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Ex Vivo Respiratory Syncytial Virus Infection Induces Host Inflammatory Response in Human and Non-Human Primate Lung Tissue Slices
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A5481 - Ex Vivo Respiratory Syncytial Virus Infection Induces Host Inflammatory Response in Human and Non-Human Primate Lung Tissue Slices
Author Block: E. S. Twisterling1, F. Dahlmann1, S. Dehmel2, P. Braubach3, D. D. Jonigk3, G. Warnecke3, K. Sewald2, A. Braun2; 1Fraunhofer ITEM, German Primate Center (DPZ), Biomedical Research in Endstage and Obstructive Lung (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany, 2Fraunhofer ITEM, Biomedical Research in Endstage and Obstructive Lung (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany, 3Hannover Medical School (MHH), Biomedical Research in Endstage and Obstructive Lung (BREATH), Member of the German Centre for Lung Research (DZL), Hannover, Germany.
Respiratory Syncytial virus (RSV) is the most common agent affecting lower airways in children with increasing evidence for higher risk of subsequent asthma. RSV infection is characterized by the cytopathic effect of syncytial formation and triggers the innate immune response to release anti-viral cytokines and chemokines, e.g., IP-10. In this work, we hypothesized that RSV could infect ex vivo lung tissue (precision-cut lung slices; PCLS) from humans and non-human primates (NHP), producing an anti-viral immune response.
PCLS containing airways were prepared from lung sections of human and non-human primates (Rhesus, Macaca mulatta). The slices were inoculated with human-RSV-A2, UV-inactivated RSV, or medium for 2h. Macaque PCLS were incubated up to 5 days post-infection (dpi) and human slices for 1 dpi. Viral load, tissue viability, and immune response assays were assessed in supernatants, lysates, or slices.
The inocula infectivity of 106 and 105 IU/ml was confirmed by plaque assay on Hep-2 cells, producing the typical cytopathic effect with syncytial formation. Virus replication was determined by immunofluorescence staining using an anti-RSV FITC-labeled monoclonal antibody in PCLS, demonstrating the presence of RSV-infected cells spread over the tissue, not only restricted to the epithelium. Live/DEAD staining and LDH assay showed a slight decrease in tissue vitality after RSV infection. Virus-induced mediator release in PCLS was assessed by human-specific enzyme-linked immunosorbent assays (ELISA). Infection of PCLS with RSV at 106 IU/ml significantly increased the release of the anti-viral chemokine IP-10 in NHP-PCLS, reaching a 4-fold increase at day 1 and 3-fold increase at day 4. At day 5, the increase was not significant. In human-PCLS, 24h incubation after RSV infection provoked a 90-fold increased IP-10 release, representing 15 times more response than in macaque-PCLS.
PCLS can be used to study RSV infection ex vivo. Although non-human primates are not a natural host for RSV, lung slices provided from rhesus macaque could be infected, presenting anti-viral immune response comparable to human PCLS. In the future, these systems can be used to further investigate RSV mechanisms, especially in the context of asthma development.
Author Block: E. S. Twisterling1, F. Dahlmann1, S. Dehmel2, P. Braubach3, D. D. Jonigk3, G. Warnecke3, K. Sewald2, A. Braun2; 1Fraunhofer ITEM, German Primate Center (DPZ), Biomedical Research in Endstage and Obstructive Lung (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany, 2Fraunhofer ITEM, Biomedical Research in Endstage and Obstructive Lung (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany, 3Hannover Medical School (MHH), Biomedical Research in Endstage and Obstructive Lung (BREATH), Member of the German Centre for Lung Research (DZL), Hannover, Germany.
Respiratory Syncytial virus (RSV) is the most common agent affecting lower airways in children with increasing evidence for higher risk of subsequent asthma. RSV infection is characterized by the cytopathic effect of syncytial formation and triggers the innate immune response to release anti-viral cytokines and chemokines, e.g., IP-10. In this work, we hypothesized that RSV could infect ex vivo lung tissue (precision-cut lung slices; PCLS) from humans and non-human primates (NHP), producing an anti-viral immune response.
PCLS containing airways were prepared from lung sections of human and non-human primates (Rhesus, Macaca mulatta). The slices were inoculated with human-RSV-A2, UV-inactivated RSV, or medium for 2h. Macaque PCLS were incubated up to 5 days post-infection (dpi) and human slices for 1 dpi. Viral load, tissue viability, and immune response assays were assessed in supernatants, lysates, or slices.
The inocula infectivity of 106 and 105 IU/ml was confirmed by plaque assay on Hep-2 cells, producing the typical cytopathic effect with syncytial formation. Virus replication was determined by immunofluorescence staining using an anti-RSV FITC-labeled monoclonal antibody in PCLS, demonstrating the presence of RSV-infected cells spread over the tissue, not only restricted to the epithelium. Live/DEAD staining and LDH assay showed a slight decrease in tissue vitality after RSV infection. Virus-induced mediator release in PCLS was assessed by human-specific enzyme-linked immunosorbent assays (ELISA). Infection of PCLS with RSV at 106 IU/ml significantly increased the release of the anti-viral chemokine IP-10 in NHP-PCLS, reaching a 4-fold increase at day 1 and 3-fold increase at day 4. At day 5, the increase was not significant. In human-PCLS, 24h incubation after RSV infection provoked a 90-fold increased IP-10 release, representing 15 times more response than in macaque-PCLS.
PCLS can be used to study RSV infection ex vivo. Although non-human primates are not a natural host for RSV, lung slices provided from rhesus macaque could be infected, presenting anti-viral immune response comparable to human PCLS. In the future, these systems can be used to further investigate RSV mechanisms, especially in the context of asthma development.
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