Interference with KCTD9 inhibits NK cell activation and ameliorates fulminant liver failure in mice

Abstract

Background: Potassium channel tetramerisation domain containing 9 (KCTD9), a member of KCTD family with a DNA-like pentapeptide repeat domain, was found to be increased particularly in NK cells of patients with HBV-induced acute-on-chronic liver failure (HBV-ACLF) and experimental viral fulminant hepatitis. Knockdown of KCTD9 in immortalized NK cells inhibits cytokines production and cytotoxicity. As NK cell activation was shown to exacerbate liver damage in viral fulminant hepatitis, we propose that target inhibition of KCTD9 may prohibit NK cells activity and thus ameliorate liver damage in viral fulminant hepatitis.

Result: Hydrodynamic delivery of plasmid expressing short-hairpin RNA against KCTD9 resulted in impaired NK cells function as demonstrated by reduced cytokine production and cytotoxicity, and ameliorated liver injury as manifested by improved liver histology and survival rate. In contrast, delivery of plasmid expressing KCTD9 led to deteriorated disease progression.

Conclusion: Interference with KCTD9 expression exert beneficial effect in viral fulminant hepatitis therapy. Such effect may be mediated by impairment of NK cell activation.

Keywords: KCTD9; Liver failure; MHV-3; NK cell; Short hairpin RNA.

Fig. 1

Elevated KCTD9 expression bothin liver tissue and hepatic NK cells in MHV-3-FHF mouse model. a KCTD9 expression in liver, heart, kidney, spleen, PBMC was determined in Balb/cJ mice with or without infection of 100 PUF of MHV3. b The expression of KCTD9 protein in liver and spleen 48 h after MHV-3 infection. Magnification: 400 X. c mKCTD9 mRNA levels in hepatic NK cell, CD4⁺ T cell, CD8⁺ T cell and hepatocyte isolated from Balb/cJ mice with or without MHV-3 infection. d The FACS assay showed that Percentage of hepatic CD4⁺ T cells and CD8⁺ T cells expressing KCTD9 in mice with or without MHV-3 infection for 24, 48, 72 and 96 h. *p < 0.05, **p < 0.01, Means ± SEM of 3 independent experiments were represented

Fig. 2

mKCTD9 shRNAs inhibited mKCTD9 expression in vitro. a Relative fold of mKCTD9 mRNA in CHO cell transfected with either shRNAs against mKCTD9, pcDNA3.1-mKCTD9, as well as scramble control. b inhibition efficiency or shRNAs against mKCTD9. c West bolt analysis of mKCTD9 expression in CHO cells transfected with pcDNA-mKCTD9, pMSCV-mKCTD9-shRNA1, pMSCV-mKCTD9-shRNA2, pMSCV-scramble shRNA (Negative Control as indicated in the figure); relative value of mKCTD9 protein levels were (from left to right): 2.25, 1.71, 1.0, 0.86, 0.96. **p < 0.01

Fig. 3

Increased survival in mice of mKCTD9 interference. a Schematic view of hydrodynamic delivery of plasmid and tissue collection. Individual plasmid were injected into mice with 200 μg plasmid in 200 μl saline by tail-vein injection at 24 h before and after MHV-3 infection. 20 PFU of MHV3 were injected intraperitoneally in each mice. Tissues were collected at indicated time point specifically at 24, 48 and 72 h post MHV3 infection. b Time of infected mice which were injected with pMSCV-mKCTD9-shRNA1, pMSCV-mKCTD9-shRNA2, pcDNA3.1-mKCTD9, Negative Control plasmid and saline, was calculated until death. Eighteen mice were pooled together in each cohort

Fig. 4

KCTD9 shRNA inhibited KCTD9 expression and ameliorates MHV-3 induced fulminant hepatitis in vivo. a ALT levels of infected mice delivered with distinct plasmid at 24 h,48 h and 108 h after MHV-3 infection. b Hematoxylin and eosin (H&E) staining of livers sections from mice 4 days post MHV-3 infection. Black arrow indicated the inflammatory cells area within black lines indicated liver necrosis. Magnification: 400 X. c Immunochemistry staining against mKCTD9 on liver sections of mice injected with various plasmid at 36 h after MHV-3 infection. Blank points to IgG stained section; The brown staining area indicates signals of KCTD9 protein. Magnification: 400 X. *p < 0.05, **p < 0.01, Means ± SEM of 5 to 7 independent experiments were represented

Fig. 5

mKCTD9 interference inhibited hepatic NK cells activation and function. a Frequency of NK cells expressing KCTD9 as well as CD69 positive NK cells in liver were measured by FACS. b The Cytotoxicity of isolated hepatic NK cells from MHV-3 infected mice which were treated with mKCTD9 shRNA expressing plasmids or negative control. Cytotoxicity of NK cells calculated as following: (Experimental ALT – Spontaneous release ALT) / (Maximum ALT – Spontaneous release ALT) * 100%. c Frequency of IFN-γ and TNF-α expressing hepatic NK cells were measured by Flow cytometry. *p < 0.05, **p < 0.01, Means ± SEM of 3 to 5 independent experiments were represented