Gene expression profiling of bovine bronchial epithelial cells exposed in vitro to bovine herpesvirus 1 and Mannheimia haemolytica

Abstract

Bovine respiratory disease (BRD) often occurs when active respiratory virus infections (BHV-1, etc.) impair resistance to Mannheimia haemolytica infection in the lower respiratory tract. The interactions that occur when the respiratory epithelium encounters these viral and bacterial pathogens are poorly understood. We used Agilent bovine gene microarray chips containing 44,000 transcripts to elucidate bovine bronchial epithelial cell (BBEC) responses following in vitro exposure to BHV-1 alone, M. haemolytica alone, or both BHV-1 and M. haemolytica. Microarray analysis revealed differential regulation (>2-fold) of 978 transcripts by BHV-1 alone, 2040 transcripts by M. haemolytica alone, and 2189 genes by BHV-1 and M. haemolytica in combination. M. haemolytica treatment produced significantly greater inductions (>10-fold) of several inflammation associated genes, such as CXCL2, IL-6, IL-1α, e-selectin, and IL-8, than to BHV-1 alone. Functional analysis of the microarray data revealed a significant upregulation of genes involved in important biological processes such as inflammation (TNF-α, IL-8, Tlr-2, IL-1, CXCL2, CSF2), vascular functions (VEGF, EDN2) and leukocyte migration (ICAM1, IL-16) during a co-infection with BHV-1 and M. haemolytica compared to either pathogen alone. This study provides evidence to support that lung epithelial cells are a source of mediators that may promote inflammatory changes observed during bovine respiratory disease.

Keywords: Bovine herpesvirus 1; Bronchial epithelial cells; Mannheimia haemolytica; Microarray.

Fig. 1

Overview of differential gene expression by bronchial epithelial cells exposed to BHV-1, M. haemolytica (MH) or BHV-1 and M. haemolytica in combination (BHV1 + MH). (A) Identification of differentially expressed features at each treatment by annotation screening (p < 0.05 and fold change ≥ 2.0). (B) There were 978, 2040, and 2189 differentially expressed transcripts identified for BHV-1, MH, and BHV-1 + MH, respectively. Three hundred and nineteen transcripts were differentially regulated by all treatments (p < 0.05, and fold change ≥ 2.0).

Fig. 2

Hierarchical clustering of the top 50 differentially expressed genes differentially regulated by BHV-1, MH and BHV-1 + MH. (A) This heat map depicts a qualitative assessment of the similarities of the gene expression of the top 50 of the 319 differentially expressed genes in response to the 3 treatments. Red indicates up-regulation while green indicates down- regulation of selected genes. (B) Multivariate pairwise correlation analysis of the BBE cell gene expression patterns elicited by BHV-1, MH, and BHV-1 + MH. This analysis provides a more quantitative examination of similarities in expression patterns of the differentially expressed genes by determining the correlation between gene expression (as fold change) and significance (p < 0.05) profiles of the genes differentially regulated by all treatments. This analysis indicates that the responses elicited by MH are highly correlated with gene expression patterns for BHV-1 + MH.

Fig. 3

One-way analysis of BBE cell gene responses elicited by BHV-1, MH or BHV-1 + MH. Total significant bovine genes responding to BHV-1, MH or BHV-1 + MH were analyzed and presented as a mean log ratio of test/control. The mean log ratio for (BHV-1 + MH), 0.023227 is greater than the sum of individual log mean ratios for MH and BHV-1, (0.009041 + 0.011703). As a result these data suggest a synergistic effect of BHV-1 and MH.

Fig. 4

Scatter plot matrix expression profiles of select differentially regulated genes. This analysis illustrates mean fold increase in expression, as compared to unstimualted cells, for selected genes of interest by BBE cell incubated with BHV-1, MH or BHV-1 + MH.

Fig. 5

Real-time PCR analysis of cytokine mRNA production by BBE cells in response to BHV-1, MH, or BHV-1 + MH. Quantitative real time PCR was used to confirm BBE cell cytokine gene expression in response to BHV-1, MH, or both BHV-1 + MH. The figure illustrates the relative expression levels (fold increase) of IFN-α, IL-1β, IL-1α, IL-8 and TNF-α (early BHV-1 transcript) by BBE cells compared to unstimulated control cells.