The Effects Of Galectin-9/Tim-3Signaling On The Function Of NK Cells

ObjectiveHepatitis B virus is a kind of hepadnavirus. HBV infection causes a series of liver inflammation, including hepatitis, liver fibrosis, liver cirrhosis, and even liver cancer.HBV infection is prone to persist into chronic infection in live, which might resulted from the immune tolerance in liver microenvironment. In the early stage of HBV infection, NK cells represent the main effector cells involved in innate immune responses to the viral infection.There are both co-stimulatory (e.g. CD28) and co-inhibitory molecules(e.g-. PD-1,2B4) on the surface of NK Cells. HBV infection leads to the up-regulation of co-inhibitory molecules on NK cells, which inhibits the functions of NK cells, relsulting in immune tolerance. It is demonstrated that the co-inhibitory molecule Tim-3on NK92cells transfected with HBV expression plasmid or primary NK cells from the liver of HBV-transgenic mouse increased. Blocking the Tim-3/Galectin-9signal pathway could restore the function of NK cells. Therefore, in the process of chronic HBV infection, Galectin-9/Tim-3signal pathway is closely related to the damaged function of NK cells. In the present study, we first observed the influence of Galectin-9on hepatocytes on the functions of NK cells.On the basis of observing the differences of Galectin-9expression on HepG2, HepG2-HBV and HepG2.2.15cell lines, the lysis of NK cells against the three types of cell lines was detected. The level of IFN-y and the apoptosis of NK cells were assayed at the same time. On the other hand, studies have reported that Tim-3was the co-receptor of NK cells. So, we next investigated the Galectin-9/Tim-3signal status on the function of NK cells. Firstly, NK cells were stimulated with different amount of recombinant human Galectin-9(rh-Galectin-9) or HepG2cells were transfected with different concentration of Galectin-9expression plasmid or transfected for different times. Secondly, the target cells were incubated with different amount of α-Lactose, which blocked Galectin-9/Tim-3signal pathway. The function of NK cells was investigated while changing the signaling status through these two aspects. Methods1. Q-PCR and FACS were applied to confirm the expression of Galectin-9on HepG2, HepG2-HBV, HepG2.2.15cells and primary hepatocytes from HBV+mouse or liver cancer patients. The expression of Galectin-9on HepG2-transfected with Galectin-9expression vector or GATA-2over-expression plasmid or HepG2.2.15cells after silencing GATA-2or GATA-3was also determined via RT-PCR and FACS. The expression of Galectin-9on HepG2and HepG2.2.15cells transfected with microRNA inhibitors or mimics respectively was also tested by FACS.2. MTT and CFSE/7AAD methods were used to test the lysis of NK cells against different types of cell lines.3. The level of IFN-y, CD107a and Perforin by NK cells were assayed by FACS.4. The apoptosis of NK cells incubated with different target cells was determined by Annexin V/PI double staining.Part ⅠHBV infection impairs the function of NK cells by up-regulating Galectin-9expression on hepatocytesResults1. The level of Galectin-9expression was higher on HepG2.2.15and HepG2-HBV cells compared with HepG2cells. The expression of Galectin-9on primary hepatocytes from HBV+mouse and liver cancer patients was also up-regulated than healthy controls.2. The cytolyticability of NK cells and the level of IFN-y secreted by NK cells decreased after incubated with higher Galectin-9expression cells HepG2.2.15and HepG2-HBV significantly, compared with low Galectin-9expression cell HepG2. 3. The Annexin V+%NK cells incubated with HBV+HepG2.2.15cells increased than with HepG2cells.4. Recombinant Galectin-9protein inhibited the cytolysis of NK cells and promoted NK cells undergo apoptosis.5. Blocking the Tim-3/Galectin-9signal pathway with α-Lactose increased the cytolyticability of NK cells and the level of IFN-γ.6. The level of transcription factors GATA-2and GATA-3was higher in HepG2.2.15cells than that in HepG2cells. Sliencing of GATA-2or GATA-3down-regulated Galectin-9expression in HepG2.2.15cells. Over-expression of GATA-2up-regulated Galectin-9expression on HepG2cells. microRNA-2, microRNA-575and microRNA-1275were not involved in the regulation of Galectin-9expression.Conclusions1. HBV infection up-regulated the expression level of Galectin-9on hepatocytes.2. The cytolyticability of NK cells and the level of IFN-γ secreted by NK cells decreased after incubated with higher Galectin-9expression cells.The target cells with higher Galectin-9expression promoted NK cells undergo apoptosis.3. GATA-2or GATA-3might be involved in the regulation of Galectin-9expression. microRNA-22, microRNA-575and microRNA1275were not the regulators of Galectin-9expression.Part ⅡThe effects of Galectin-9/Tim-3signaling status on the function of NK cellsResults1. FACS results indicated that the Galectin-9was expressed at a low, moderate and high level on Raji, HL-60and Jurkat cells, respectively. 2. The cytolyticability of NK cells against Raji, HL-60and Jurkat cells with different expresson level of Galectin-9was similar.3. The Galectin-9expression plasmid was constructed successfully.4. The lysis of NK cells increased after incubated with HEK-293cells transfected with Galectin-9expression plasmid.5. With the increasing concentration of rh-Galectin-9to stimulate NK cells, the cytolyticability of NK cells against HepG2cells was increasingly elevated, and then dropped.6. When the doses of α-Lactose used was lower, the lysis of NK cells against HCT-116and HL-60cells increased, and then dropped; when the doses were higher, the lysis of NK cells decreased.7. Galectin-9expression on HepG2cells was increasingly elevated accompanied by different concentration of over-expression plasmid transfected. Meanwhile the cells displayed increasingly high sensitivity to NK cytolysis, and then dropped. On the other hand, after the over-expression plasmid was transfected into HepG2cells for24h,48h, and72h, Galectin-9expression was gradually increased then dropped, especially at the lowest level at24h. Notely, the HepG2cells showed the highest sensitivity to NK cytolysis.Conclusions1.The strength of Galectin-9/Tim-3signaling displayed different influence on the cytolyticability of NK cells.2. Specifically, the cytolytic ability was enhanced when the signaling was weak, while decreased as the signaling was strong.3. A weak Tim-3signaling might be necessary for NK cell activation and cytolytic ability; however, a strong Tim-3signaling might lead to dysfunction or apoptosis of NK cells.