FIGURE 4.

Short propionate pretreatment did not alter NO production by infected macrophages but reduces anaerobic L. monocytogenes infection. (A–C) RAW264.7 macrophages were incubated with or without 10 mM of propionate for 3 h prior to infection (gray bars) or during infection (black bars) by aerobically or anaerobically grown L. monocytogenes. Infected macrophages were lysed at 2 and 6 hpi to enumerate intracellular CFU. Percent input intracellular CFU (A) was calculated by comparing intracellular CFU at 2 hpi to CFU in the infection inoculum. Immunofluorescence microscopy was performed at 4 hpi (B) where filamentous actin and L. monocytogenes were differentially stained and quantified for co-localization. A minimum of 200 L. monocytogenes cells were counted from each replicate and condition over 2 independent experiments performed in duplicate. Averages were plotted with error bars representing standard errors of the mean (B). Fold increase intracellular CFU (C) was calculated by comparing intracellular CFU between 2 and 6 hpi. Averages from 4 independent experiments, each performed with triplicate, were plotted with error bars representing standard errors of the mean (A,C). Nitrite concentrations in culture supernatant were quantified for RAW264.7 macrophages treated with or without activation by LPS and IFNγ overnight and with propionate for 3 h prior to infections. Overnight aerobically or anaerobically grown L. monocytogenes were used to infect macrophages for 30 min where supernatant nitrite levels (D) and intracellular CFUs (E) were measured at 24 hpi. Averages are from at least 3 independent experiments, each performed in triplicate, with error bars representing standard errors of the mean (D,E). Asterisks denote statistical significance between pair-wise comparisons with ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001.