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TNF-α/calreticulin dual signaling induced NLRP3 inflammasome activation associated with HuR nucleocytoplasmic shuttling in rheumatoid arthritis

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Abstract

Objective

The present study was undertaken to validate whether TNF-α and calreticulin (CRT) serve as dual signaling to activate nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasome in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and HUVECs. The effect of human antigen R (HuR) in NLRP3 inflammasome activation was also explored in RA FLS.

Methods

Immunofluorescence was used to determine the expression of NLRP3 and adaptor protein apoptosis associated speck-like protein containing a CARD (ASC) in RA synovial tissue and HuR location in RA FLS. Western blot and quantitative real-time PCR were employed to measure the priming effect of NLRP3 inflammasome in cells and HuR expression in synovial tissue. The concentrations of IL-1β and IL-18 were detected by enzyme linked immunosorbent assay. Immunohistochemistry was used to visualize the expression of HuR in synovial tissue. HuR knockdown in RA FLS was achieved by siRNA-mediated gene silencing.

Results

Higher expression of NLRP3 and ASC in RA synovial tissue than those in osteoarthritis was detected. The staining of NLRP3, ASC and cleaved IL-1β were observed in FLS and vascular endothelial cells in RA synovium. Expression of NLRP3 and pro-IL-1β in RA FLS and HUVECs treated with TNF-α was increased. The pro-IL-18 expression was also enhanced in HUVECs, but not in RA FLS. TNF-α/CRT dual stimulation of cells gave rise to caspase-1 p20 expression and the secretion of IL-1β. The secreted IL-18 was also elevated in HUVECs but not in RA FLS. HuR expression was significantly elevated in RA synovial tissue. TNF-α initiated the nucleocytoplasmic shuttling of HuR in both FLS and HUVECs. The knockdown of HuR in FLS incubated with TNF-α led to reduced caspase-1 p20 protein expression and further resulted in decreased secretion of IL-1β in the presence of CRT.

Conclusions

TNF-α/CRT dual signaling induced NLRP3 inflammasome activation, which could be suppressed by HuR knockdown presumably due to the block of HuR translocating from nucleus to cytoplasma.

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Acknowledgements

This work was supported by the Natural Science Foundation of Tianjin City (no. 14JCYBJC25600).

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Author notes

  1. Yixin Liu and Wei Wei contributed equally to this work.

Authors and Affiliations

  1. Department of Clinical Immunology, School of Medical Laboratory, Tianjin Medical University, Tianjin, 300203, China

    Yixin Liu, Yang Wang, Yingyu Bai, Xuguo Sun & Fang Zheng

  2. Department of Rheumatology, General Hospital, Tianjin Medical University, Tianjin, 300052, China

    Wei Wei

  3. Center of Joint Surgery, Tianjin Hospital, Tianjin, 300210, China

    Chunyou Wan

  4. Department of Health Statistics, College of Public Health, Tianjin Medical University, Tianjin, 300070, China

    Jun Ma

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  1. Yixin Liu
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Liu, Y., Wei, W., Wang, Y. et al. TNF-α/calreticulin dual signaling induced NLRP3 inflammasome activation associated with HuR nucleocytoplasmic shuttling in rheumatoid arthritis. Inflamm. Res. 68, 597–611 (2019). https://doi.org/10.1007/s00011-019-01244-w

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