Transcription analysis of the porcine alveolar macrophage response to Mycoplasma hyopneumoniae

Abstract

Mycoplasma hyopneumoniae is considered the major causative agent of porcine respiratory disease complex, occurs worldwide and causes major economic losses to the pig industry. To gain more insights into the pathogenesis of this organism, the high throughput cDNA microarray assays were employed to evaluate host responses of porcine alveolar macrophages to M. hyopneumoniae infection. A total of 1033 and 1235 differentially expressed genes were identified in porcine alveolar macrophages in responses to exposure to M. hyopneumoniae at 6 and 15 hours post infection, respectively. The differentially expressed genes were involved in many vital functional classes, including inflammatory response, immune response, apoptosis, cell adhesion, defense response, signal transduction, protein folding, protein ubiquitination and so on. The pathway analysis demonstrated that the most significant pathways were the chemokine signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway, nucleotide-binding oligomerization domains (Nod)-like receptor signaling pathway and apoptosis signaling pathway. The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR. The expression kinetics of chemokines was further analyzed. The present study is the first to document the response of porcine alveolar macrophages to M. hyopneumoniae infection. The data further developed our understanding of the molecular pathogenesis of M. hyopneumoniae.

Figure 1. Clustering and characterization of the differential expression of genes.

(A) The DE genes showing clear functional annotation at 6 HPI have been selected for cluster analysis which is described in methods. Each row represents a separate gene, and each column represents a experiment sample. Color legend is on the left, the color scale ranges from saturated green for log ratios −3.0 and above to saturated red for log ratios 3.0 and above. Red indicates increased gene expression levels; green indicates decreased levels compared with normal samples. (B) Categories of annotated genes based on biological process GO term at 6 HPI. (C) The genes showing clear functional annotation at 15 HPI have been selected for cluster analysis. (D) Categories of annotated genes based on biological process GO term at 15 HPI.

Figure 2. STRING analysis of the relationship between DE genes.

The DE genes in PAM cells infected M. hyopneumoniae were analyzed using the STRING database. The network nodes represent the proteins encoded by the DE genes. Seven different colored lines link a number of nodes and represent seven types of evidence used in predicting associations. A red line indicates the presence of fusion evidence; a green line represents neighborhood evidence; a blue line represents coocurrence evidence; a purple line represents experimental evidence; a yellow line represents textmining evidence; a light blue line represents database evidence and a black line represents co-expression evidence.

Figure 3. Expression kinetics of chemokines in PAM.

PAMs were mock-infected, infected with M. hyopneumoniae. Cells were collected at the indicated time points, and subjected to real-time PCR to analyze the expression of CCL4 (A), CCL8 (B), CXCL2 (C) and CXCL10 (D).