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iNOS activity is critical for the clearance of Burkholderia mallei from infected RAW 264.7 murine macrophages.

Brett PJ, Burtnick MN, Su H, Nair V, Gherardini FC - Cell. Microbiol. (2007)

Bottom Line: Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of < or = 1.Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-alpha, IL-6, IL-10, GM-CSF, RANTES and IFN-beta when infected at an moi of 10.In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Zoonotic Pathogens, RTB, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT 59840, USA.

ABSTRACT
Burkholderia mallei is a facultative intracellular pathogen that can cause fatal disease in animals and humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of < or = 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-alpha, IL-6, IL-10, GM-CSF, RANTES and IFN-beta when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells.

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E. coli LPS stimulates the clearance of B. mallei from RAW 264.7 cells infected at an moi of 1. Infected monolayers were incubated with either AG (200 μg ml−1), LPS (100 ng ml−1), or AG (200 μg ml−1) and LPS (100 ng ml−1) at 1 h post infection. A. Uptake (white bars) and intracellular survival (black bars) were determined at 3 and 24 h post infection respectively. Values represent the means ± SD of three independent experiments. B. Culture supernatants harvested 24 h post infection from mock-infected and B. mallei-infected monolayers were assayed for the production of NO. Values represent the means ± SD of three independent experiments assayed in triplicate.
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fig08: E. coli LPS stimulates the clearance of B. mallei from RAW 264.7 cells infected at an moi of 1. Infected monolayers were incubated with either AG (200 μg ml−1), LPS (100 ng ml−1), or AG (200 μg ml−1) and LPS (100 ng ml−1) at 1 h post infection. A. Uptake (white bars) and intracellular survival (black bars) were determined at 3 and 24 h post infection respectively. Values represent the means ± SD of three independent experiments. B. Culture supernatants harvested 24 h post infection from mock-infected and B. mallei-infected monolayers were assayed for the production of NO. Values represent the means ± SD of three independent experiments assayed in triplicate.

Mentions: Toll-like receptor (TLR) signalling is an important means by which phagocytic cells are activated by PAMPs (Carpenter and O'Neill, 2007). Numerous studies have demonstrated that E. coli LPS is a potent activator of murine macrophages (Weisz et al., 1994; Zughaier et al., 2005; Okemoto et al., 2006). In particular, RAW 264.7 cells stimulated with E. coli LPS are known to produce high levels of NO (Weisz et al., 1994; Zughaier et al., 2005). To confirm the relationship between activated RAW 264.7 cells, iNOS activity and bacterial clearance, monolayers infected with B. mallei at an moi of 1 were incubated with various combinations of AG (200 μg ml−1) and LPS (100 ng ml−1). Results of these assays demonstrated that uptake levels did not appear to be influenced by the AG and LPS treatments (Fig. 8A). Quantification of intracellular survival at 24 h post infection demonstrated that B. mallei was cleared by RAW 264.7 cells treated with LPS alone. In contrast, B. mallei was shown to survive in AG-treated macrophages even in the presence of LPS (Fig. 8A). In order to correlate intracellular survival with iNOS activity, culture supernatants harvested at 24 h post infection were assayed for NO production. Consistent with predicted results, high levels of NO were only produced by infected monolayers in the absence of AG (Fig. 8B). Taken together, these findings indicated that when infected at a subcritical moi (≤ 1), RAW 264.7 cells could be stimulated by LPS to clear B. mallei in an iNOS-dependent manner.


iNOS activity is critical for the clearance of Burkholderia mallei from infected RAW 264.7 murine macrophages.

Brett PJ, Burtnick MN, Su H, Nair V, Gherardini FC - Cell. Microbiol. (2007)

E. coli LPS stimulates the clearance of B. mallei from RAW 264.7 cells infected at an moi of 1. Infected monolayers were incubated with either AG (200 μg ml−1), LPS (100 ng ml−1), or AG (200 μg ml−1) and LPS (100 ng ml−1) at 1 h post infection. A. Uptake (white bars) and intracellular survival (black bars) were determined at 3 and 24 h post infection respectively. Values represent the means ± SD of three independent experiments. B. Culture supernatants harvested 24 h post infection from mock-infected and B. mallei-infected monolayers were assayed for the production of NO. Values represent the means ± SD of three independent experiments assayed in triplicate.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2228653&req=5

fig08: E. coli LPS stimulates the clearance of B. mallei from RAW 264.7 cells infected at an moi of 1. Infected monolayers were incubated with either AG (200 μg ml−1), LPS (100 ng ml−1), or AG (200 μg ml−1) and LPS (100 ng ml−1) at 1 h post infection. A. Uptake (white bars) and intracellular survival (black bars) were determined at 3 and 24 h post infection respectively. Values represent the means ± SD of three independent experiments. B. Culture supernatants harvested 24 h post infection from mock-infected and B. mallei-infected monolayers were assayed for the production of NO. Values represent the means ± SD of three independent experiments assayed in triplicate.
Mentions: Toll-like receptor (TLR) signalling is an important means by which phagocytic cells are activated by PAMPs (Carpenter and O'Neill, 2007). Numerous studies have demonstrated that E. coli LPS is a potent activator of murine macrophages (Weisz et al., 1994; Zughaier et al., 2005; Okemoto et al., 2006). In particular, RAW 264.7 cells stimulated with E. coli LPS are known to produce high levels of NO (Weisz et al., 1994; Zughaier et al., 2005). To confirm the relationship between activated RAW 264.7 cells, iNOS activity and bacterial clearance, monolayers infected with B. mallei at an moi of 1 were incubated with various combinations of AG (200 μg ml−1) and LPS (100 ng ml−1). Results of these assays demonstrated that uptake levels did not appear to be influenced by the AG and LPS treatments (Fig. 8A). Quantification of intracellular survival at 24 h post infection demonstrated that B. mallei was cleared by RAW 264.7 cells treated with LPS alone. In contrast, B. mallei was shown to survive in AG-treated macrophages even in the presence of LPS (Fig. 8A). In order to correlate intracellular survival with iNOS activity, culture supernatants harvested at 24 h post infection were assayed for NO production. Consistent with predicted results, high levels of NO were only produced by infected monolayers in the absence of AG (Fig. 8B). Taken together, these findings indicated that when infected at a subcritical moi (≤ 1), RAW 264.7 cells could be stimulated by LPS to clear B. mallei in an iNOS-dependent manner.

Bottom Line: Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of < or = 1.Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-alpha, IL-6, IL-10, GM-CSF, RANTES and IFN-beta when infected at an moi of 10.In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Zoonotic Pathogens, RTB, Rocky Mountain Laboratories, NIAID, NIH, Hamilton, MT 59840, USA.

ABSTRACT
Burkholderia mallei is a facultative intracellular pathogen that can cause fatal disease in animals and humans. To better understand the role of phagocytic cells in the control of infections caused by this organism, studies were initiated to examine the interactions of B. mallei with RAW 264.7 murine macrophages. Utilizing modified kanamycin-protection assays, B. mallei was shown to survive and replicate in RAW 264.7 cells infected at multiplicities of infection (moi) of < or = 1. In contrast, the organism was efficiently cleared by the macrophages when infected at an moi of 10. Interestingly, studies demonstrated that the monolayers only produced high levels of TNF-alpha, IL-6, IL-10, GM-CSF, RANTES and IFN-beta when infected at an moi of 10. In addition, nitric oxide assays and inducible nitric oxide synthase (iNOS) immunoblot analyses revealed a strong correlation between iNOS activity and clearance of B. mallei from RAW 264.7 cells. Furthermore, treatment of activated macrophages with the iNOS inhibitor, aminoguanidine, inhibited clearance of B. mallei from infected monolayers. Based upon these results, it appears that moi significantly influence the outcome of interactions between B. mallei and murine macrophages and that iNOS activity is critical for the clearance of B. mallei from activated RAW 264.7 cells.

Show MeSH
Related in: MedlinePlus