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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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Proteasome inhibitors block processing and release of IL-1ß.A) LPS-primed WT BMDM were incubated in control medium or medium containing 10 ng/ml ricin or 25 µg/ml cycloheximide for 4 h. MG-132 (30 µM) or Bortezimib (0.5 µM) was included as indicated. Secreted IL-1ß was measured by ELISA in triplicate wells. B) LPS-primed WT BMDM were incubated in the presence or absence of MG-132 for 4 hours, in the presence or absence of inhibitors of protein synthesis, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting. C) LPS-primed or unprimed WT BMDM were or exposed to MSU, MG-132, or both for 4 h, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting.
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pone-0036044-g006: Proteasome inhibitors block processing and release of IL-1ß.A) LPS-primed WT BMDM were incubated in control medium or medium containing 10 ng/ml ricin or 25 µg/ml cycloheximide for 4 h. MG-132 (30 µM) or Bortezimib (0.5 µM) was included as indicated. Secreted IL-1ß was measured by ELISA in triplicate wells. B) LPS-primed WT BMDM were incubated in the presence or absence of MG-132 for 4 hours, in the presence or absence of inhibitors of protein synthesis, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting. C) LPS-primed or unprimed WT BMDM were or exposed to MSU, MG-132, or both for 4 h, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting.

Mentions: Previously we showed that inhibitors of proteasome activity reduce the ricin-mediated activation of the NLRP3 inflammasome in LPS-primed BMDM [22]. Others have reported that proteasome inhibitors reduce the activity of the NLRP1 inflammasome but not the accumulation of pro-IL-1ß by anthrax lethal toxin [40]. To determine if proteasome inhibitors would block the ability of translation inhibitors to activate the NLRP3 inflammasome, we employed two proteasome inhibitors, MG-132 and bortezimib. IL-1ß release mediated by cycloheximide or ricin was strongly suppressed in LPS-primed BMDM co-treated with either bortezimib or MG-132 (Fig. 6A). Inclusion of MG-132 blocked the ability of each of the translation inhibitors to induce the processing of pro-IL-1ß and the release of IL-1ß from cells (Fig. 6B). MG-132 similarly suppressed the release of IL-1ß from cells exposed to MSU (Fig. 6C) and poly I:C (Fig. 5A), suggesting that proteasomes may participate more generally in activation of the NLRP3 inflammasome.


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

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

Proteasome inhibitors block processing and release of IL-1ß.A) LPS-primed WT BMDM were incubated in control medium or medium containing 10 ng/ml ricin or 25 µg/ml cycloheximide for 4 h. MG-132 (30 µM) or Bortezimib (0.5 µM) was included as indicated. Secreted IL-1ß was measured by ELISA in triplicate wells. B) LPS-primed WT BMDM were incubated in the presence or absence of MG-132 for 4 hours, in the presence or absence of inhibitors of protein synthesis, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting. C) LPS-primed or unprimed WT BMDM were or exposed to MSU, MG-132, or both for 4 h, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351443&req=5

pone-0036044-g006: Proteasome inhibitors block processing and release of IL-1ß.A) LPS-primed WT BMDM were incubated in control medium or medium containing 10 ng/ml ricin or 25 µg/ml cycloheximide for 4 h. MG-132 (30 µM) or Bortezimib (0.5 µM) was included as indicated. Secreted IL-1ß was measured by ELISA in triplicate wells. B) LPS-primed WT BMDM were incubated in the presence or absence of MG-132 for 4 hours, in the presence or absence of inhibitors of protein synthesis, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting. C) LPS-primed or unprimed WT BMDM were or exposed to MSU, MG-132, or both for 4 h, as indicated. Cell lysates (cell) and culture medium (medium) were examined by immunoblotting.
Mentions: Previously we showed that inhibitors of proteasome activity reduce the ricin-mediated activation of the NLRP3 inflammasome in LPS-primed BMDM [22]. Others have reported that proteasome inhibitors reduce the activity of the NLRP1 inflammasome but not the accumulation of pro-IL-1ß by anthrax lethal toxin [40]. To determine if proteasome inhibitors would block the ability of translation inhibitors to activate the NLRP3 inflammasome, we employed two proteasome inhibitors, MG-132 and bortezimib. IL-1ß release mediated by cycloheximide or ricin was strongly suppressed in LPS-primed BMDM co-treated with either bortezimib or MG-132 (Fig. 6A). Inclusion of MG-132 blocked the ability of each of the translation inhibitors to induce the processing of pro-IL-1ß and the release of IL-1ß from cells (Fig. 6B). MG-132 similarly suppressed the release of IL-1ß from cells exposed to MSU (Fig. 6C) and poly I:C (Fig. 5A), suggesting that proteasomes may participate more generally in activation of the NLRP3 inflammasome.

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