Oxysterols protect bovine endometrial cells against pore-forming toxins from pathogenic bacteria

Abstract

Many species of pathogenic bacteria secrete toxins that form pores in mammalian cell membranes. These membrane pores enable the delivery of virulence factors into cells, result in the leakage of molecules that bacteria can use as nutrients, and facilitate pathogen invasion. Inflammatory responses to bacteria are regulated by the side-chain-hydroxycholesterols 27-hydroxycholesterol and 25-hydroxycholesterol, but their effect on the intrinsic protection of cells against pore-forming toxins is unclear. Here, we tested the hypothesis that 27-hydroxycholesterol and 25-hydroxycholesterol help protect cells against pore-forming toxins. We treated bovine endometrial epithelial and stromal cells with 27-hydroxycholesterol or 25-hydroxycholesterol, and then challenged the cells with pyolysin, which is a cholesterol-dependent cytolysin from Trueperella pyogenes that targets these endometrial cells. We found that treatment with 27-hydroxycholesterol or 25-hydroxycholesterol protected both epithelial and stomal cells against pore formation and the damage caused by pyolysin. The oxysterols limited pyolysin-induced leakage of potassium and lactate dehydrogenase from cells, and reduced cytoskeletal changes and cytolysis. This oxysterol cytoprotection against pyolysin was partially dependent on reducing cytolysin-accessible cholesterol in the cell membrane and on activating liver X receptors. Treatment with 27-hydroxycholesterol also protected the endometrial cells against Staphylococcus aureus α-hemolysin. Using mass spectrometry, we found 27-hydroxycholesterol and 25-hydroxycholesterol in uterine and follicular fluid. Furthermore, epithelial cells released additional 25-hydroxycholesterol in response to pyolysin. In conclusion, both 27-hydroxycholesterol and 25-hydroxycholesterol increased the intrinsic protection of bovine endometrial cells against pore-forming toxins. Our findings imply that side-chain-hydroxycholesterols may help defend the endometrium against pathogenic bacteria.

Keywords: cattle; cholesterol; cytoprotection; liver X receptor; uterus.

FIGURE 1

27‐hydroxycholesterol protects endometrial cells against pyolysin‐induced damage. Leakage of lactate dehydrogenase (LDH) into supernatants and viable cells determined by MTT assay for endometrial epithelial (A) and stromal cells (B) challenged for 2 h with control medium () or the indicated amounts of pyolysin (). Data are presented as mean (SEM) using cells from 4 independent animals; statistical significance is determined using ANOVA. Leakage of LDH and viability of epithelial (C) and stromal cells (D) treated for 24 h with vehicle (V) or the indicated concentrations of 27‐hydroxycholesterol, and then challenged for 2 h with control medium () or pyolysin (, epithelium 200 HU, stroma 25 HU). Data are presented as mean (SEM) using cells from ≥3 independent animals; statistical significance is determined using two‐way ANOVA and p values reported for the effect of 27‐hydroxycholesterol on the pyolysin challenge. Percent viable epithelial (E) and stromal cells (F) treated for the indicated times with 25 ng/ml 27‐hydroxycholesterol, and then challenged for 2 h with control medium or pyolysin. Dots represent individual data points using cells from 4 independent animals, and Pearson correlation coefficients are reported (r). Fluorescent microscope images of epithelial (G) and stromal cells (H) treated for 24 h with vehicle or 25 ng/ml 27‐hydroxycholesterol (27HC), challenged for 2 h with control medium or pyolysin, and then stained with Alexa Fluor 555‐conjugated phalloidin to visualize actin (white; nuclei are red; scale bars are 20 μm). Images are representative of cells from 4 independent animals

FIGURE 2

Side‐chain‐hydroxycholesterols protect endometrial cells against pyolysin. Leakage of LDH into supernatants and viable epithelial (A) and stromal cells (B) treated with vehicle (V) or the indicated concentrations of 25‐hydroxycholesterol for 24 h, and then challenged for 2 h with control medium () or pyolysin (, epithelium 200 HU, stroma 25 HU). Data are presented as mean (SEM) using cells from 4 independent animals; statistical significance is determined using ANOVA, and p values are reported for the effect of 25‐hydroxycholesterol treatment on pyolysin challenge. Potassium in supernatants of epithelial (C) and stromal cells (D) treated with vehicle, 25 ng/ml 27‐hydroxycholesterol (27HC), 5 ng/ml 25‐hydroxycholesterol (25HC) or 0.5 mM methyl‐β‐cyclodextrin (MβCD) for 24 h, and then challenged for 5 min with control medium (○) or pyolysin (●). Data are presented as mean (SEM) using cells from ≥4 independent animals; statistical significance is determined using one‐way ANOVA and Bonferroni's post hoc test. Leakage of LDH and viability of epithelial (E) and stromal cells (F) treated with vehicle, 25 ng/ml 27‐hydroxycholesterol (27HC) or 5 ng/ml 25‐hydroxycholesterol (25HC) for 24 h, and then challenged for 24 h with control medium () or pyolysin (). Data are presented as mean (SEM) using cells from ≥3 independent animals; statistical significance is determined using one‐way ANOVA and Bonferroni's post hoc test. Leakage of LDH into supernatants and viable epithelial (G) and stromal cells (H) treated with vehicle (V) or the indicated concentrations of 7β‐hydroxycholesterol or 25 ng/ml 27‐hydroxycholesterol (27HC) for 24 h, and then challenged for 2 h with control medium () or pyolysin (, epithelium 200 HU, stroma 25 HU). Data are presented as mean (SEM) using cells from 3 independent animals; statistical significance is determined using one‐way ANOVA, and p values reported for the effect of 7β‐hydroxycholesterol treatment on pyolysin challenge

FIGURE 3

Steroid hormones do not protect endometrial cells against pyolysin. Leakage of LDH and viability of epithelial (A) and stromal cells (B) treated with vehicle (V) or the indicated concentrations of estradiol, progesterone, or hydrocortisone for 24 h, and then challenged for 2 h with control medium () or pyolysin (, epithelium 200 HU, stroma 25 HU). Data are presented as mean (SEM) using cells from 3 or 4 independent animals; statistical significance was determined using two‐way ANOVA and p values reported for the effect of each treatment on the pyolysin challenge. Leakage of LDH and viability of epithelial (C) and stromal cells (D) cultured for 1 h in serum‐free medium with or without the indicated concentrations of ICI 182,780, which is an estrogen receptor antagonist, then treated with vehicle or 25 ng/ml 27‐hydroxycholesterol (in the continuing presence or absence of the antagonist) for 24 h, and then challenged for 2 h with control medium () or pyolysin (). Data are presented as mean (SEM) using cells from 3 independent animals; statistical significance is determined using one‐way ANOVA and p‐values reported for the effect of the estrogen receptor inhibitor on the oxysterol cytoprotection

FIGURE 4

Oxysterols reduce pyolysin binding and cytolysin‐accessible cholesterol in cell membranes. Epithelial (A) and stromal cells (B to D) are cultured for 24 h in control serum‐free medium or medium containing 25 ng/ml 27‐hydroxycholesterol (27HC), 5 ng/ml 25‐hydroxycholesterol (25HC), or 0.5 mM methyl‐β‐cyclodextrin (MβCD). Cellular cholesterol is quantified and normalized to protein concentration (A, B), or total cell fluorescence was quantified (C) from confocal microscope images (D) of cells stained using filipin III to visualize cholesterol (white, scale bars are 20 µm). Data are presented as mean (SEM) using cells from 3 or 4 independent animals; statistical significance is determined using one‐way ANOVA and Dunnett's post hoc test. Viability of epithelial (E) and stromal cells (F) cultured in complete culture medium containing 10% serum and treated with vehicle (V) or the indicated concentrations of 27‐hydroxycholesterol or 25‐hydroxycholesterol for 24 h, and then challenged for 2 h with control medium () or pyolysin (, epithelium 200 HU, stroma 25 HU). Data are presented as mean (SEM) using cells from ≥3 independent animals; statistical significance is determined using ANOVA and p values reported for the effect of treatment on pyolysin challenge. (G) Representative western blots of pyolysin binding and α‐tubulin for stromal cells treated with vehicle, 50 nM progesterone, 0.1 nM estradiol, 10 µM hydrocortisone, 25 ng/ml 27‐hydroxycholesterol or 0.5 mM MβCD for 24 h, and then incubated with 25 HU pyolysin for 2 h. Left panel, image representative of 3 experiments; right panel, densitometry measurement of pyolysin binding normalized to α‐tubulin, with statistical significance determined using ANOVA and Dunnett's post hoc test. Leakage of LDH and viability of epithelial (H) and stromal cells (I) cultured for 16 h in serum‐free medium with or without 10 µM SZ5‐035 ACAT inhibitor, then treated with vehicle, 25 ng/ml 27‐hydroxycholesterol, or 25 nM T0901317 (in the continuing presence or absence of SZ5‐035) for 24 h, and then challenged for 2 h with control medium () or 25 HU pyolysin (). Data are presented as mean (SEM) using cells from 4 independent animals; statistical significance is determined using two‐way ANOVA and Tukey's post hoc test

FIGURE 5

LXR agonist cytoprotection against pyolysin. Leakage of LDH and viability of epithelial (A) and stromal cells (B) treated with vehicle (V) or the indicated concentrations of LXR agonists T0901317 or GW3965 for 24 h, and then challenged for 2 h with control medium () or pyolysin (, epithelium 200 HU, stroma 25 HU). Data are presented as mean (SEM) using cells from ≥3 independent animals; statistical significance is determined using two‐way ANOVA and p values reported for the effect of treatment on the pyolysin challenge. Potassium in supernatants from epithelial (C) and stromal cells (D) treated with vehicle, 25 nM T0901317 or 250 nM GW3965 for 24 h, and then challenged for 5 min with control medium (○) or pyolysin (●). Data are presented as mean (SEM) using cells from 4 independent animals; statistical significance is determined using ANOVA and Bonferroni's post hoc test. Representative western blot of ABCA1 and α‐tubulin for epithelial (E) and stromal cells (F) treated with vehicle, 25 ng/ml 27‐hydroxycholesterol (27HC) or 25 nM T0901317 for 24 h. Densitometry data are normalized to α‐tubulin and presented as mean (SEM) using cells from 3 independent animals; statistical significance is determined using one‐way ANOVA and p values reported for Dunnett's post hoc test

FIGURE 6

Oxysterol cytoprotection depends partly on NR1H3 and NR1H2. Leakage of LDH and viability of epithelial (A) and stromal (B) cells transfected for 48 h with scramble siRNA or siRNA targeting NR1H3, NR1H2, or both NR1H3 and NR1H2; treated with vehicle or 25 ng/ml 27‐hydroxycholesterol for 24 h; and, then challenged for 2 h with control medium () or pyolysin (, epithelium 200 HU, stroma 25 HU). Data are presented as mean (SEM) using cells from 4 independent animals; statistical significance is determined using two‐way ANOVA and Tukey's post hoc test

FIGURE 7

Side‐chain‐hydroxycholesterols protect endometrial cells against Staphylococcus aureus α‐hemolysin. Leakage of LDH and viability of epithelial (A) and stromal (B) cells treated with vehicle or 25 ng/ml 27‐hydroxycholesterol (27HC) for 24 h, and then challenged for 24 h with control medium () or 8 µg α‐hemolysin (). Data are presented as mean (SEM) using cells from 4 independent animals; statistical significance is determined using ANOVA and Dunnett's multiple comparison post hoc test, *p < .05; **p < .01. Potassium in supernatants from epithelial (C) and stromal cells (D) treated with vehicle or 25 ng/ml 27‐hydroxycholesterol for 24 h, and then challenged for 15 min with control medium (○) or 8 µg α‐hemolysin (●). Data are presented as mean (SEM) using cells from 4 independent animals; statistical significance is determined using ANOVA and Dunnett's post hoc test. Fluorescent microscope images of epithelial (E) and stromal cells (F) treated with vehicle or 25 ng/ml 27‐hydroxycholesterol (27HC) for 24 h, challenged for 24 h with control medium or 8 µg α‐hemolysin, and then stained with Alexa Fluor 555‐conjugated phalloidin to visualize actin (white; nuclei are red; scale bars are 20 μm). Images are representative of cells from 3 animals

FIGURE 8

Oxysterols in the bovine reproductive tract. Chromatographic separation (m/z 534.4054 ± 5 ppm) of indicated oxysterols from (A) dominant ovarian follicular fluid sample derivatized with [²H₀] Girard P reagent, and (B) supernatant of epithelial cells challenged for 24 h with 5 HU pyolysin. Concentrations of selected oxysterols in supernatants from epithelial (C) and stromal cells (D) challenged for 24 h with control medium (), or medium containing 1 µg/ml LPS () or 5 HU pyolysin (). Data are presented as mean (SEM) using cells from 3 independent animals; statistical significance is determined using two‐way ANOVA and Dunnett's multiple comparison post hoc test, with p‐values reported for difference from control