The Role Of Endoplasmic Reticulum Stress In Brucella-infected Goat Endometrial And Trophoblast Cells In Vitro

Pregnant domestic animals abortion is the most common complication caused by Brucella infection. Trophoblast cells are the main target cells of Brucella except monocyte-macrophages, and endometrial epithelial cells are the structure- and function-related cells with trophoblast cells. Previous study confirmed that Brucella infects and proliferates in association with the endoplasmic reticulum(ER). The ER can be used as the proliferation site by Brucella to escape from immune-mediated killing. ER also is the main place for various protein synthesis, transport, secretion and plays an important role for the maintenance of cellular homeostasis. When the cell homeostasis is broken in pathogenic bacteria infections, ER stress and the unfolded protein response of host cell is activated to restore the normal function of host cells. The formation of ERA-BCV(Endoplasmic reticulum associated-Brucella containing vacuoles) and the inhibition of host cell apoptosis are key for Brucella immune escape, survival and proliferation in Brucella-infected cells. The ER stress caused by the unbalance of ER homeostasis is one of the pathways of cell apoptosis. However, the mechanism of ER stress in the process of Brucella infecting domestic animals uterus and inducing pregnancy animal abortion is still unclear.In this study, Brucella suis strain 2(B.suis S2) was used to infect goat trophoblast cells(GTC) and endometrial epithelial cells(EEC), inhibitors/activators of ER stress and multiple methods including immunofluorescence, gene over-expression and gene silence, q RT-PCR, FCM, Western Blot were use to explore:(1) The infectious ability of B.suis S2 to goat trophoblast cells and endometrial epithelial cells;(2) The apoptosis of goat trophoblast cells and endometrial epithelial cells in B.suis S2 infections;(3) The activation of ER stress in B.suis S2-infected goat trophoblast cells and endometrial epithelial cells;(4) The proliferation of B.suis S2 in goat trophoblast cells under the ER stress is controled;(5) The bioactivity of goat trophoblast cells and endometrial epithelial cells in B.suis S2 infections.1. According to our laser confocal microscopy and colony counting results, we found that B.suis S2 can infect not only goat trophoblast cells but also endometrial epithelial cells, increasing along with prolonged time.2. Here we provided the evidence that B.suis S2 has different extent of damage to different kind of host cells. B.suis S2 can induce cell apoptosis and the expression of apoptosis related genes in B.suis S2-infected GTC. However, B.suis S2 did not induce cell apoptosis and remarkable change of apoptosis related genes in B.suis S2-infected EEC by FCM, immunofluorescence, Western Blot and q RT-PCR. So the damage to trophoblast cells in Brucella infections maybe is one of the primary causes of abortion.3. According to our immunofluorescence, Western Blot and q RT-PCR results, we found that ER stress is activated not only in B.suis S2-infected GTC but also in B.suis S2-infected EEC along with prolonged time. However, different UPR pathways are activated, the most likely reason is that different host cells responded to Brucella infection with a distinct resistance.4. By means of immunofluorescence, Western Blot and q RT-PCR, manipulating the ER stress induced host cell apoptosis and autophagy response can affect the proliferation of B.suis S2 in GTC with ER stress activator(Tm), inhibitor(4-PBA), IRE1 inhibitor(Irestatin 9389), autophagy inhibitors(chloroquine) and GRP78 gene over-expression and silence. However, the proliferation of B.suis S2 was not significantly influenced under the conditions of IRE1α protein expression manipulated alone.5. The goat pregnant uterus model was constructed with goat trophoblast cells and endometrial epithelial cells in combination with progesterone in vitro. In the process of B.suis S2 infecting the model in vitro, we found that pregnancy goat abortion and premature delivery were mainly caused by destroying the endocrine balance of goat trophoblast cells and the receptivity of endometrial epithelial cells rather than damaging the migration and invasion of trophoblast cells. ER stress also activated along with B.suis S2 infecting the model in vitro.Taken togehter, ER stress plays an important role in the proliferation of Brucella in Brucella-infected GTC and EEC. Different UPR pathways are activated when Brucella proliferating in different host cells. ER stress related apoptosis and autophagy affects the proliferation of B.suis S2 in GTC. Furthermore, not only the endocrine balance of trophoblast cells under normal growth conditions but also endometrial receptivity was defective, which may be a reason for the abortion and premature delivery induced by Brucella infection. This study explored the mechanisms of abortion in Brucella infection from the viewpoint of pathogen invasion, ER stress and reproductive endocrinology. Our study will provide new theoretical basis for perfecting the regulation mechanism of Brucella infections and the mechanism of the infertility and miscarriage in Brucella infections...