Molecular Mechanism For The Effects Of E. Coli Heat-labile Enterotoxin On Stability Of Mouse Embryo

Escherichia coli (E. coli) heat-labile enterotoxin (LT), an intestinal toxin secreted by enterotoxigenic E. coli (ETEC), is an important pathogenic factor for animal enteritis and diarrhea, composed of one A subunit (LTA) and five B subunits (LTB). The A subunit has ADP-ribosylation activity and five B subunits can bind GM1 ganglioside on eukaryotic cell membrane. In addition to its cytotoxity, LT also has strong immunomodulatory functions, which can stimulate cytokine productions and therefore is a potent mucosal immune adjuvant. The study showed that ETEC-induced diarrhoea was often accompanied by abortion in pregnant animals. The pathogenic factors affecting embryo stability are not only involved in endotoxin (LPS), but involved in other pathogenic factors. Based on an established theory of embryo stability depending on the balance of immune regulation and LT functions of immunoregulation and cytotoxic effect, we speculated that E. coli-induced abortion was related to disturbance of embryo stability by LT. In order to investigate the effects of LT on animal embryo stability, in this study, we prepared recombinant LT (including wide-type LT and detoxified-mutated LTK63) using E. coli expression system, and treated pregnant mice in vivo and mouse macrophages and two-cell embryos in vitro with the LT to investigate the effects of LT on embryo stability in the following aspects: immunoregulation, inflammasome activity and toxicity on embryo, respectively.The results showed that the recombinant LTA, LTB and detoxified mutant LTK63 were successfully prepared using E. coli expression system. The LT has a powerful cytotoxicity, the LTB possesses the specific binding activity to GM1 ganglioside. As expected, the detoxified mutant LTK63 lacked of cytotoxicity. These results suggest that the LT has the biological activity.To investigate LT effects on the embryo stability and immunoregulation of uterine placental tissue, we treated the pregnant mice with LT and found that LT significantly decreased mouse embryo survival rate. The levels of Th1 cytokines (IFN-γ, IL-2) in uterine placental tissue were significantly increased, but Th2 cytokines (IL-4, IL-10) were not changed, which indicate that LT can shift Th1/Th2 immune balance to Th1 immune response, which was considered as a contributor to embryo instability. The results also showed that LT could enhance IL-1βproduction in uterine placental tissue, which suggest that LT can activate inflammasome, the later promotes pro-IL-1βmaturation and secretion from monocyte/macrophage, resulting in inflammatory reaction in uterine placental tissue. Immunohistochemistry results showed that LT significantly reduced CD8~+ T cells and inceased CD4~+ T cells and macrophage in uterine placental tissue. All these results indicate that LT affects the embryo stability by disturbing Th1/Th2 and CD4~+ T/CD8~+ T immune balance and activating inflammasome.To further verify inflammasome activation by LT, we stimulated LPS-primed mouse macrophage (B6 cells) in vitro with LT and LTK63, respectively, and found that both LT and LTK63 could increase IL-1βsecretion, which suggests that the high level of IL-1βpresented in uterine placental tissue upon LT treatment is related to inflammasome activation in mouse macrophage. By comparing ability of activating imflammasome for LT different subunits (LTA and LTB), only LTA was proved to be able to activate inflammasome.To elucidate the LT-induced inflammasome activation pathway, the two gene-knockout mouse macrophage cell line, the NALP3 (one of the inflammasome sensors)-knockout macrophage and caspase1(cleavage of pro-IL-1β)-knockout macrophage, were used for analysis of inflammasome activation upon LT treatment. The results showed that LT could not elicit IL-1βsecretion from both LPS-primed NALP3-knockout cells and caspase1-knockout cells. These results suggest that LT activates in?ammasome by activating NLRP3/caspase-1 signal pathway.To study the effects of LT on early embryo deveopment, two-cell mouse embryos were treated with LT in vitro, we found that low level of LT did not affect the development of morula and blastocyst. However, the high level of LT resulted in advanced development of morula and blastocyst. The detoxified mutant LTK63 did not affect development of early embryos. Those results indicate that the cytotoxicity of LT may be an important factor affecting the early embryo development.In conclusion, LT can significantly decrease embryo survival rate, enhancing Th1 cytokines expression, decreasing the number of CD8~+T cells in uterine placental tissue, shifting Th1/ Th2 and CD4~+T/CD8~+T immune banlance to the direction which does not benifit to embryonic stability. LT also can activate inflammasome and promote mature IL-1βsecretion in uterine placental tissue, causing inflammatory reaction of the tissue. All of these contribute to disturbance of mouse embryo stability.This study will enrich and refine our knowledge of pathogenic mechanism of E. coli-induced abortion and provide a theoretical basis for prevention and treatment of E. coli-induced abortion.