Lamb model of respiratory syncytial virus-associated lung disease: insights to pathogenesis and novel treatments

Abstract

Preterm birth is a risk factor for respiratory syncytial virus (RSV) bronchiolitis and hospitalization. The pathogenesis underlying this is not fully understood, and in vivo studies are needed to better clarify essential cellular features and molecular mechanisms. Such studies include analysis of lung tissue from affected human infants and various animal models. The preterm and newborn lamb lung has developmental, structural, cellular, physiologic, and immunologic features similar to that of human infants. Also, the lamb lung is susceptible to various strains of RSV that infect infants and cause similar bronchiolar lesions. Studies in lambs suggest that viral replication in airways (especially bronchioles) is extensive by 4 days after infection, along with bronchiolitis characterized by degeneration and necrosis of epithelial cells, syncytial cell formation, neutrophil infiltration, epithelial cell hypertrophy and hyperplasia, and innate and adaptive immune responses. RSV bronchiolitis greatly affects airflow and gaseous exchange. RSV disease severity is increased in preterm lambs compared with full-term lambs; similar to human infants. The lamb is conducive to experimental assessment of novel, mechanistic therapeutic interventions such as delivery of vascular endothelial growth factor and enhancement of airway epithelial oxidative responses, Club (Clara) cell protein 10, and synthesized compounds such as nanobodies. In contrast, exposure of the fetal ovine lung in vivo to ethanol, a risk factor for preterm birth, reduces pulmonary alveolar development and surfactant protein A expression. Because the formalin-inactivated RSV vaccination enhances some inflammatory responses to RSV infection in lambs, this model has the potential to assess mechanisms of formalin-inactivated RSV enhanced disease as well as newly developed vaccines.

Keywords: bronchiolitis; infants; lambs; pneumonia; preterm; respiratory syncytial virus (RSV).

Figure 1

Respiratory syncytial virus (RSV) strain A2 causes bronchiolitis in lambs (Olivier et al. 2009). (A) Lung from a control lamb not infected with RSV that contains a bronchiole and alveoli. (B) Lung from a lamb 6 days after inoculation with RSV with a bronchiole (outlined) containing epithelial cells admixed with neutrophils (*). Alveoli around the bronchiole are collapsed with accumulation of degenerate neutrophils and areas of necrosis (arrow). (C and D) Immunohistochemical detection of RSV antigen in lung from a lamb 6 days after inoculation with RSV, in which RSV viral antigen is present within the bronchi, epithelial cells lining the bronchi, and the syncytial cells in areas of alveolar consolidation. Bars = 25 µm.

Figure 2

Pretreatment with exogenous recombinant human vascular endothelial growth factor (rhVEGF) decreases histologic consolidation score of lambs infected with human respiratory syncytial virus (RSV) A2 strain (Olivier et al. 2011). Lambs received either media followed by RSV A2 (no vascular endothelial growth factor [VEGF]) or rhVEGF followed by RSV A2 and were assessed for clinical signs, viral lesions, and mRNA levels. At 6 days after RSV A2 infection, VEGF-treated lambs had reduced histologic consolidation scores (an overall score of bronchiolitis and lung inflammation), indicating a decreased in alveolar consolidation and inflammatory cell infiltration (*p < 0.05). Exogenous rhVEGF treatment also reduced RSV mRNA levels at 4 and 6 days after RSV inoculation (not shown). Previous studies have demonstrated that VEGF treatment also reduces the severity of bovine RSV in newborn lambs (not shown).

Figure 3

Club (Clara) cell protein 10 (CC10) reduces respiratory syncytial virus (RSV) gross lesions and RSV mRNA levels. Two lambs received recombinant human CC10 (rhCC10) intravenously (1.5 mg/kg) twice daily 1 day before RSV inoculation (6 ml; 107 PFU/ml by nebulizer) and each day thereafter; another group of lambs (n = 6) received RSV alone and no rhCC10; a third group lacked rhCC10 and were nebulized with media (control). rhCC10-treated lambs had reduced gross lesions and decreased RSV mRNA levels by quantitative polymerase chain reaction (qPCR). ***p < 0.05.

Figure 4

Respiratory syncytial virus (RSV) Memphis 37 (M37) strain causes bronchiolitis in lambs that is reduced with enhanced airway oxidative responses. (A) Lung from a lamb infected with RSV M37 strain 4 days after inoculation in which there is erosion of the epithelium with accumulation of cell debris in the airway lumen and formation of a syncytial cell. (B) Lung from a lamb 6 days after inoculation with RSV M37 strain in which viral antigen is detected by immunohistochemistry and viral antigen is present in epithelial cells, macrophages, and in cell debris of the airway lumen. There is moderate thickening of the epithelium due to hyperplasia. (C) Enhancement of the airway oxidative response (dual oxidases/lactoperoxidase oxidative system) by administration of potassium iodide (KI) reduces RSV M37 mRNA levels (as well as viral titers, antigen, and lesions; not shown) in RSV M37–inoculated lambs treated with KI (M37 + KI) compared with lambs inoculated with RSV M37 but not receiving KI (M37). Inhibition of the oxidative defense system with dapsone (DAP) in a group of lambs resulted in significantly enhanced RSV M37 RNA levels (DAP + M37 + KI) despite KI treatment (Derscheid, Van Geelen, Gallup et al. 2013).

Figure 5

Maternal ethanol consumption reduces lung maturation through hyxpoxia inducible factor (HIF) and its downstream gene vascular endothelial growth factor (VEGF) (Lazic et al. 2011). (A) Relative mRNA expression of HIF-1α, -2α, and -3α in the neonatal ovine lung. Exposure to ethanol in utero reduced mRNA expression of HIF-1α and HIF-2α in the lungs of preterm (PT) lambs when compared with the control lambs of the same age group. Such changes are not observed in the lungs of full-term (FT) lambs exposed to ethanol in utero. No significant changes are seen with HIF-3α mRNA expression levels in both PT and FT lambs. (*p < 0.10; significance was established at 90% confidence interval). (B) Relative mRNA expression of vascular endothelial growth factor (panVEGF) in the neonatal ovine lung. Exposure to ethanol in utero reduces mRNA expression of panVEGF in the lungs of PT lambs when compared with control lambs of the same age group. Such changes are not observed in the lungs of FT lambs exposed to ethanol in utero. (*p < 0.10; significance was established at 90% confidence interval). (C) Relative mRNA expression of vascular endothelial growth factor receptor 1 and 2 (VEGFR-1 and VEGFR-2, respectively) in the neonatal ovine lung. Exposure to ethanol in utero reduces mRNA expression of VEGFR-1 and VEGFR-2 in the lungs of PT lambs when compared with the control lambs of the same age group. Such changes are not observed in the lungs of FT lambs exposed to ethanol in utero. (*p < 0.10; significance was established at 90% confidence interval). (D) Periodic acid-Schiff (PAS) stain for glycogen granules in the type II pneumocytes (ATII). Glycogen granules in the ATII are more abundant in the PT lambs exposed to ethanol in utero (B) when compared with control lambs of the same age group (A). Glycogen content in the ATII is similar in the FT lambs exposed to ethanol in utero (D) and the control lambs of the same age group (C).