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Toll-Like Receptor 9 Is Involved in NLRP3 Inflammasome Activation and IL-1β Production Through Monosodium Urate-Induced Mitochondrial DNA

Abstract

The NLR family pyrin domain-containing 3 (NLRP3) inflammasome is a cytoplasmic multimolecular complex that generates interleukin (IL)-1β and is considered a main pathogenic mechanism for uric acid-induced inflammation. Whether toll-like receptor 9 (TLR9) is responsible for uric acid-induced NLRP3 inflammasome activation remains unclear. Thus, the aim of this study was to identify the role of TLR9 in NLRP3 inflammasome activation through monosodium urate (MSU) crystal-induced mitochondrial DNA. RAW 264.7 cells treated with MSU crystals, CpG oligonucleotides (ODNs), or a combination of both were used to assess nuclear factor (NF)-κB signaling, NLRP3 inflammasome components such as NLRP3, ASC, and caspase-1, and IL-1β. Real-time polymerase chain reaction (RT-PCR), Western blotting, DNA fragmentation assay, mitochondrial DNA copy number assay, and immunofluorescence were used in the in vitro study. RAW 264.7 cells treated with CpG-ODN stimulated the activation of NF-κB signaling, the NLRP3 inflammasome components NLRP3, ASC, and caspase-1, and IL-1β gene and protein expression. DNA fragmentation assay showed that MSU crystals induced cellular apoptosis. Fragmented DNA prompted by MSU crystals induced TLR9 expression. RAW 264.7 cells treated with CpG-ODN or MSU crystals and both increased expression of mitochondrial DNA relative to nuclear DNA. CpG-ODN and MSU crystals augmented the activation of NLRP3 inflammasome components and IL-1β expression, which was significantly suppressed in RAW 264.7 cells transfected with TLR9 siRNA. This study suggests that TLR9 activated by MSU crystal-mediated mitochondrial DNA contributes to the activation of NLRP3 inflammasomes and IL-1β production.

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Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2019R1F1A1061098).

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Correspondence to Seong-Kyu Kim.

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Kim, SK., Park, KY. & Choe, JY. Toll-Like Receptor 9 Is Involved in NLRP3 Inflammasome Activation and IL-1β Production Through Monosodium Urate-Induced Mitochondrial DNA. Inflammation 43, 2301–2311 (2020). https://doi.org/10.1007/s10753-020-01299-6

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  • DOI: https://doi.org/10.1007/s10753-020-01299-6

KEY WORDS

  • monosodium urate
  • toll-like receptor 9
  • interleukin-1β
  • NLRP3 inflammasome
  • NF-κB