IL-17A increases TNF-α-induced COX-2 protein stability and augments PGE2 secretion from airway smooth muscle cells: impact on ß2 -adrenergic receptor desensitization.

BACKGROUND: IL-17A plays an important role in respiratory disease and is a known regulator of pulmonary inflammation and immunity. Recent studies have linked IL-17A with exacerbation in asthma and COPD. We have shown that the enzyme cyclooxygenase-2 (COX-2) and its prostanoid products, prostaglandin E2 (PGE2 ) in particular, are key contributors in in vitro models of infectious exacerbation; however, the impact of IL-17A was not known. METHODS AND RESULTS: We address this herein and show that IL-17A induces a robust and sustained upregulation of COX-2 protein and PGE2 secretion from airway smooth muscle (ASM) cells. COX-2 can be regulated at transcriptional, post-transcriptional and/or post-translational levels. We have elucidated the underlying molecular mechanisms responsible for the sustained upregulation of TNF-α-induced COX-2 by IL-17A in ASM cells and show that is not via increased COX-2 gene expression. Instead, TNF-α-induced COX-2 upregulation is subject to regulation by the proteasome, and IL-17A acts to increase TNF-α-induced COX-2 protein stability as confirmed by cycloheximide chase experiments. In this way, IL-17A acts to amplify the COX-2-mediated effects of TNF-α and greatly enhances PGE2 secretion from ASM cells. CONCLUSION: As PGE2 is a multifunctional prostanoid with diverse roles in respiratory disease, our studies demonstrate a novel function for IL-17A in airway inflammation by showing for the first time that IL-17A impacts on the COX-2/PGE2 pathway, molecules known to contribute to disease exacerbation.

Effect of Plantago ovata (psyllium) husk and seeds on sterol metabolism: studies in normal and ileostomy subjects.

The diet of six normal and five ileostomy subjects was supplemented with 10 g/d Plantago ovata psyllium husk for 3 wk while six normal and four ileostomy subjects received 10 g/d psyllium seed. Fecal and ileostomy output, sterol excretion, serum cholesterol, and triglycerides were measured before and after supplementation. The husk had no effect on cholesterol or triglyceride concentrations in either normal or ileostomy subjects. Total and high-density-lipoprotein-cholesterol concentrations were reduced on average by 6.4% and 9.3%, respectively, in the normal group after seed supplementation. No effect on fecal bile acid excretion in the normal subjects was found after both regimes. Ileostomy bile acids were increased (on average 25%) after seed supplementation, whereas no effect on cholesterol concentrations was found. These results suggest that psyllium seed might be more effective than the husk in reducing serum cholesterol, that this cholesterol-lowering effect is not mediated by increased fecal bile acid losses, and increased ileal losses of bile acids might be compensated for by enhanced reabsorption in the colon.

Reproducibility of the breath hydrogen measurement after a low and high fibre meal.

OBJECTIVE: To assess the intra-subject variability of the breath hydrogen (H2) response to a low and a high fibre test meal. DESIGN: Six week trial, consisting of three phases: screening (1 weeks), baseline period (1 week) and high fibre period (4 weeks). SETTING: Western General Hospital, Edinburgh, Scotland. SUBJECTS: Sixteen subjects from the hospital staff and student population were screened for breath methane (CH4) production. Seven non-CH4 producers, four males and three females, were included in the trial. All completed the study successfully. INTERVENTION: Breath H2 and CH4 production were measured over a 12h period on duplicate test days after a low and high fibre meal. The high fibre meal was consumed for 4 weeks before the high fibre test days to allow for bacterial adaptation. The effect of the high fibre meal on stool output and whole gut transit time was furthermore assessed. RESULTS: Individual differences in breath H2 area-under-the-curve between duplicate test days ranged from 4% to 39% after the low fibre meal and from 4% to 37% after the high fibre meal. Considerable variation was found to be inherent in the breath collection method. In two subjects, breath CH4 was detected in response to the test meals. Stool output and whole gut transit time remained unchanged. CONCLUSION: Considerable differences were found between duplicate breath H2 responses to standard test meals. The variation inherent in the collection procedure emphasizes the need to collect breath samples at least in duplicate. The data presented here can be useful in future sample size calculations for similar studies.

Taurocholic acid adsorption during non-starch polysaccharide fermentation: an in vitro study.

The association of radiolabelled taurocholic acid with the solid fraction of a faecal fermentation mixture was measured. A human faecal inoculum was incubated with [24-14C]taurocholic acid and several non-starch polysaccharide sources (pectin, wheat bran, ispaghula (Plantago ovata) husk and seed), glucose or a substrate-free control. Portions of fermentation mixture were taken at 0, 3, 6, 21 and 24 h and centrifuged to acquire a supernatant fraction and a pellet containing the fermentation residue. 14C was measured in supernatant fractions and pellets at all time points. Volatile fatty acids (VFA) were measured at 0 and 24 h to confirm bacterial growth. Radioactivity in the pellet increased over time for all substrates. Glucose resulted in the greatest incorporation of taurocholic acid into the pellet, followed by pectin. At 24 h the proportion of the total radioactivity found in the pellet was 92% for glucose, 79% for pectin, 60% for wheat bran, 59% for ispaghula seed, 53% for ispaghula husk and 26% for the control (mean of duplicates). Glucose and pectin produced the greatest quantity of VFA at 24 h. VFA production was highly correlated with radioactivity in the pellet (r0.976, P < 0.005). These results suggest that the bile acid binding capacity of a faecal culture mixture may be strongly influenced by the fermentability of the available substrate and hence related to bacterial metabolic activity.

Oxysterol efflux from macrophage foam cells: the essential role of acceptor phospholipid.

Oxidized forms of cholesterol (oxysterols) are present in atherosclerotic lesions and may play an active role in lesion development. For example, 7-ketocholesterol (7KC) inhibits cholesterol efflux from macrophage foam cells induced by apolipoprotein A-I (apoA-I). Such oxysterols may promote foam cell formation in atherosclerotic lesions by preventing effective clearance of excess cholesterol. ApoA-I also induces phospholipid (PL) export from foam cells and it has been suggested that cholesterol efflux is dependent upon PL association with the apolipoprotein. In the current study, the effect of oxysterol enrichment of foam cells on phospholipid efflux was measured. Export of cellular PL to apoA-I from 7KC-enriched foam cells was inhibited to the same extent as cholesterol, indicating that the reduced cholesterol export may be a consequence of a decline in the capacity of the foam cells to generate PL/apoA-I particles capable of accepting cellular cholesterol. Incubation of foam cells with pre-formed PL/apoA-I discs increased cholesterol export from 7KC-enriched cells to levels seen in 7KC-free cells. Foam cells produced by uptake of oxidized LDL, which contain similar amounts of 7KC plus other oxidation products, expressed a more profound inhibition of PL export to apoA-I. Cholesterol efflux from these cells improved only partially by provision of PL-containing acceptors. Efflux of 7KC from both foam cell types occurred to PL/apoA-I discs but was only minimal to lipid-free apoA-I, indicating that export of this oxysterol is more dependent than cholesterol upon the presence of extracellular phospholipid.

Cholesterol and oxysterol metabolism and subcellular distribution in macrophage foam cells. Accumulation of oxidized esters in lysosomes.

Cholesterol- and cholesteryl ester-rich macrophage foam cells, characteristic of atherosclerotic lesions, are often generated in vitro using oxidized low density lipoprotein (OxLDL). However, relatively little is known of the nature and extent of sterol deposition in these cells or of its relationship to the foam cells formed in atherosclerotic lesions. The purpose of this study was to examine the content and cellular processing of sterols in OxLDL-loaded macrophages, and to compare this with macrophages loaded with acetylated LDL (AcLDL; cholesteryl ester-loaded cells containing no oxidized lipids) or 7-ketocholesterol-enriched acetylated LDL (7KCAcLDL; cholesteryl ester-loaded cells selectively supplemented with 7-ketocholesterol (7KC), the major oxysterol present in OxLDL). Both cholesterol and 7KC and their esters were measured in macrophages after uptake of these modified lipoproteins. Oxysterols comprised up to 50% of total sterol content of OxLDL-loaded cells. Unesterified 7KC and cholesterol partitioned into cell membranes, with no evidence of retention of either free sterol within lysosomes. The cells also contained cytosolic, ACAT-derived, cholesteryl and 7-ketocholesteryl esters. The proportion of free cholesterol and 7KC esterified by ACAT was 10-fold less in OxLDL-loaded cells than in AcLDL or 7KCAcLDL-loaded cells. This poor esterification rate in OxLDL-loaded cells was partly caused by fatty acid limitation. OxLDL-loaded macrophages also contained large (approximately 40-50% total cell sterol content) pools of oxidized esters, containing cholesterol or 7KC esterified to oxidized fatty acids. These were insensitive to ACAT inhibition, very stable and located in lysosomes, indicating resistance to lysosomal esterases. Macrophages loaded with OxLDL do not accumulate free sterols in their lysosomal compartment, but do accumulate lysosomal deposits of OxLDL-derived cholesterol and 7-ketocholesterol esterified to oxidized fatty acids. The presence of similar deposits in lesion foam cells would represent a pool of sterols that is particularly resistant to removal.

Sterol efflux is impaired from macrophage foam cells selectively enriched with 7-ketocholesterol.

The aim of the present study was to investigate whether impairment of cholesterol efflux previously found from mouse peritoneal macrophages loaded with oxidized low density lipoprotein (OxLDL) could be ascribed to the presence of oxysterols in these cells. 7-Ketocholesterol (7KC), the major oxysterol present in OxLDL-loaded cells, was selectively incorporated into unoxidized LDL, which was subsequently acetylated to produce a high uptake form. Mouse macrophages incubated with 7KC-enriched acetylated LDL (7kAcLDL) did not reveal cytotoxicity judged by cell protein and trypan blue exclusion. A large proportion of cellular 7KC was esterified, indicating that it is a substrate for acyl CoA:cholesterol acyltransferase. Cholesterol efflux from mouse macrophages loaded with 7kAcLDL, using apoA-I as a sterol acceptor, was impaired in cells containing >50 nmol of 7KC/mg of cell protein compared with cells loaded with oxysterol-free acetylated LDL. Thus impairment of cholesterol efflux could be reproduced in cells loaded with 7kAcLDL containing similar proportions of 7KC as OxLDL. 7KC itself was exported very poorly, even when the levels of 7KC in the cells were low. These results suggest that oxysterols present in foam cells in vitro can affect reverse sterol transport and may be potentially important in foam cell formation in vivo.

Apolipoprotein J (clusterin) induces cholesterol export from macrophage-foam cells: a potential anti-atherogenic function?

Apolipoprotein J (apo J) is a secreted glycoprotein of which the exact function remains a matter for speculation. Apo J has been implicated in such diverse processes as sperm maturation, regulation of complement activation, programmed cell death, tissue remodelling and lipid transport. In this study a possible role for apo J in lipid transport was explored. Mouse peritoneal macrophages were incubated with acetylated low-density lipoprotein (AcLDL) to produce foam cells containing cholesterol and cholesteryl esters. Incubation of the foam cells with physiological concentrations of purified apo J led to a dose-dependent export of cholesterol. The appearance of cholesterol in the medium was associated predominantly with a decline in intracellular cholesteryl esters rather than intracellular free cholesterol. The kinetics of cholesterol release to apo J were similar to apo A-I, an established promoter of cholesterol efflux. Apo J was also shown to induce phospholipid efflux from cells, whereas the cholesterol exported to the medium was associated with the apo J. Studies using foam cells from apo E-null mice showed that the cholesterol exported to the medium was independent of apo E production by the cells. These results present the first evidence that apo J can promote cholesterol efflux from foam cells and indicates that it might have a function in cellular cholesterol homoeostasis in both normal and pathological situations.