Microarray Analysis of mRNA Levels from RAW264.7 Macrophages Infected with Brucella abortus. Mus musculus

Identification of host responses at the gene transcription level provides a molecular profile of the events that occur following infection. Brucella abortus is a facultative intracellular pathogen of macrophages that induces chronic infection in humans and domestic animals. Using microarray technology, the response of macrophages 4 hours following B. abortus infection was analyzed to identify early intracellular infection events that occur in macrophages. Of the more than 6,000 genes, we identified over 140 genes that were reproducibly differentially transcribed. First, an increase in the transcription of a number of pro-inflammatory cytokines and chemokines, such as TNF-α, IL-1β, IL-1α, and members of the SCY family of proteins, was evident that may constitute a general host recruitment of antibacterial defenses. Alternatively, Brucella may subvert newly arriving macrophages for additional intracellular infection. Second, transcription of receptors and cytokines associated with antigen presentation, e.g., MHC class II and IL-12p40, were not evident at this 4 hour period of infection. Third, Brucella inhibited transcription of various host genes involved in apoptosis, cell cycling, and intracellular vesicular trafficking. Identification of macrophage genes whose transcription was inhibited suggests that Brucella utilizes specific mechanisms to target certain cell pathways. In conclusion, these data suggest that B. abortus can alter macrophage pathways to recruit additional macrophages for future infection while simultaneously inhibiting apoptosis and innate immune mechanisms within the macrophage permitting intracellular survival of the bacterium. These results provide insights into the pathogenic strategies used by Brucella to survive long-term within a hostile environment. Keywords: Macrophage, intracellular pathogen, Brucella abortus, inflammatory immune response Overall design: Five murine U74A gene chips (Affymetrix) were independently hybridized with cRNA from uninfected macrophage samples (2 independent samples) and macrophages infected 4 hrs with B. abortus (3 independent samples). Washing and staining with streptavidin phycoerythrin was done using a GeneChip Fluidics station 400 (Affymetrix). Scanning was performed with an Affymetrix GeneArray. GeneChip Expression Analysis software (Affymetrix) was used to scan and analyze data. Output was stored in an Excel spreadsheet. Difference calls were assigned the following values: increased = 2, marginally increased = 1, no change = 0, marginally decrease = -1 and decreased = -2. The sum of the six difference calls from the inter-group comparisons of 2 uninfected and 3 Brucella infected arrays (2 x 3) was calculated. A sum of ≥8 or ≥ -8 was the cutoff values for increase/decrease, respectively. Expressed sequence tag (EST) sequences were eliminated from the analysis.