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 in 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 infection^3,4. 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

Note: Experiments with HRSV must be performed in a biological safety cabinet in a biosafety level 2 laboratory. Collect all viral waste and material that has been in contact with the virus for proper disposal due to biological hazards. Special precaution must be taken when using potentially infected human blood. It is crucial to use endotoxin-free reagents throughout all protocols. 1. Culturing of HRSV A2 …

Representative Results

The HRSV A2 cultivation and purification method described here will give a yield between 2 x 107 and 2 x 108 FITC-50 quantified infectious viral particles/ml. Results of microscopic analysis, where FITC-stained infected cells were counted (results not shown), were compared to results of quantification by FACS (Figure 1). Both methods resulted in similar titers, although the FACS method had a much lower standard deviation between different experiments and was less laborious…

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…

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
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			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