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NLRP3 promotes autophagy of urate crystals phagocytized by human osteoblasts.

Allaeys I, Marceau F, Poubelle PE - Arthritis Res. Ther. (2013)

Bottom Line: Simultaneously, MSU decreases phosphorylation of the protein kinases TOR (target of rapamycin) and p70S6K.MSU activates the cleavage of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, and MSU microcrystals are coated with GFP-tagged LC3.MSU does not increase death and late apoptosis of OBs, but reduces their proliferation in parallel to decreasing their competence for mineralization and to increasing their matrix metalloproteinase activity.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Monosodium urate (MSU) microcrystals present in bone tissues of chronic gout can be ingested by nonprofessional phagocytes like osteoblasts (OBs) that express NLRP3 (nucleotide-binding domain and leucine-rich repeat region containing family of receptor protein 3). MSU is known to activate NLRP3 inflammasomes in professional phagocytes. We have identified a new role for NLRP3 coupled to autophagy in MSU-stimulated human OBs.

Methods: Normal human OBs cultured in vitro were investigated for their capacity for phagocytosis of MSU microcrystals by using confocal microscopy. Subsequent mineralization and matrix metalloproteinase activity were evaluated, whereas regulatory events of phagocytosis were deciphered by using signaling inhibitors, phosphokinase arrays, and small interfering RNAs. Statistics were carried out by using paired or unpaired t tests, and the one-way ANOVA, followed by multiple comparison test.

Results: Most of the OBs internalized MSU in vacuoles. This process depends on signaling via PI3K, protein kinase C (PKC), and spleen tyrosine kinase (Syk), but is independent of Src kinases. Simultaneously, MSU decreases phosphorylation of the protein kinases TOR (target of rapamycin) and p70S6K. MSU activates the cleavage of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, and MSU microcrystals are coated with GFP-tagged LC3. However, MSU-stimulated autophagy in OBs absolutely requires the phagocytosis process. We find that MSU upregulates NLRP3, which positively controls the formation of MSU-autophagosomes in OBs. MSU does not increase death and late apoptosis of OBs, but reduces their proliferation in parallel to decreasing their competence for mineralization and to increasing their matrix metalloproteinase activity.

Conclusions: MSU microcrystals, found locally encrusted in the bone matrix of chronic gout, activate phagocytosis and NLRP3-dependent autophagy in OBs, but remain intact in permanent autophagosomes while deregulating OB functions.

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Related in: MedlinePlus

Phagocytosis of monosodium urate by osteoblasts. Confluent human OBs, previously stained with CellTracker Orange CMTMR, were cultured with vehicle (A) or with 0.5 mg MSU/106 cells (B) for 48 hours at 37°C, and then analyzed with confocal microscopy by using Olympus Fluoview microscope with helium-neon 543-nm lasers (left panel), differential interference contrast (DIC; middle panel), and overlap (right panel); ×400 magnification. Arrows indicate vacuoles and crystals of MSU. Representative of independent experiments with OB from three different donors.
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Figure 1: Phagocytosis of monosodium urate by osteoblasts. Confluent human OBs, previously stained with CellTracker Orange CMTMR, were cultured with vehicle (A) or with 0.5 mg MSU/106 cells (B) for 48 hours at 37°C, and then analyzed with confocal microscopy by using Olympus Fluoview microscope with helium-neon 543-nm lasers (left panel), differential interference contrast (DIC; middle panel), and overlap (right panel); ×400 magnification. Arrows indicate vacuoles and crystals of MSU. Representative of independent experiments with OB from three different donors.

Mentions: To evaluate the fate of MSU in the presence of OBs, live confluent primary human OBs were cultured with graded concentrations of MSU during 7 days. OBs that phagocytized MSU showed, after 48 hours of incubation, consistent morphologic changes, as studied with confocal microscopy. OBs dose-dependently internalized MSU from 0.1 to 1 mg/106 cells with an optimal effect at 0.5 mg/106 cells, followed by a plateau (data not shown). More than 90% of OBs had MSU internalized in large and fluid-filled vacuoles, each containing a single microcrystal (Figure 1). Volume and shape of vacuoles depend on crystal size. Vacuoles were individualized with light microscopy after, at least, 24 hours of incubation. Numbers of vacuoles with MSU averaged 30 per OB. Most of MSU were completely internalized in cells, but some crystals remained partially engulfed or alongside the membrane. After 7 days of culture, phagocytosis of 0.5 mg MSU/106 OBs was associated with unchanged vacuoles (data not shown). These data suggest a prolonged process that could partly detoxify the cells by retaining MSU microcrystals in permanent phagosomes with a final noncapacity of OB to eliminate MSU-containing vacuoles.


NLRP3 promotes autophagy of urate crystals phagocytized by human osteoblasts.

Allaeys I, Marceau F, Poubelle PE - Arthritis Res. Ther. (2013)

Phagocytosis of monosodium urate by osteoblasts. Confluent human OBs, previously stained with CellTracker Orange CMTMR, were cultured with vehicle (A) or with 0.5 mg MSU/106 cells (B) for 48 hours at 37°C, and then analyzed with confocal microscopy by using Olympus Fluoview microscope with helium-neon 543-nm lasers (left panel), differential interference contrast (DIC; middle panel), and overlap (right panel); ×400 magnification. Arrows indicate vacuoles and crystals of MSU. Representative of independent experiments with OB from three different donors.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061723&req=5

Figure 1: Phagocytosis of monosodium urate by osteoblasts. Confluent human OBs, previously stained with CellTracker Orange CMTMR, were cultured with vehicle (A) or with 0.5 mg MSU/106 cells (B) for 48 hours at 37°C, and then analyzed with confocal microscopy by using Olympus Fluoview microscope with helium-neon 543-nm lasers (left panel), differential interference contrast (DIC; middle panel), and overlap (right panel); ×400 magnification. Arrows indicate vacuoles and crystals of MSU. Representative of independent experiments with OB from three different donors.
Mentions: To evaluate the fate of MSU in the presence of OBs, live confluent primary human OBs were cultured with graded concentrations of MSU during 7 days. OBs that phagocytized MSU showed, after 48 hours of incubation, consistent morphologic changes, as studied with confocal microscopy. OBs dose-dependently internalized MSU from 0.1 to 1 mg/106 cells with an optimal effect at 0.5 mg/106 cells, followed by a plateau (data not shown). More than 90% of OBs had MSU internalized in large and fluid-filled vacuoles, each containing a single microcrystal (Figure 1). Volume and shape of vacuoles depend on crystal size. Vacuoles were individualized with light microscopy after, at least, 24 hours of incubation. Numbers of vacuoles with MSU averaged 30 per OB. Most of MSU were completely internalized in cells, but some crystals remained partially engulfed or alongside the membrane. After 7 days of culture, phagocytosis of 0.5 mg MSU/106 OBs was associated with unchanged vacuoles (data not shown). These data suggest a prolonged process that could partly detoxify the cells by retaining MSU microcrystals in permanent phagosomes with a final noncapacity of OB to eliminate MSU-containing vacuoles.

Bottom Line: Simultaneously, MSU decreases phosphorylation of the protein kinases TOR (target of rapamycin) and p70S6K.MSU activates the cleavage of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, and MSU microcrystals are coated with GFP-tagged LC3.MSU does not increase death and late apoptosis of OBs, but reduces their proliferation in parallel to decreasing their competence for mineralization and to increasing their matrix metalloproteinase activity.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Monosodium urate (MSU) microcrystals present in bone tissues of chronic gout can be ingested by nonprofessional phagocytes like osteoblasts (OBs) that express NLRP3 (nucleotide-binding domain and leucine-rich repeat region containing family of receptor protein 3). MSU is known to activate NLRP3 inflammasomes in professional phagocytes. We have identified a new role for NLRP3 coupled to autophagy in MSU-stimulated human OBs.

Methods: Normal human OBs cultured in vitro were investigated for their capacity for phagocytosis of MSU microcrystals by using confocal microscopy. Subsequent mineralization and matrix metalloproteinase activity were evaluated, whereas regulatory events of phagocytosis were deciphered by using signaling inhibitors, phosphokinase arrays, and small interfering RNAs. Statistics were carried out by using paired or unpaired t tests, and the one-way ANOVA, followed by multiple comparison test.

Results: Most of the OBs internalized MSU in vacuoles. This process depends on signaling via PI3K, protein kinase C (PKC), and spleen tyrosine kinase (Syk), but is independent of Src kinases. Simultaneously, MSU decreases phosphorylation of the protein kinases TOR (target of rapamycin) and p70S6K. MSU activates the cleavage of microtubule-associated protein light chain 3 (LC3)-I into LC3-II, and MSU microcrystals are coated with GFP-tagged LC3. However, MSU-stimulated autophagy in OBs absolutely requires the phagocytosis process. We find that MSU upregulates NLRP3, which positively controls the formation of MSU-autophagosomes in OBs. MSU does not increase death and late apoptosis of OBs, but reduces their proliferation in parallel to decreasing their competence for mineralization and to increasing their matrix metalloproteinase activity.

Conclusions: MSU microcrystals, found locally encrusted in the bone matrix of chronic gout, activate phagocytosis and NLRP3-dependent autophagy in OBs, but remain intact in permanent autophagosomes while deregulating OB functions.

Show MeSH
Related in: MedlinePlus