Análisis de la respuesta innata mucosal desencadenada por agonistas de receptores tipo toll (TLR) : Evaluación de su relevancia en la interacción B. pertussis-huésped

El objetivo general de esta tesis es generar conocimiento sobre los aspectos claves de la respuesta inicial frente a una infección respiratoria para aplicarlo en la formulación de estrategias de inmunoprofilaxis de la enfermedad. Este trabajo puede servir de prueba de concepto para contrastar las hipótesis enunciadas. Con este fin, emplearemos el modelo murino de infección por B. pertussis, analizando la contribución de distintos receptores TLR en la inducción de la defensa mucosal frente a la infección para luego evaluar cómo la activación de dichas vías en protocolos de inmunización intranasal son capaces de dar origen a respuestas protectivas frente al desafío intranasal con B. pertussis.Facultad de Ciencias Exacta

The stimulated innate resistance event in Bordetella pertussis infection is dependent on reactive oxygen species production

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 μg) was added before (48h or 24h), after (24h), or simultaneously with the B. pertussis challenge (107 CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.Facultad de Ciencias ExactasLaboratorio de Investigaciones del Sistema Inmun

The stimulated innate resistance event in Bordetella pertussis infection is dependent on reactive oxygen species production

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 μg) was added before (48h or 24h), after (24h), or simultaneously with the B. pertussis challenge (107 CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.Facultad de Ciencias ExactasLaboratorio de Investigaciones del Sistema Inmun

The stimulated innate resistance event in Bordetella pertussis infection is dependent on reactive oxygen species production

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can effi- ciently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 g) was added before (48 h or 24 h), after (24 h), or simultaneously with the B. pertussis challenge (107 CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.Fil: Zurita, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Moreno, Griselda Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; ArgentinaFil: Errea, Agustina Juliana. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ormazabal, Maximiliano Emanuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; ArgentinaFil: Rumbo, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Departamento de Ciencias Biológicas. Laboratorio de Investigaciones del Sistema Inmune; ArgentinaFil: Hozbor, Daniela Flavia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Biotecnología y Biología Molecular. Universidad Nacional de la Plata. Facultad de Ciencias Exactas. Instituto de Biotecnología y Biología Molecular; Argentin

Transgenic mouse model harboring the transcriptional fusion ccl20-luciferase as a novel reporter of pro-inflammatory response

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.Fil: Crispo, Martina. Unidad de Animales Transgénicos y de Experimentación – Institut Pasteur de Montevideo; UruguayFil: Van Maele, Laurye. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; FranciaFil: Tabareau, Julien. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; FranciaFil: Cayet, Delphine. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; FranciaFil: Errea, Agustina Juliana. Universidad Nacional de la Plata. Facultad de Cs.exactas. Departamento de Cs.biologicas. Laboratorio de Invest.del Sistema Inmune; ArgentinaFil: Ferreira, Ana María. Cátedra de Inmunologíaa. Facultad de Ciencias/Facultad de Química. Universidad de la República; UruguayFil: Rumbo, Martin. Universidad Nacional de la Plata. Facultad de Cs.exactas. Departamento de Cs.biologicas. Laboratorio de Invest.del Sistema Inmune; ArgentinaFil: Sirard, Jean Claude. Institut Pasteur de Lille. Centre d’Infection et d’Immunite de Lille; Franci

Transgenic mouse model harboring the transcriptional fusion Ccl20-luciferase as a novel reporter of pro-inflammatory response

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.Laboratorio de Investigaciones del Sistema InmuneFacultad de Ciencias Exacta

A method for the purification of bacterial flagellin that allows simple upscaling

There is a growing interest in enterobacterial flagellins that may result in a demand to produce flagellin on an industrial scale for possible applications as an adjuvant, immunomodulatory agent or vaccine antigen. Traditionally, small-scale production of flagellin has occurred in the laboratory by flagellar shearing of bacterial surfaces and subsequent ultracentrifugation. The main drawback of this method is the need to use low-agitation cultures to avoid the loss of flagella due to shearing during culture. In the present work, we describe a scalable protocol for the production of flagellin with higher yields than traditional laboratory-scale protocols. The use of cross-flow filtration to concentrate bacterial cultures combines extensive shearing of flagella with a reduction in volume, greatly simplifying downstream processing. This technique also allows the use of highly-agitated culture conditions because any sheared flagella are retained in the bacterial concentrate. Flagella obtained with this procedure showed in vivo and in vitro innate activating capacities similar to those of flagella produced at laboratory scale. This procedure is flexible, allowing an increase in production scale, an enhancement of flagellin yield and no requirement for expensive equipment.Facultad de Ciencias ExactasLaboratorio de Investigaciones del Sistema Inmun

Characterization of structural and immunological properties of a fusion protein between flagellin from Salmonella and lumazine synthase from Brucella

Aiming to combine the flexibility of Brucella lumazine synthase (BLS) to adapt different protein domains in a decameric structure and the capacity of BLS and flagellin to enhance the immunogenicity of peptides that are linked to their structure, we generated a chimeric protein (BLS-FliC131) by fusing flagellin from Salmonella in the N-termini of BLS. The obtained protein was recognized by anti-flagellin and anti-BLS antibodies, keeping the oligomerization capacity of BLS, without affecting the folding of the monomeric protein components determined by circular dichroism. Furthermore, the thermal stability of each fusion partner is conserved, indicating that the interactions that participate in its folding are not affected by the genetic fusion. Besides, either in vitro or in vivo using TLR5-deficient animals we could determine that BLS-FliC131 retains the capacity of triggering TLR5. The humoral response against BLS elicited by BLS-FliC131 was stronger than the one elicited by equimolar amounts of BLS + FliC. Since BLS scaffold allows the generation of hetero-decameric structures, we expect that flagellin oligomerization on this protein scaffold will generate a new vaccine platform with enhanced capacity to activate immune responsesFil: Hiriart, Yanina. Inmunova S.A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Rossi, Andrés Hugo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Biedma, Marina Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Errea, Agustina Juliana. Universidad Nacional de La Plata; Argentina. Universidad Nacional de La Plata; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Moreno, Griselda Noemí. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Cayet, D.. Universidad Nacional de La Plata; ArgentinaFil: Rinaldi, Jimena Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Blancá, Bruno Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; ArgentinaFil: Sirard, J.C.. Centre National de la Recherche Scientifique; FranciaFil: Goldbaum, Fernando Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Berguer, Paula Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Rumbo, Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Estudios Inmunológicos y Fisiopatológicos. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Instituto de Estudios Inmunológicos y Fisiopatológicos; Argentin

Mucosal innate response stimulation induced by lipopolysaccharide protects against Bordetella pertussis colonization

Non-specific enhancement of the airways innate response has been shown to impair lung infections in several models of infection such diverse as influenza A, Streptococcus pneumoniae, and Aspergillus niger. Our aim was to evaluate whether a similar event could operate in the context of Bordetella pertussis respiratory infection, not only to enrich the knowledge of host-bacteria interaction but also to establish immunological basis for the development of new control strategies against the pathogen. Using a B. pertussis intranasal infection model and coadministration of different TLR agonists at the moment of the infection, we observed that the enhancement of innate response activation, in a TLR4-dependent way, could efficiently impair B. pertussis colonization (P < 0.001). While LPS from different microbial sources were equally effective in promoting this effect, flagellin and poly I:C coadministration, in spite of inducing expression of innate response markers TNFalpha, CXCL2, CXCL10 and IL6, was not effective to prevent B. pertussis colonization. Our results indicate that during the early stage of infection, specific anti-microbial mechanisms triggered by TLR4 stimulation are able to impair B. pertussis colonization. These findings could complement our current view of the role of TLR4-dependent processes that contribute to anti-pertussis immunity.Instituto de Biotecnología y Biología MolecularLaboratorio de Investigaciones del Sistema Inmun

The stimulated innate resistance event in Bordetella pertussis infection is dependent on reactive oxygen species production

The exacerbated induction of innate immune responses in airways can abrogate diverse lung infections by a phenomenon known as stimulated innate resistance (StIR). We recently demonstrated that the enhancement of innate response activation can efficiently impair Bordetella pertussis colonization in a Toll-like receptor 4 (TLR4)-dependent manner. The aim of this work was to further characterize the effect of lipopolysaccharide (LPS) on StIR and to identify the mechanisms that mediate this process. Our results showed that bacterial infection was completely abrogated in treated mice when the LPS of B. pertussis (1 μg) was added before (48h or 24h), after (24h), or simultaneously with the B. pertussis challenge (107 CFU). Moreover, we detected that LPS completely cleared bacterial infection as soon as 2 h posttreatment. This timing suggests that the observed StIR phenomenon should be mediated by fast-acting antimicrobial mechanisms. Although neutrophil recruitment was already evident at this time point, depletion assays using an anti-GR1 antibody showed that B. pertussis clearance was achieved even in the absence of neutrophils. To evaluate the possible role of free radicals in StIR, we performed animal assays using the antioxidant N-acetyl cysteine (NAC), which is known to inactivate oxidant species. NAC administration blocked the B. pertussis clearance induced by LPS. Nitrite concentrations were also increased in the LPS-treated mice; however, the inhibition of nitric oxide synthetases did not suppress the LPS-induced bacterial clearance. Taken together, our results show that reactive oxygen species (ROS) play an essential role in the TLR4-dependent innate clearance of B. pertussis.Facultad de Ciencias ExactasLaboratorio de Investigaciones del Sistema Inmun