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NLRP3 mediates NF-κB activation and cytokine induction in microbially induced and sterile inflammation.

Kinoshita T, Imamura R, Kushiyama H, Suda T - PLoS ONE (2015)

Bottom Line: To clarify the physiological relevance of this latter function, we examined the effect of NLRP3 on NF-κB activation and cytokine induction in RNA-interference-based NLRP3-knockdown cell lines generated from the human monocytic cell line THP-1.Knocking down NLRP3 reduced NF-κB activation and cytokine induction in the early stages of Staphylococcus aureus infection.Expression of cytokine genes induced by Staphylococcus aureus was not inhibited by a caspase-1 inhibitor, and did not occur through an autocrine mechanism in response to newly synthesized cytokines.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kakumamachi, Kanazawa, Ishikawa, Japan.

ABSTRACT
Nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain containing 3 (NLRP3) has recently emerged as a central regulator of innate immunity and inflammation in response to both sterile inflammatory and microbial invasion signals. Although its ability to drive proteolytic procaspase-1 processing has drawn more attention, NLPR3 can also activate NF-κB. To clarify the physiological relevance of this latter function, we examined the effect of NLRP3 on NF-κB activation and cytokine induction in RNA-interference-based NLRP3-knockdown cell lines generated from the human monocytic cell line THP-1. Knocking down NLRP3 reduced NF-κB activation and cytokine induction in the early stages of Staphylococcus aureus infection. Expression of cytokine genes induced by Staphylococcus aureus was not inhibited by a caspase-1 inhibitor, and did not occur through an autocrine mechanism in response to newly synthesized cytokines. We also demonstrated that NLRP3 could activate NF-κB and induce cytokines in response to sterile signals, monosodium urate crystals and aluminum adjuvant. Thus, NLRP3 mediates NF-κB activation in both sterile and microbially induced inflammation. Our findings show that not only does NLRP3 activate caspase-1 post-translationally, but it also induces multiple cytokine genes in the innate immune system.

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NLRP3 mediates NF-κB activation downstream of lysosomes.(A) After being cultured with or without Dox for 5 d, miCtrl and miNLRP3 cells were incubated with 500 nM bafilomycin A1 for 30 min and then infected with S. aureus at an MOI of 2 for 85 min. The total RNA was extracted and analyzed for TNF-α mRNA by RT-PCR. (B) ELISA analysis of TNF-α released from the indicated cell lines treated with or without bafilomycin A1 (bafilo) and then infected with S. aureus at an MOI of 4 for 120 min. (C) ELISA analysis of TNF-α released from the indicated Dox-treated or -untreated cell lines treated with or without CA-074Me (20 μM) for 30 min, and then infected with S. aureus at an MOI of 4 for 100 min. (D and E) ELISA analysis of TNF-α released from THP-1 cells treated with or without cytochalasin D (0.25 μM), bafilomycin A1 (0.5 μM), or DPI (2 μM), and then stimulated with MSU for 3 h. All results are representative of three independent experiments. **P<0.01.
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pone.0119179.g005: NLRP3 mediates NF-κB activation downstream of lysosomes.(A) After being cultured with or without Dox for 5 d, miCtrl and miNLRP3 cells were incubated with 500 nM bafilomycin A1 for 30 min and then infected with S. aureus at an MOI of 2 for 85 min. The total RNA was extracted and analyzed for TNF-α mRNA by RT-PCR. (B) ELISA analysis of TNF-α released from the indicated cell lines treated with or without bafilomycin A1 (bafilo) and then infected with S. aureus at an MOI of 4 for 120 min. (C) ELISA analysis of TNF-α released from the indicated Dox-treated or -untreated cell lines treated with or without CA-074Me (20 μM) for 30 min, and then infected with S. aureus at an MOI of 4 for 100 min. (D and E) ELISA analysis of TNF-α released from THP-1 cells treated with or without cytochalasin D (0.25 μM), bafilomycin A1 (0.5 μM), or DPI (2 μM), and then stimulated with MSU for 3 h. All results are representative of three independent experiments. **P<0.01.

Mentions: To explore the signaling pathway by which NLRP3 activates NF-κB, we examined a proposed phagolysosome destabilization model for procaspase-1 activation [29, 30]. Pre-incubating control and knockdown cells with bafilomycin A1, which neutralizes the lysosomal pH and prevents lysosomal protease maturation, completely blocked the TNF-α mRNA induction and cytokine release (Fig. 5A and Fig. 5B) in the early stages of S. aureus infection. The cytokine release was markedly reduced by pre-incubating control and knockdown cells with the cathepsin B inhibitor CA-074Me (Fig. 5C, CA-074Me). Similarly, the MSU-induced TNF-α secretion was markedly reduced by pre-incubating cells with cytochalasin D, bafilomycin A1, or the NADPH oxidase inhibitor DPI (Fig. 5D and 5E). These results indicate that NLRP3 mediates NF-κB activation downstream of phagolysosome pathways and suggest that, in both sterile and microbially induced immune responses, NF-κB and inflammasomes may be activated concurrently.


NLRP3 mediates NF-κB activation and cytokine induction in microbially induced and sterile inflammation.

Kinoshita T, Imamura R, Kushiyama H, Suda T - PLoS ONE (2015)

NLRP3 mediates NF-κB activation downstream of lysosomes.(A) After being cultured with or without Dox for 5 d, miCtrl and miNLRP3 cells were incubated with 500 nM bafilomycin A1 for 30 min and then infected with S. aureus at an MOI of 2 for 85 min. The total RNA was extracted and analyzed for TNF-α mRNA by RT-PCR. (B) ELISA analysis of TNF-α released from the indicated cell lines treated with or without bafilomycin A1 (bafilo) and then infected with S. aureus at an MOI of 4 for 120 min. (C) ELISA analysis of TNF-α released from the indicated Dox-treated or -untreated cell lines treated with or without CA-074Me (20 μM) for 30 min, and then infected with S. aureus at an MOI of 4 for 100 min. (D and E) ELISA analysis of TNF-α released from THP-1 cells treated with or without cytochalasin D (0.25 μM), bafilomycin A1 (0.5 μM), or DPI (2 μM), and then stimulated with MSU for 3 h. All results are representative of three independent experiments. **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356585&req=5

pone.0119179.g005: NLRP3 mediates NF-κB activation downstream of lysosomes.(A) After being cultured with or without Dox for 5 d, miCtrl and miNLRP3 cells were incubated with 500 nM bafilomycin A1 for 30 min and then infected with S. aureus at an MOI of 2 for 85 min. The total RNA was extracted and analyzed for TNF-α mRNA by RT-PCR. (B) ELISA analysis of TNF-α released from the indicated cell lines treated with or without bafilomycin A1 (bafilo) and then infected with S. aureus at an MOI of 4 for 120 min. (C) ELISA analysis of TNF-α released from the indicated Dox-treated or -untreated cell lines treated with or without CA-074Me (20 μM) for 30 min, and then infected with S. aureus at an MOI of 4 for 100 min. (D and E) ELISA analysis of TNF-α released from THP-1 cells treated with or without cytochalasin D (0.25 μM), bafilomycin A1 (0.5 μM), or DPI (2 μM), and then stimulated with MSU for 3 h. All results are representative of three independent experiments. **P<0.01.
Mentions: To explore the signaling pathway by which NLRP3 activates NF-κB, we examined a proposed phagolysosome destabilization model for procaspase-1 activation [29, 30]. Pre-incubating control and knockdown cells with bafilomycin A1, which neutralizes the lysosomal pH and prevents lysosomal protease maturation, completely blocked the TNF-α mRNA induction and cytokine release (Fig. 5A and Fig. 5B) in the early stages of S. aureus infection. The cytokine release was markedly reduced by pre-incubating control and knockdown cells with the cathepsin B inhibitor CA-074Me (Fig. 5C, CA-074Me). Similarly, the MSU-induced TNF-α secretion was markedly reduced by pre-incubating cells with cytochalasin D, bafilomycin A1, or the NADPH oxidase inhibitor DPI (Fig. 5D and 5E). These results indicate that NLRP3 mediates NF-κB activation downstream of phagolysosome pathways and suggest that, in both sterile and microbially induced immune responses, NF-κB and inflammasomes may be activated concurrently.

Bottom Line: To clarify the physiological relevance of this latter function, we examined the effect of NLRP3 on NF-κB activation and cytokine induction in RNA-interference-based NLRP3-knockdown cell lines generated from the human monocytic cell line THP-1.Knocking down NLRP3 reduced NF-κB activation and cytokine induction in the early stages of Staphylococcus aureus infection.Expression of cytokine genes induced by Staphylococcus aureus was not inhibited by a caspase-1 inhibitor, and did not occur through an autocrine mechanism in response to newly synthesized cytokines.

View Article: PubMed Central - PubMed

Affiliation: Division of Immunology and Molecular Biology, Cancer Research Institute, Kanazawa University, Kakumamachi, Kanazawa, Ishikawa, Japan.

ABSTRACT
Nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain containing 3 (NLRP3) has recently emerged as a central regulator of innate immunity and inflammation in response to both sterile inflammatory and microbial invasion signals. Although its ability to drive proteolytic procaspase-1 processing has drawn more attention, NLPR3 can also activate NF-κB. To clarify the physiological relevance of this latter function, we examined the effect of NLRP3 on NF-κB activation and cytokine induction in RNA-interference-based NLRP3-knockdown cell lines generated from the human monocytic cell line THP-1. Knocking down NLRP3 reduced NF-κB activation and cytokine induction in the early stages of Staphylococcus aureus infection. Expression of cytokine genes induced by Staphylococcus aureus was not inhibited by a caspase-1 inhibitor, and did not occur through an autocrine mechanism in response to newly synthesized cytokines. We also demonstrated that NLRP3 could activate NF-κB and induce cytokines in response to sterile signals, monosodium urate crystals and aluminum adjuvant. Thus, NLRP3 mediates NF-κB activation in both sterile and microbially induced inflammation. Our findings show that not only does NLRP3 activate caspase-1 post-translationally, but it also induces multiple cytokine genes in the innate immune system.

Show MeSH
Related in: MedlinePlus