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.
Highlights
Parturition is accompanied by bacterial infection of the uterus, and disruption of the protective epithelium of the endometrium, which exposes the underlying stroma
These pores lead to leakage of potassium ions from cells within minutes, leakage of cytosolic proteins such as lactate dehydrogenase (LDH) within an hour, changes in the cytoskeleton, and cytolysis.9,16–1 9 S. aureus secretes an α-h emolysin that binds to cell membranes, with seven molecules oligomerizing to form a 1.4 nm internal diameter β-b arrel transmembrane pore, which leads to leakage of cytosolic molecules and cytolysis.[20,21]
These oxysterols act via liver X receptor transcription factors, LXRα and LXRβ (NR1H2), which are both expressed in the ovary and uterus,[25,26] and by activating acetyl-CoA acetyltransferase (ACAT) to catalyze cholesterol esterification.[27,28]
Summary
Parturition is accompanied by bacterial infection of the uterus, and disruption of the protective epithelium of the endometrium, which exposes the underlying stroma. Cell membrane cholesterol homeostasis is controlled by several mechanisms, including the action of side-chain-h ydroxycholesterols, such as 27-h ydroxycholesterol and 25-hydroxycholesterol.[24,25] These oxysterols act via liver X receptor transcription factors, LXRα (encoded by NR1H3) and LXRβ (NR1H2), which are both expressed in the ovary and uterus,[25,26] and by activating acetyl-CoA acetyltransferase (ACAT) to catalyze cholesterol esterification.[27,28] side- chain-hydroxycholesterols regulate immunity and inflammation.4–6,29,30 We considered whether these oxysterols might alter the intrinsic protection of endometrial cells against pore-forming toxins. We measured the abundance of oxysterols in samples from the bovine reproductive tract
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