Uma<em> In vitro</em> Modelo para estudar respostas imunes de sangue periférico humano células mononucleares para Vírus Respiratório Sincicial Humano Infecção
Summary
Human respiratory syncytial virus (HRSV) can cause severe bronchiolitis in young infants. Part of the pathogenesis of severe HRSV disease is caused by the host immune response. Stimulation of primary human immune cells with HRSV provides a fast and reproducible model system to study activation of inflammatory pathways and infection.
Abstract
O vírus sincicial respiratório humano (HRSV) infecções apresentar um largo espectro de gravidade da doença, variando desde as infecções suaves a bronquiolite com risco de vida. Uma parte importante da patogênese da doença grave é uma resposta imune melhorada levando a imunopatologia.
Aqui, nós descrevemos um protocolo usado para investigar a resposta imune de células do sistema imunológico humano a uma infecção HRSV. Em primeiro lugar, descrevemos métodos utilizados para a cultura, purificação e quantificação de HRSV. Subsequentemente, foi descrito um modelo humano de vitro, em que as células mononucleares do sangue periférico (PBMC) são estimuladas com HRSV vivo. Este sistema modelo pode ser usado para estudar vários parâmetros que podem contribuir para a gravidade da doença, incluindo a resposta imune inata e adaptativa. Estas respostas podem ser medido ao nível da transcrição e da tradução. Além disso, a infecção virai de células pode ser facilmente medida utilizando citometria de fluxo. Tomadojuntos, a estimulação de PBMC com HRSV vivo fornece um sistema modelo rápido e reprodutível para examinar os mecanismos envolvidos na doença induzida por HRSV.
Introduction
Human Respiratory Syncytial Virus (HRSV) is the most common cause of lower respiratory tract infections in children. Each year, over 33 million children under the age of five are infected with HRSV, leading to over three million hospitalizations and almost 200,000 deaths1. A growing body of evidence suggests that HRSV also poses a significant threat to the elderly and adults with underlying chronic illnesses2.
The majority of HRSV infections in young children presents with mild symptoms, comparable to the common cold, and do not require clinical intervention. However, a small proportion of patients require hospitalization and mechanical ventilation due to severe bronchiolitis.
Part of the pathogenesis of HRSV disease is the host's overexuberant and inadequate immune response to infection3,4. This is illustrated by several observations. The period of maximal illness is often preceded by the peak of viral infection and coincides better with cellular infiltration of infected tissues and the release of inflammatory cytokines3. Another line of evidence comes from the formalin inactivated HRSV (FI-RSV) trials in the 1960s. Instead of inducing protection in young infants, the vaccine resulted in an exaggerated immune response resulting in enhanced respiratory disease and higher morbidity and mortality.
Several models have been developed to study the pathogenesis of HRSV infections. Continuous cell lines, like HEp-2 and A549, have been used extensively to study HRSV infection in vitro. Epithelial cells are the primary targets of HRSV infection5, therefore a lot of focus has been on these cell types. However, the in vivo situation is much more complex and not limited to one cell type. In order to examine these complex interactions, several animal infection models have been developed to study HRSV pathogenesis. Thus far, two different strategies have been employed, focusing either on heterologous (nonhuman) models as well as cognate host-pneumovirus models. Examples of heterologous models for HRSV include chimpanzees, sheep, cotton rats and mice. RSV infection in its natural host has been studied in cattle and in mice, using bovine RSV and pneumovirus, respectively.
Whilst each of these models has provided important insights into disease pathogenesis, HRSV is highly adapted to humans. Therefore, as an addition to the cell lines and animal models currently used, we propose to use human primary PBMCs as a model for stimulation with HRSV. Human PBMCs can be obtained from a range of individuals to address specific research questions, including young children6, immunocompromised individuals as well as healthy adults7 or elderly individuals8. PBMCs can be used to study innate aspects of infection as well as the adaptive response to the virus. Activated inflammatory pathways important for the pathogenesis of HRSV disease can be studied at the mRNA and protein level. Further, viral infection of (subsets of) immune cells can be determined by flow cytometry. We have used this model previously to identify the synergistic effects of HRSV infection together with the bacterial ligand muramyl dipeptide (MDP) on the induction of proinflammatory cytokines7. We propose to use HRSV stimulation of human primary PBMCs as a robust, easy and fast in vitro model to study inflammatory pathways involved in the pathogenesis of HRSV disease.
Protocol
Representative Results
Discussion
In this study, we demonstrate that infection of PBMC with HRSV is a fast and reliable model system in which inflammatory pathways can be studied. In order to stimulate PBMCs an HRSV stock has to be prepared and quantified.
Multiple cell lines are susceptible to HRSV infection. Cell lines most used for HRSV culturing are HEp-2, HeLa, and Vero cells. However, we would not recommend the usage of Vero cells for culturing of HRSV because it has been shown that culturing of HRSV in Vero cells r…
Divulgations
The authors have nothing to disclose.
Acknowledgements
RSV A2 was kindly provided by Dr. R. de Swart (Erasmus MC, Rotterdam, The Netherlands). MV and GF are supported by the Virgo consortium, funded by the Dutch government project number FES0908, and by the Netherlands Genomics Initiative (NGI) project number 050-060-452. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Materials
| Reagent | |||
| PBS | Lonza | BE17-516F | PBS without Ca2+ Mg2+ or phenol red |
| HBSS | Invitrogen | 14025-100 | HBSS, Calcium, Magnesium, no Phenol Red |
| DMEM | Invitrogen | 31966-047 | DMEM, High Glucose, GlutaMAX, Pyruvate |
| RPMI | Invitrogen | 72400054 | RPMI 1640 Medium, GlutaMAX, HEPES |
| Reduced Serum Medium (Opti-MEM) | Invitrogen | 51985-026 | Opti-MEM I Reduced Serum Medium, GlutaMAX |
| FCS | Greiner Bio-one | FBS (Fetal Bovine Serum) | |
| BSA | Sigma Aldrich | A7030 | Albumin from bovine serum |
| P/S | Invitrogen | 15140-122 | Penicillin-Streptomycin, liquid |
| Trypsin | Invitrogen | 25300-054 | 0.05% Trypsin-EDTA (1x), Phenol Red |
| HeLa cells | ATCC | CCL-2 | |
| A549 cells | ATCC | CCL-185 | |
| Sucrose | Sigma Aldrich | S7903 | Sucrose BioXtra, ≥99.5% |
| IgG2 anti-mouse-FITC | BD Pharmingen | 555057 | Ms IgG2b K FITC isotype control |
| Anti-NP-RSV-FITC | Abcam | ab25849 | Anti-Respiratory Syncytial Virus antibody [671] (FITC) |
| Density gradient medium (Lymphoprep) | Axis-Shield | 1114545 | |
| EDTA tubes | BD Biosciences | 367525 | |
| Acetone | Merck Millipore | 1000141000 | Acetone for analysis |
| [header] | |||
| Material | |||
| Centrifuge Tubes | Beckman Coulter | 326823 | Thinwall, Polyallomer, 38.5 ml, 25 mm x 89 mm |
| SureSpin 630 Rotor with 36 ml buckets | Sorvall | 79368 | Swinging bucket titanium rotor |
| Sorvall WX80 Ultracentrifuge | Sorvall | 46900 | |
| CB 150l CO2 Incubator | Binder |
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