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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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TLRs are involved in activating NF-κB in early S. aureus infection.(A and B) Control cells (miCtrl) and MyD88 miRNA-introduced cells (miMyD88) were cultured with or without 2 ng/ml Dox for 5 d. The total RNA was analyzed for MyD88 mRNA by RT-PCR (A) and real-time PCR (B); numbers below lanes indicate each band's intensity relative to a Dox-untreated control. (C) ELISA analysis of TNF-α released from each Dox-treated or -untreated cell line infected with S. aureus at an MOI of 4 for 120 min. Values represent the averages of duplicate wells, and error bars represent the range. All results are representative of three independent experiments. *P<0.05, **P<0.01.
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pone.0119179.g002: TLRs are involved in activating NF-κB in early S. aureus infection.(A and B) Control cells (miCtrl) and MyD88 miRNA-introduced cells (miMyD88) were cultured with or without 2 ng/ml Dox for 5 d. The total RNA was analyzed for MyD88 mRNA by RT-PCR (A) and real-time PCR (B); numbers below lanes indicate each band's intensity relative to a Dox-untreated control. (C) ELISA analysis of TNF-α released from each Dox-treated or -untreated cell line infected with S. aureus at an MOI of 4 for 120 min. Values represent the averages of duplicate wells, and error bars represent the range. All results are representative of three independent experiments. *P<0.05, **P<0.01.

Mentions: When TLRs recognize bacterial ligands, they activate NF-κB to provoke the release of proinflammatory cytokines. To examine whether the TLR pathway is also involved in activating NF-κB and releasing cytokines in the early stages of infection, we generated MyD88-knockdown cell lines based on a Tet-on system. MyD88 mRNA was slightly reduced in the MyD88 miRNA-introduced (miMyD88) cells even before Dox treatment, and was reduced further by Dox treatment (Fig. 2A and Fig. 2B). Following S. aureus infection in the Dox-treated MyD88-knockdown cells, TNF-α release evaluated by ELISA (Fig. 2C) and NF-κB activation evaluated by EMSA (data not shown) were lower than in Dox-untreated cells. Thus, the NLPR3 and TLR signaling pathways seem to mediate NF-κB activation redundantly or cooperatively in the early stages of S. aureus infection.


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)

TLRs are involved in activating NF-κB in early S. aureus infection.(A and B) Control cells (miCtrl) and MyD88 miRNA-introduced cells (miMyD88) were cultured with or without 2 ng/ml Dox for 5 d. The total RNA was analyzed for MyD88 mRNA by RT-PCR (A) and real-time PCR (B); numbers below lanes indicate each band's intensity relative to a Dox-untreated control. (C) ELISA analysis of TNF-α released from each Dox-treated or -untreated cell line infected with S. aureus at an MOI of 4 for 120 min. Values represent the averages of duplicate wells, and error bars represent the range. All results are representative of three independent experiments. *P<0.05, **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4356585&req=5

pone.0119179.g002: TLRs are involved in activating NF-κB in early S. aureus infection.(A and B) Control cells (miCtrl) and MyD88 miRNA-introduced cells (miMyD88) were cultured with or without 2 ng/ml Dox for 5 d. The total RNA was analyzed for MyD88 mRNA by RT-PCR (A) and real-time PCR (B); numbers below lanes indicate each band's intensity relative to a Dox-untreated control. (C) ELISA analysis of TNF-α released from each Dox-treated or -untreated cell line infected with S. aureus at an MOI of 4 for 120 min. Values represent the averages of duplicate wells, and error bars represent the range. All results are representative of three independent experiments. *P<0.05, **P<0.01.
Mentions: When TLRs recognize bacterial ligands, they activate NF-κB to provoke the release of proinflammatory cytokines. To examine whether the TLR pathway is also involved in activating NF-κB and releasing cytokines in the early stages of infection, we generated MyD88-knockdown cell lines based on a Tet-on system. MyD88 mRNA was slightly reduced in the MyD88 miRNA-introduced (miMyD88) cells even before Dox treatment, and was reduced further by Dox treatment (Fig. 2A and Fig. 2B). Following S. aureus infection in the Dox-treated MyD88-knockdown cells, TNF-α release evaluated by ELISA (Fig. 2C) and NF-κB activation evaluated by EMSA (data not shown) were lower than in Dox-untreated cells. Thus, the NLPR3 and TLR signaling pathways seem to mediate NF-κB activation redundantly or cooperatively in the early stages of S. aureus infection.

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