mTORC1-S6K Activation by Endotoxin Contributes to Cytokine Up-Regulation and Early Lethality in Animals

Background: mTORC1 (mammalian target of rapamycin complex 1) activation has been demonstrated in response to endotoxin challenge, but the mechanism and significance are unclear. We investigated the effect of mTORC1 suppression in an animal model of endotoxemia and in a cellular model of endotoxin signaling. Methodology/Principal Findings: Mice were treated with the mTORC1 inhibitor rapamycin or vehicle prior to lethal endotoxin challenge. Mortality and cytokine levels were assessed. Cultured macrophage-like cells were challenged with endotoxin with or without inhibitors of various pathways known to be upstream of mTORC1. Activated pathways, including downstream S6K pathway, were assessed by immunoblots. We found that mTORC1-S6K suppression by rapamycin delayed mortality of mice challenged with lethal endotoxin, and was associated with dampened circulating levels of VEGF, IL-1b, IFN-c and IL-5. Furthermore, in vitro cellular studies demonstrated that LPS (lipopolysaccharide) activation of mTORC1-S6K still occurs in the presence of PI3K-Akt inhibition alone, but can be suppressed by concurrent inhibition of PI3K-Akt and MEK-ERK pathways. Conclusions/Significance: We conclude that cellular activation of mTORC1-S6K contributes to cytokine up-regulation an

Rapamycin suppression of mTORC1-S6K enhanced early survival of endotoxemic mice.

<p>Kaplan-Meier curves of LPS-challenged mice treated with vehicle control (N = 10) or rapamycin (N = 9) are shown here. Rapamycin-treated mice had significantly better survival compared to vehicle-treated (P = 0.033, logrank test). However, all mice had died by 72 hr after LPS challenge.</p

Concurrent inhibition of PI3K and MEK-ERK pathways suppresses LPS-induced mTORC1-S6K activation in macrophages.

<p>Representative immunoblots of RAW cells (A) and murine peritoneal macrophages (B) pretreated with vehicle control, 20 nM rapamycin (Rapa), 5 µM MEK1/2 inhibitor U0216 (U), 100 nM wortmannin (Wort), or combination of U0216 and wortmannin (U–W) 30 min prior to LPS stimulation. Cells were harvested 2 hr post LPS. Only the combined Inhibition of MEK1/2-ERK and PI3K-Akt significantly suppressed mTORC1-S6K activation by LPS, as indicated by pS6 levels. The same doses of pharmacologic inhibitors were applied to Tsc2-null MEFs (C), and did not suppress mTORC1-S6K activity as reflected by pS6, suggesting the effects of these compounds are dependent upon Tsc2.</p

mTORC1-S6K activation in endotoxemic TNFR-null mice.

<p>Transgenic mice deficient in TNF-α receptors (TNFR) were given LPS and sacrificed at 2, 6, and 24 hr later for organ extraction. Representative immunoblots probed for pS6 (S240/244), a downstream target of mTORC1-S6K are shown here. mTORC1-S6K activation could be observed in the livers, lungs and kidneys of these mice similar to wildtype mice.</p

Rapamycin suppression of mTORC1-S6K was associated with lower cytokine levels.

<p>Following LPS treatment in vivo, circulating cytokine levels of LPS-challenged mice pre-treated with vehicle (solid bar) or rapamycin (open bar) at 0 (no LPS), 2, 6, and 24 hr post LPS are shown here. Data are expressed as mean of three mice in each group at each time point, with error bars representing standard deviations. Statistical analysis was performed using Repeated Measures ANOVA, and p values<0.05 are considered as significant (*). mTORC1-S6K suppression by rapamycin led to decreased circulation levels of IL1-β, VEGF, IFN-γ and IL-5. There was a trend toward lower TNF-α levels with a p value of 0.058.</p

IKKβ was not required for LPS-induced mTORC1-S6K activation.

<p>IKKβ was suppressed in RAW cells by siRNA (IK) 48 hr prior to LPS stimulation. Nonsense siRNA constructs (NS) served as controls. Representative immunoblots show that IKKβ suppression had no effect on mTORC1-S6K activation in LPS-treated RAW cells as reflected by pS6 levels.</p

TNF-α secretion of LPS-treated macrophages occurred after mTORC1-S6K activation.

<p>Conditioned media and cell lysates of RAW cells treated with 100 ng/ml of LPS were collected at various time points. The immunoblots probing for pS6(S240/244) (A.), and the averages of TNF-α levels in the conditioned media from triplicate experiments with error bars representing standard deviations (B.) are shown here. mTORC1-S6K activation occurred as soon as 30 min after LPS when TNF-α levels were not detectably increased at that time point.</p

Rapamycin treatment effectively suppressed mTORC1-S6K signaling.

<p>Following LPS challenge in vivo, mice were treated with vehicle or 6 mg/kg rapamycin 30 min prior to 25 mg/kg LPS challenge. Organs were harvested from mice at 2, 6, 24 hr post challenge. Immunoblots of organs extracted from 3 mice from each treatment group probed for pS6 (S240/244), a downstream target of mTORC1-S6K are shown here. Unchallenged mice with or without rapamycin served as controls (time 0 hr). Rapamycin treatment successfully suppressed mTORC1-S6K up-regulation in endotoxemic mice as reflected by the near-absent pS6 signals.</p

mTORC1-S6K up-regulation in the livers, lungs, and kidneys of mice treated with endotoxin.

<p>Representative immunoblots of normalized organ protein extracts probed for pS6 (S240/244), a downstream target of mTORC1-S6K are shown here. Mice were subjected to 25 mg/kg of LPS i.p. and sacrificed at 2,6, and 24 hr later. Untreated mice served as time 0 hr controls. mTORC1-S6K activation could be seen as early as 2 hr after LPS challenge in the livers and lungs of endotoxemic mice. mTORC1-S6K was also activated in the kidneys at 24 hr post challenge. Increased pAkt (S473) was seen only in the lung extracts post LPS challenged, occurring at 6 hr post LPS, after mTORC1-S6K activation, and was not seen in either liver or kidney.</p