Article Figures & Data
Figures
- FIGURE 1.
Induction of AA-Mø by Ft LVS. A, Peritoneal macrophages from C57BL/6 WT or TLR2−/− mice were infected with Ft LVS (MOI = 5). Total RNA was extracted from the macrophage cultures analyzed by real-time PCR for genes associated with AA-Mø. Gene expression is reported as relative gene expression compared with peritoneal macrophages exposed to medium only. All treatments were performed in triplicate, and data are presented as means ± SEM. Data are represented as a single experiment (n = 2). B, Peritoneal macrophages from C57BL/6 mice were treated, in triplicate, with medium only or rIL-4 (40 ng/ml), or exposed to Ft LVS (MOI = 5) for 48 h. Cells were harvested and stained for protein expression of FIZZ1, arginase-1, or mannose receptor by FACS analysis. Data presented are histograms from a single representative experiment (n = 4).
- FIGURE 2.
Ft LVS redirects macrophage differentiation from CA-Mø to AA-Mø over time. Peritoneal macrophages from C57BL/6 mice were treated with medium only, IFN-γ (20 ng/ml) plus LPS (10 ng/ml), or rIL-4 (40 ng/ml), or infected with Ft LVS (MOI = 5) for either 24 (A) or 48 (B) h. Cells were harvested and simultaneously stained for FIZZ1 and IL-12 p70. Protein expression was determined by FACS analysis. The numbers in the quadrants indicate the percentage of cells within that quadrant and have been rounded to the nearest one-tenth of a percent. All treatments were performed in triplicate, and data shown are from a single representative experiment (n = 3).
- FIGURE 3.
In vivo Ft LVS infection results in AA-Mφ. C57BL/6 mice were administered saline or Ft LVS (10,000 CFU) i.p. Three days later, the mice were sacrificed and peritoneal macrophages were harvested and simultaneously stained for FIZZ1 and F4/80. Protein expression was determined by FACS analysis. The numbers in the quadrants indicate the percentage of cells within that quadrant and have been rounded to the nearest one-tenth of a percent. Six mice were used for each treatment, and data shown are from a single representative mouse.
- FIGURE 4.
Ft LVS induction of AA-Mφ results in intracellular survival. Peritoneal macrophages from C57BL/6 mice were treated with medium only, rIFN-γ (20 ng/ml) plus LPS (10 ng/ml), or rIL-4 (40 ng/ml) for 48 h. The cells were then infected with Ft LVS (MOI = 5) for either 1 or 24 h. Intracellular Ft LVS was determined by (A) colony counts from the plated lysates or (B) real-time PCR amplification of the Ft LVS 16S rRNA in the same samples as analyzed in A. All treatments were performed in triplicate, and data are presented as means ± SEM (the SEMs are too small to be seen). Data are derived from a single representative experiment (n = 4).
- FIGURE 5.
Failure to induce AA-Mφ results in prolonged inflammatory response by the host. A, Peritoneal macrophages from WT BALB/cByJ, IL-4Rα−/−, or STAT6−/− mice were treated with medium only or rIL-4 (40 ng/ml), or exposed to Ft LVS (MOI = 5) for 0–72 h. Total RNA was extracted and analyzed by real-time PCR for genes associated with AA-Mφ and CA-Mφ. Gene expression is reported as relative gene expression normalized to untreated control samples. All treatments were performed in triplicate, and data are presented as means ± SEM. B, Peritoneal macrophages from WT BALB/cByJ, IL-4Rα−/−, or STAT6−/− mice were treated with medium only or rIL-4 (40 ng/ml), or exposed to Ft LVS (MOI = 5) for 48 h. Cells were harvested and stained for expression of either FIZZ1 or IL-12 p70 protein and subjected to FACS analysis. All treatments were performed in triplicate, and data are presented as means ± SEM. Data are derived from a single representative experiment (n = 3). C, Real-time PCR for Ft LVS 16S rRNA was conducted using the same samples as A. Gene expression is reported as relative gene expression.
- FIGURE 6.
Neutralization of IL-4 and IL-13 during Ft LVS infection results in prolonged macrophage classical activation. A, Peritoneal macrophages were cultured in medium, treated with rIL-4 (40 ng/ml) or rIL-13 (40 ng/ml), or infected with Ft LVS (MOI = 5). Cultures were also treated with medium only or an isotype-matched IgG control Ab or neutralizing anti-IL-4 (100 μg), anti-IL-13 (100 μg), or both Abs for 48 h. Cells were harvested and stained for expression of FIZZ1 and IL-12 p70 protein and subjected to FACS analysis. All treatments were performed in triplicate, and data are presented as means ± SEM. Data are represented as a single representative experiment (n = 3). B, Peritoneal macrophages were cultured in medium alone, or infected with Ft LVS. Cultures were treated with either medium only; an isotype control IgG; or neutralizing anti-IL-4, anti-IL-13, or both for 48 h. Intracellular Ft LVS was determined by colony counts from plated lysates. C, Peritoneal macrophages were treated the same as in A. Total RNA was extracted and analyzed by real-time PCR for IL-10. Gene expression is reported as fold induction normalized to untreated control samples. All treatments were performed in triplicate, and data are presented as means ± SEM. Statistical significance for comparison between one neutralizing Ab and control samples is represented as *, whereas statistical significance for treatment with both neutralizing Abs is represented as #.
- FIGURE 7.
Production of IL-4 and IL-13 promotes the AA-Mφ phenotype while attenuating the CA-Mφ phenotype during Ft LVS infection. Peritoneal macrophages were treated with medium alone or infected with Ft LVS (MOI = 5). A, Cell supernatants were harvested at the indicated times and analyzed by ELISA for production of IL-4, IL-13, IL-1β, and IFN-γ. B, Total RNA was extracted and analyzed by real-time PCR for arginase-1 and iNOS mRNA. Gene expression is reported as fold induction normalized to untreated control samples. All treatments were performed in triplicate, and data are presented as means ± SEM of four individual experiments. C, BALB/cByJ WT and IL-4Rα−/− mice were i.p. inoculated with either saline or Ft LVS (10,000 CFU). Survival was monitored for up to 10 days. Data are pooled from four separate experiments with four to five mice/treatment/experiment. Statistical significance for comparison between Ft LVS-infected WT and Ft LVS-infected IL-4Rα−/− mice is represented as * (p < 0.001).
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