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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 by dsRNA and inhibition of IL-1ß processing by MG-132.A) BMDM were treated with or without 4 h of LPS priming, as indicated. Cells were then rinsed in fresh medium and treated with either LipofectAMINE 2000 or LipofectAMINE 2000-poly I:C complex for 4 h, in the presence or absence of 30 µM MG-132, as indicated. Cell lysates (cell) or media (medium) samples were subjected to immunoblotting with the antibodies indicated. B) BMDM were treated with either LipofectAMINE 2000 alone or with LipofectAMINE 2000-dsRNA complex for the times indicated. Fifteen minutes before each time-point, 1 µCi of [3H]-leucine was added, and leucine incorporation was terminated by trichloroacetic acid. Each treatment was conducted in triplicate wells, and values are shown as mean ± S.D. Percent incorporation of [3H]-leucine at each point was calculated as the [3H]-leucine incorporated into cells exposed to LipofectAMINE 2000-dsRNA complex/[3H]-leucine incorporated into cells exposed to LipofectAMINE 2000 alone×100.
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pone-0036044-g005: Inhibition of protein synthesis by dsRNA and inhibition of IL-1ß processing by MG-132.A) BMDM were treated with or without 4 h of LPS priming, as indicated. Cells were then rinsed in fresh medium and treated with either LipofectAMINE 2000 or LipofectAMINE 2000-poly I:C complex for 4 h, in the presence or absence of 30 µM MG-132, as indicated. Cell lysates (cell) or media (medium) samples were subjected to immunoblotting with the antibodies indicated. B) BMDM were treated with either LipofectAMINE 2000 alone or with LipofectAMINE 2000-dsRNA complex for the times indicated. Fifteen minutes before each time-point, 1 µCi of [3H]-leucine was added, and leucine incorporation was terminated by trichloroacetic acid. Each treatment was conducted in triplicate wells, and values are shown as mean ± S.D. Percent incorporation of [3H]-leucine at each point was calculated as the [3H]-leucine incorporated into cells exposed to LipofectAMINE 2000-dsRNA complex/[3H]-leucine incorporated into cells exposed to LipofectAMINE 2000 alone×100.

Mentions: Many forms of cellular stresses, including exposure to dsRNA and accumulation of unfolded proteins [39], activate pathways that lead to the phosphorylation of the translation initiation factor eIF-2alpha on serine 51. Phosphorylation of eIF-2alpha on serine 51 results in global, frequently transient, inhibition of protein synthesis [39]. Intracellular poly I:C, an analog of dsRNA employed experimentally to mimic the effects of viral dsRNA in cells, has been shown to activate the NLRP3 inflammasome in BMDM [6]. Indeed, exposure of BMDM to poly I:C induced the release of IL-1ß from primed BMDM as well as the phosphorylation of eIF-2alpha on serine 51 (Fig. 5A). The release of IL-1ß from primed BMDM induced by poly I:C was accompanied by inhibition of protein synthesis (Fig. 5B).


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 by dsRNA and inhibition of IL-1ß processing by MG-132.A) BMDM were treated with or without 4 h of LPS priming, as indicated. Cells were then rinsed in fresh medium and treated with either LipofectAMINE 2000 or LipofectAMINE 2000-poly I:C complex for 4 h, in the presence or absence of 30 µM MG-132, as indicated. Cell lysates (cell) or media (medium) samples were subjected to immunoblotting with the antibodies indicated. B) BMDM were treated with either LipofectAMINE 2000 alone or with LipofectAMINE 2000-dsRNA complex for the times indicated. Fifteen minutes before each time-point, 1 µCi of [3H]-leucine was added, and leucine incorporation was terminated by trichloroacetic acid. Each treatment was conducted in triplicate wells, and values are shown as mean ± S.D. Percent incorporation of [3H]-leucine at each point was calculated as the [3H]-leucine incorporated into cells exposed to LipofectAMINE 2000-dsRNA complex/[3H]-leucine incorporated into cells exposed to LipofectAMINE 2000 alone×100.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351443&req=5

pone-0036044-g005: Inhibition of protein synthesis by dsRNA and inhibition of IL-1ß processing by MG-132.A) BMDM were treated with or without 4 h of LPS priming, as indicated. Cells were then rinsed in fresh medium and treated with either LipofectAMINE 2000 or LipofectAMINE 2000-poly I:C complex for 4 h, in the presence or absence of 30 µM MG-132, as indicated. Cell lysates (cell) or media (medium) samples were subjected to immunoblotting with the antibodies indicated. B) BMDM were treated with either LipofectAMINE 2000 alone or with LipofectAMINE 2000-dsRNA complex for the times indicated. Fifteen minutes before each time-point, 1 µCi of [3H]-leucine was added, and leucine incorporation was terminated by trichloroacetic acid. Each treatment was conducted in triplicate wells, and values are shown as mean ± S.D. Percent incorporation of [3H]-leucine at each point was calculated as the [3H]-leucine incorporated into cells exposed to LipofectAMINE 2000-dsRNA complex/[3H]-leucine incorporated into cells exposed to LipofectAMINE 2000 alone×100.
Mentions: Many forms of cellular stresses, including exposure to dsRNA and accumulation of unfolded proteins [39], activate pathways that lead to the phosphorylation of the translation initiation factor eIF-2alpha on serine 51. Phosphorylation of eIF-2alpha on serine 51 results in global, frequently transient, inhibition of protein synthesis [39]. Intracellular poly I:C, an analog of dsRNA employed experimentally to mimic the effects of viral dsRNA in cells, has been shown to activate the NLRP3 inflammasome in BMDM [6]. Indeed, exposure of BMDM to poly I:C induced the release of IL-1ß from primed BMDM as well as the phosphorylation of eIF-2alpha on serine 51 (Fig. 5A). The release of IL-1ß from primed BMDM induced by poly I:C was accompanied by inhibition of protein synthesis (Fig. 5B).

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