Simultaneous knockdown of multiple ligands of innate receptor NKG2D prevents natural killer cell–mediated fulminant hepatitis in mice

Authors

  • Mei Huang,

    1. Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, China
    2. Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China
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  • Rui Sun,

    Corresponding author
    1. Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, China
    2. Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China
    • School of Life Sciences, University of Science and Technology of China, #443 Huangshan Road, Hefei 230027, China
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    • Fax: (86)-551-3606783

  • Haiming Wei,

    1. Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, China
    2. Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China
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  • Zhigang Tian

    Corresponding author
    1. Department of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, China
    2. Hefei National Laboratory for Physical Sciences at Microscale, Hefei, China
    • School of Life Sciences, University of Science and Technology of China, #443 Huangshan Road, Hefei 230027, China
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  • Potential conflict of interest: Nothing to report.

  • Supported by the Ministry of Science & Technology of China (973 Basic Science Project 2010CB911901); Natural Science Foundation of China grants 91029303, 30911120480, and 31021061; and National Science & Technology Major Projects 2012ZX10002014 and 2012ZX10002006.

Abstract

NKG2D activation plays an important role in initiating and maintaining liver inflammation, and blockade of NKG2D recognition becomes a promising approach to alleviate liver inflammation. Treatment by silencing NKG2D ligands on hepatocytes, but not NKG2D on circulating immune cells, is more liver-specific, and simultaneous knockdown of multiple NKG2D ligands on hepatocytes will be more efficient in liver disease intervention. Here, we constructed a single vector that could simultaneously express multiple short hairpin RNAs (shRNAs) against all murine NKG2D ligands including Rae1, Mult1, and H60. After hydrodynamic injection of plasmid containing the three shRNA sequences (shRae1-shMult1-shH60), also called pRNAT-shRMH, we found the expression of all three NKG2D ligands on hepatocytes was downregulated both on messenger RNA and protein levels. Moreover, natural killer (NK) cell–mediated NKG2D-dependent fulminant hepatitis of the mice was alleviated, along with inactivation of hepatic NK cells, by pRNAT-shRMH if compared with its counterpart RNA interference vectors against single or double ligands. The therapeutic efficacy of pRNAT-shRMH was equivalent to that of injecting three monoclonal antibodies against Rae1, Mult1, and H60. For better in vivo application, we constructed a recombinant adenovirus containing pRNAT-shRMH (called Ad-RMH) with efficient hepatotropic infection capacity and observed that Ad-RMH intravenous injection exerted a similar therapeutic efficiency as plasmid pRNAT-shRMH hydrodynamic injection. Noticeably, simultaneous knockdown of multiple human NKG2D ligands (MICA/B, ULBP2, and ULBP3) also significantly attenuated NK cell cytolysis against human NKG2D ligand-positive hepatocyte L-02 cells, suggesting a possible translation into human settings. Conclusion: Simultaneous knockdown of multiple ligands of NKG2D prevents NK cell–mediated fulminant hepatitis and is a potential therapeutic approach to treat liver diseases. (HEPATOLOGY 2013)

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