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

Hulme SD, Barrow PA, Foster N - Interdiscip Perspect Infect Dis (2012)

Bottom Line: Methods.A macrophages via an intact phoP regulon.This correlated with increased survival and reduced iNOS expression.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Science, The University of Nottingham, Sutton Bonington Campus, Sutton Bonington, Leicestershire, Nottingham NG7 2NR, UK.

ABSTRACT
Background. There are no reported data to explain how Salmonella suppress nitrite ion production in macrophages or whether this phenomenon is unique to typhoidal or non-typhoidal serovars. The aims of this study were, therefore, to investigate these phenomena. Methods. We measured survival of S. typhimurium 14028 and its phoP mutant in murine J774 macrophages, cultured with or without interferon gamma. We compared expression of inducible nitric oxide synthase (iNOS) mRNA and protein, and nitrite ion production and also examined binding of nuclear factor κB (NFκB) and activator protein 1 (AP-1) to macrophage DNA. Results. S. typhimurium 14028 inhibited binding of NFκB and AP-1 to DNA in murine J774. A macrophages via an intact phoP regulon. This correlated with increased survival and reduced iNOS expression. Suppression of NFκB activity was ameliorated in macrophages cultured with IFN-γ and this correlated with increased expression of iNOS mRNA and nitrite ion production, although IFN-γ had no effect on AP-1/DNA interaction. We show, that with one exception, suppression of iNOS is unique to typhoidal serovars. Conclusion. S. typhimurium inhibit NFκB and AP-1 interaction with macrophage DNA via the PhoP regulon, this reduces nitrite ion production and is principally associated with typhoidal serovars.

No MeSH data available.


Related in: MedlinePlus

Wild type S. typhimurium suppress NFκB/DNA interaction in J774.2 cells 2 and 12 h afterculture via phoP. (a) 2 h postculture. (b) 12 h post-culture. Controls include, uninfected J774.2 cells, manufacturers kit positive control (HeLa cell nuclear lysate with oligonucleotide), and manufacturers kit negative control (HeLa cell nuclear lysate without oligonucleotide). Data is representative of EMSAa performed on 3 separate occasions.
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fig3: Wild type S. typhimurium suppress NFκB/DNA interaction in J774.2 cells 2 and 12 h afterculture via phoP. (a) 2 h postculture. (b) 12 h post-culture. Controls include, uninfected J774.2 cells, manufacturers kit positive control (HeLa cell nuclear lysate with oligonucleotide), and manufacturers kit negative control (HeLa cell nuclear lysate without oligonucleotide). Data is representative of EMSAa performed on 3 separate occasions.

Mentions: Our results show that mutation in the Salmonella phoP regulon increased the amount of NFκB and AP-1 bound to macrophage DNA after 2 h, when compared to wild type 14028 (Figure 3(a)). When compared to a positive control, NFκB/DNA interaction following wild type 14028 infection was reduced by a mean of 68% but when infected macrophages were co-cultured with IFN-γ (100 U/mL), NFκB/DNA interaction was reduced by 42% (Figure 3(a)). In comparison, when macrophages were infected with 14028 phoP mutants, NFκB/DNA interaction was only reduced by a mean of 22% (relative to the positive control) and this remained constant following co-culture of infected cells with IFN-γ (Figure 3(a)). After macrophages were cultured with wild type 14028 for 12 h, NFκB/DNA interaction was reduced by a mean of 66% but when infected macrophages were co-cultured with IFN-γ, NFκB/DNA interaction was reduced by 76% (Figure 3(b)). when macrophages were infected for 12 h with 14028 phoP mutants, NFκB/DNA interaction was only reduced by 40% (relative to the positive control) and this also remained constant following co-culture of infected cells with IFN-γ (Figure 3(b)).


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

Hulme SD, Barrow PA, Foster N - Interdiscip Perspect Infect Dis (2012)

Wild type S. typhimurium suppress NFκB/DNA interaction in J774.2 cells 2 and 12 h afterculture via phoP. (a) 2 h postculture. (b) 12 h post-culture. Controls include, uninfected J774.2 cells, manufacturers kit positive control (HeLa cell nuclear lysate with oligonucleotide), and manufacturers kit negative control (HeLa cell nuclear lysate without oligonucleotide). Data is representative of EMSAa performed on 3 separate occasions.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3405670&req=5

fig3: Wild type S. typhimurium suppress NFκB/DNA interaction in J774.2 cells 2 and 12 h afterculture via phoP. (a) 2 h postculture. (b) 12 h post-culture. Controls include, uninfected J774.2 cells, manufacturers kit positive control (HeLa cell nuclear lysate with oligonucleotide), and manufacturers kit negative control (HeLa cell nuclear lysate without oligonucleotide). Data is representative of EMSAa performed on 3 separate occasions.
Mentions: Our results show that mutation in the Salmonella phoP regulon increased the amount of NFκB and AP-1 bound to macrophage DNA after 2 h, when compared to wild type 14028 (Figure 3(a)). When compared to a positive control, NFκB/DNA interaction following wild type 14028 infection was reduced by a mean of 68% but when infected macrophages were co-cultured with IFN-γ (100 U/mL), NFκB/DNA interaction was reduced by 42% (Figure 3(a)). In comparison, when macrophages were infected with 14028 phoP mutants, NFκB/DNA interaction was only reduced by a mean of 22% (relative to the positive control) and this remained constant following co-culture of infected cells with IFN-γ (Figure 3(a)). After macrophages were cultured with wild type 14028 for 12 h, NFκB/DNA interaction was reduced by a mean of 66% but when infected macrophages were co-cultured with IFN-γ, NFκB/DNA interaction was reduced by 76% (Figure 3(b)). when macrophages were infected for 12 h with 14028 phoP mutants, NFκB/DNA interaction was only reduced by 40% (relative to the positive control) and this also remained constant following co-culture of infected cells with IFN-γ (Figure 3(b)).

Bottom Line: Methods.A macrophages via an intact phoP regulon.This correlated with increased survival and reduced iNOS expression.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Science, The University of Nottingham, Sutton Bonington Campus, Sutton Bonington, Leicestershire, Nottingham NG7 2NR, UK.

ABSTRACT
Background. There are no reported data to explain how Salmonella suppress nitrite ion production in macrophages or whether this phenomenon is unique to typhoidal or non-typhoidal serovars. The aims of this study were, therefore, to investigate these phenomena. Methods. We measured survival of S. typhimurium 14028 and its phoP mutant in murine J774 macrophages, cultured with or without interferon gamma. We compared expression of inducible nitric oxide synthase (iNOS) mRNA and protein, and nitrite ion production and also examined binding of nuclear factor κB (NFκB) and activator protein 1 (AP-1) to macrophage DNA. Results. S. typhimurium 14028 inhibited binding of NFκB and AP-1 to DNA in murine J774. A macrophages via an intact phoP regulon. This correlated with increased survival and reduced iNOS expression. Suppression of NFκB activity was ameliorated in macrophages cultured with IFN-γ and this correlated with increased expression of iNOS mRNA and nitrite ion production, although IFN-γ had no effect on AP-1/DNA interaction. We show, that with one exception, suppression of iNOS is unique to typhoidal serovars. Conclusion. S. typhimurium inhibit NFκB and AP-1 interaction with macrophage DNA via the PhoP regulon, this reduces nitrite ion production and is principally associated with typhoidal serovars.

No MeSH data available.


Related in: MedlinePlus