TWEAK-mediated Modulation And Underlying Molecular Mechanism Of UNK Cytotoxic Function By LPS In Mouse

Uterine NK cells(uNK) are the most abundant lymphocytes at the maternal-fetal interface during early pregnancy. Uterine NK cells are closely associated with the early embryonic blood vessel formation and maternal-fetal immune tolerance, which plays an important role in establishing and maintaining a normal pregnancy. Tumour necrosis factor-like weak inducer of apoptosis(TWEAK) can involve in preventing local cytotoxicity and counterbalancing the cytotoxic function of uNK cells through the interaction with its receptor fibroblast growth factor-inducible molecule 14(Fn14). In order to study the regulation and the underlying mechanism of TWEAK/Fn14 mediated innate immunity in the uterus during early pregnancy, we estabished a lipopolysaccharide(LPS)-induced abortion mouse model. Pathomorphology of the uterus was observed with the use of HE staining; the dynamic distribution changes of uNK cells were detected with the DBA lectin; IHC was used to detect the expression and distribution of TWEAK/Fn14; IF staining was used to detect the colocalazition of TWEAK/Fn14, NKG2 D, TNFα and uNK cells; Western blot experiments were used to evaluate the protein expression of TWEAK and Fn14. Meanwhile, immune magnetic bead separation(MACS) technology was used to separate and purify uNK cells; RT-PCR and Western blot were used to study TWEAK/Fn14-mediated molecular regulation mechanism of uNK cytotoxic function and secretory activity. The results are as follows:1. We injected a dose of 2 ug LPS into each mouse at gestation day(GD) 3, Then the abortion mouse exhibited embryonic loss at GD 9.5. The result of HE staining showed that the uterine tissue of the abortion mouse appeared edema, inflammatory cells infiltration, tissue necrosis, and bleeding. DBA lectin staining revealed at GD 9.5 the number of uNK cells in normal pregengy mouse markedly increased and reached the peak, and the number of uNK cells in LPS treated group was significantly lower than the control group at GD 9.5(P<0.05). IHC method was used to study the localization of the TWEAK and Fn14, we found that TWEAK was mainly detected in the lamina propria and gland of endometrium, and Fn14 was mainly expressed in the luminal and glandular cells. At GD 9.5,we observed that there were very weak TWEAK staining and strong Fn14 staining in the uterus of the LPS-treated mice. Western blot analysis revealed that the protein levels of both TWEAK and Fn14 were in accord with the results of immunohistochemistry. The immunofluorescence assays revealed that TWEAK/Fn14 could be expressed in uNK cells and TWEAK was mainly expressed in uNK cells. The immunofluorescent localization aslo found that the proportion of the TNFα +and NKG2D+ uNK cells in the total uNK cells was obviously increased(P<0.05).2. In order to further study the impact of TWEAK/Fn14 pathway on the molecular regulation of uNK cell toxicity function and secretory activity induced by LPS, we isolated and purified the uNK cells of the mouse uterine in normal pregnancy and LPS-treated group by using DBA lectin-coated magnetic beads. The results of the western blot and RT-PCR showed that the expression level of TWEAK and Fn14 protein in uNK cells was consistent with the uterine tissue. The level of NKG2 D and TNFα was increased in the uNK cells in LPS-treated group compared with the control group. In order to study the impact of TWEAK on the expression modulation of NKG2 D and TNFα in uNK cells, we used the isolated uNK cells treated with the different concentration of recombinant TWEAK protein and TWEAK-special antibody. Western Blot analysis did not show any significant difference in NKG2 D and TNFα expression treated with any concentration of recombinant TWEAK protein, however, the TWEAK-special antibody resulted in a significant dose-dependent up-regulation of NKG2 D and TNFα expression. Based on the above research results and related research reports, we chose the JAK2-STAT3 signaling pathway for further research. Compared to the control group, we found that the expression of pSTAT3 was all obviously decreased in WP1066 group, anti-TWEAK group, WP1066 and anti-TWEAK group, and WP1066 and TWEAK group(P<0.05). Meanwhile, Western Blot detected that the negative feedback mechanism exsited in both the expression of pSTAT3 and the expression of NKG2 D and TNFα.To sum up, the research studied the TWEAK-mediated regulation and molecular mechanism of uNK cytotoxic function and secretory activity by establishing the LPS-induced abortion mouse model, isolating and purifying uNK cells. The study illustrated the TWEAK/Fn14 molecular regulation mechanism in the abortion induced by LPS, namely, the level of endogenous TWEAK regulated the level of phosphorylation of STAT3 and then led to the transcription regulation of NKG2 D, which changed the cytotoxic function of uNK cells that increased TNFα secretion level. And the accumulation of TNFα induced the occurrence of abortion in normal pregnancy mouse.