Bluetongue virus infection activates bovine monocyte-derived macrophages and pulmonary artery endothelial cells

Abstract

Bluetongue virus (BTV) is the cause of bluetongue (BT), an emerging, arthropod-transmitted disease of ungulates. The cellular tropism of BTV in ruminants includes macrophages, dendritic cells and endothelial cells (ECs), and fulminant infection is characterized by lesions consistent with those of so-called viral hemorrhagic fevers. Specifically, BT is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema. To further investigate the pathogenesis of vascular injury in BT, we evaluated the responses of cultured bovine pulmonary artery EC (bPAEC) and monocyte-derived macrophages (bMDM) to BTV infection by measuring transcript levels of genes encoding molecules important in mediating EC activation and/or endothelial barrier dysregulation. The data confirm that BTV infection of bPAEC resulted in increased transcription of genes encoding chemokine ligand 2 (CCL2) and E-selectin, and BTV infection of bMDM resulted in increased transcription of genes encoding TNF-alpha, IL-1beta, IL-8, and inducible nitric oxide synthase (iNOS). The data from these in vitro studies provide further evidence that cytokines and other vasoactive substances produced in macrophages potentially contribute to vascular injury in BTV-infected ruminants, along with direct effects of the virus itself on ECs.

Fig. 1

Immunofluorescence staining of VP7 in BTV infected (8hr) bMDM and bPAEC. Granular, cytoplasmic staining in (A) bMDM and (B) bPAEC. (400X).

Fig. 2

Relative quantification of endothelial cell mRNA of genes encoding (A) proinflammatory mediators CCL2 and IL-8 and (B) adhesion molecules E-selectin, VCAM1 and ICAM1 from BTV infected bPAEC, as measured by real time quantitative PCR. Values are the means ±SD of three replicate experiments performed in duplicate. Expression of genes encoding CCL-2 and E-selectin was significantly increased (p < 0.05). (*) indicates genes with significant increase in mRNA expression.

Fig. 3

Relative quantification of macrophage mRNA of genes encoding (A) proinflammatory and chemokine mediators TNF-α, IL-1 and IL-8 and (B) immune related and anti-viral molecules (iNOS and MX1) from BTV infected bMDM, as measured by real time quantitative PCR. Values are the means ±SD of 5 replicate experiments (5 individual cows) performed in duplicate. Expression of genes encoding TNF-α, IL-1β, IL-8, iNOS, and MX-1 was significantly increased (p < 0.05). (*) indicates genes with significant increase in mRNA expression.