Limits...
Inhibition of anthrax lethal toxin-induced cytolysis of RAW264.7 cells by celastrol.

Chapelsky S, Batty S, Frost M, Mogridge J - PLoS ONE (2008)

Bottom Line: Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin.We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro.This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized.

Methodology/principal findings: Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation.

Conclusions/significance: This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.

Show MeSH

Related in: MedlinePlus

Celastrol prevents LeTx-mediated activation of IL-18 processing.RAW264.7 or J774A.1 cells were treated for 2 h with LeTx (10−8 M PA, 5×10−10 M LF) in the absence or presence of 3 µΜ celastrol, as indicated. Cellular lysates were prepared and probed for IL-18 and α-tubulin by Western blotting. Cellular supernatants were collected and probed for IL-18 by Western blotting. Representative results of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2170518&req=5

pone-0001421-g005: Celastrol prevents LeTx-mediated activation of IL-18 processing.RAW264.7 or J774A.1 cells were treated for 2 h with LeTx (10−8 M PA, 5×10−10 M LF) in the absence or presence of 3 µΜ celastrol, as indicated. Cellular lysates were prepared and probed for IL-18 and α-tubulin by Western blotting. Cellular supernatants were collected and probed for IL-18 by Western blotting. Representative results of three independent experiments are shown.

Mentions: Since it has been reported previously that inhibition of proteasome activity prevents activation of the Nalp1b inflammasome [38], [39], we sought to determine whether celastrol inhibits processing of IL-18 by the Nalp1b inflammasome. RAW264.7 cells were incubated with LeTx in the absence or presence of celastrol for 2 h and cellular lysates and supernatants were subjected to Western blotting using an antibody raised against IL-18. The 18 kDa mature form of IL-18 was observed in lysates derived from cells treated with LeTx and a reduced amount was present in lysates derived from cells treated with both LeTx and celastrol; no IL-18 was detected in the cell supernatants in these conditions (Fig. 5). J774A.1 cells that were treated with LeTx had a reduced amount of the 24 kDa form of IL-18 in cell lysates; the processed form was observed in the supernatants. The stimulation of IL-18 processing by LeTx was reduced in cells that were co-treated with celastrol (Fig. 5). These results are consistent with celastrol inhibiting an intoxication step upstream of Nalp1b inflammasome activation.


Inhibition of anthrax lethal toxin-induced cytolysis of RAW264.7 cells by celastrol.

Chapelsky S, Batty S, Frost M, Mogridge J - PLoS ONE (2008)

Celastrol prevents LeTx-mediated activation of IL-18 processing.RAW264.7 or J774A.1 cells were treated for 2 h with LeTx (10−8 M PA, 5×10−10 M LF) in the absence or presence of 3 µΜ celastrol, as indicated. Cellular lysates were prepared and probed for IL-18 and α-tubulin by Western blotting. Cellular supernatants were collected and probed for IL-18 by Western blotting. Representative results of three independent experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2170518&req=5

pone-0001421-g005: Celastrol prevents LeTx-mediated activation of IL-18 processing.RAW264.7 or J774A.1 cells were treated for 2 h with LeTx (10−8 M PA, 5×10−10 M LF) in the absence or presence of 3 µΜ celastrol, as indicated. Cellular lysates were prepared and probed for IL-18 and α-tubulin by Western blotting. Cellular supernatants were collected and probed for IL-18 by Western blotting. Representative results of three independent experiments are shown.
Mentions: Since it has been reported previously that inhibition of proteasome activity prevents activation of the Nalp1b inflammasome [38], [39], we sought to determine whether celastrol inhibits processing of IL-18 by the Nalp1b inflammasome. RAW264.7 cells were incubated with LeTx in the absence or presence of celastrol for 2 h and cellular lysates and supernatants were subjected to Western blotting using an antibody raised against IL-18. The 18 kDa mature form of IL-18 was observed in lysates derived from cells treated with LeTx and a reduced amount was present in lysates derived from cells treated with both LeTx and celastrol; no IL-18 was detected in the cell supernatants in these conditions (Fig. 5). J774A.1 cells that were treated with LeTx had a reduced amount of the 24 kDa form of IL-18 in cell lysates; the processed form was observed in the supernatants. The stimulation of IL-18 processing by LeTx was reduced in cells that were co-treated with celastrol (Fig. 5). These results are consistent with celastrol inhibiting an intoxication step upstream of Nalp1b inflammasome activation.

Bottom Line: Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin.We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro.This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT

Background: Bacillus anthracis is the bacterium responsible for causing anthrax. The ability of B. anthracis to cause disease is dependent on a secreted virulence factor, lethal toxin, that promotes survival of the bacteria in the host by impairing the immune response. A well-studied effect of lethal toxin is the killing of macrophages, although the molecular mechanisms involved have not been fully characterized.

Methodology/principal findings: Here, we demonstrate that celastrol, a quinone methide triterpene derived from a plant extract used in herbal medicine, inhibits lethal toxin-induced death of RAW264.7 murine macrophages. Celastrol did not prevent cleavage of mitogen activated protein kinase kinase 1, a cytosolic target of the toxin, indicating that it did not inhibit the uptake or catalytic activity of lethal toxin. Surprisingly, celastrol conferred almost complete protection when it was added up to 1.5 h after intoxication, indicating that it could rescue cells in the late stages of intoxication. Since the activity of the proteasome has been implicated in intoxication using other pharmacological agents, we tested whether celastrol blocked proteasome activity. We found that celastrol inhibited the proteasome-dependent degradation of proteins in RAW264.7 cells, but only slightly inhibited proteasome-mediated cleavage of fluorogenic substrates in vitro. Furthermore, celastrol blocked stimulation of IL-18 processing, indicating that celastrol acted upstream of inflammasome activation.

Conclusions/significance: This work identifies celastrol as an inhibitor of lethal toxin-mediated macrophage lysis and suggests an inhibitory mechanism involving inhibition of the proteasome pathway.

Show MeSH
Related in: MedlinePlus