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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 triggers secretion of IL-1ß from primed BMDM.A) WT BMDM were stimulated or not with 50 ng/ml LPS for 4 h followed by exposure to 10 µg/ml ricin, 25 µg/ml cycloheximide, 10 µg/ml emetine, 75 µg/ml puromycin, 0.2 µg/ml pactamycin, or 10 µg/ml anisomycin for 4 h prior to harvesting. Cell extracts were analyzed by immunoblotting for p38 MAPK (loading control) and pro-IL-1ß (upper panel). Media supernatants were analyzed by ELISA for released IL-1ß (lower panel). ELISA data are represented as mean ± SEM from triplicate dishes. B) LPS-primed WT BMDM were stimulated for 4 h with increasing doses of cycloheximide (from right to left: 0.03, 0.1, 0.3, 1.0, 3.0, 10, 30, and 100 µg/ml). Media supernatants were analyzed by ELISA for released IL-1ß (upper panel). Cell extracts were analyzed by immunoblotting for p38 MAPK (loading control) and pro-IL-1ß (lower panel). ELISA data are represented as mean ± SEM from triplicate dishes. C) Release of IL-1ß triggered by inhibitors of translation was determined by immunoblotting of cell extracts (cell) or culture medium (medium) from LPS-primed BMDM obtained from WT mice or mice deficient in NLRP3, ASC, or caspase-1, as indicated. P38 MAPK was loading control.
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pone-0036044-g001: Inhibition of protein synthesis triggers secretion of IL-1ß from primed BMDM.A) WT BMDM were stimulated or not with 50 ng/ml LPS for 4 h followed by exposure to 10 µg/ml ricin, 25 µg/ml cycloheximide, 10 µg/ml emetine, 75 µg/ml puromycin, 0.2 µg/ml pactamycin, or 10 µg/ml anisomycin for 4 h prior to harvesting. Cell extracts were analyzed by immunoblotting for p38 MAPK (loading control) and pro-IL-1ß (upper panel). Media supernatants were analyzed by ELISA for released IL-1ß (lower panel). ELISA data are represented as mean ± SEM from triplicate dishes. B) LPS-primed WT BMDM were stimulated for 4 h with increasing doses of cycloheximide (from right to left: 0.03, 0.1, 0.3, 1.0, 3.0, 10, 30, and 100 µg/ml). Media supernatants were analyzed by ELISA for released IL-1ß (upper panel). Cell extracts were analyzed by immunoblotting for p38 MAPK (loading control) and pro-IL-1ß (lower panel). ELISA data are represented as mean ± SEM from triplicate dishes. C) Release of IL-1ß triggered by inhibitors of translation was determined by immunoblotting of cell extracts (cell) or culture medium (medium) from LPS-primed BMDM obtained from WT mice or mice deficient in NLRP3, ASC, or caspase-1, as indicated. P38 MAPK was loading control.

Mentions: We tested the ability of a panel of well-characterized inhibitors of translation (cycloheximide, emetine, puromycin, pactamycin, and anisomycin) to stimulate IL-1ß release from BMDM via the NLRP3 inflammasome. When added at doses that suppress incorporation of [3H]-leucine by at least 90%, each inhibitor led to release of IL-1ß from LPS-primed BMDM by 4 h (Fig. 1A). Conversion of pro-IL-1ß to IL-1ß induced by cycloheximide proceeded in a dose-dependent manner (Fig. 1B), suggesting that the degree of translational impairment correlated well with the amount of IL-1ß that was released. As expected, there was a reciprocal relationship between the amount of pro-IL-1ß present in the cell lysate (Fig. 1A, upper panel) and the amount of IL-1ß detected in the culture medium by ELISA (Fig. 1A, lower panel). Cells remained strongly adherent to culture dishes at the termination of the experiments, and did not show morphological signs of apoptosis or activation of caspase-3 (not shown). To gain insight into the mechanism of IL-1ß release triggered by translational inhibitors, we exposed BMDM from mice deficient in NLRP3, ASC, or caspase-1 to agents that inhibited translation by a variety of mechanisms and compared their responses to those of wild-type (WT) cells. When BMDM deficient in NLRP3, ASC, or caspase-1 were exposed to the inhibitors, the cells secreted substantially reduced amounts of IL-1ß compared with WT BMDM exposed to the same inhibitors (Fig. 1C), suggesting that processing and release of IL-1ß were mediated through formation 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)

Inhibition of protein synthesis triggers secretion of IL-1ß from primed BMDM.A) WT BMDM were stimulated or not with 50 ng/ml LPS for 4 h followed by exposure to 10 µg/ml ricin, 25 µg/ml cycloheximide, 10 µg/ml emetine, 75 µg/ml puromycin, 0.2 µg/ml pactamycin, or 10 µg/ml anisomycin for 4 h prior to harvesting. Cell extracts were analyzed by immunoblotting for p38 MAPK (loading control) and pro-IL-1ß (upper panel). Media supernatants were analyzed by ELISA for released IL-1ß (lower panel). ELISA data are represented as mean ± SEM from triplicate dishes. B) LPS-primed WT BMDM were stimulated for 4 h with increasing doses of cycloheximide (from right to left: 0.03, 0.1, 0.3, 1.0, 3.0, 10, 30, and 100 µg/ml). Media supernatants were analyzed by ELISA for released IL-1ß (upper panel). Cell extracts were analyzed by immunoblotting for p38 MAPK (loading control) and pro-IL-1ß (lower panel). ELISA data are represented as mean ± SEM from triplicate dishes. C) Release of IL-1ß triggered by inhibitors of translation was determined by immunoblotting of cell extracts (cell) or culture medium (medium) from LPS-primed BMDM obtained from WT mice or mice deficient in NLRP3, ASC, or caspase-1, as indicated. P38 MAPK was loading control.
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pone-0036044-g001: Inhibition of protein synthesis triggers secretion of IL-1ß from primed BMDM.A) WT BMDM were stimulated or not with 50 ng/ml LPS for 4 h followed by exposure to 10 µg/ml ricin, 25 µg/ml cycloheximide, 10 µg/ml emetine, 75 µg/ml puromycin, 0.2 µg/ml pactamycin, or 10 µg/ml anisomycin for 4 h prior to harvesting. Cell extracts were analyzed by immunoblotting for p38 MAPK (loading control) and pro-IL-1ß (upper panel). Media supernatants were analyzed by ELISA for released IL-1ß (lower panel). ELISA data are represented as mean ± SEM from triplicate dishes. B) LPS-primed WT BMDM were stimulated for 4 h with increasing doses of cycloheximide (from right to left: 0.03, 0.1, 0.3, 1.0, 3.0, 10, 30, and 100 µg/ml). Media supernatants were analyzed by ELISA for released IL-1ß (upper panel). Cell extracts were analyzed by immunoblotting for p38 MAPK (loading control) and pro-IL-1ß (lower panel). ELISA data are represented as mean ± SEM from triplicate dishes. C) Release of IL-1ß triggered by inhibitors of translation was determined by immunoblotting of cell extracts (cell) or culture medium (medium) from LPS-primed BMDM obtained from WT mice or mice deficient in NLRP3, ASC, or caspase-1, as indicated. P38 MAPK was loading control.
Mentions: We tested the ability of a panel of well-characterized inhibitors of translation (cycloheximide, emetine, puromycin, pactamycin, and anisomycin) to stimulate IL-1ß release from BMDM via the NLRP3 inflammasome. When added at doses that suppress incorporation of [3H]-leucine by at least 90%, each inhibitor led to release of IL-1ß from LPS-primed BMDM by 4 h (Fig. 1A). Conversion of pro-IL-1ß to IL-1ß induced by cycloheximide proceeded in a dose-dependent manner (Fig. 1B), suggesting that the degree of translational impairment correlated well with the amount of IL-1ß that was released. As expected, there was a reciprocal relationship between the amount of pro-IL-1ß present in the cell lysate (Fig. 1A, upper panel) and the amount of IL-1ß detected in the culture medium by ELISA (Fig. 1A, lower panel). Cells remained strongly adherent to culture dishes at the termination of the experiments, and did not show morphological signs of apoptosis or activation of caspase-3 (not shown). To gain insight into the mechanism of IL-1ß release triggered by translational inhibitors, we exposed BMDM from mice deficient in NLRP3, ASC, or caspase-1 to agents that inhibited translation by a variety of mechanisms and compared their responses to those of wild-type (WT) cells. When BMDM deficient in NLRP3, ASC, or caspase-1 were exposed to the inhibitors, the cells secreted substantially reduced amounts of IL-1ß compared with WT BMDM exposed to the same inhibitors (Fig. 1C), suggesting that processing and release of IL-1ß were mediated through formation 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