Cytokine induction in murine macrophages infected with virulent and avirulent Rhodococcus equi

Abstract

To look for a possible correlation between the virulence of Rhodococcus equi and its cytokine-inducing capacity, we evaluated intracellular survival and measured cytokine induction by mouse macrophages infected with a virulent strain containing an 85-kb plasmid and expressing VapA (103+), its avirulent plasmid-cured derivative (103-), and heat-killed 103+ (HK). After incubation with similar numbers of bacteria, macrophages infected with 103- contained significantly more organisms than those infected with 103+ or HK. The number of bacteria in the macrophages infected with 103- and HK decreased progressively, whereas the 103+ numbers remained constant over 48 h. Interleukin 1beta (IL-1beta), IL-6, IL-10, IL-12 p40, and tumor necrosis factor alpha (TNF-alpha) mRNA induction peaked at 4 h and returned to baseline between 12 and 48 h postinfection. IL-1beta, IL-6, IL-10, and TNF-alpha concentrations assessed by enzyme-linked immunosorbent assay generally agreed well with mRNA expression; IL-12 could, however, not be detected. For all the cytokines detected, mean concentrations in the supernatants were consistently higher in the 103(-)-infected monolayers than in those infected with 103+, although, with the exception of IL-1beta, the differences were not statistically significant. R. equi HK was a poor inducer of cytokine production. In conclusion, virulent and avirulent R. equi strains induced similar levels of cytokine synthesis. The slightly greater induction of most cytokines observed following infection with 103- is likely secondary to greater uptake by macrophages rather than to a direct role of VapA or another plasmid-encoded product in downregulating cytokine induction.

FIG. 1

Uptake and intracellular survival of R. equi within murine resident peritoneal macrophages. (A) The number of bacteria associated with 200 macrophages was determined at 0, 4, 12, 24, and 48 h by visual counting, with light microscopy. Macrophages containing 10 or more bacteria were scored as having 10 organisms. (B) The number of macrophages containing 10 or more bacteria (200 macrophages were counted) was recorded at various times postinfection. Values are shown as the means ± standard errors of the means of three independent experiments. ∗, P ≤ 0.05 compared with results for 103⁺.

FIG. 2

IL-1β, IL-6, IL-10, IL-12 p40, and TNF-α mRNA expression at various time points in murine resident peritoneal macrophages infected in vitro with R. equi 103⁺ (solid bars), 103⁻ (shaded bars), and HK (striped bars). mRNA expression was quantified by competitive reverse transcriptase PCR. The results are expressed as the fold of cytokine mRNA expression above that of noninfected cells cultured under the same conditions. The results shown were measured from a pool of macrophages obtained from two independent experiments.

FIG. 2

IL-1β, IL-6, IL-10, IL-12 p40, and TNF-α mRNA expression at various time points in murine resident peritoneal macrophages infected in vitro with R. equi 103⁺ (solid bars), 103⁻ (shaded bars), and HK (striped bars). mRNA expression was quantified by competitive reverse transcriptase PCR. The results are expressed as the fold of cytokine mRNA expression above that of noninfected cells cultured under the same conditions. The results shown were measured from a pool of macrophages obtained from two independent experiments.

FIG. 3

IL-1β, IL-6, IL-10, and TNF-α concentrations at various time points in the supernatants of murine resident peritoneal macrophages infected in vitro with R. equi 103⁺ (diamonds), 103⁻ (circles), and HK (stars). Noninfected macrophages cultured under the same conditions (open squares) were used as a baseline for cytokine production. Values are shown as the means of three independent experiments. Letters differing between two groups indicate a statistically significant difference in cytokine production over a 48-h period (P ≤ 0.05). When at least one letter is common between two groups, the difference is not statistically significant.