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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: 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.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.

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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Cellular integrity of RAW 264.7 cells infected with B. mallei and B. pseudomallei. Monolayers were infected with B. mallei at moi 0.1 (Bm 0.1), moi 1 (Bm 1), moi 10 (Bm 10) and B. pseudomallei at moi 10 (Bp 10). Per cent cytotoxicity was determined by assaying for LDH release in culture supernatants at 24 h post infection. Values represent the means ± SD of three independent experiments assayed in duplicate.
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fig03: Cellular integrity of RAW 264.7 cells infected with B. mallei and B. pseudomallei. Monolayers were infected with B. mallei at moi 0.1 (Bm 0.1), moi 1 (Bm 1), moi 10 (Bm 10) and B. pseudomallei at moi 10 (Bp 10). Per cent cytotoxicity was determined by assaying for LDH release in culture supernatants at 24 h post infection. Values represent the means ± SD of three independent experiments assayed in duplicate.

Mentions: Several studies have shown that B. pseudomallei and B. cenocepacia have the ability to cause significant morphological changes in RAW 264.7 cell monolayers following infection (Utaisincharoen et al., 2001; 2004; Lamothe et al., 2007). To exclude the possibility that our inability to recover B. mallei from infected RAW 264.7 cells at an moi of 10 was due to monolayer destruction, fixed cells were examined using light and confocal microscopy at 24 h post infection. In comparison with control cells, monolayers infected with B. mallei at moi ranging from 0.1 to 10 appeared generally healthy and intact (Fig. 2A–C,E). Interestingly, sporadic multinucleated giant cell (MNGC) formation and evidence of actin-based motility were observed in monolayers infected at an moi of 1 but not at an moi of 10 (Fig. 2C–D). In stark contrast, monolayers infected with B. pseudomallei at an moi of 10 for control purposes, demonstrated significant MNGC formation and monolayer sloughing (Fig. 2F). Consistent with these observations, cytotoxicity assays also demonstrated that B. pseudomallei caused the RAW 264.7 cells to release significantly more lactate dehydrogenase (LDH) than B. mallei at all moi tested (Fig. 3). Taken together, these findings indicated that, in contrast to other closely related Burkholderia species, RAW 264.7 cells appeared to be capable of killing B. mallei when infected at a critical moi (≥ 10).


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)

Cellular integrity of RAW 264.7 cells infected with B. mallei and B. pseudomallei. Monolayers were infected with B. mallei at moi 0.1 (Bm 0.1), moi 1 (Bm 1), moi 10 (Bm 10) and B. pseudomallei at moi 10 (Bp 10). Per cent cytotoxicity was determined by assaying for LDH release in culture supernatants at 24 h post infection. Values represent the means ± SD of three independent experiments assayed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2228653&req=5

fig03: Cellular integrity of RAW 264.7 cells infected with B. mallei and B. pseudomallei. Monolayers were infected with B. mallei at moi 0.1 (Bm 0.1), moi 1 (Bm 1), moi 10 (Bm 10) and B. pseudomallei at moi 10 (Bp 10). Per cent cytotoxicity was determined by assaying for LDH release in culture supernatants at 24 h post infection. Values represent the means ± SD of three independent experiments assayed in duplicate.
Mentions: Several studies have shown that B. pseudomallei and B. cenocepacia have the ability to cause significant morphological changes in RAW 264.7 cell monolayers following infection (Utaisincharoen et al., 2001; 2004; Lamothe et al., 2007). To exclude the possibility that our inability to recover B. mallei from infected RAW 264.7 cells at an moi of 10 was due to monolayer destruction, fixed cells were examined using light and confocal microscopy at 24 h post infection. In comparison with control cells, monolayers infected with B. mallei at moi ranging from 0.1 to 10 appeared generally healthy and intact (Fig. 2A–C,E). Interestingly, sporadic multinucleated giant cell (MNGC) formation and evidence of actin-based motility were observed in monolayers infected at an moi of 1 but not at an moi of 10 (Fig. 2C–D). In stark contrast, monolayers infected with B. pseudomallei at an moi of 10 for control purposes, demonstrated significant MNGC formation and monolayer sloughing (Fig. 2F). Consistent with these observations, cytotoxicity assays also demonstrated that B. pseudomallei caused the RAW 264.7 cells to release significantly more lactate dehydrogenase (LDH) than B. mallei at all moi tested (Fig. 3). Taken together, these findings indicated that, in contrast to other closely related Burkholderia species, RAW 264.7 cells appeared to be capable of killing B. mallei when infected at a critical moi (≥ 10).

Bottom Line: 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.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.

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