Comparison of cytokine transcript levels in cultured macrophages infected with hyperinfectious and conventionally virulent salmonellae.

<p>Innate immune cytokine transcript levels were examined from cultured RAW264.7 murine macrophages infected with hyperinfectious <i>S.</i> Choleraesuis χ3246, <i>S.</i> Bovismorbificans 158 or conventionally virulent <i>S.</i> Typhimurium reference strain 14028 grown under permissive (LPM pH 5.5; dotted lines) or nonpermissive (LB; solid lines) conditions for the hypervirulent phenotype. (A) IFN-β; (B) IL-1β; (C) IL-6; (D) iNOS; (E) IL-10. Bacterial cells derived from stationary phase cultures containing either LB or LPM pH 5.5 medium were used to infect cultured RAW 264.7 murine macrophage cells as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002647#s2" target="_blank"><i>Materials and Methods</i></a>. The bacteria were centrifuged onto cultured monolayers at 1,000× <i>g</i> for 10 min at room temperature, after which they were incubated for 30 min at 37°C in a 5% CO₂ incubator (<i>t</i> = 0 time point). The coculture was washed once and incubated for 45 min with gentamicin (100 µg/ml) at 37°C in a 5% CO₂ incubator, washed once with pre-warmed cell culture medium, and incubated with gentamicin (10 µg/ml) to the time points indicated (2, 5 and 8 hr). Total RNA was isolated from infected cultured RAW 264.7 murine macrophage cells, and from mock-infected controls as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002647#s2" target="_blank"><i>Materials and Methods</i></a>. RNA samples were analyzed by reverse transcription and real-time qPCR for: IFN-β; IL-1β; IL-6; iNOS; and IL-10 expression as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002647#s2" target="_blank"><i>Materials and Methods</i></a>. Relative target gene transcripts were normalized to the level of the GAPDH gene, relative to the average of the normalized values obtained for uninfected RAW 264.7 cells. Values given were obtained from triplicate wells SE <22%. Although reduced stimulation of all cytokine transcripts tested was observed upon infection with both hyperinfectious and conventionally virulent strains grown in LPM pH 5.5 medium relative to that exhibited in LB medium (<i>P</i><0.05), only hyperinfectious strains exhibited a significant reduced stimulation of IFN-β, IL-1β and IL-6 transcript levels at the 2 h infection time point (2.5- to 3.5- fold; <i>P</i><0.05). ^*Designates statistical significance for those measures that are specific to hypervirulent strains after growth in LPM pH 5.5 medium relative to that exhibited in LB medium (<i>P</i><0.05).