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Suppression of ribosomal function triggers innate immune signaling through activation of the NLRP3 inflammasome.

Vyleta ML, Wong J, Magun BE - PLoS ONE (2012)

Bottom Line: Processing and release of IL-1ß was reduced or absent from cells deficient in NLRP3, ASC, or caspase-1, demonstrating the role of the NLRP3 inflammasome.For agents that inhibit translation through decreased cellular potassium, the application of high extracellular potassium restores protein translation and suppresses activation of the NLRP inflammasome.For agents that inhibit translation through mechanisms that do not involve loss of potassium, high extracellular potassium suppresses IL-1ß processing through a mechanism that remains undefined.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, United States of America.

ABSTRACT
Some inflammatory stimuli trigger activation of the NLRP3 inflammasome by inducing efflux of cellular potassium. Loss of cellular potassium is known to potently suppress protein synthesis, leading us to test whether the inhibition of protein synthesis itself serves as an activating signal for the NLRP3 inflammasome. Murine bone marrow-derived macrophages, either primed by LPS or unprimed, were exposed to a panel of inhibitors of ribosomal function: ricin, cycloheximide, puromycin, pactamycin, and anisomycin. Macrophages were also exposed to nigericin, ATP, monosodium urate (MSU), and poly I:C. Synthesis of pro-IL-ß and release of IL-1ß from cells in response to these agents was detected by immunoblotting and ELISA. Release of intracellular potassium was measured by mass spectrometry. Inhibition of translation by each of the tested translation inhibitors led to processing of IL-1ß, which was released from cells. Processing and release of IL-1ß was reduced or absent from cells deficient in NLRP3, ASC, or caspase-1, demonstrating the role of the NLRP3 inflammasome. Despite the inability of these inhibitors to trigger efflux of intracellular potassium, the addition of high extracellular potassium suppressed activation of the NLRP3 inflammasome. MSU and double-stranded RNA, which are known to activate the NLRP3 inflammasome, also substantially inhibited protein translation, supporting a close association between inhibition of translation and inflammasome activation. These data demonstrate that translational inhibition itself constitutes a heretofore-unrecognized mechanism underlying IL-1ß dependent inflammatory signaling and that other physical, chemical, or pathogen-associated agents that impair translation may lead to IL-1ß-dependent inflammation through activation of the NLRP3 inflammasome. For agents that inhibit translation through decreased cellular potassium, the application of high extracellular potassium restores protein translation and suppresses activation of the NLRP inflammasome. For agents that inhibit translation through mechanisms that do not involve loss of potassium, high extracellular potassium suppresses IL-1ß processing through a mechanism that remains undefined.

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Inhibition of protein synthesis fails to elicit efflux of potassium from cells.A) Intracellular potassium in WT BMDM was analyzed by ICP-MS after priming with LPS for 4 h and exposure to emetine, ATP, and nigericin for indicated times. Data are represented as mean ± SEM from triplicate dishes. Measurements of potassium release by cells exposed to ATP and nigericin were terminated at 10 min and 60 min, respectively, as a result of cytotoxicity and detachment of cells from the dishes. Cells exposed to emetine showed no signs of cytotoxicity or detachment by 4 h. B) Elevated potassium blocks emetine-mediated release of IL-1ß and processing of caspase-1. LPS-primed BMDM were either exposed or not exposed to 10 µg/ml emetine for 4 h, at which time proteins were precipitated from the media with TCA and analyzed by Western blotting as shown. C) BMDM in triplicate wells were pulse-labeled in medium containing [3H]-leucine for 15 min prior to harvest at the indicated times and the amount of [3H]-leucine incorporation was measured.
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pone-0036044-g003: Inhibition of protein synthesis fails to elicit efflux of potassium from cells.A) Intracellular potassium in WT BMDM was analyzed by ICP-MS after priming with LPS for 4 h and exposure to emetine, ATP, and nigericin for indicated times. Data are represented as mean ± SEM from triplicate dishes. Measurements of potassium release by cells exposed to ATP and nigericin were terminated at 10 min and 60 min, respectively, as a result of cytotoxicity and detachment of cells from the dishes. Cells exposed to emetine showed no signs of cytotoxicity or detachment by 4 h. B) Elevated potassium blocks emetine-mediated release of IL-1ß and processing of caspase-1. LPS-primed BMDM were either exposed or not exposed to 10 µg/ml emetine for 4 h, at which time proteins were precipitated from the media with TCA and analyzed by Western blotting as shown. C) BMDM in triplicate wells were pulse-labeled in medium containing [3H]-leucine for 15 min prior to harvest at the indicated times and the amount of [3H]-leucine incorporation was measured.

Mentions: To determine whether inhibition of translation would trigger efflux of potassium from macrophages, we exposed LPS-primed BMDM to emetine, a potent inhibitor of translation [31], [32] that irreversibly inhibits the elongation cycle of translation by greater than 99% within 1 minute [33] and produces release of IL-1ß within 4 h. Intracellular potassium was measured by inductively coupled plasma mass spectroscopy (ICP-MS). Nigericin and ATP, agents that have been shown to reduce intracellular potassium [7], [24], produced a 50% decrease in intracellular potassium concentration by 15 min and 45 min, respectively (Fig. 3A). BMDM exposed to nigericin and ATP detached from the culture dishes by 1 h. By contrast, cells exposed to emetine maintained normal levels of intracellular potassium for at least 4 h (Fig. 3A). Ricin and cycloheximide similarly failed to cause release of cellular potassium (not shown). These data suggest that in BMDM the inhibition of translation promoted the activation of the NLRP3 inflammasome but failed to induce leakage of cellular potassium in the interval of time (4 h) that preceded activation of the inflammasome and release of IL-1ß.


Suppression of ribosomal function triggers innate immune signaling through activation of the NLRP3 inflammasome.

Vyleta ML, Wong J, Magun BE - PLoS ONE (2012)

Inhibition of protein synthesis fails to elicit efflux of potassium from cells.A) Intracellular potassium in WT BMDM was analyzed by ICP-MS after priming with LPS for 4 h and exposure to emetine, ATP, and nigericin for indicated times. Data are represented as mean ± SEM from triplicate dishes. Measurements of potassium release by cells exposed to ATP and nigericin were terminated at 10 min and 60 min, respectively, as a result of cytotoxicity and detachment of cells from the dishes. Cells exposed to emetine showed no signs of cytotoxicity or detachment by 4 h. B) Elevated potassium blocks emetine-mediated release of IL-1ß and processing of caspase-1. LPS-primed BMDM were either exposed or not exposed to 10 µg/ml emetine for 4 h, at which time proteins were precipitated from the media with TCA and analyzed by Western blotting as shown. C) BMDM in triplicate wells were pulse-labeled in medium containing [3H]-leucine for 15 min prior to harvest at the indicated times and the amount of [3H]-leucine incorporation was measured.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351443&req=5

pone-0036044-g003: Inhibition of protein synthesis fails to elicit efflux of potassium from cells.A) Intracellular potassium in WT BMDM was analyzed by ICP-MS after priming with LPS for 4 h and exposure to emetine, ATP, and nigericin for indicated times. Data are represented as mean ± SEM from triplicate dishes. Measurements of potassium release by cells exposed to ATP and nigericin were terminated at 10 min and 60 min, respectively, as a result of cytotoxicity and detachment of cells from the dishes. Cells exposed to emetine showed no signs of cytotoxicity or detachment by 4 h. B) Elevated potassium blocks emetine-mediated release of IL-1ß and processing of caspase-1. LPS-primed BMDM were either exposed or not exposed to 10 µg/ml emetine for 4 h, at which time proteins were precipitated from the media with TCA and analyzed by Western blotting as shown. C) BMDM in triplicate wells were pulse-labeled in medium containing [3H]-leucine for 15 min prior to harvest at the indicated times and the amount of [3H]-leucine incorporation was measured.
Mentions: To determine whether inhibition of translation would trigger efflux of potassium from macrophages, we exposed LPS-primed BMDM to emetine, a potent inhibitor of translation [31], [32] that irreversibly inhibits the elongation cycle of translation by greater than 99% within 1 minute [33] and produces release of IL-1ß within 4 h. Intracellular potassium was measured by inductively coupled plasma mass spectroscopy (ICP-MS). Nigericin and ATP, agents that have been shown to reduce intracellular potassium [7], [24], produced a 50% decrease in intracellular potassium concentration by 15 min and 45 min, respectively (Fig. 3A). BMDM exposed to nigericin and ATP detached from the culture dishes by 1 h. By contrast, cells exposed to emetine maintained normal levels of intracellular potassium for at least 4 h (Fig. 3A). Ricin and cycloheximide similarly failed to cause release of cellular potassium (not shown). These data suggest that in BMDM the inhibition of translation promoted the activation of the NLRP3 inflammasome but failed to induce leakage of cellular potassium in the interval of time (4 h) that preceded activation of the inflammasome and release of IL-1ß.

Bottom Line: Processing and release of IL-1ß was reduced or absent from cells deficient in NLRP3, ASC, or caspase-1, demonstrating the role of the NLRP3 inflammasome.For agents that inhibit translation through decreased cellular potassium, the application of high extracellular potassium restores protein translation and suppresses activation of the NLRP inflammasome.For agents that inhibit translation through mechanisms that do not involve loss of potassium, high extracellular potassium suppresses IL-1ß processing through a mechanism that remains undefined.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, United States of America.

ABSTRACT
Some inflammatory stimuli trigger activation of the NLRP3 inflammasome by inducing efflux of cellular potassium. Loss of cellular potassium is known to potently suppress protein synthesis, leading us to test whether the inhibition of protein synthesis itself serves as an activating signal for the NLRP3 inflammasome. Murine bone marrow-derived macrophages, either primed by LPS or unprimed, were exposed to a panel of inhibitors of ribosomal function: ricin, cycloheximide, puromycin, pactamycin, and anisomycin. Macrophages were also exposed to nigericin, ATP, monosodium urate (MSU), and poly I:C. Synthesis of pro-IL-ß and release of IL-1ß from cells in response to these agents was detected by immunoblotting and ELISA. Release of intracellular potassium was measured by mass spectrometry. Inhibition of translation by each of the tested translation inhibitors led to processing of IL-1ß, which was released from cells. Processing and release of IL-1ß was reduced or absent from cells deficient in NLRP3, ASC, or caspase-1, demonstrating the role of the NLRP3 inflammasome. Despite the inability of these inhibitors to trigger efflux of intracellular potassium, the addition of high extracellular potassium suppressed activation of the NLRP3 inflammasome. MSU and double-stranded RNA, which are known to activate the NLRP3 inflammasome, also substantially inhibited protein translation, supporting a close association between inhibition of translation and inflammasome activation. These data demonstrate that translational inhibition itself constitutes a heretofore-unrecognized mechanism underlying IL-1ß dependent inflammatory signaling and that other physical, chemical, or pathogen-associated agents that impair translation may lead to IL-1ß-dependent inflammation through activation of the NLRP3 inflammasome. For agents that inhibit translation through decreased cellular potassium, the application of high extracellular potassium restores protein translation and suppresses activation of the NLRP inflammasome. For agents that inhibit translation through mechanisms that do not involve loss of potassium, high extracellular potassium suppresses IL-1ß processing through a mechanism that remains undefined.

Show MeSH
Related in: MedlinePlus