Myeloperoxidase and serum amyloid A contribute to impaired in vivo reverse cholesterol transport during the acute phase response but not group IIA secretory phospholipase A(2).

Atherosclerosis is linked to inflammation. HDL protects against atherosclerotic cardiovascular disease, mainly by mediating cholesterol efflux and reverse cholesterol transport (RCT). The present study aimed to test the impact of acute inflammation as well as selected acute phase proteins on RCT with a macrophage-to-feces in vivo RCT assay using intraperitoneal administration of [(3)H]cholesterol-labeled macrophage foam cells. In patients with acute sepsis, cholesterol efflux toward plasma and HDL were significantly decreased (P < 0.001). In mice, acute inflammation (75 microg/mouse lipopolysaccharide) decreased [(3)H]cholesterol appearance in plasma (P < 0.05) and tracer excretion into feces both within bile acids (-84%) and neutral sterols (-79%, each P < 0.001). In the absence of systemic inflammation, overexpression of serum amyloid A (SAA, adenovirus) reduced overall RCT (P < 0.05), whereas secretory phospholipase A(2) (sPLA(2), transgenic mice) had no effect. Myeloperoxidase injection reduced tracer appearance in plasma (P < 0.05) as well as RCT (-36%, P < 0.05). Hepatic expression of bile acid synthesis genes (P < 0.01) and transporters mediating biliary sterol excretion (P < 0.01) was decreased by inflammation. In conclusion, our data demonstrate that acute inflammation impairs cholesterol efflux in patients and macrophage-to-feces RCT in vivo in mice. Myeloperoxidase and SAA contribute to a certain extent to reduced RCT during inflammation but not sPLA(2). However, reduced bile acid formation and decreased biliary sterol excretion might represent major contributing factors to decreased RCT in inflammation.

. The sPLA 2 -transgenic line has been backcrossed to the C57BL/6J genetic background lacking the endogenous murine sPLA 2 enzyme due to a frameshift mutation for >14 generations. The animals were caged in animal rooms with alternating 12 h periods of light (from 7 AM to 7 PM) and dark (from 7 PM to 7 AM) with ad libitum access to water and mouse chow diet (Arie Blok, Woerden, The Netherlands). Animal experiments were performed in conformity with Public Health Service policy and in accordance with the national laws. All protocols were approved by the responsible ethics committees of the University of Groningen and the Landesamt für Gesundheit, Ernährung und techni sche Sicherheit Berlin (LAGETSI).

Construction of recombinant adenoviruses
The human SAA1 cDNA was amplifi ed from IL-6-treated HepG2 cells (ATCC via LGC Promochem, Teddington, UK) by PCR using specifi c primers according to the human sequence (NM_000331, GenBank) and subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA). Recombinant adenovirus (AdhSAA) was generated using the Adeno-X kit (Clontech, Mountain View, CA) according to the manufacturer's instructions. The mouse CE/J SAA expressing adenovirus was kindly provided by Dr. Nancy Webb, University of Kentucky, Lexington, KY. An empty adenovirus (AdNull) was used as control ( 16 ). Recombinant adenoviruses were amplifi ed and purifi ed as described previously ( 17 ). For mouse experiments, a dose of 1 × 10E11 particles/mouse of each of the respective adenoviruses was used.

In vivo reverse cholesterol transport studies
Wild-type C57BL/6J donor mice were injected intraperitoneally with 0.5 ml of 10% Brewer thioglycollate medium (Becton Dickinson, Le Point de Claix, France). On day 4 after thioglycollate injection, peritoneal macrophages were harvested as described ( 18 ). Macrophages were plated in RPMI 1640 medium (Invitrogen) supplemented with 10% FBS (HyClone, Logan, UT) and 1% penicillin/streptomycin (Invitrogen) and were allowed to adhere for 4 h at 37°C under 5% CO 2 humidifi ed air. Then nonadherent cells were removed by washing twice with PBS followed by loading of the macrophages with 50 g/ml acetylated LDL and 3 Ci/ml [ 3 H]cholesterol (Perkin Elmer, Boston, MA) for 24 h. After washing twice with PBS, the macrophages were equilibrated for 18 h in RPMI 1640 medium containing 1% penicillin/streptomycin and 2% BSA (Sigma, St. Louis, MO). Immediately before injection, cells were harvested and resuspended in RPMI 1640 medium. As general procedure for in vivo macrophage-to-feces RCT studies, [ 3 H]cholesterol-loaded macrophage foam cells were injected intraperitoneally into individually housed recipient mice. Plasma was collected at the indicated time points after macophage injection by retroorbital puncture and for the fi nal blood draw by heart puncture. At the end of the experimental period, livers were harvested, snapfrozen in liquid nitrogen, and stored at Ϫ 80°C until further use. Feces were collected continuously as indicated up to 48 h. Counts in plasma were assessed directly by liquid scintillation counting (Packard 1600CA Tri-Carb, Packard, Meriden, CT). To determine uptake of tracer into the liver and secretion into the feces, respective samples were processed as indicated below before scintillation counting. All obtained counts were expressed relative to the administered tracer dose.
Specifi cally, to study the effects of the APR on RCT, C57BL/6J mice were injected intraperitoneally with 75 g lipopolysaccharide (LPS, Escherichia coli O111:B4, Sigma) in PBS, while controls received PBS injections shortly before receiving the labeled macrophage foam cells. LPS injection reduced food intake by 28% and feces production by 34%. For the subsequent experiment as-(APPs) have been shown to possess a predictive value for future cardiovascular events ( 8,9 ). Recently, it was also demonstrated that eliminating a large part of the hepatic acute phase response (APR) by knocking out the gp130 receptor on hepatocytes confers signifi cant protection from atherosclerosis, indicating that APP might mechanistically contribute to the atherosclerotic process ( 10 ). Furthermore, infl ammation impacts on HDL metabolism resulting in accelerated HDL catabolism and substantial remodeling of the HDL particle (11)(12)(13), making it likely that these changes might translate into altered in vivo RCT.
Therefore, the aim of the present study was to assess whether induction of the APR affects in vivo RCT and to dissect out the role of important APP that have been established to regulate HDL metabolism during an infl ammatory response. Our data demonstrate that acute infl ammation transiently reduces cholesterol effl ux to plasma in patients with an APR and impairs macrophageto-feces RCT in vivo in mice. Group IIA secretory phospholipase A 2 (sPLA 2 ) and myeloperoxidase (MPO), but not serum amyloid A (SAA), contribute to reduced cholesterol effl ux toward plasma. However, although expression of sPLA 2 in the absence of generalized infl ammation did not affect macrophage-to-feces RCT, SAA overexpression and MPO infusion decreased in vivo RCT signifi cantly.

Patients
Consecutive patients admitted with acute sepsis to the intensive care unit (ICU) at Charité-Campus Benjamin Franklin in Berlin, Germany were included if they met established criteria for septic shock ( 14 ). Patients under 18 years of age, pregnant women, and patients with preexisting endocrine disorders were excluded. A fi rst blood sample was obtained during a routine blood draw at admittance (d0) and a second blood sample was drawn at day 21 following complete clinical recovery, defi ned as SAPSII score below 35, no clinical signs of systemic infection, and no necessity for treatment at the ICU. Blood was immediately placed on ice and plasma was obtained and stored at Ϫ 80°C until further analysis. Ten patients were included, eight matched healthy volunteers served as controls (for clinical details, see Table 1 ). The origin of infection in the patients was lungs (n = 5), urinary tract (n = 4), and gallbladder (n = 1). Six patients had gram-negative and 2 gram-positive infection; no bacteria could be isolated in two patients. Organ dysfunction included respiratory (n = 8), cardiovascular (n = 7), renal (n = 4), central nervous system (n = 3), liver (n = 1), and hematologic (n = 1) failure. All patients were receiving inotropic drugs or vasopressors and required mechanical ventilation and hydrocortisone during the acute phase of sepsis. The study was approved by the local Ethics Committee and written informed consent for blood sampling was obtained from patients or their proxies.

Animals
C57BL/6J control mice were purchased from Charles River (Sulzfeld, Germany). The human group IIA sPLA 2 -transgenic mice expressing the human transgene under the control of the endogenous human promoter have been described previously blanks to determine effl ux not due to serum or HDL addition, and these values were subtracted from the respective experimental values. Effl ux is given as the percentage of counts recovered from the medium in relation to the total counts present on the plate (sum of medium and cells).

In vitro cholesterol uptake assays
HDL isolated from patient plasma by sequential ultracentrifugation (1.063 < d < 1.21) was labeled with the nonhydrolyzable trap label [ 3 H]cholesteryl ether (Perkin Elmer) essentially as previously described ( 15 ). Cholesteryl ether behaves metabolically as cholesteryl ester ( 20 ); however, by use of the ether bond resecretion by the cells is prevented. HepG2 cells were used as an established model for human hepatocytes and were cultured as detailed above except that 24 h before adding the labeled HDL preparations (50 µg HDL cholesterol/ml), 10% FBS was substituted by 10% lipoprotein-depleted serum. Labeled HDL preparations were then added to the cells in serum-free DMEM and incubations were continued for 6 h. Supernatants and cells were processed as detailed above for macrophages, and radioactivity within medium and cells was determined. Cellular uptake is expressed as the percentage of counts recovered from the cells in relation to the total counts present on the plate (sum of medium and cells). Additional experiments were performed, in which HepG2 cells were treated for 24 h with a mixture of tumor necrosis factor-␣ (25 ng/ml), interleukin-1 ␤ (10 ng/ml) and interleukin-6 (500 pg/ml) (all from R and D Systems, Minneapolis, MN) and tracer uptake from healthy control HDL was determined essentially as outlined above.

Western blotting
Expression of human SAA was checked by Western blot performed on plasma. Equal amounts of plasma were resolved by SDS-PAGE electrophoresis and blotted onto nitrocellulose (GE Healthcare Biosciences Corp., Piscataway, NJ). SAA was visualized using a commercially available monoclonal mouse anti-human SAA antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) followed by the appropriate HRP-conjugated secondary antibody (GE Healthcare, Chalfont St Giles, UK). Western blots for different hepatic transport proteins involved in the biliary secretion process were carried out on liver plasma membranes prepared essentially as described ( 21 ). Protein concent rations were determined with the bicinchoninic acid assay (Pierce Biotechnology, Inc., Rockford, IL). Equal amounts of protein were resolved by SDS-PAGE electrophoresis and blotted onto nitrocellulose. Abcg5 was visualized using a rabbit anti-mouse Abcg5 antibody (kindly provided by Dr. Albert Groen, University Medical Center Groningen, The Netherlands) and Abcb11 (Bsep) was detected using a rabbit anti-rat Abcb11 antibody (a kind gift from Dr Bruno Stieger, University Hospital Zurich, Switzerland), followed by the appropriate HRP-conjugated secondary antibody (GE Healthcare). HRP was detected using chemiluminescence (ECL, GE Healthcare). Blots were quantitated using the freely available ImageJ software adjusting the background for the area size of each band and subtracting it from the respective bands. Results were normalized for the average of the control mice.

Determination of plasma SAA, sPLA 2 , and MPO levels
Commercially available ELISA kits were used according to the manufacturer's instructions to respectively determine human SAA (Biosupply, Bradford, UK), mouse SAA (BioSource, Camarillo, CA), sPLA 2 (Cayman Chemicals, Ann Arbor, MI), mouse MPO (Hycult Biotechnology b.v., Uden, The Netherlands), and human MPO (R and D Systems) levels in plasma. Please note that although plasma MPO levels obtained with the human and sessing the impact of SAA on macrophage-to-feces RCT, C57BL/6J mice were injected with labeled macrophage foam cells on day 3 after receiving AdSAA or the control adenovirus AdNull. To investigate the role of sPLA 2 in RCT, sPLA 2 -transgenic mice were compared with C57BL/6J controls. To assess the impact of MPO on RCT, C57BL/6J mice received intravenously three consecutive injections in 6 h intervals with either PBS (controls) or 50 µg/ mouse purifi ed MPO in 200 µl of 1 mM H 2 O 2 ( 19 ).

Plasma lipid and lipoprotein analysis
Plasma total cholesterol and triglycerides were measured enzymatically using commercially available reagents (Wako Pure Chemical Industries, Neuss, Germany). To determine plasma HDL cholesterol levels, apolipoprotein (apo)B-containing lipoproteins were precipitated using 0.36% phosphotungstic acid (Sigma) and cholesterol content in the supernatant was determined as described above. HDL for effl ux and cellular cholesterol uptake studies described below was isolated from patient plasma obtained at d0 and d21 by sequential ultracentrifugation (1.063 < d < 1.21) as described previously ( 15 ). Pooled plasma samples from mice of the same experimental group were subjected to fast protein liquid chromatography (FPLC) gel fi ltration using a Superose 6 column (GE Healthcare, Uppsala, Sweden) as described ( 20 ). Samples were chromatographed at a fl ow rate of 0.5 ml/min, and fractions of 500 l each were collected. Individual fractions were assayed for cholesterol concentrations as described above.

Determination of counts recovered within liver and feces
Counts within liver were determined following solubilization of the tissue (Solvable, Packard) exactly as previously reported ( 15 ). Counts recovered from a respective piece of liver were backcalculated to total liver mass. Fecal samples were dried, weighed, and thoroughly ground. Aliquots were separated into bile acid and neutral sterol fractions as previously published ( 16 ). Briefl y, samples were fi rst heated for 2 h at 80°C in alkaline methanol and then extracted three times with petroleum ether. In the top layer, counts within the neutral sterol fraction were determined by liquid scintillation counting, while counts incorporated into bile acids were assessed from the bottom layer. Counts recovered from the respective aliquots were related to the total amount of feces produced over the whole experimental period.

In vitro effl ux assays
Two sets of experiments were performed using either THP-1 cells or primary mouse peritoneal macrophages (see above). THP-1 human monocytes (ATCC via LGC Promochem) were grown in suspension culture in RPMI 1640 medium supplemented with 10% FBS, 50 µM ␤ -mercaptoethanol, and 1% penicillin/streptomycin until differentiation into macrophages by the addition of 100 ng/ml phorbol myristate acetate (PMA, Sigma). Differentiated THP-1 macrophages were loaded with 50 g/ml acetylated LDL and 3 Ci/ml [ 3 H]cholesterol for 24 h followed by equilibration for 18 h as detailed above for peritoneal macrophages. Then cells were washed with PBS, and either 1% serum from patients and controls (THP-1 macrophages) or 50 µg protein/ml of isolated HDL (peritoneal macrophages) diluted in RPMI 1640 was added. After 24 h, the supernatant was taken off and radioactivity within the medium was determined by liquid scintillation counting. The cell layer was washed twice with PBS, then 0.1 M NaOH was added, plates were incubated for 30 min at room temperature, and the radioactivity remaining within the cells was assessed by liquid scintillation counting. Wells incubated with RPMI but without added serum or HDL were used as teins related to HDL metabolism were measured. Plasma SAA ( P < 0.001), sPLA 2 ( P < 0.001), and MPO ( P < 0.05) were signifi cantly higher in sepsis patients than in controls ( Table 1) . After recovery from sepsis, plasma levels of sPLA 2 and MPO returned to control values, whereas plasma levels of SAA at day 21 remained still slightly elevated in patients compared with controls ( P < 0.05, Table 1 ).

Acute infl ammation decreases macrophage-to-feces RCT in vivo in mice
Next we aimed to determine whether an acute infl ammatory response would also translate into reduced RCT in vivo, using an established macrophage-to-feces RCT assay ( 23 ). LPS injection increased plasma levels of SAA (24 h: 3685 ± 133, 48 h: 40 ± 3 g/ml vs. 15 ± 2 g/ml in the PBS controls) as well as MPO (48 h: 67 ± 4 vs. 145 ± 20 ng/ml, P < 0.01). Consistent with previous results ( 24 ), induction of the APR in mice did not result in signifi cant changes in plasma total cholesterol (102 ± 7 vs. 112 ± 8 mg/dl, n.s.), HDL cholesterol (76 ± 3 vs. 75 ± 10 mg/dl, n.s.) and non-HDL cholesterol (26 ± 3 vs. 37 ± 12 mg/dl, n.s.) levels 48 h after LPS injection, refl ected by only minor shifts in the plasma lipoprotein profi le as determined by FPLC analysis ( Fig. 2A ). On the other hand, plasma triglyceride levels increased signifi cantly after LPS injection (40 ± 2 vs. 145 ± 36 mg/dl, P < 0.05). A single dose of LPS signifi cantly decreased the movement of [ 3 H]cholesterol toward plasma by 33% at 6 h ( P < 0.05, Fig. 2B ) and by 27% at 24 h after injection ( P < 0.05), whereas counts within plasma were not different at 48 h compared with saline controls. [ 3 H] cholesterol tracer within liver was similar in both experimental groups ( Fig. 2C ). In contrast, tracer recovery within feces was markedly reduced by LPS injection, both within the bile acid ( P < 0.001) and the neutral sterol fraction ( P < 0.001, Fig. 2D ). These data demonstrate, consistent with a previous report ( 25 ) that acute infl ammation results in impaired RCT from macrophages to feces in vivo.

Overexpression of human and mouse SAA has divergent effects on macrophage-to-feces RCT in vivo
SAA is an APP with apolipoprotein properties that preferentially associates with HDL ( 13,24,26 ). To investigate whether SAA contributes to decreased RCT during the APR, human and mouse SAA were specifi cally overexpressed in the absence of generalized infl ammation using recombinant adenoviruses. Human SAA overexpression did not impact on the plasma lipoprotein profi le as revealed by FPLC analysis and also left plasma [ 3 H]cholesterol counts, liver counts, and tracer excretion into the feces essentially unchanged (supplementary Fig. I ). Plasma levels of human SAA following recombinant adenovirus administration were 1020 ± 284 g/dl at the 48 h time point. In contrast, mouse SAA overexpression increased LDL cholesterol in plasma ( Fig. 3A ). Although plasma (F ig. 3B ) and liver counts ( Fig. 3C ) were not signifi cantly affected, fecal [ 3 H]cholesterol tracer excretion was significantly decreased compared with control adenovirusinjected mice, indicating lower overall RCT in response to SAA overexpression ( P < 0.05, Fig. 3D ). Plasma mouse SAA mouse MPO-specifi c systems are comparable, this is not given for the mouse versus the human SAA-specifi c ELISA systems.

MPO purifi cation
Human MPO was isolated as described previously ( 22 ). Briefl y, isolated human neutrophils were dissolved in cetylmethyl ammonium bromide (Sigma) and sonicated. The extract was absorbed to a concanavalin A sepharose gel (Pharmacia, Uppsala, Sweden) and eluted with ␣ -methyl-D-mannoside (Sigma). The eluate was further purifi ed on a Sephadex G100 gel (Pharmacia). The fi nal human MPO preparation had an optical density ratio 428/280 of 0.78 and was not contaminated with proteinase 3, elastase or lactoferrin as determined by antigen specifi c ELISAs. Analysis by gel electrophoresis showed specifi c bands for human MPO at 15, 38, and 58 kDa.

Analysis of gene expression by real-time quantitative PCR
Total RNA from mouse livers was isolated using Trizol (Invitrogen) and quantifi ed with a NanoDrop ND-100 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, DE). cDNA synthesis was performed from 1 g of total RNA. Real-time quantitative PCR was carried out on an ABI-Prism 7700 (Applied Biosystems, Foster City, CA) sequence detector with the default settings ( 21 ). PCR primers and fl uorogenic probes were designed with the Primer Express Software (Applied Biosystems) and synthesized by Eurogentec (Seraing, Belgium). The mRNA expression levels presented were calculated relative to the average of the housekeeping gene cyclophilin and further normalized to the relative expression levels of the respective controls.

Statistical analysis
Statistical analysis was carried out using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL). Values are expressed as means ± SEM. Paired or unpaired Student's t -test was used as appropriate to assess statistical differences between groups. Statistical signifi cance for all comparisons was assigned at P < 0.05.

Acute infl ammation impairs macrophage cholesterol effl ux to plasma and HDL in sepsis patients
To assess the impact of acute infl ammation in human patients on cholesterol effl ux from macrophage foam cells, the fi rst step in RCT, in vitro effl ux experiments were performed. Blood samples were collected from sepsis patients on the day of admittance to the ICU (d0) and on day 21 following clinical recovery. Cholesterol effl ux toward plasma was reduced by 73% in sepsis patients compared with matched healthy controls ( P < 0.001, Fig. 1A ). After recovery, cholesterol effl ux in the same patients improved signifi cantly ( P < 0.001) but was still lower than in controls ( P < 0.05, Fig. 1A ). Because plasma HDL cholesterol levels are considerably low in sepsis patients ( Table 1 ), we next isolated HDL from patients and controls to delineate whether reduced effl ux toward plasma might refl ect decreased HDL levels or is due to altered acceptor properties of the HDL particle. Cholesterol effl ux toward isolated HDL was also markedly impaired by acute sepsis compared with HDL from the same patients after recovery ( P < 0.001, Fig. 1B ). Next, plasma levels of several acute phase pro- There was no signifi cant difference in the recovery of [ 3 H] cholesterol within liver between sPLA 2 transgenic mice and C57BL/6J controls ( Fig. 4C ). Fecal tracer excretion within the bile acid fraction was lower in sPLA 2 overexpressing mice ( P < 0.05, Fig. 4D ), whereas tracer excretion within the neutral sterol fraction and total RCT remained essentially unchanged. These results indicate that sPLA 2 expression might contribute to decreased cholesterol effl ux in response to acute infl ammation; however, sPLA 2 has no effect on overall macrophage-to-feces RCT during the APR.

MPO decreases macrophage-to-feces RCT in vivo
Plasma MPO levels increase during the APR, and MPOmediated oxidative modifi cations of HDL apolipoproteins, especially apoA-I, have been shown to decrease macrophage cholesterol effl ux ( 28,29 ). First, the effect of genetic MPO defi ciency on RCT under conditions of acute infl ammation was tested. RCT in LPS-injected MPO knockout mice tended to be increased, but did not differ significantly from RCT in LPS-injected controls (supplementary Fig. II ). However, murine neutrophils have only 10-20% of the MPO present in the respective human cells ( 30,31 ), so that, especially in the context of a full-blown APR, the relative contribution of MPO might be too small to result in a signifi cant effect. Therefore, we next tested the impact of injecting purifi ed human MPO on RCT. Human MPO administration in the absence of an APR increased plasma levels (527 ± 41 ng/ml, P < 0.001), resulted in discrete changes of the plasma lipoprotein profi le ( Fig. 5A ), and in the formation of higher molecular mass aggregates of apoA-I upon SDS-PAGE electrophoresis consistent with oxidative modifi cation of the HDL particle (data not shown) ( 32 ). Although plasma total cholesterol levels de-levels in response to AdmSAA injection were 404 ± 96 g/ ml at the 48 h time point and 4.1-fold higher at the start of the experiment (0 h).

sPLA 2 overexpression reduces cholesterol effl ux toward plasma, but does not affect macrophage-to-feces RCT in vivo
Similar to SAA, plasma sPLA 2 levels are strongly elevated during human infl ammation and sPLA 2 has been shown to increase HDL catabolism in transgenic mice ( 15,27 ). To explore the effects of sPLA 2 on RCT, we used transgenic mice specifi cally overexpressing human sPLA 2 in the absence of systemic infl ammation. These mice had on average 4.7-fold higher plasma sPLA 2 levels compared with the septic patients, and were on the C57BL/6J genetic background that lacks endogenous expression of the murine enzyme due to a frameshift mutation ( 15 ). Consistent with previously published data ( 15 ), HDL cholesterol levels were lower in sPLA 2 transgenic mice as revealed by FPLC analysis 3 H]labeled cholesterol and acetylated LDL toward HDL (50 g/ml) isolated by sequential ultracentrifugation. Experiments were performed as described under Materials and Methods. Data are given as means ± SEM. n = 10 for patients and n = 8 for controls. * indicates statistically signifi cant differences from healthy controls (at least P < 0.05), and # indicates statistically signifi cant differences from patients at day 21 ( P < 0.001) as assessed by Student's t -test. Day 0, patients with acute sepsis at the day of admittance to the ICU; day 21, the same patients following full recovery from sepsis 21 days after admittance. does not fully explain decreased RCT during the APR, we next assessed whether hepatic cholesterol delivery from acute phase HDL might be impaired. Uptake of [ 3 H]cholesteryl ether from acute phase HDL isolated from sepsis patients (d0) into HepG2 cells was not signifi cantly different compared with HDL obtained after recovery (d21) (8.1 ± 0.7 vs. 7.2 ± 0.5%, n.s.). On the other hand, [ 3 H] cholesteryl ether uptake from control HDL into HepG2 cells treated with a mixture of several pro-infl ammatory cytokines was signifi cantly increased (7.7 ± 0.3 vs. 9.8 ± 0.4%, P < 0.05). Because these data suggested that the hepatic cholesterol uptake step might not be impaired during infl ammation, we next assessed the impact of the APR on the expression levels of key enzymes involved in conversion of cholesterol into bile acids as well as of transporters critical for the biliary secretion process. Hepatic mRNA expression of Cyp27a1, the initial enzyme in the alternative pathway of bile acid synthesis, was decreased by 67% in response to LPS ( P < 0.001) whereas the expression of two other enzymes involved in bile acid synthesis, namely Cyp7a1, the rate-limiting enzyme for the classic sterol-regulated pathway, and Cyp8b1, the key creased signifi cantly in MPO-injected mice (90 ± 2 vs. 80 ± 2 mg/dl, P < 0.05), levels of HDL cholesterol (54 ± 2 vs. 48 ± 5 mg/dl) as well as non-HDL cholesterol (21 ± 1 vs. 20 ± 2 mg/dl) did not differ signifi cantly between groups. Cholesterol tracer recovery in plasma was reduced in MPO-injected mice at 6 h ( Fig. 5B ) and 24 h ( P < 0.05). Liver counts were not altered following MPO administration ( Fig. 5C ). However, total [ 3 H]cholesterol excretion into feces was signifi cantly decreased in the mice injected with MPO compared with controls ( P < 0.05, Fig. 5D ). This was mainly due to a decreased tracer recovery within fecal neutral sterols ( P < 0.05), whereas tracer within fecal bile acids was not different between the experimental groups. These data show that MPO reduces macrophage-to-feces RCT in vivo, although quantitatively this effect is still substantially lower than the impact of the APR on RCT.

Hepatic cholesterol uptake from acute phase HDL is not altered, but the hepatic gene expression profi le in response to LPS indicates decreased biliary secretion
RCT comprises cholesterol effl ux toward HDL in the periphery followed by hepatic uptake and excretion into bile. Because the action of three major secreted APPs

DISCUSSION
The results of this study demonstrate that acute infl ammation impairs cholesterol effl ux to plasma and HDL in patients and macrophage-to-feces RCT in vivo in mice. sPLA 2 and MPO but not SAA contribute to reduced cholesterol effl ux toward plasma. Although sPLA 2 does not represent the underlying basis for the diminished macrophage-to-feces RCT during infl ammation, mouse SAA and MPO cause a limited though signifi cant decrease in overall RCT.
Infl ammation is closely linked to atherosclerotic CVD on several levels. Chronic infl ammatory diseases such as rheumathoid arthritis are typically associated with an increased CVD risk in patients ( 4,33 ), and chronic infl ammation causally contributes to atherogenesis in vivo in mouse models ( 10 ). On the other hand, plasma levels of several APPs are elevated in patients with atherosclerotic CVD and have been shown to possess a predictive value for future cardiovascular events ( 8,9 ). In addition, acute infl ammation impacts on lipid metabolism resulting, in humans, in characteristic changes that resemble a enzyme responsible for synthesis of cholic acid, was decreased, however, not signifi cantly ( Table 2 ). Although Sr-b1 expression was not different between LPS-treated mice and saline controls, hepatic mRNA expression of Abcg5 ( P < 0.05) and Abcg8 ( P < 0.001), major transport proteins mediating biliary cholesterol secretion, was signifi cantly decreased ( Table 2 ). In addition, expression of Abcb11, an important bile acid export pump, was significantly reduced in response to LPS ( P < 0.001), whereas the expression of Abcb4, which mediates biliary phospholipid secretion, was unaffected ( Table 2 ). By Western blot (supplementary Fig. III ) we confi rmed that decreased hepatic mRNA expression during the APR also translates into reduced protein expression of Abcg5 by 65% ( P < 0.05) and Abcb11 by 36% ( P < 0.05).
Taken together, these data indicate that coinciding with a substantial reduction in RCT acute infl ammation significantly decreases the hepatic expression levels of key enzymes for bile acid synthesis as well as major transport proteins mediating the biliary secretion of bile acids and cholesterol. study was published confi rming the basic fi nding of reduced RCT in response to infl ammatory stimuli ( 35 ). With regard to the impact of infl ammation on cholesterol effl ux from macrophages, it also has been shown previously that LPS and pro-infl ammatory cytokines downregulate the expression of the two key cholesterol effl ux transporters ABCA1 and ABCG1 in macrophages ( 36,37 ). Furthermore, acute phase HDL was demonstrated to not be able to support cholesterol effl ux as well as normal HDL (38)(39)(40). However, the differential contribution of distinct components of the APR to these results has not been addressed thus far.
In an attempt to delineate the impact of several APPs on RCT in vivo, we fi rst investigated the effect of isolated overexpression of human and mouse SAA in the absence of an APR using recombinant adenoviruses. In plasma, SAA is primarily associated with HDL and can comprise the main apolipoprotein of acute-phase HDL ( 41,42 ). Although earlier reports suggested that SAA might displace apoA-I from the HDL particle ( 12,43 ), thereby potentially accelerating the HDL catabolic rate, overexpression of SAA in vivo by means of a recombinant adenovirus showed no effect on HDL catabolism ( 24 ). However, SAA might change pro-atherogenic lipoprotein profi le as it is found in CVD patients, namely reduced plasma HDL cholesterol levels and increased plasma triglycerides ( 12 ). In mice, the drop in plasma HDL levels in response to an infl ammatory stimulus does not occur as pronounced as in humans and we previously demonstrated using lipoprotein kinetics that the catabolic rates of HDL apolipoproteins as well as HDL cholesteryl ester remain largely unaffected by acute infl ammation ( 11 ). However, in wild-type mice, infl ammation results in altered tissue uptake of HDL and its components, namely signifi cantly increased HDL holoparticle catabolism by the liver and increased selective uptake of HDL cholesteryl ester by the adrenals ( 11 ). The adrenals use HDL-derived cholesterol for the production of steroid hormones, which represent an important contributing factor to increased survival during an acute infection ( 34,35 ). Therefore, redistribution of HDL cholesterol during the APR might have proven benefi cial during evolution and several APPs might have evolved to aid in directing this process. In addition, the strong decrease in RCT that we observed in response to LPS injection might serve the general purpose of preventing cholesterol loss from the body. While our experiments were in progress, another ceptor BI (SR-BI) mediated selective uptake from SAA-enriched HDL is reduced and lipid-free SAA competes with HDL for SR-BI binding, also resulting in decreased selective uptake rates in vitro ( 47 ). On the other hand, selective uptake from acute phase HDL containing high levels of SAA was also reported to increase ( 38 ). As exemplifi ed in our study, human and mouse SAA might possess different metabolic properties in the context of a murine HDL particle. Although overexpression of human SAA had no impact on tracer appearance in plasma or RCT in vivo, overexpression of mouse SAA decreased overall RCT. sPLA 2 is an APP with a major effect on HDL catabolism. Transgenic overexpression of human sPLA 2 in C57BL/6J mice, a strain lacking the endogenous murine sPLA 2 enzyme due to a frameshift mutation, enhances the catabolic rates of HDL cholesteryl ester as well as HDL apoA-I resulting in decreased plasma HDL levels ( 11,15,27 ). In addition, sPLA 2 appears to be a key factor mediating decreased HDL cholesterol levels in response to infl ammation. Although LPS injection into wild-type or human apoA-I transgenic mice lacking the endogenous murine sPLA 2 enzyme has relatively little effects on HDL cholesterol levels, the functional properties of the HDL particle. Although consistent with the apolipoprotein structure of SAA, this APP is able to mobilize cholesterol from cells ( 44,45 ); SAA in the context of the HDL particle might reduce cholesterol effl ux from macrophages ( 38,46 ). Scavenger re-  loss of sterols from the body. This is, on the one hand, achieved by downregulation of the enzymes required for the conversion of cholesterol to bile acids ( 58,59 ) and on the other hand, by decreasing the expression of Abcg5/g8 and Abcb11, responsible for the largest part of biliary secretion of cholesterol and bile acids, respectively. The decreased expression of Abcg5 and Abcb11 on the mRNA and protein level observed in our study is consistent with previously published fi ndings ( 35,37 ) and translates also into an actual reduction in biliary secretion rates of cholesterol and bile acids during the APR in mice (unpublished observations). In summary, our data demonstrate that reduced cholesterol effl ux observed in patients with acute infl ammation could at least partially be attributed to increased plasma levels of sPLA 2 and MPO. Although in mice, sPLA 2 did not infl uence overall RCT, murine SAA and MPO injection resulted in a limited decrease in RCT from macrophages to feces in vivo. However, also reduced biliary secretion of bile acids and cholesterol by the liver appears to be a major contributing factor to decreased macrophage-to-feces RCT during the APR.
LPS administration to the respective models expressing a human sPLA 2 transgene that is responsive to infl ammatory stimuli results in a pronounced decrease in plasma HDL cholesterol ( 11,26 ). Furthermore, we have also previously established that sPLA 2 signifi cantly enhances the redistribution of HDL cholesterol during infl ammation, namely causing a strong increase in cholesteryl ester uptake into the adrenals ( 11 ), most likely due to increased susceptibility of HDL cholesteryl ester within sPLA 2 -modifi ed HDL toward SR-BI-mediated selective uptake ( 21 ). Although our data indicate that sPLA 2 reduces cholesterol effl ux toward plasma and might thereby explain the fi nding of decreased effl ux toward plasma from sepsis patients, sPLA 2 overexpression does not affect overall in vivo RCT. These results are especially interesting given that transgenic overexpression of human sPLA 2 in mice accelerates atherosclerotic lesion development, even on a chow diet ( 18,48 ), and that sPLA 2 represents a strong CVD risk factor (49)(50)(51).
MPO is an enzyme released from activated neutrophils and monocytes during an infl ammatory response and has been implicated in the oxidative modifi cation of HDL apolipoproteins resulting in dysfunctional HDL particles ( 52,53 ). MPO-modifi ed apoA-I and HDL have a reduced ability to elicit cholesterol effl ux from macrophages ( 28,29 ), consistent with the decrease in tracer appearance in plasma in response to MPO injection observed in our current study. In addition, MPO decreases the ability of apoA-I to activate LCAT ( 54 ). On the other hand, hypochlorite modifi cation of HDL mimicking the enzymatic activity of MPO has been shown to also decrease SR-BI-mediated selective uptake from oxidized HDL ( 32 ). These MPOinduced changes have been suggested to translate into decreased RCT; however, to our knowledge, the present study is the fi rst to demonstrate signifi cantly reduced RCT in response to MPO using an integrated physiologically relevant in vivo RCT assay. In humans, reduced RCT might therefore conceivably represent a contributing factor to the tight association between plasma MPO levels and risk of atherosclerotic CVD in several population studies (53)(54)(55). However, in mice the absence of MPO did not affect RCT in response to an APR, pointing toward a different mechanistic basis for decreased RCT during LPS-induced infl ammation in the murine model.
Of note, mice do not express the cholesteryl ester transfer protein that plays an important role in human lipoprotein metabolism and also in RCT. Cholesteryl ester transfer protein expression is downregulated by infl ammation ( 56,57 ), which conceivably might represent an additional factor impacting on RCT in humans that was not addressed in the present study.
As a general scheme, our results suggest that plasma APPs contribute to a certain extent to decreased RCT during an APR but do not fully explain it. Rather, APPs appear to be responsible for preserving the cholesterol content of peripheral tissues by reducing effl ux and for mediating the redistribution of tissue cholesterol uptake from lipoproteins, especially HDL. The liver apparently serves in the setting of the APR as a gatekeeper to prevent