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

Many species of pathogenic bacteria damage tissue cells by secreting toxins that form pores in plasma membranes. Here we show that glucocorticoids increase the intrinsic protection of tissue cells against pore-forming toxins. Dexamethasone protected several cell types against the cholesterol-dependent cytolysin, pyolysin, from Trueperella pyogenes. Dexamethasone treatment reduced pyolysin-induced leakage of potassium and lactate dehydrogenase, limited actin cytoskeleton alterations, reduced plasma membrane blebbing, and prevented cytolysis. Hydrocortisone and fluticasone also protected against pyolysin-induced cell damage. Furthermore, dexamethasone protected HeLa and A549 cells against the pore-forming toxins streptolysin O from Streptococcus pyogenes, and alpha-hemolysin from Staphylococcus aureus. Dexamethasone cytoprotection was not associated with changes in cellular cholesterol or activating mitogen-activated protein kinase (MAPK) cell stress responses. However, cytoprotection was dependent on the glucocorticoid receptor and 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGCR). Collectively, our findings imply that glucocorticoids could be exploited to limit tissue damage caused by pathogens secreting pore-forming toxins.

Oxysterols Protect Epithelial Cells Against Pore-Forming Toxins.

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 7ß-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.

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

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.

Bisphosphonate inhibitors of squalene synthase protect cells against cholesterol-dependent cytolysins.

Certain species of pathogenic bacteria damage tissues by secreting cholesterol-dependent cytolysins, which form pores in the plasma membranes of animal cells. However, reducing cholesterol protects cells against these cytolysins. As the first committed step of cholesterol biosynthesis is catalyzed by squalene synthase, we explored whether inhibiting this enzyme protected cells against cholesterol-dependent cytolysins. We first synthesized 22 different nitrogen-containing bisphosphonate molecules that were designed to inhibit squalene synthase. Squalene synthase inhibition was quantified using a cell-free enzyme assay, and validated by computer modeling of bisphosphonate molecules binding to squalene synthase. The bisphosphonates were then screened for their ability to protect HeLa cells against the damage caused by the cholesterol-dependent cytolysin, pyolysin. The most effective bisphosphonate reduced pyolysin-induced leakage of lactate dehydrogenase into cell supernatants by >80%, and reduced pyolysin-induced cytolysis from >75% to <25%. In addition, this bisphosphonate reduced pyolysin-induced leakage of potassium from cells, limited changes in the cytoskeleton, prevented mitogen-activated protein kinases cell stress responses, and reduced cellular cholesterol. The bisphosphonate also protected cells against another cholesterol-dependent cytolysin, streptolysin O, and protected lung epithelial cells and primary dermal fibroblasts against cytolysis. Our findings imply that treatment with bisphosphonates that inhibit squalene synthase might help protect tissues against pathogenic bacteria that secrete cholesterol-dependent cytolysins.

Role of hypoxia inducible factor 1α in cobalt nanoparticle induced cytotoxicity of human THP-1 macrophages.

Cobalt is one of the main components of metal hip prostheses and cobalt nanoparticles (CoNPs) produced from wear cause inflammation, bone lyses and cytotoxicity at high concentrations. Cobalt ions mimic hypoxia in the presence of normal oxygen levels, and activate hypoxic signalling by stabilising hypoxia inducible transcription factor 1α (HIF1α). This study aimed to assess in vitro the functional role of HIF1α in CoNP induced cellular cytotoxicity. HIF1α, lysosomal pH, tumour necrosis factor α and interleukin 1ß expression were analysed in THP-1 macrophages treated with CoNP (0, 10 and 100 µg/mL). HIF1α knock out assays were performed using small interfering RNA to assess the role of HIF1α in CoNP-induced cytotoxicity. Increasing CoNP concentration increased lysosomal activity and acidity in THP-1 macrophages. Higher doses of CoNP significantly reduced cell viability, stimulated caspase 3 activity and apoptosis. Reducing HIF1αactivity increased the pro-inflammatory activity of tumour necrosis factorαand interleukin 1ß,but had no significant impact on cellular cytotoxicity. This suggests that whilst CoNP promotes cytotoxicity and cellular inflammation, the apoptotic mechanism is not dependent on HIF1α.

Glutamine supports the protection of tissue cells against the damage caused by cholesterol-dependent cytolysins from pathogenic bacteria.

Pathogenic bacteria often damage tissues by secreting toxins that form pores in cell membranes, and the most common pore-forming toxins are cholesterol-dependent cytolysins. During bacterial infections, glutamine becomes a conditionally essential amino acid, and glutamine is an important nutrient for immune cells. However, the role of glutamine in protecting tissue cells against pore-forming toxins is unclear. Here we tested the hypothesis that glutamine supports the protection of tissue cells against the damage caused by cholesterol-dependent cytolysins. Stromal and epithelial cells were sensitive to damage by the cholesterol-dependent cytolysins, pyolysin and streptolysin O, as determined by leakage of potassium and lactate dehydrogenase from cells, and reduced cell viability. However, glutamine deprivation increased the leakage of lactate dehydrogenase and reduced the viability of cells challenged with cholesterol-dependent cytolysins. Without glutamine, stromal cells challenged with pyolysin leaked lactate dehydrogenase (control vs. pyolysin, 2.6 ± 0.6 vs. 34.4 ± 4.5 AU, n = 12), which was more than three-fold the leakage from cells supplied with 2 mM glutamine (control vs. pyolysin, 2.2 ± 0.3 vs. 9.4 ± 1.0 AU). Glutamine cytoprotection did not depend on glutaminolysis, replenishing the Krebs cycle via succinate, changes in cellular cholesterol, or regulators of cell metabolism (AMPK and mTOR). In conclusion, although the mechanism remains elusive, we found that glutamine supports the protection of tissue cells against the damage caused by cholesterol-dependent cytolysins from pathogenic bacteria.

Akt and STAT5 mediate naïve human CD4+ T-cell early metabolic response to TCR stimulation.

Metabolic pathways that regulate T-cell function show promise as therapeutic targets in diverse diseases. Here, we show that at rest cultured human effector memory and central memory CD4+ T-cells have elevated levels of glycolysis and oxidative phosphorylation (OXPHOS), in comparison to naïve T-cells. Despite having low resting metabolic rates, naive T-cells respond to TCR stimulation with robust and rapid increases in glycolysis and OXPHOS. This early metabolic switch requires Akt activity to support increased rates of glycolysis and STAT5 activity for amino acid biosynthesis and TCA cycle anaplerosis. Importantly, both STAT5 inhibition and disruption of TCA cycle anaplerosis are associated with reduced IL-2 production, demonstrating the functional importance of this early metabolic program. Our results define STAT5 as a key node in modulating the early metabolic program following activation in naive CD4+ T-cells and in turn provide greater understanding of how cellular metabolism shapes T-cell responses.

ARF6-dependent regulation of P2Y receptor traffic and function in human platelets.

Adenosine diphosphate (ADP) is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors, the P2Y(1) and P2Y(12) purinoceptors. Recently, we demonstrated that P2Y(1) and P2Y(12) purinoceptor activities are rapidly and reversibly modulated in human platelets, revealing that the underlying mechanism requires receptor internalization and subsequent trafficking as an essential part of this process. In this study we investigated the role of the small GTP-binding protein ADP ribosylation factor 6 (ARF6) in the internalization and function of P2Y(1) and P2Y(12) purinoceptors in human platelets. ARF6 has been implicated in the internalization of a number of GPCRs, although its precise molecular mechanism in this process remains unclear. In this study we show that activation of either P2Y(1) or P2Y(12) purinoceptors can stimulate ARF6 activity. Further blockade of ARF6 function either in cell lines or human platelets blocks P2Y purinoceptor internalization. This blockade of receptor internalization attenuates receptor resensitization. Furthermore, we demonstrate that Nm23-H1, a nucleoside diphosphate (NDP) kinase regulated by ARF6 which facilitates dynamin-dependent fission of coated vesicles during endocytosis, is also required for P2Y purinoceptor internalization. These data describe a novel function of ARF6 in the internalization of P2Y purinoceptors and demonstrate the integral importance of this small GTPase upon platelet ADP receptor function.

ADP-ribosylation factor 6 expression and activation are reduced in myometrium in complicated pregnancies.

BACKGROUND: ARF6 (ADP-ribosylation factor 6) small GTP binding protein plays critical roles in actin cytoskeleton rearrangements and membrane trafficking, including internalisation of G protein coupled receptors (GPCR). ARF6 operates by cycling between GDP-bound (inactive) and GTP-bound (active) forms and is a potential regulator of GPCR-mediated uterine activity during pregnancy and labour. ARF6 contains very low intrinsic GTP binding activity and depends on GEFs (guanine nucleotide exchange factors) such as CYTH3 (cytohesin 3) to bind GTP. ARF6 and CYTH3 were originally cloned from human placenta, but there is no information on their expression in other reproductive tissues. METHODS: The expression of ARF6, ARF1, and CYTH1-4 was investigated by measuring mRNA (using RT-PCR) and protein levels (using immunoblotting) in samples of myometrium obtained from non-pregnant women, and women with normal pregnancies, before or after the spontaneous onset of labour. We also analysed myometrial samples from women with spontaneous preterm labour and from women with complicated pregnancies requiring emergency preterm delivery. The GST)-effector pull down assay was used to study the presence of active ARF6 and ARF1 in all myometrial extracts. RESULTS: ARF6, ARF1 and CYTH3 but not CYTH1, CYTH2 and CYTH4 were expressed in all samples and the levels did not change with pregnancy or labour. However, ARF6 and CYTH3 but not ARF1 levels were significantly reduced in complicated pregnancies. The alterations in the expression of ARF6 and its GEF in human myometrium indicate a potential involvement of this signalling system in modulating the response of myometrial smooth muscle in complicated pregnancies. The levels of ARF6-GTP or ARF1-GTP did not change with pregnancy or labour but ARF6-GTP levels were significantly decreased in women with severe complications of pregnancy. CONCLUSIONS: We have demonstrated a functional ARF6 system in human myometrium and a correlation between ARF6 level and activity in uterine and abnormal pregnancy.