N 2O 3 enhances the nitrosative potential of IFN γ-primed macrophages in response to Salmonella

Abstract

We show here that the nitric oxide (NO)-detoxifying Hmp flavohemoprotein increases by 3-fold the transcription of the Salmonella pathogenicity island 2 (SPI2) in macrophages expressing a functional inducible NO synthase (iNOS). However, Hmp does not prevent NO-related repression of SPI2 transcription in IFN γ-primed phagocytes, despite preserving intracellular transcription of sdhA sdhB subunits of Salmonella succinate dehydrogenase within both control and IFN γ-primed phagocytes. To shed light into the seemingly paradoxical role that Hmp plays in protecting intracellular SPI2 expression in various populations of macrophages, N 2O 3 was quantified as an indicator of the nitrosative potential of Salmonella-infected phagocytes in different states of activation. Hmp was found to prevent the formation of 300 nM N 2O 3/h/bacteria in IFN γ-primed macrophages, accounting for about a 60% reduction of the nitrosative power of activated phagocytes. Utilization of the vacuolar ATPase inhibitor bafilomycin indicates that a fourth of the ∼200 nM N 2O 3/h sustained by IFN γ-primed macrophages is generated in endosomal compartments via condensation of HNO 2. In sharp contrast, control macrophages infected with wild-type Salmonella produce as little N 2O 3 as iNOS-deficient controls. Collectively, these findings indicate that the NO-metabolizing activity of Salmonella Hmp is functional in both control and IFN γ-primed macrophages. Nonetheless, a substantial amount of the NO generated by IFN γ-primed macrophages gives rise to N 2O 3, a species that not only enhances the nitrosative potential of activated phagocytes but also avoids detoxification by Salmonella Hmp.