Simultaneous Knockdown Of Multiple Ligands Of Innate Receptor NKG2D Prevents Natural Killer Cells Mediated Fulminant Hepatitis In Mice

NKG2D, a lectin-like type-2transmembrane protein, is reported to be a stimulatory immunoreceptor. It is expressed by many kinds of cells, including NK (natural killer) cells, NK1.1+T cells, γδT cells, CD8+T cells and some myeloid cells. NKG2D recognizes several various ligands, which are distantly related to MHC class I molecules, including MICA/B, ULBPs in human, and Rael, Multl and H60in mice. The unique feature of NKG2D ligands is that they are not constitutively expressed, but could be stress-induced, such as viral infection, DNA damage or malignant transformation. Expression of NKG2D ligands, renders target cells susceptible to NK cells cytolysis and induces IFN-y production by NK cells. It was reported that NKG2D was involved in NK cells-mediated allergic airway inflammation,and blocking antibody for NKG2D receptor delayed airway allergy. In renal ischaemia model, NKG2D-ligands recognition induced local inflammation and leucocyte accumulation. Blockade of NKG2D signaling is beneficial in cell transfer-induced colitis in SCID mice and ameliorates collage-induced arthritis by administration of a non-depleting anti-NKG2D mAb (CX5). A few years ago, we and other group reported that blockade of NKG2D-ligand interaction could attenuated NKG2D-mediated acute hepatitis in hepatitis B viral infected subjects. So, NKG2D activation plays an important role in initiating and maintaining inflammation, and blockade of NKG2D recognition becomes a potential approach to treat a variety of tissue injury.Since treatment by silencing NKG2D ligands on hepatocytes, but not NKG2D on circulating immune cells, will be more liver-specific, and considering short life-time of antibody in vivo (that’s why repeat administrations) and thereafter the expensive cost, we chose gene knockdown approach via RNA interference (RNAi) to suppress expression of multiple NKG2D ligands on hepatocytes. In the context of NKG2D ligands diversity, we constructed a single vector that could simultaneously express multiple shRNAs, targeting various NKG2D ligands. Different shRNA transcripts (Rae1, Mult1and H60or negative control) were amplified, including H1.1promoter, shRNA cassette and terminator, then ligated with each other, and further combinated into pRNAT-H1.1/shuttle. We construct a serial of plasmids targeting all murine NKG2D ligands, including triple shRNA-expressing vector, double, single shRNA-expressing vectors and negative control vector. We identified the function of these multiple shRNA expressing vectors against murine NKG2D ligands on poly I:C/D-GalN-induced NK cell-mediated hepatitis model and ConA induced NKG2D-dependent acute hepatitis in hepatitis B viral infected subjects. Hepatocytes were isolated for quantitative PCR and FACs analysis of mRNA and surface expression of NKG2D ligands; serum were harvested for transaminase ALT, cytokine IFN-γ and TNF-α levels determination, and liver tissue section were analyzed for pathologic changes; we separated liver lymphocytes for numbers counting, and phenotype analysis and cytokine secretion by FACs, to investigate the molecular mechanism of multiple shRNA expressing plasmids protection from liver damage. A triple shRNA expressing vector against all human NKG2D ligands was constructed, and transfected into human NKG2D ligands-positive hepatocytes L-02, which were then incubated with human NKG cells for51Cr release cytotoxicity assay and CD107a expression. The major findings are shown as followed:1. Construction of a shRNA vector (pRNAT-shRMH) for knockdown of multiple NKG2D ligandsWe selected RNAi target sequences for silencing murine NKG2D ligands genes (Rae1, Mult1and H60), using siRNA designer software based on various algorithms, as reported previously. These RNAi target sequences were efficient in silencing surface expression of Rae1, Multl and H50, respectively, after delivery of siRNA or shRNA-expressing vector into corresponaing positive expression cell lines Raw264.7cells and NIH/3T3cells. Three single-shRNA expressing vectors against murine NKG2D ligands Rae1, Mult1and H60(called as pRNAT-shRael, pRNAT-shMultl and pRNAT-shH60) were constructed by subcloning chemically synthesized oligonucleotides into pRNAT-H1.1/shuttle, a shuttle plasmid for Adenovirus. With the constructed shRNA expressing vectors as PCR templates, the shRNA expression cassettes, including H1.1promoter, RNAi oligonucleotids and terminator, were amplified together with designed restricted enzyme sites. These shRNA expression cassettes were finally inserted with pRNAT-H1.1/shuttle to be multiple shRNA expressing vectors. A serial of multiple shRNA expressing vectors were constructed, including single, double or triple shRNA expressing vector against murine NKG2D ligands. The shRNA expression sequences of Rae1(400bp), Mult1(200bp), H60(200bp) and negative control (400bp) were amplified from corresponding single shRNA expressing vectors by PCR primers with designed restricted enzyme sites. These shRNA expression cassettes were then sequentially ligated to be a large shRNA fragment of1.4kb showed which was further inserted into pRNAT-H1.1/shuttle.2. Multiple expressions of NKG2D ligands and their simultaneous knockdown by a pRNAT-shRMH vector in vivoIn our previous report, poly I:C in D-GalN-sensitized mice caused severe liver injury in an NK cell-dependent NKG2D-mediated manner. In order to test the efficacy of pRNAT-shRMH vector on simultaneous knockdown of multiple NKG2D ligands, we examined expression of murine NKG2D ligands in this mouse model and found poly I:C and D-GalN coadministration markedly stimulated multiple expression of NKG2D ligands (Rael, Multl and H60) on hepatocytes, indicating NK cells may interact with hepatocytes through multiple NKG2D-ligands recognition. We hydrodynamically administrated the multiple shRNA-expressing plasmids in order to efficiently deliver the gene into hepatocytes in vivo, and found that expression of all three NKG2D ligands was simultaneously inhibited in hepatocytes on both mRNA and protein levels by injection with a triple shRNA expressing vector pRNAT-shRMH3days prior to Poly (I:C)/D-GalN coadministration. pRNAT-shRMH vector simultaneously markedly reduced mRNA expression of Rae1, Multl and H60, the efficiency of which was almost equivalent to a single-or double-RNAi vector targeting a common ligand, demonstrating the specificity of RNAi against each ligand. In consistent with knockdown of mRNA expression, pRNAT-shRMH inhibited the surface expression of Rael, Multl and H60on hepatocytes, respectively. Overall, triple shRNA expressing vector (pRNAT-shRMH) simultaneously suppressed expression of three kinds of murine NKG2D ligands, whereas single or double shRNA expressing vectors only suppressed one or two kinds of corresponding ligands, further proving that simultaneous knockdown by pRNAT-shRMH was ligand-specific.3. Simultaneous knockdown of multiple ligands of mouse NKG2D high efficiently prevents polyI:C/D-GalN induced fulminant hepatitis in vivoCoadministration of Poly (I:C) and D-GalN induced severe liver injury with significant elevation of serum ALT level, as reported previously by us. We hydrodynamically injected multiple shRNA expressing vectors (against murine NKG2D ligands)3days before Poly (I:C)/D-GalN coadministration. Mice treated with shRNA expressing vectors against single, double, or triple murine NKG2D ligands, showed decrease in ALT levels and alleviation of liver injury, if compared to control vector pRNAT-shNNN treated mice; however, the most efficient protective effect occurred only in pRNAT-shRMH vector treated mice. Levels of inflammatory cytokines, including IFN-γ and TNF-α, was consistent with ALT levels and histopathological results of liver tissues. Serum IFN-γ and TNF-α dramatically increased when Poly (I:C)/D-GalN was administrated. However, when multiple shRNA expressing vectors were injected3days before Poly (I:C)/D-GalN injection, IFN-γ and TNF-α decreased to different extents. Additionally, among three single shRNA expressing vectors, pRNAT-shRNN (only against Rael) more greatly alleviated liver inflammation if compared with other two single shRNA expressing vectors (against Mult1or H60, respectively), suggesting that Rael plays a more important role in Poly (I:C)/D-GalN induced fulminant hepatitis. When one more ligand of NKG2D was simultaneously silenced, the inflammation was further inhibited. For example, pRNAT-shRMN (against both Rael and Mult1) and pRNAT-shRNH (against both Rael and F60) exerted more strong effects on alleviating liver injury if compared with those of pRNAT-shRNN group. Moreover, triple shRNA expressing vector pRNAT-shRMH exhibited most dramatic alleviation on liver inflammation, and survival rate was extremely increased from0%to100%by treatment with pRNAT-shRMH when mice was administrated with lethal dose of Poly (I:C)/D-GalN.The protective effects of simultaneous knockdown of multiple ligands of NKG2D in liver injury were almost same as blocking antibody against NKG2D or multiple NKG2D ligands. NKG2D antibody or Rael, Multl, H60antibody treatment alleviated liver injury induced by Poly (I:C)/D-GalN injection, showing the decrease in serum ALT, IFN-γ, TNF-α levels and histopathological score of liver tissue. Though each antibody of NKG2D ligands, including Rael, Mult1or H60antibody, alleviated liver inflammation to varying degrees (Rael> H60> Multl) and did not reach the level of anti-NKG2D antibody, the mixture cocktail of Rael, Multl and H60antibodies reached the therapeutic level of anti-NKG2D antibody, further implying all NKG2D ligands participate in NK cell-mediated NKG2D-dependent hepatitis. Noticeably, blocking antibody kept its protective effect for less than24hours for its protein property, whereas RNAi therapy by using shRNA-expressing vector could last for7days, indicating RNAi gene therapy has advantages over blocking antibody in long-lasting in vivo efficiency.Although there was no difference in recruitment of MNCs and NK cells to liver and NKG2D expression on NK cells after Poly (I:C)/D-GalN injection among pRNAT-shNNN-, pRNAT-shRNN-or pRNAT-shRMH-treated mice, we found intracellular IFN-γ, one of critical cytokines produced by activated NK cells, decreased in pRNAT-shRMH-treated mice by flowcytometry, from18.28%to2.81%, indicating NKG2D-mediated NK cell activation was inhibited after knockdown of its ligands on hepatocytes.4. Multiple shRNA-expressing recombinant adenovirus were constructed and found efficient to protect mice from polyI:C/D-GalN induced fulminant hepatitisAfter verification of high efficient protective effect of simultaneous knockdown of multiple NKG2D ligands on liver immune injury by hydrodynamics-based procedure, we packaged the shRNA-expressing plasmids with clinical grad recombinant adenovirus for its hepatotropism and high infection efficiency(Wilson,1996). Triple shRNA-expressing vector pRNAT-shRMH and control vector pRNAT-shNNN were further reconstructed with Adeno-X Expression System, forming Adeno-shRMH (called Ad-RMH) and Adeno-shNNN (called Ad-NNN). Similarly, recombinant adenovirus Ad-RMH prevented mice from the fulminant hepatitis either by hydrodynamic injection or by i.v. infection, when administrated3days prior to Poly (I:C)/D-GalN injection. Ad-RMH-treated mice showed significant alleviation of liver immune injury, showing decrease in serum ALT levels. Surprisingly, this protective effect could last more than10days, since when mice were i.v. infected with Ad-RMH at10d,7d or3d before Poly (I:C)/D-GalN injection, the protection could sustain.5. Simultaneous knockdown of multiple ligands of mouse NKG2D prevents ConA induced fulminant hepatitis in HBsAg-Tg miceIn addition to the Poly (I:C)/D-GalN-induced hepatitis, we wondered if the multiple shRNA expressing vector is also effective in other inflammatory models. It was previously reported that NKG2D recognition of hepatocytes by NK cells plays critical role in oversensitive liver injury during chronic HBV infection. HBsAg-Tg mice were more sensitive to acute liver injury after ConA stimulation, compared to C57BL/6mice, shown by the levels of serum ALT. It was found that NKG2D ligands Rael and Multl up-regulated on hepatocytes, but not H60. We hydrodynamically injected multiple shRNA expressing vectors3days before ConA stimulation, and excitedly found that the multiple shRNA expressing vectors had similar effect in this inflammatory model. HBsAg-Tg mice showed decreased serum ALT levels and alleviation of liver injury when Rael and Multl were both inhibited, suggesting in vivo application potential of the multiple shRNA expressing vector.6. Simultaneous knockdown of multiple ligands of human NKG2D high efficiently prevents NK cell cytolysis against human hepatocytesIn order to verify whether knockdown of multiple ligands of human NKG2D high efficiently prevents NK cell-mediated liver inflammation in human liver diseases, we tried to construct multiple shRNA expressing vectors against human NKG2D ligands, including MICA/B, ULBP2and ULBP3molecules. Triple (called shMica-shUlbp2/3) and single shRNA expressing vectors against human NKG2D ligands were constructed, and transfected into hepatocytes L-02cells which constitutively expressed surface MICA/B, ULBP2and ULBP3. Stable shRNA-expressing L-02cells were generated and showed the downregulated expression of three NKG2D ligands MICA/B, ULBP2and ULBP3. For example, triple knockdown of three NKG2D ligands led to down-expression of MICA/B, ULBP2and ULBP3, whereas single knockdown of NKG2D ligands led the silence of one corresponding ligand. Furthermore, when incubated with NK cell line NKG cells which highly express human NKG2D, triple shRNA-treated L-02cells became less sensitive to cytolysis of NKG cells than control shRNA-treated L-02, and correlated to lower CD107a expression of NKG cells when co-cultured these NKG cells with NKG2D ligands-silenced L-02cells, indicating inactivation of NK cells by knockdown of NKG2D ligand on hepatocytes may protect against immune injury of human liver.