Lactobacillus rhamnosus GR-1 ameliorates Trueperella pyogenes–induced barrier dysfunction of bovine endometrial epithelial cells

Trueperella pyogenes is a common opportunistic pathogen which is one of the main causes of postpartum endometritis in dairy cows. As a substitute for antibiotics, the probiotic Lactobacillus rhamnosus GR-1 has been used in a wide range of clinical treatments. Our experiments were designed to establish a model of anti-damage which LGR-1 was used to protect bovine endometrial epithelial cells (BEECs) from inflammatory damage and cell destruction caused by T. pyogenes. Increased expression of NLRP3 inflammasomes and cytokines was observed following T. pyogenes challenge, but this increase was relieved by LGR-1 pretreatment. Immunofluorescence and Western blot analyses revealed that T. pyogenes infection also results in the damage of tight junction proteins in BEECs. The expression levels of Claudin-1, Occludin, and ZO-1 were decreased in cells only infected with T. pyogenes but not in cells pretreated with LGR-1. Moreover, the detection of the anti-apoptotic protein Bcl-2 and apoptotic proteins BAX, cytochrome c, as well as the activating effector caspase-3 revealed that T. pyogenes induced apoptosis of BEECs, which was also confirmed by DAPI staining to observe the morphological changes of the nuclei of cell apoptosis and by TUNEL staining to locate the cells undergoing apoptosis. Our data indicate that LGR-1 ameliorates the T. pyogenes–induced barrier dysfunction of BEECs and pre-application of LGR-1 could be an effective strategy for controlling T. pyogenes infection.

preventing and treating a variety of intestinal disorders through several regulation mechanisms [46].
The probiotic Lactobacillus rhamnosus GG (LGG) may be an adjunct therapy to attenuate and prevent the course of infection, which will allow more judicious use of prophylactic antibiotics and hopefully reduce overall usage [47]. Our previous study showed that probiotic Lactobacillus rhamnosus GR-1 ameliorates E. coli-induced inflammatory damage via attenuation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-independent NLRP3 inflammasome activation in primary bovine mammary epithelial cells (PBMCs) [48]. However, the use of LGR-1 as a probiotic treatment in protecting cows from postpartum endometritis is rarely reported and the mechanism is still unknown. Using LGR-1 to cure endometritis caused by bacteria provides a significant advantage and requires more energy to study its mechanisms.
The aim of the study is to establish a model of endometrial epithelial cells invaded by clinically isolated T. pyogenes in order to explore the processes and mechanisms of the barrier dysfunction and cell apoptosis and to investigate the potential mechanisms of LGR-1 against T. pyogenes infection.

Bacteria
The 8 clinical T. Pyogenes isolates were collected from uterine lavage fluid using the uterine infusion pipette with an inflated balloon and stainless-steel pins from dairy cows within 15 days postpartum with clinical endometritis. The bacteria were recovered from sheep blood plate medium and a single colony was cultivated in 10 ml brain heart infusion (BHI) broth (Aobox, Beijing, China) at 37 ℃ overnight under aerobic conditions. All of the T. pyogenes were harvested by centrifugation and re-suspended twice with Dulbecco's phosphate-buffered saline (PBS; Solarbio, Beijing, China) and counted under the optical microscope.
LGR-1 ATCC 55826 was purchased from the American Type Culture Collection (Manassas, VA, United States) and grown in De Man, Rogosa, and Sharpe (MRS) broth (Aobox, Beijing, China) for 24 h at 37 ℃ under microaerophilic conditions [48]. LGR-1 was inoculated and grown in fresh MRS broth for 8 h at 37 ℃ with the beginning ratio of 1:100 after passing the mid-log phase. The LGR-1 was harvested, re-suspended, and counted as the same method as T. pyogenes. The concentration of LGR-1 was used as 5 × 10 7 CFU/ml.

Bovine Cell Culture
For cell culture, BEECs were obtained from the uterine horn and processed as the method previously described [49]. The BEECs were cultured with Dulbecco's Modified Eagle medium/Ham's F-12 medium (1:1) and supplied with 10% heat-inactivated fetal horse serum, 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, United States), respectively. After flowing evenly into cell flasks, the BEECs were cultured at 37 ℃ in an incubator with 5% CO2 atmosphere.

Bacterial Growth Curves
To examine different growth rates of all clinical T. pyogenes isolates and the standard strain, bacterial growth curves were measured for all of 9 T. pyogenes, respectively. Sterilized turbidity tubes were added 1 ml BHI broth and bacterial solution was dropped until its turbidity is 0.2. 198 l Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 October 2020 doi:10.20944/preprints202010.0185.v1 of BHI broth was added to each well into a 96-well plate and then 2 l of bacterial solution with a turbidity of 0.5 was added to each well. Each T. pyogenes was repeated three times. The plate was placed in the microplate reader and measured OD600 every hour for a continuously 30 h measurement.

Adhesion Assay
BEECs were harvested onto six-well cell culture plates. After growing to 80% full, cells were washed three times with PBS. Cells were pre-incubated with LGR-1 (5 × 10 7 CFU/ml) for 3 h.
LGR-1 was washed for three times with PBS and cells were then exposed to T. pyogenes (10 7 CFU/ml) for 9 h. Cells were washed three times with PBS to eliminate non-adherent bacteria and treated with 0.05% Triton X-100 for 15 min at room temperature. After harvested by centrifugation for 10 min, cell lysate and cell supernatant were diluted by 10-fold into 4 concentration gradients and 10 l of each gradient of cell supernatant and lysate were evenly spread on blood plates and for three repeats. Colonies were counted after incubation in a 37 ℃ incubator for 48 h.

Lactate Dehydrogenase (LDH) Assay
Different treatments of cells were evaluated with CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, United States) following the instructions of manufacturer. Cells were challenged by T. pyogenes for 9 h which MOI was 5 for each challenged well. The results were calculated by the following formula: % cytotoxixity = (sample-control)/(max lysis-correction volume-control+background).

Western Blotting
Cells were pre-incubated with LGR-1 for 3 h and then exposed with T. pyogenes for 9 h in six-well cell culture plates as the method mentioned above. After T. pyogenes challenged for 9 h, cells were washed for three times with sterilized PBS and simultaneously extracted for western blot assay. The primary antibodies were rabbit polyclonal anti-ASC (1:500 dilution, 10500-1-AP), rabbit polyclonal anti-claudin-  (5´-AGA GTT TGA TCM TGG CTC AG-3´) and 1492R (5´-TAC   GGY TAC CTT GTT ACG ACT T-3´

Immunofluorescence
The supernatant was discarded from a 24-well plate with a cell fusion rate of 80% growing on glass coverslips. With PBS washing three time gently, LGR-1 was pre-applied for 3 h and cells were then exposed to T. pyogenes for 9 h. Bacteria were washed for three times with PBS. After washing as mentioned above, 500 μL of 4% PFA was added to each well and cells were fixed at room temperature for 10 min. For the next step, 500 μL of 1% Triton X-100 was dropped to per well at room temperature for 15 minutes and washed three times with PBS following with 2% BSA blocking at room temperature for 1 h. For tight junction experiment, primary antibody (ZO-1, 1:100 dilution) was applied and incubated at 4 ℃ overnight and Alexa Fluor 488 goat anti-rabbit as the secondary antibody was applied and incubated in dark at room temperature for 1 h.
For apoptosis experiment, nuclei of cells were stained with DAPI at room temperature for 10 min. Glass coverslips was face down on slides with the anti-fluorescence quencher drip-proof and mounted with neutral resin. Slides were observed and pictured with Nikon A1 confocal laser scanning microscope.

TUNEL
The BEECs were cultivated on the 24-well cell slides. After the cells were ready for use, the LGR-1 was applied for 3 h in advance after washing three times gently with PBS. Then, TP1804 was infected the BEECs for 9 h. The apoptosis cells were marked with TUNEL kit from Beyotime (Cat. No.C1806; Beijing, China) following the instructions provided by the manufacturer after the cells were treated as the same method as the immunofluorescence. The Nikon A1 confocal laser scanning microscope was employed for images.

Statistical Analysis
Every experiment was repeated at least for three times which obtained similar outcomes. All data were analyzed by GraphPad Prism 8. The western blotting results were preformed by ImagineJ software and then analyzed by GraphPad Prism 8. All data were preformed with statistical methods of one-way ANOVA and t-test. Multiple corrections were applied by Tukey's honestly significant difference for post hoc test. Data are presented as the mean ± SEM which were calculated from three similar parallel experiments. A P value of < 0.05 was considered statistically significant.

Screening clinical isolates of T. pyogenes based on bacterial growth characteristics
The obvious hemolytic rings and needle-like white colonies were exhibited on a sheep blood medium after the isolated and purified T. pyogenes was cultured at a 37 ℃ incubator for 48 h ( Figure   1C). T. pyogenes was observed with strong mobility and high moving speed and the bacteria were short rod-shaped under light microscopy. The length of T. pyogenes is 2-3 m and the diameter is The growth curves of 8 isolates of T. pyogenes clinical strains from TP1801 to TP1808 and a standard strain of T. pyogenes ATCC19411 were measured from 0 h to 30 h ( Figure 1A). The clinical strains TP1804 and TP1806 grew fastest. TP1806 reached its mid-log phase at about 7 h and entered the plateau phase, while TP1804 passed its mid-log phase at 9 h and its peak was slightly higher than its of TP1806. The growth curves of the four strains, the standard strain, TP1801, TP1802, and TP1803, were relatively consistent and the growth rates were slow to pass the mid-log phase values which were much lower than the values of TP1804 and TP1806 in approximately 18 hours. The mid-log phase values of TP1805 and TP1807 were still not reached within 27 hours and the mid-log phase value was very low. TP1808 basically had no obvious growth.
The six strains, TP1801 to TP1806, were detected to equip all six virulence genes. Only FimA was not detected in the standard strains, and FimC was not detected in TP1807 and TP1808. The carrying percentage of PLO, fimE, nanH, and nanP in these 9 strains was 100%; the carrying percentage of fimA was 88.9%, and the carrying percentage of fimC was 77.8% (Table 3). Quantitative measurement of lactate dehydrogenase (LDH) method was used to determine the cell mortality after 3 h, 6 h, and 9 h of challenge of T. pyogenes ( Figure 1B). The average mortality rates were about 10%, 30%, and 40% at 3 h, 6 h, and 9 h, respectively. Comparing 3 h, 6 h, and 9 h, it is not difficult to find out that the cell death rates of the standard strain and TP1801 to TP1805 have significantly increased with the extension of time. The mortality of cells challenged by TP1806-TP1808 increased significantly at 6 h, but there was no significant change between 6 h and 9 h. The mortality of TP1806-TP1808 increased significantly at 6 h, but there was no significant change between 6 h and 9 h. In each time period, TP1804 had a particularly strong virulence causing the highest cell death rate among all T. pyogenes strains. The cell death rates of the standard strains, TP1801, TP1802, and TP1805 were at an intermediate level among all strains and TP1803 caused the the lowest cell mortality. Figure 1E is exhibited as an evidence that TP1804 infection increased the cell death rate significantly, which LGR-1 protection had decreased the cell death rate.
However, there was statistic difference between control cells and LGR-1 protected cells.
Pre-incubation LGR-1 before 3 h of TP1804 challenge demonstrated, compared with TP1804 treated alone, the adhesion rate of TP1804 was reduced nearly 60%, which is indicating that LGR-1 significantly inhibited a certain extent of adhesion of T. pyogenes to BEECs.

Lactobacillus rhamnosus GR-1 attenuates the BEECs damage caused by T. pyogenes
In order to observe the changes in cell morphological structure and the degree of 10 protein was also significantly increased in the TP1804 challenged cells (P < 0.001) ( Figure 3C). In contrast, the expression level of caspase-1 p 10 protein in the cells protected by LGR-1 was considerately reduced (P <0.001) and was slightly higher than that in control cells.
In order to investigate whether LGR-1 is capable to ameliorate the inflammatory responses LGR-1 had a significant decrease and were higher than these factors in control cells except for the CXCL-1/2 factor which was lower than that in control cells without statistical difference ( Figure 3F).
With the TP1804 challenge, the expression levels of TNF-α and CXCL-3 in BEECs increased compared with control cells after 9 h treatment. However, the expression of these two factors in cells challenged by TP1804 did not show statistically significant modulated compared with these factors in control cells ( Figure 3H, I).
3.1.5 Pre-treatment of Lactobacillus rhamnosus GR-1 reduces the disruption of tight junctions in

BEECs infected by T. pyogenes
In order to investigate whether LGR-1 is bale to improve the damage of tight junction structures caused by T. pyogenes, western blotting was used to quantitatively analyze the expression of tight junction proteins in BEECs, including ZO-1, Claudin-1, and Occludin ( Figure 4A-C). As shown in Figure 4A, the expression of Occludin in the BEECs challenged by TP1804 was significantly lower than that in control cells in 9 h after challenge (P <0.01).
LGR-1 alone treated cells had a slight decrease compared with control cells. Compared with the cells challenged by TP1804, the expression of Occludin in the LGR-1 protected cells increased significantly, but it was still lower than that control cells.
The expression of Claudin-1 in BEECs infected by TP1804 was significantly lower than that in control cells after 9 h challenge (P <0.05) ( Figure 4B). Compared with control cells, the expression of Claudin-1 in the cells treated by LGR-1 alone showed a slight increase without significant difference.
The expression of Claudin-1 in the LGR-1 protected cells was significantly higher than that in the cells infected by TP1804, and was slightly higher than that in control cells.
As shown in the Figure

Pre-treatment of Lactobacillus rhamnosus GR-1 decreases apoptosis of BEECs infected by T. pyogenes
In the study of apoptosis caused by T. pyogenes to cells, western blotting was executed to detect proteins that are closely related to apoptosis, including BAX, Bcl-2, caspase-3, and cytochrome c. In Figure 5A, the expression of BAX in the TP1804 challenged cells was obviously up-regulated compared with control cells and LGR-1 protected cells with statistically significant difference. In Figure 5B, the expression of Bcl-2 in the TP1804 challenged cells was down-regulated significantly compared with control cells. The relative amount of Bax is higher than Bcl-2. However, the expression of LGR-1 protected cells was higher than that in the cells challenged by TP1804 without statistically significant difference. Moreover, the expression levels of the caspase-3 p 19 protein and cytochrome c were increased significantly distinguished between the TP1804 treated cells and control cells. The LGR-1 had down-regulated the effects of apoptosis caused by T. pyogenes with P-value < 0.01. However, LGR-1 had no significant influences on the expression of the caspase-3 p 19 protein caused by T. pyogenes ( Figure 5C-E).
The pictures are exhibited the DAPI staining of nuclei of BEECs ( Figure 5F). In the TP1804 infected cells, the nuclei were shrank and deformed to form an irregular shape with empty spots compared with control cells. The TUNNEL staining was applied for confirming the BEECs apoptosis caused by T. pyogenes and the effects of LGR-1 against the T. pyogenes on attenuating the cell apoptosis ( Figure 5G). The photos show that the apparent apoptosis in the TP1804 infected cells which nuclei are bright and irregular due to the treatment of T. pyogenes.  immunofluorescence photographs show comparison of tight junction protein ZO-1 in T. pyogenes destruction and Lactobacillus rhamnosus GR-1 protection. T. pyogenes was exposed to BEECs for 9 h and Lactobacillus rhamnosus GR-1 was applied 3 h in advance in the protected cells. The mean ± SEM of data was shown from three independent repeat. (*P < 0.05, **P < 0.01, ***P < 0.001) through negative feedback regulation [51]. TNF-α and CXCL-3 showed a slight increase with no significant change in expression after TP1804 challenge. In vitro [16[ and in vivo [52] studies provided evidence that CXCL3 plays a integrative role in uterine inflammation in cows [53].

Figures and Tables
Nevertheless, the biological significance of CXCL3 is not fully understood, as CXCL3 is constitutively secreted by epithelial cells also in the absence of inflammation [54].
NLRP3 is activated by a wide variety of stimuli, including pore-forming toxins, extracellular adenosine triphosphate, RNA-DNA hybrid molecules, and pathogens [55]. Caspase-1 is thought to be activated by a proximity-induced dimerization and autoproteolytic process in the NLRP3/ASC complex platform [56]. Active caspase-1 cleaves pro-IL-lβ and pro-IL-18 into mature IL-lβ and IL-18, which are essential for coordination of immune responses to pathogen infection through allograft neutrophil sequestration, mononuclear phagocyte recruitment, and T-cell activation [57]. In this study, we also found the high connectivity of inflammatory proteins and inflammatory factors. The previous studies in our laboratory have shown that Lactobacillus rhamnosus GR-1 inhibited ASC-dependent NLRP3 activation, thereby improving inflammatory damage caused by E. coli [58].
A major challenge is to identify as early as possible the precise molecular mechanisms that regulate normal inflammation in healthy animals and those which signal pathological inflammation in cattle susceptible to clinical endometritis [2]. In this study, the expression of ASC, NLRP3, and caspase-1 quantitatively detected by western blotting was increased significantly after 9 h of TP1804 challenge. provides an effectively protection on apoptosis. In addition, the members of the Bcl-2 family are a group of crucial regulatory factors in apoptosis [65].

Conclusions
In conclusion, T. pyogenes is able to inhibit the expression of tight junction proteins ZO-1, Claudin-1, and Occludin, damage the tight junction structure between cells, destroy the integrity of BEECs. In addition, apoptosis induced by T. pyogenes was found after TP1804 infection. The application of LGR-1 relieves the destruction of BEECs caused by T. pyogenes and attenuates the degree of apoptosis induced by T. pyogenes. Our findings suggest that LGR-1 might be used as a new strategy for prevention and treatment of bovine postpartum endometritis. The determination of the method of administration of LGR-1 (uterine catheter administration, oral administration, etc.), dosage, and administration time requires further in vivo and in vitro studies in conjunction with the reproductive cycle of dairy cows.

Conflicts of Interest:
The authors declare the statement of interests with no conflict.