Tolerant peritoneal macrophages from LPS-injected <i>Aoah^−/−</i> mice have distinctive characteristics.

<p>(<b>A–C</b>) <i>Aoah^+/+</i> or <i>Aoah^−/−</i> mice were injected i.p. with PBS or 10 µg <i>E. coli</i> O14 LPS. Fourteen days later, their peritoneal cells were harvested; the F4/80+ cells (macrophages) were identified using flow cytometry and their SSC (<b>A</b>), surface expression of F4/80 (<b>B</b>) and CD86 (<b>C</b>) were measured. Data shown are from 2 independent experiments. n = 6–8. ***, p<0.001. When compared with the other groups, LPS-exposed <i>Aoah^−/−</i> macrophages have lower SSC and express less surface F4/80 and CD86. Blue bars, <i>Aoah^+/+</i>; Red bars, <i>Aoah^−/−</i>; diagonal markings indicate i.p. LPS injection. (<b>D–F</b>) <b>Macrophage IL-6 production following LPS re-stimulation correlates with surface marker expression. </b><i>Aoah^+/+</i> or <i>Aoah^−/−</i> mice were injected with 0, 0.016, 0.08, 0.4, 2 or 10 µg <i>E. coli</i> O14 LPS i.p. Each LPS dose was given to 3–5 <i>Aoah</i>^+/+ and <i>Aoah^−/−</i> mice. Fourteen days later, their peritoneal cells were removed. Some were used to measure surface markers F4/80, CD86 and SSC by flow cytometry. Others were cultured for 18 hours, the floating cells were washed away and the adherent macrophages were re-stimulated with <i>E. coli</i> O111 LPS (1 µg/ml) for 6 hours. Medium IL-6 levels were measured by ELISA and correlated with SSC, F4/80 and CD86 expression on macrophages from the same mice. Each dot represents data from one mouse. Secreted TNF concentrations also positively correlated with these markers (<b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003339#ppat.1003339.s001" target="_blank">Fig. S1</a></b>). MFI; mean fluorescence intensity. Geo MFI; geometric mean fluorescence intensity.