Inhibited Production of iNOS by Murine J774 Macrophages Occurs via a phoP-Regulated Differential Expression of NFκB and AP-1

Figure 2

S. typhimurium 14028 phoP regulon suppresses iNOS mRNA and protein expression in murine macrophages but fails to suppress iNOS when co-cultured with IFN-γ. (a) PCR showing iNOS mRNA expression is reduced in macrophages cultured with 14028 wild type compared to 14028 phoP mutants. The addition of IFN-γ to culture media increased iNOS mRNA expression in macrophages infected with either wild type or phoP mutant. (b) Intracellular iNOS protein cannot be detected by immunocytochemistry in uninfected (control) macrophages. (c) Suppression of intra-cellular iNOS protein observed in macrophages cultured with wild type 14028 compared to macrophages cultured with phoP mutants (d). Addition of IFN-γ to cell culture media also increased intra-cellular iNOS in macrophages cultured with wild type 14028 (e) or phoP mutants (f). Arrows show iNOS positive cells, scale bar bottom left: 20 μm. (g) Western blot of iNos protein in whole cell preparations stimulated with wild type 14028 or 14028 phoP with or without IFN-γ. Lane 1, molecular weight marker: 130 kDa. Lane 7: Unstimulated J774 cells (negative control). All analyses are representative of data obtained on 3 separate occasions.