macrophages and also TLR-dependent CD40 upregulation in bone marrow derived dendritic in a dose-dependent manner. Butyrate and propionate have been effective at concentrations of 1 to 5mM whereas acetate and lactate produced modulatory effects at concentrations higher than 20-50mM in different assays. Our results indicate that in concentrations similar to found in large bowel lumen, all SCFAs tested and lactate can modulate activity of relevant sentinel cell types activated by TLR signals. Modulatory activity was not inhibited by pertussis toxin treatment indicating that the effects are not related to Gi signaling. The use of these molecules in combined or separately as intervention strategy in conditions where epithelial or myeloid cells are main triggers of the inflammatory situation seems appropriate.
Asunto(s)
Bacterias/inmunología , Regulación hacia Abajo/inmunología , Ácidos Grasos/inmunología , Mucosa Intestinal/inmunología , Ácido Láctico/inmunología , Células Mieloides/inmunología , Animales , Antígenos CD40/inmunología , Células CACO-2 , Células Epiteliales/inmunología , Femenino , Humanos , Mucosa Intestinal/microbiología , Ratones , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 5/inmunologíaRESUMEN
The chemokine CCL20, the unique ligand of CCR6 functions as an attractant of immune cells. Expression of CCL20 is induced by Toll-like Receptor (TLR) signaling or proinflammatory cytokine stimulation. However CCL20 is also constitutively produced at specific epithelial sites of mucosa. This expression profile is achieved by transcriptional regulation. In the present work we characterized regulatory features of mouse Ccl20 gene. Transcriptional fusions between the mouse Ccl20 promoter and the firefly luciferase (luc) encoding gene were constructed and assessed in in vitro and in vivo assays. We found that liver CCL20 expression and luciferase activity were upregulated by systemic administration of the TLR5 agonist flagellin. Using shRNA and dominant negative form specific for mouse TLR5, we showed that this expression was controlled by TLR5. To address in situ the regulation of gene activity, a transgenic mouse line harboring a functional Ccl20-luc fusion was generated. The luciferase expression was highly concordant with Ccl20 expression in different tissues. Our data indicate that the transgenic mouse model can be used to monitor activation of innate response in vivo.
Asunto(s)
Quimiocina CCL20/genética , Genes Reporteros/genética , Inflamación/genética , Luciferasas de Luciérnaga/genética , Proteínas Recombinantes de Fusión/genética , Transcripción Genética , Animales , Secuencia de Bases , Humanos , Ratones , Ratones Transgénicos , Imagen Molecular , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genéticaRESUMEN
Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn's disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb (-/-)) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm(avir); invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb (-/-) mice are efficiently infected by S.Tm(avir) and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb (-/-) Myd88 (-/-) mice indicated that the S.Tm(avir)-inflicted disease in Cybb (-/-) mice hinges on CD11c(+)CX3CR1(+) monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm(avir) in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.
Asunto(s)
Bacteriemia/microbiología , Sistemas de Secreción Bacterianos , Colitis/microbiología , NADPH Oxidasas/deficiencia , Salmonelosis Animal/complicaciones , Salmonella typhimurium/fisiología , Animales , Bacteriemia/enzimología , Bacteriemia/inmunología , Antígenos CD11/metabolismo , Colitis/enzimología , Colitis/inmunología , Regulación Enzimológica de la Expresión Génica , Mucosa Intestinal/microbiología , Ratones , Monocitos/inmunología , Monocitos/metabolismo , Mutación , Factor 88 de Diferenciación Mieloide/metabolismo , NADPH Oxidasas/metabolismo , Salmonella typhimurium/genéticaRESUMEN
Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium) causes diarrhea and acute inflammation of the intestinal mucosa. The pro-inflammatory cytokines IL-17A and IL-17F are strongly induced in the infected mucosa but their contribution in driving the tissue inflammation is not understood. We have used the streptomycin mouse model to analyze the role of IL-17A and IL-17F and their cognate receptor IL-17RA in S. Typhimurium enterocolitis. Neutralization of IL-17A and IL-17F did not affect mucosal inflammation triggered by infection or spread of S. Typhimurium to systemic sites by 48 h p.i. Similarly, Il17ra(-/-) mice did not display any reduction in infection or inflammation by 12 h p.i. The same results were obtained using S. Typhimurium variants infecting via the TTSS1 type III secretion system, the TTSS1 effector SipA or the TTSS1 effector SopE. Moreover, the expression pattern of 45 genes encoding chemokines/cytokines (including CXCL1, CXCL2, IL-17A, IL-17F, IL-1α, IL-1ß, IFNγ, CXCL-10, CXCL-9, IL-6, CCL3, CCL4) and antibacterial molecules was not affected by Il17ra deficiency by 12 h p.i. Thus, in spite of the strong increase in Il17a/Il17f mRNA in the infected mucosa, IL-17RA signaling seems to be dispensable for eliciting the acute disease. Future work will have to address whether this is attributable to redundancy in the cytokine signaling network.