Oxysterols Protect Epithelial Cells Against Pore-Forming Toxins

Abstract

Many species of bacteria produce toxins such as cholesterol-dependent cytolysins that form pores in cell membranes. Membrane pores facilitate infection by releasing nutrients, delivering virulence factors, and causing lytic cell damage - cytolysis. Oxysterols are oxidized forms of cholesterol that regulate cellular cholesterol and alter immune responses to bacteria. Whether oxysterols also influence the protection of cells against pore-forming toxins is unresolved. Here we tested the hypothesis that oxysterols stimulate the intrinsic protection of epithelial cells against damage caused by cholesterol-dependent cytolysins. We treated epithelial cells with oxysterols and then challenged them with the cholesterol-dependent cytolysin, pyolysin. Treating HeLa cells with 27-hydroxycholesterol, 25-hydroxycholesterol, 7α-hydroxycholesterol, or 7β-hydroxycholesterol reduced pyolysin-induced leakage of lactate dehydrogenase and reduced pyolysin-induced cytolysis. Specifically, treatment with 10 ng/ml 27-hydroxycholesterol for 24 h reduced pyolysin-induced lactate dehydrogenase leakage by 88%, and reduced cytolysis from 74% to 1%. Treating HeLa cells with 27-hydroxycholesterol also reduced pyolysin-induced leakage of potassium ions, prevented mitogen-activated protein kinase cell stress responses, and limited alterations in the cytoskeleton. Furthermore, 27-hydroxycholesterol reduced pyolysin-induced damage in lung and liver epithelial cells, and protected against the cytolysins streptolysin O and Staphylococcus aureus α-hemolysin. Although oxysterols regulate cellular cholesterol by activating liver X receptors, cytoprotection did not depend on liver X receptors or changes in total cellular cholesterol. However, oxysterol cytoprotection was partially dependent on acyl-CoA:cholesterol acyltransferase (ACAT) reducing accessible cholesterol in cell membranes. Collectively, these findings imply that oxysterols stimulate the intrinsic protection of epithelial cells against pore-forming toxins and may help protect tissues against pathogenic bacteria.

Glucocorticoids increase tissue cell protection against pore-forming toxins from pathogenic bacteria

“…5 ). Instead, glucocorticoid treatment prevented pyolysin-induced phosphorylation of ERK1/2, p38 and JNK, which is consistent with glucocorticoid suppression of MAPK 24 , 25 , or with preventing pyolysin-induced damage as evidenced by using the cholesterol-depleting agent methyl-β-cyclodextrin 36 . Furthermore, dexamethasone remained cytoprotective when cells were challenged with pyolysin in high-potassium buffer to limit potassium efflux (Supplementary Fig 5a ), or in calcium-free conditions to limit calcium influx (Supplementary Fig 5b ).…”

A concerted mechanism involving ACAT and SREBPs by which oxysterols deplete accessible cholesterol to restrict microbial infection

“…Having determined how ACAT-activating oxysterols trigger both rapid and sustained depletion of accessible cholesterol from the PM, we next sought to determine if these mechanisms are responsible for the previously reported immunological effects of 25HC. Recent studies have indicated that 25HC protects cells from pore-forming activities of bacterial cholesterol-dependent cytolysins (CDCs) that exploit accessible PM cholesterol ( Zhou et al, 2020 ; Ormsby et al, 2022 ; Ormsby et al, 2021 ). We used ACAT1 KO CHO-K1 cells as a model to test whether ACAT activation is the primary mechanism for 25HC’s protection from pore formation by two CDCs, Anthrolysin O (ALO) ( Mosser and Rest, 2006 ; Shannon et al, 2003 ) and Perfringolysin O (PFO) ( Shimada et al, 2002 ; Tweten et al, 2015 ).…”

A Concerted Mechanism Involving ACAT and SREBPs By which Oxysterols Deplete Accessible Cholesterol To Restrict Microbial Infection

“…Having determined how ACAT-activating oxysterols trigger both rapid and sustained depletion of accessible cholesterol from the PM, we next sought to determine if these mechanisms are responsible for the previously reported immunological effects of 25HC. Recent studies have indicated that 25HC protects cells from pore-forming activities of bacterial cholesterol-dependent cytolysins (CDCs) that exploit accessible PM cholesterol (7, 52, 53). We used ACAT1 -/- CHO-K1 cells as a model to test whether ACAT activation is the primary mechanism for 25HC’s protection from pore formation by two CDCs, Anthrolysin O (ALO) (54, 55) and Perfringolysin O (PFO) (56, 57).…”