Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of extracellular potassium on IL-1 processing and release.A) Bone marrow-derived macrophages were plated in triplicate wells in 12-well plates and primed with 50 ng/ml of LPS for 4 h. Cells were then incubated in medium containing 130 mM NaCl/5 mM KCl or 5 mM KCl/130 mM NaCl in the presence of absence of 0.01 µg/mL ricin, 25 µg/mL cycloheximide, 10 µg/mL, 10 µg/mL emetine, 75 µg/mL puromycin, 0.2 µg/mL pactamycin, 10 µg/mL anisomycin, 3.4 µM nigericin, or 5 mM ATP for 4 h. Medium was collected, and p17 IL-1 was determined by ELISA. B) Macrophages were plated, primed with LPS and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 5 mM ATP or 3.4 µM nigericin for 4 h. Proteins were precipitated from the media with TCA and analyzed by Western blotting. C) Macrophages were plated, primed with LPS, and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 10 ng/mL ricin, 10 µg/mL emetine, or 25 µg/mL cycloheximide for 4 h. Proteins were precipitated with TCA and analyzed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3351443&req=5

pone-0036044-g002: Effect of extracellular potassium on IL-1 processing and release.A) Bone marrow-derived macrophages were plated in triplicate wells in 12-well plates and primed with 50 ng/ml of LPS for 4 h. Cells were then incubated in medium containing 130 mM NaCl/5 mM KCl or 5 mM KCl/130 mM NaCl in the presence of absence of 0.01 µg/mL ricin, 25 µg/mL cycloheximide, 10 µg/mL, 10 µg/mL emetine, 75 µg/mL puromycin, 0.2 µg/mL pactamycin, 10 µg/mL anisomycin, 3.4 µM nigericin, or 5 mM ATP for 4 h. Medium was collected, and p17 IL-1 was determined by ELISA. B) Macrophages were plated, primed with LPS and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 5 mM ATP or 3.4 µM nigericin for 4 h. Proteins were precipitated from the media with TCA and analyzed by Western blotting. C) Macrophages were plated, primed with LPS, and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 10 ng/mL ricin, 10 µg/mL emetine, or 25 µg/mL cycloheximide for 4 h. Proteins were precipitated with TCA and analyzed by Western blotting.

Mentions: Previous studies have described a requirement for potassium efflux by a variety of agents reported to activate the NLRP3 inflammasome, including nigericin and ATP [11]. Here, substitution of 130 mM Na+/5 mM K+ by 5 mM Na+/130 mM K+ in the culture medium suppressed the appearance of mature IL-1ß by translation inhibitors to a similar extent as nigericin and ATP, as detected by ELISA (Fig. 2A). Immunoblotting of the high potassium culture medium revealed undetectable amounts of mature IL-1ß in the culture medium of LPS-primed cells exposed to nigericin or ATP (Fig. 2B), or ricin, emetine, or cycloheximide (Fig. 2C).


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

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

Effect of extracellular potassium on IL-1 processing and release.A) Bone marrow-derived macrophages were plated in triplicate wells in 12-well plates and primed with 50 ng/ml of LPS for 4 h. Cells were then incubated in medium containing 130 mM NaCl/5 mM KCl or 5 mM KCl/130 mM NaCl in the presence of absence of 0.01 µg/mL ricin, 25 µg/mL cycloheximide, 10 µg/mL, 10 µg/mL emetine, 75 µg/mL puromycin, 0.2 µg/mL pactamycin, 10 µg/mL anisomycin, 3.4 µM nigericin, or 5 mM ATP for 4 h. Medium was collected, and p17 IL-1 was determined by ELISA. B) Macrophages were plated, primed with LPS and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 5 mM ATP or 3.4 µM nigericin for 4 h. Proteins were precipitated from the media with TCA and analyzed by Western blotting. C) Macrophages were plated, primed with LPS, and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 10 ng/mL ricin, 10 µg/mL emetine, or 25 µg/mL cycloheximide for 4 h. Proteins were precipitated with TCA and analyzed by Western blotting.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351443&req=5

pone-0036044-g002: Effect of extracellular potassium on IL-1 processing and release.A) Bone marrow-derived macrophages were plated in triplicate wells in 12-well plates and primed with 50 ng/ml of LPS for 4 h. Cells were then incubated in medium containing 130 mM NaCl/5 mM KCl or 5 mM KCl/130 mM NaCl in the presence of absence of 0.01 µg/mL ricin, 25 µg/mL cycloheximide, 10 µg/mL, 10 µg/mL emetine, 75 µg/mL puromycin, 0.2 µg/mL pactamycin, 10 µg/mL anisomycin, 3.4 µM nigericin, or 5 mM ATP for 4 h. Medium was collected, and p17 IL-1 was determined by ELISA. B) Macrophages were plated, primed with LPS and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 5 mM ATP or 3.4 µM nigericin for 4 h. Proteins were precipitated from the media with TCA and analyzed by Western blotting. C) Macrophages were plated, primed with LPS, and incubated in medium containing 130 mM NaCl/5 mM KCl (MEM-Na) or 5 mM NaKCl/130 mM KCl (MEM-K) in the presence or absence 10 ng/mL ricin, 10 µg/mL emetine, or 25 µg/mL cycloheximide for 4 h. Proteins were precipitated with TCA and analyzed by Western blotting.
Mentions: Previous studies have described a requirement for potassium efflux by a variety of agents reported to activate the NLRP3 inflammasome, including nigericin and ATP [11]. Here, substitution of 130 mM Na+/5 mM K+ by 5 mM Na+/130 mM K+ in the culture medium suppressed the appearance of mature IL-1ß by translation inhibitors to a similar extent as nigericin and ATP, as detected by ELISA (Fig. 2A). Immunoblotting of the high potassium culture medium revealed undetectable amounts of mature IL-1ß in the culture medium of LPS-primed cells exposed to nigericin or ATP (Fig. 2B), or ricin, emetine, or cycloheximide (Fig. 2C).

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