# Innate immune response triggered by triacyl lipid A is dependent on phospholipid transfer protein (PLTP) gene expression

Thomas Gautier, Catherine Paul, Valerie Deckert, Catherine M Desrumaux, Alexis Klein, Jéôme Labbé, Naïg Le Guern, Anne Athias, Serge Monier, Arlette Hammann, et al.

<figure><img src="image_1.png" /></figure>

## To cite this version:

Thomas Gautier, Catherine Paul, Valerie Deckert, Catherine M Desrumaux, Alexis Klein, et al. "Innate immune response triggered by triacyl lipid A is dependent on phospholipid transfer protein (PLTP) gene expression." FASEB Journal, 2010, 24, pp.3544 - 3554. 10.1096/fj.09-152876. hal-04200323

HAL Id: hal-04200323

https://hal.science/hal-04200323v1

Submitted on 8 Sep 2023

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>
WILEY

# Frontiers in Flow Cytometry™

24 hour Virtual Event

September 13th, 2023

Frontiers in Flow Cytometry™ is for researchers across the globe looking for an opportunity to share and learn about current developments in flow cytometry. This 24 hour virtual event will feature keynote presentations by industry colleagues, webinars, demos, live networking opportunities and more.

## Key topics include:

* Spectral and conventional flow cytometry

* Immunophenotyping and Standardization

* Panel design and optimization

* Cancer Biology and Auto-immune Diseases

* Infectious diseases

* Advances in flow cytometry technology

Register Now

This event is sponsored by

**ThermoFisher**
SCIENTIFIC
# Innate immune response triggered by triacyl lipid A is dependent on phospholipid transfer protein (PLTP) gene expression

Thomas Gautier,*,<fnref n="1" /> Catherine Paul,*,†,<fnref n="1" /> Valérie Deckert,* Catherine Desrumaux,* Alexis Klein,* Jérôme Labbé,*,† Naig Le Guern,* Anne Athias,* Serge Monier,* Arlette Hammann,* Ali Bettaieb,*,† Jean-François Jeannin,*,† and Laurent Lagrost*,<fnref n="2" />

*Université de Bourgogne, INSERM UMR866, Dijon, France; and †EPHE, Laboratoire d'Immunologie et Immunothérapie des Cancers, Dijon, France

**ABSTRACT** Hexaacyl lipopolysaccharide (LPS) aggregates in aqueous media, but its partially deacylated lipid A moiety forms monomers with weaker toxicity. Because plasma phospholipid transfer protein (PLTP) transfers hexaacyl LPS, its impact on metabolism and biological activity of triacyl lipid A in mice was addressed. Triacyl lipid A bound readily to plasma high-density lipoproteins (HDLs) when active PLTP was expressed [HDL-associated lipid A after 4.5 h: 59.1±16.0% of total in wild-type (WT) vs. 32.5±10.3% in PLTP-deficient mice, *P*<0.05]. In the opposite to hexaacyl LPS, plasma residence time of lipid A was extended by PLTP, and proinflammatory cytokines were produced in higher amounts in WT than PLTP⁻/⁻ mice (remaining lipid A after 8 h: 53±12 vs. 35±7%, and IL6 concentration after 4.5 h: 45.5±5.9 vs. 14.6±7.8 ng/ml, respectively; *P*<0.05 in all cases). After 1 wk, onset of B16-induced melanoma was observed in only 30% of lipid A-treated WT mice, whereas >80% of the untreated WT, untreated PLTP-deficient, or lipid A-treated PLTP-deficient animals bore tumors (*P*<0.05 in all cases). It is concluded that PLTP is essential in mediating the association of triacyl lipid A with lipoproteins, leading to extension of its residence time and to magnification of its proinflammatory and anticancer properties.—Gautier, T., Paul, C., Deckert, V., Desrumaux, C., Klein, A., Labbé, J., Le Guern, N., Athias, A., Monier, S., Hammann, A., Bettaieb, A., Jeannin, J.-F., Lagrost, L. Innate immune response triggered by triacyl lipid A is dependent on phospholipid transfer protein (PLTP) gene expression. *FASEBJ.* 24, 3544–3554 (2010). www.fasebj.org

*Key Words:* lipopolysaccharide · inflammation · lipoprotein · lipid transfer protein · cancer

LIPOPOLYSACCHARIDES (LPS) ARE amphipathic molecules that are localized in the outer leaflet of the outer membranes of gram-negative bacteria. They activate the innate immune system through a complex process involving Toll-like receptors (TLRs) and the MD-2, CD14, and lipopolysaccharide-binding protein (LBP)

accessory proteins (1). In the first step of endotoxic shock, LPS binds to plasma LBP and then complexes with CD14 antigen, which can behave either as a membrane-anchored or a soluble protein. After the TLR4-MD2-CD14 complex has formed, cell signaling results in the production of proinflammatory cytokines (TNFα, IL-6, IFNγ, etc.) and nitric oxide, which leads to shock-like symptoms (1). A common feature of LPS is a hydrophilic polysaccharide (O chain) attached to a hydrophobic endotoxic principle (lipid A). Although the O side of LPS confers the antigenic properties, the lipid A moiety has therapeutic potential because of its adjuvant and antitumoral properties (2). These beneficial properties have given rise to a growing interest in lipid A derivatives and has led to the emergence of several partially deacylated lipid A analogs, which retain the immunomodulatory properties of bacterial LPS, but with a tremendous decrease in their toxicity (3). The different behaviors of LPS and partially deacylated lipid A may rely at least in part on the ability of these amphipathic compounds to form preferentially large aggregates and monomers/small micelles in aqueous media, respectively (4–7).

Besides their key role in the transport of lipids in extracellular media, circulating lipoproteins [e.g., chylomicrons, very low-density lipoproteins (VLDLs), low-density lipoproteins (LDLs), high-density lipoproteins (HDLs)] were found to bind LPS, resulting in substantial modulation of their endotoxic and immunostimulatory bioactivities (5–8). In this context, peculiar attention was given to the formation of HDL-LPS complexes because they were found to neutralize LPS proinflammatory activity (9), to block contact-mediated activation of monocytes by T lymphocytes (10), to reduce the production rate of IL-1β and TNFα, and to impair the cytokine-mediated expression of adhesion

<footnote n="1">¹ These authors contributed equally to this work.</footnote>

<footnote n="2">² Correspondence: Centre de Recherche INSERM UMR866, Université de Bourgogne, Faculté de Médecine, 7 boulevard Jeanne d'Arc, BP87900, 21079 Dijon Cedex, France. E-mail: laurent.lagrost@u-bourgogne.fr</footnote>

<footnote>doi: 10.1096/fj.09-152876</footnote>
molecules on endothelial cells (11). The LPS-carrying property of lipoproteins is actually governed both by the amphipathic nature of derivates and by lipoprotein-associated proteins, including LBP and the related phospholipid transfer protein (PLTP). LBP, which circulates mainly in complexes with apoB-containing lipoproteins (LDLs and VLDLs) (12, 13), is a key component in driving the LPS-mediated inflammatory response. Unlike LBP, PLTP is mostly, if not exclusively, bound to the HDL fraction (14). HDL is the main lipoprotein fraction in mouse plasma, and recent studies in PLTP-deficient mice demonstrated that PLTP plays a physiologically relevant role in the disaggregation, binding, and transfer of bacterial LPS to lipoproteins (15). Interestingly, the specificity of PLTP for amphipatic compounds is rather broad because, in addition to LPS, it can bind and transfer phospholipids, unesterified cholesterol, α-tocopherol, and diacylglycerides (16).

Among bacterial lipopolysaccharide derivatives that were obtained by removing acyl chains, polysaccharide side groups, and phosphates (2, 17), OM-174 triacyl lipid A seems to be particularly interesting. This partially deacylated product of *Escherichia coli* hexaacyl lipid A is a TLR2/4 agonist, which was found to be a highly effective treatment in a model of colon cancer in rats when injected either intraperitoneally or intravenously (18, 19). As compared with untreated control mice, OM-174-treated mice showed significant increases in the production of various proinflammatory cytokines and immune cell recruitment. In mice bearing B16 melanoma pulmonary metastasis, OM-174 triacyl lipid A was associated with a significant reduction in tumor growth and with a significant increase in survival (2, 20). Notably, and in contrast to the natural hexaacyl parent LPS, no toxic or side effects were observed in animals treated with the OM-174 triacyl lipid A derivative (2). Finally, whether PLTP binds to and transfers therapeutic lipid A derivatives remains to be established, and the consequences of PLTP expression on the inflammatory and anticancer properties of lipid A are unknown.

In the present study, wild-type (WT) and PLTP-knockout mice were treated or not by intraperitoneal or intravenous injections of the OM-174 triacyl lipid A analog. PLTP expression was found to favor the association of triacyl lipid A with lipoproteins, increasing its residence time and proinflammatory effect. In addition, the antitumor property of triacyl lipid A was found to rely strictly on PLTP gene expression.

# MATERIALS AND METHODS

## Lipid A and LPS compounds

The triacyl lipid A derived from *E. coli*, OM-174, was obtained from OM Pharma (Meyrin/Geneva, Switzerland). Hexaacylated LPS with a complete oligosaccharide chain (*E. coli*, serotype 055:B5) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Both compounds were dissolved in either apyrogen

sodium chloride (0.15 M) or phosphate-buffered saline (PBS) as appropriate, heated to 37°C, and vigorously mixed for 15 min before use. All materials were apyrogen or depyrogenated by overnight heating at 150°C, and all the reagents used were of “endotoxin-free” grade. As assessed by 3-hydroxymyristate quantitation (see below), only trace amounts of LPS were detected in experimental media that were not supplemented with exogenous lipid A.

## Animals

PLTP-deficient mice were previously generated by Dr. X. C. Jiang and colleagues (21). Briefly, these mice were obtained by replacing exon 2 (containing the translation start site) with a neo gene (21). The animals were from a homogenous C57BL/6 background. Female PLTP<sup>-/-</sup> homozygous mice, PLTP<sup>+/-</sup> heterozygous mice, and WT/nontargeted C57BL/6 mice (2-3 mo old) were fed a standard chow diet (A03 diet, Safe, Augy, France). The animals had free access to water and food. All experiments involving the animals were performed in accordance with institutional guidelines and approved by the University of Burgundy's Ethics Committee on the Use of Laboratory Animals (protocol number 5707).

## Cell lines

Murine B16-F10 melanoma cells (B16) and EMT6 mammary cancer cells were purchased from the American Type Culture Collection (Manassas, VA, USA), and they were grown in RPMI 1640 medium supplemented with 10% FBS.

## In vitro macrophage stimulation

Peritoneal macrophages were harvested from the peritoneal cavity of the WT or PLTP<sup>-/-</sup> mice 3 d after the intraperitoneal injection of 0.5 ml of 10% thioglycolate. The cells from 4-5 animals were pooled, washed, and plated on 6-well plates (1×10<sup>6</sup> macrophages/well) in EMEM medium (Biowhittaker, Basel, Switzerland) and supplemented with 10% apyrogen fetal calf serum (Biowest, Nueille, France), L-glutamine (Biowhittaker), and penicillin-streptomycin (Invitrogen, Paisley, UK). The cells were left to attach to the plates for 4 h before renewal of the medium and then cultured for an additional 24 h period prior to treatment. The cells were then treated for 24 h with increasing amounts of LPS or triacyl lipid A, which were dissolved in apyrogen PBS (Biowhittaker). At the end of treatment, the culture medium was collected, cell debris were spun down, and medium aliquots were stored for subsequent analysis.

## Cytokine and chemokine measurement

Cytokines (IL-10, TNFα, IFNγ, IL-6) and chemokines (monocyte chemoattractant protein-1, MCP-1) in mouse plasma and macrophage culture medium were assayed by cytometric bead array (CBA) using a commercially available kit (CBA mouse inflammation kit; BD Biosciences, San Diego, CA, USA) according to the manufacturer's instructions and analyzed on a Galaxy (Dako, Glostrup, Denmark) or a Guava (Millipore, Billerica, MA, USA) flow cytometer. Cytokine concentrations in samples that were successively analyzed with the Galaxy and the Guava systems appeared to correlate significantly.

## Quantitation of adherent cells

The impact of supernatants from the WT or PLTP<sup>-/-</sup> macrophages treated with LPS or triacyl lipid A on cell
adhesion was assessed as the ability of EMT-6 cells to adhere to plastic according to the general procedure previously described (22). Briefly, EMT-6 cells (10⁴ cells/well) were grown for 24 h in 96-well plates and then incubated with macrophage supernatant for another 24 h period. The cells were washed with PBS, fixed in methanol, stained by 5% methylene blue, and dissolved in 0.1 M HCl. Absorbance was measured at 590 nm in a microplate reader UVM-340 (Asys, Eugendorf, Austria). Results are expressed as the percentage of adherent cells; 100% adhesion corresponded to the control value obtained in the absence of LPS or OM-174 triacyl lipid A.

### In vitro assessment of metabolic activity of tumor cells

The metabolic activity of EMT6 and B16-F10 cells was assessed by using a commercially available kit of 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) reduction assay (Cell Proliferation XTT kit; Biological Industries, Beit Haemek, Israel) measuring the activity of mitochondrial dehydrogenases. Briefly, EMT6 and B16 cells (10⁴ cells/well) were grown for 24 h in 96-well plates prior to incubation with macrophage supernatants for another 24 h period. They were then incubated in the presence of XTT reagent for 1 h at 37°C, and absorbance at 490 nm was measured in a UVM-340 microplate reader (Asys). Results were expressed as percentage of the metabolic activities of cells treated with control supernatants.

### Quantitation of cell death by flow cytometry

Apoptosis was evaluated by quantitation of phosphatidylyserine exposure using a commercially available Annexin V-fluorescein isothiocyanate kit according to the manufacturer's instructions (Bender MedSystems, San Bruno, CA, USA). Necrosis was determined using the 7-aminoactinomycin (7-AAD) agent (BD Biosciences). Fluorescence was monitored on a FACSCalibur Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Acquisition was performed on 10⁴ events/sample, and data analysis was conducted using the FlowJo Software (Tree Star, Ashland, OR, USA).

### Lipid A quantitation

The amounts of triacyl lipid A in total serum or isolated lipoprotein fractions were assayed using the direct quantitation of 3-hydroxymyristic acid (3-OH) by gas chromatography-mass spectrometry (GC-MS) (23). The quantitation of 3-OH was performed as described previously, with 3-hydroxypentadecanoic acid used as an internal standard (15). GC-MS analysis of 3-OH was performed using the selected ion monitoring mode as described previously (15).

### Triacyl lipid A binding to HDLs in vitro

Native HDLs were obtained from the WT and PLTP⁻/⁻ mice under apyrogenic conditions. Briefly, plasma samples were obtained by cardiac puncture on anesthetized animals after a 4 h fast. HDLs were isolated by sequential ultracentrifugation as the 1.063 < *d* < 1.21 g/ml fractions after 2 successive 16-h runs at 8 × 10⁴ rpm in a TLA-120 rotor on an Optima ultracentrifuge (Beckman, Palo Alto, CA, USA). Isolated HDLs from WT and PLTP⁻/⁻ mice showed similar relative contents of protein, cholesterol, triglyceride, and phospholipid. PLTP activity in the isolated lipoprotein fractions was measured using a fluorescent assay (Roar Biomedical, Calverton, NY, USA) as described elsewhere (15). In the WT

animals, ~75% of total plasma PLTP activity was recovered in the HDL fraction, with the rest of the activity being detected in the lipoprotein-free fraction and no activity in the VLDL-LDL fraction. HDLs from the PLTP⁻/⁻ mice displayed no PLTP activity.

For association studies, free triacyl lipid A (final concentration 1 µg/ml) was added to the incubation medium containing PLTP-bearing HDLs from the WT mice or PLTP-free HDLs from the PLTP⁻/⁻ mice (50 µg protein in apyrogen PBS at 37°C). Aliquots were successively drawn from the incubation media after 0, 10, 30, and 60 min and were immediately mixed with a precooled KBr solution (final density, 1.21 g/ml) and ultracentrifuged for 4 h at 10⁵ rpm at 4°C in a TLA-120 rotor on an Optima ultracentrifuge (Beckman). Triacyl lipid A concentration in supernatants (HDL-bound triacyl lipid A) and infranatants (unbound triacyl lipid A) was finally determined by 3-OH quantitation (see above).

### Triacyl lipid A distribution in mouse plasma

The WT, PLTP⁺/⁻, and PLTP⁻/⁻ mice (*n*=8/group) were injected intraperitoneally with triacyl lipid A (8 mg/kg). For blood sampling, the mice were briefly anesthetized with isoflurane, and blood was drawn at the indicated times by retroorbital puncture and collected in endotoxin-free heparin-containing tubes. Plasma was obtained by blood centrifugation (10 min, 2000 g at 4°C), and lipoproteins were further isolated from the plasma by sequential ultracentrifugation as described previously (15). The *d* > 1.21 g/ml fraction constituted the lipoprotein-free fraction. At the end of the kinetics measurements, the animals were killed by cardiac puncture under anesthesia with isoflurane. All samples were stored at −80°C until 3-OH quantitation. Areas under the curves (AUC) in total plasma were calculated from kinetic curves over the entire period studied.

### Triacyl lipid A clearance in vivo

The WT and PLTP⁻/⁻ mice (*n*=9/group) received a single intravenous injection of triacyl lipid A (8 mg/kg) via the tail vein. Blood samples were drawn at the indicated times over a 24 h period. For blood sampling, the mice were briefly anestethized with isoflurane and blood was drawn at the indicated times by retroorbital puncture and collected in endotoxin-free heparin-containing tubes. Triacyl lipid A concentration was determined by 3-OH quantitation as described above. Plasma 3-OH concentration measured 5 min after triacyl lipid A injection was considered as the initial pool. Clearance rates were calculated from decay curves with Prism 5.0 software (GraphPad, La Jolla, CA, USA) using a nonlinear top-to-bottom regression model according to the following formula: $Y = (\text{top} - \text{bottom}) \times e^{(-K \times X)} + \text{bottom}$. Results are expressed as the fraction of the initial 3-OH pool cleared per hour.

### Tumor growth experiment

Three days before starting lipid A treatment, 20 WT mice and 20 PLTP⁻/⁻ littermates were injected subcutaneously in the left flank with B16 melanoma cells (2×10⁴ cells/mouse). For each genotype, the mice were subsequently divided into 2 groups: one received OM-174 triacyl lipid A, while the other received the vehicle only (sterile apyrogen sodium chloride). The treatment was started 3 d after injection of the tumor cells and was continued over a 20 d period with 5 intraperitoneal injections of OM-174 (8 mg/kg) or vehicle on d 0, 5, 10, 15, and 20. Early tumors were detected by palpation, and tumor growth was monitored every 2 d with a caliper rule, and
tumor volume (mm³) was calculated using the formula
$$ \text{length} \times \text{width}^2 \times 0.5. $$

## Statistical analyses

Results are expressed as means ± SE. The statistical significance of differences between 2 data means was determined with the nonparametric Mann-Whitney test. The statistical significance of differences between 3 or more groups was determined by multiple analysis with ANOVA and the PLSD Fisher test. Tumor appearance was analyzed by the Kaplan-Meier method and compared using the χ² test.

## RESULTS

### Formation of HDL-triacyl lipid A complexes is impaired in PLTP-deficient mice

PLTP-containing or -deficient HDLs were isolated from total plasma of WT and PLTP<sup>-/-</sup> mice, respectively. They were subsequently incubated with triacyl lipid A (final concentration, 1 µg/ml) for 10, 30, or 60 min prior to recovery of the lipoprotein-bound and unbound fractions by ultracentrifugation. As shown in Fig. 1A, over the 60-min period studied, triacyl lipid A associated more readily with PLTP-containing HDLs from WT mice than with PLTP-deficient HDLs from PLTP<sup>-/-</sup> mice, at the later time point with HDL-bound to free ratios of 56.2 and 12.4, respectively (P<0.05).

To gain insights into the effect of PLTP on metabolism and lipoprotein association of triacyl lipid A *in vivo*, mice were injected intraperitoneally with triacyl lipid A (at a single dose of 8 mg/kg), and the concentration of triacyl lipid A in ultracentrifugally isolated lipoprotein fractions was determined by the quantitation of 3-hydroxymyristic acid over a 9-h period. Although the low amounts of triacyl lipid A detected in VLDL, LDL, and plasma lipoprotein-free fraction did not differ significantly regardless of whether the mice expressed PLTP, triacyl lipid A associated in larger amounts with circulating HDLs in WT than in PLTP<sup>-/-</sup> mice (Fig. 1B–E). A similar tendency was observed earlier with hexaacyl LPS (15). The ratio of HDL-bound to unbound triacyl lipid A in WT mice was ~3 times higher than in PLTP<sup>-/-</sup> mice (Fig. 1F).

In complementary experiments, PLTP<sup>-/-</sup> mice showed concentrations of 3-hydroxymyristate in total plasma that were approximately half those in WT counterparts (P<0.05 at times 1.5, 9, and 24 h; P<0.05 for the AUC value corresponding to the entire 24 h period) (Fig. 2). As for the PLTP<sup>-/-</sup> homozygous mice, the concentration of 3-hydroxymyristate in total plasma from PLTP<sup>+/-</sup> heterozygotes tended to be lower than in WT counterparts, here with the statistical significance level being reached at the 24 h time point (P<0.05 at time 24 h, and P=0.054 for the AUC value; Fig. 2). Unlike PLTP<sup>-/-</sup> homozygotes in which complete deficiency in plasma phospholipid transfer activity is accompanied

A

<figure><img src="image_3.png" /></figure>

B

<figure><img src="image_4.png" /></figure>

D

<figure><img src="image_5.png" /></figure>

E

<figure><img src="image_6.png" /></figure>

C

<figure><img src="image_7.png" /></figure>

F

<figure><img src="image_8.png" /></figure>

**Figure 1.** PLTP promotes the association of triacyl lipid A to HDLs. A) At 10-, 30-, and 60-min time points, triacyl lipid A associates readily with PLTP-bearing HDLs but to a much lower extent with PLTP-deficient HDLs *in vitro* (P<0.05). WT mice (solid squares; n=6) and PLTP<sup>-/-</sup> mice (open squares; n=6) were injected intraperitoneally with a single dose of triacyl lipid A (8 mg/kg of body weight). B–E) Blood was collected at indicated times under anesthesia, and the triacyl lipid A content of ultracentrifugally isolated fractions (VLDL, B; LDL, C; d>1.21 g/ml fraction, D; HDL, E) was measured by 3-OH determination as described in Materials and Methods. F) Calculated HDL-bound to free triacyl lipid A ratio was significantly higher in PLTP-expressing mice than in PLTP<sup>-/-</sup> mice at 4.5 h. See Materials and Methods for additional experimental details. Error bars = SEM. \*P < 0.05 vs. WT mice.
<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

**Figure 2.** Effect of PLTP on triacyl lipid A accumulation in mouse plasma. WT (solid squares), PLTP<sup>+/−</sup> heterozygous (shaded squares), and PLTP<sup>−/−</sup> homozygous (open squares) mice (n=8 in each group) were injected intraperitoneally with a single dose of triacyl lipid A (8 mg/kg). A) Blood was collected at the indicated times under anesthesia, and triacyl lipid A levels in total plasma were assessed by 3-OH determination as described in Materials and Methods. B) Triacyl lipid A AUC in total plasma was calculated from kinetic curves measured over 24 h. Error bars = SEM. <sup>a</sup>P < 0.05 for PLTP<sup>−/−</sup> vs. WT mice; <sup>b</sup>P < 0.05 for PLTP<sup>+/−</sup> vs. WT mice; <sup>c</sup>P = 0.054 for PLTP<sup>−/−</sup> vs. WT mice. Experiment was done twice with similar results.

by a concomitant decrease in plasma cholesterol con-
centration, PLTP<sup>+/−</sup> heterozygotes display an ~40%
decrease in plasma phospholipid transfer activity with
no significant change in their plasma lipid and lipopro-
tein levels as compared with WT mice (21). Thus, these
observations come in support of a direct role of PLTP,
above its effect on plasma cholesterol, in determining
triacyl lipid A kinetics *in vivo*.

Triacyl lipid A clearance is faster in PLTP-deficient mice

WT and PLTP<sup>-/-</sup> mice were injected intravenously
with one single dose of triacyl lipid A (8 mg/kg), and
total plasma concentrations were determined over a
24 h period. As shown in **Fig. 3**, the injected triacyl lipid
A was rapidly cleared in PLTP<sup>-/-</sup> mice, with only 35%
of the initial dose remaining in plasma after 8 h. The
PLTP-mediated association of triacyl lipid A with HDLs

significantly increased its plasma residence time in WT
mice, with 53% of the initial dose remaining after 8 h
(*P*<0.05 vs. PLTP<sup>-/-</sup> mice). The clearance rate of
triacyl lipid A in WT mice was approximately half that
in PLTP<sup>-/-</sup> mice (Fig. 3B). The data with triacyl lipid
A are in sharp contrast with earlier studies from this
group in which hexaacyl LPS was more rapidly cleared
when associated with lipoproteins than when remain-
ing in its aggregated form in the plasma lipoprotein-
free fraction (15).

Triacyl lipid A has different effects on plasma
cytokine levels in WT and PLTP-deficient mice

The immunoinflammatory response to triacyl lipid A
was first evaluated using a specific ELISA measuring
plasma levels of IL-6 after one intraperitoneal injection
of triacyl lipid A (8 mg/kg). In both PLTP<sup>-/-</sup> and WT
mice, levels of IL-6 gradually increased and reached a
peak after 4.5 h. Levels then fell gradually and were

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

**Figure 3.** Kinetic analysis of plasma triacyl lipid A clearance in WT and PLTP<sup>-/-</sup> mice. A) WT mice (solid squares) and PLTP<sup>-/-</sup> mice (open squares) (n=9/group) received one single intraveinous injection of triacyl lipid A (8 mg/kg), and plasma 3-OH was assayed at 5-min, 90-min, 8-h, and 24-h time points. B) Clearance rate was expressed as fraction of the initial 3-OH pool cleared per hour. Error bars = SEM. \*P < 0.05 vs. WT mice. Experiment was done twice with similar results.
almost undetectable after 9 h (Fig. 4A). In the PLTP<sup>-/-</sup> mice, however, the levels at 1.5 and 4.5 h were 74 and 68% lower than those in the WT mice, respectively (*P*<0.05 in both cases; Fig. 4A). Over the entire 9-h period, PLTP<sup>-/-</sup> mice produced half as much IL-6 as did the WT mice. Consistent observations were made with regard to cytokines, which were assayed this time using a multiplex cytometric bead array following a single intravenous injection of 8 mg/kg of triacyl lipid A (Fig. 4B–F). In the latter case, triacyl lipid A was shown to produce transient increases in circulating levels of IL-6, IL-10, TNFα, IFNγ, and MCP-1. At the 1.5 h time point, increases in IL-6, IL-10, and TNFα were 35, 64, and 33% lower in PLTP<sup>-/-</sup> than in WT mice, respectively (*P*<0.05 in all cases, Fig. 4B–D).

In the opposite, and in complementary, experiments with isolated macrophages, the production of IL-6 and MCP-1 in the presence of the 500 ng/ml dose of triacyl lipid A was 95 ± 34 and 42 ± 2% lower in WT, PLTP-expressing than in PLTP-deficient cells (*P*<0.05 in both cases; **Fig. 5A, C**). Similarly, at the 10 ng/ml dose of hexaacyl LPS, production of IL-6 and MCP-1 by WT macrophages was 50 ± 5 and 40 ± 18% lower than by PLTP<sup>-/-</sup> macrophages, respectively (*P*<0.05 in both cases; Fig. 5B, D). Noticeably, TNFα production was similar in WT and PLTP-deficient macrophages treated with triacyl lipid A or LPS (Fig. 5E, F). In bioassays for the quantitation of either the metabolic activity of EMT-6 and B16-F10 cells (**Fig. 6A, B**) or of adherent EMT-6 cell quantitation (Fig. 6C), significantly lower values were observed with supernatants of LPS- or triacyl-lipid A-activated macrophages, and similar trends were observed when supernatants came from macrophages expressing PLTP or not. Finally, by

using flow cytometry analysis of annexin V- and 7-AAD-positive cells, there was no evidence for apoptosis or necrosis of EMT-6 and B16 cells when exposed to supernatants of triacyl lipid A-treated macrophages, in all cases with <15% of dead cells regardless of whether supernatants were from macrophages expressing PLTP (Fig. 6D). TNFα production, which is the main effector of macrophage-mediated cytotoxicity, was identical regardless of whether macrophages expressed PLTP (Fig. 5E). It may explain why low/similar cytotoxicity was observed with macrophage supernatants despite significant changes in IL-6 and MCP-1 production.

Overall, the latter observations indicate that differences in the intrinsic responsiveness of PLTP<sup>-/-</sup> vs. WT macrophages were unlikely to account for the observed differences in circulating levels of proinflammatory cytokines in triacyl lipid A-treated WT mice as compared with triacyl lipid A-treated PLTP<sup>-/-</sup> mice.

**Triacyl lipid A prevents the growth of B16 tumors in WT mice, but not in PLTP-deficient mice**

WT and PLTP<sup>-/-</sup> mice, aged 2–3 mo, were injected subcutaneously with B16 melanoma cells, and they were treated for 20 d with repeated intraperitoneal injections of either triacyl lipid A or control vehicle. Triacyl lipid A treatment (dose, 8 mg/kg) was found to be optimal when administered at 5 d intervals, with much weaker effects on tumor size when injected at 1 or 3 d intervals (**Fig. 7**). Thus, triacyl lipid A injections were systematically started at d 3, and applied at 5-d intervals throughout the study.

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

**Figure 4.** Time course of triacyl lipid A-induced cytokine and chemokine release in WT mice and PLTP-deficient mice. WT mice (n=6; solid squares) and PLTP<sup>-/-</sup> mice (n=6; open squares) were injected intraperitoneally (A) or intravenously (B–F) with a single dose of triacyl lipid A (8 mg/kg of body weight). Blood was collected at indicated times under anesthesia and plasma levels of cytokines/chemokines (IL-6, A–B; IL-10, C; TNFα, D; IFNγ, E; and MCP-1, F) were measured by ELISA (A) or cytometric bead array (B–F). Error bars = SEM. \* *P* < 0.05 vs. PLTP<sup>-/-</sup> mice. Experiment was done twice with similar results.
<figure><img src="image_19.png" /><figcaption>**Figure 5.** Effect of triacyl lipid A and LPS on proinflammatory response in isolated WT and PLTP<sup>-/-</sup> macrophages. Thioglycolate-elicited macrophages from WT mice (solid squares, *n*=5) and PLTP<sup>-/-</sup> mice (open squares, *n*=5) were incubated for 24 h in the presence of 5–500 ng/ml of triacyl lipid A (*A*, *C*, *E*) or 0.1–10 ng/ml bacterial LPS (*B*, *D*, *F*). Cytokine (IL-6, *A*–*B*; MCP-1, *C*, *D*; TNFα, *E*, *F*) contents of supernatants were measured by a cytometric bead array as described in Materials and Methods. Error bars = SEM; 3 independent experiments. \*P < 0.05 vs. WT mice.</figcaption></figure>

As shown in **Fig. 8A, B**, early tumors could be detected by palpation at the same time point (from d 7) in both PLTP<sup>-/-</sup> and WT mice. Intraperitoneal treatment of WT mice with triacyl lipid A produced a significant delay in tumor formation, after 7 d with only 30% of triacyl lipid A-treated WT mice bearing tumors against >80% of the untreated WT, untreated PLTP-deficient, or lipid A-treated PLTP-deficient animals (*P*<0.05 in all cases; Fig. 8A, B). When focusing on tumor volumes exceeding 4 mm³ (24) (Fig. 8C, D), triacyl lipid A treatment was even more clearly associated with a delay in tumor occurrence, an effect that was again fully abolished by PLTP deficiency.

Tumor size tended to increase less rapidly in untreated PLTP<sup>-/-</sup> than in untreated WT mice (Fig. 9). This might relate at least in part to the hypocholesterolemic trait of PLTP<sup>-/-</sup> mice (21), because tumor growth is known to be dependent on cholesterol availability, and weight of subcutaneous tumors was reported earlier to be positively correlated with plasma cholesterol (25). After 15 and 17 d, 70 and 69% reductions in tumor size were observed in lipid A-treated compared with untreated WT mice, respectively (*P*<0.05 in both cases; Fig. 9A). In contrast to its effect on WT mice, triacyl lipid A was not effective in the PLTP-deficient genetic background, and B16 tumors developed in similar ways regardless of whether animals were treated (Figs. 8B and 9B).

Overall, these results indicate that triacyl lipid A is able to delay significantly B16 tumor growth in mice, but only when they express active PLTP.

DISCUSSION

The production of cytokines by leukocytes is recognized as the hallmark of lipid A treatments (2). As observed with other lipid A derivatives, OM-174 triacyl lipid A (*i.e.*, a dual TLR2/TLR4 agonist) was shown to increase TNFα, IL-6, IL-8, and IL-10 and to a lesser extent IFNγ and IL-1β in rats (2, 19). Similarly, in the present study and in agreement with previous work (20), triacyl lipid A, whether it was injected intraperitoneally or intravenously, was associated with a transient increase in circulating levels of cytokines. The absence of efficacy of the triacyl lipid A treatment in PLTP-deficient mice was associated with lower levels of plasma TNFα, IL-6, and IL-10 as compared with treated WT mice. PLTP is a secreted protein that is highly expressed in the mouse (26) and is produced in large amounts by macrophages (27, 28). In the present study, a local effect of macrophage PLTP could be excluded since the triacyl lipid A-mediated production of cytokines tended to be higher for IL-6 and MCP-1 or unchanged for TNFα in PLTP-deficient cells compared with PLTP-expressing cells. Because LPS and triacyl lipid A stimulation led to the production of similar levels of TNFα (*i.e.*, the main macrophage-derived antitumor molecule) by WT and PLTP<sup>-/-</sup> macrophages (2, 29), it was predictable that both genotypes elicit similar effects on cultured target EMT6 and B16-F10 tumor cells. Based on previous observations of the exposure of mouse splenocytes to LPS (15), *in vitro* modulation of inflammation by locally produced PLTP
A

XTT, EMT6 target cells

<figure><img src="image_20.png" /></figure>

B

XTT, B16-F10 target cells

<figure><img src="image_21.png" /></figure>

C

cell adhesion, EMT6 target cells

<figure><img src="image_22.png" /></figure>

D

EMT6
target
cells

B16-F10
target
cells

Wild-type
macrophages

<figure><img src="image_23.png" /></figure>

PLTP-/-
macrophages

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

**Figure 6.** Metabolic activity, adhesion, and PS externalization of target tumor cells exposed to supernatants from WT and PLTP<sup>-/-</sup> macrophages incubated with high doses of triacyl lipid A or LPS. Thioglycolate-elicited macrophages from WT mice (solid bars, *n*=5) and PLTP<sup>-/-</sup> (open bars, *n*=5) mice were incubated for 24 h in the presence of 500 ng/ml of triacyl lipid A or 10 ng/ml LPS. Cell supernatants were recovered, and their effects on respiratory function (XTT test) (A, B), adhesion (C), necrosis, and PS externalization (D) of EMT-6 or B16-F10 target cells were determined as described in Materials and Methods. A–C) Bars are means ± SE of 3 independent experiments. D) Cytometric analysis of annexin-V-labeled EMT-6 and B16-F10 cells after treatment with supernatants from triacyl lipid A-treated macrophages. <sup>a</sup>P < 0.05 *vs.* control WT macrophages; <sup>b</sup>P < 0.05 *vs.* control PLTP<sup>-/-</sup> macrophages.

is dependent on the presence of extracellular lipoproteins that most likely sequestered triacyl lipid A in the extracellular culture medium at the expense of cell receptors. In support of this mechanism, the transfer of cell-bound LPS to lipoproteins by soluble CD14 was

also found to decrease cellular responses (5). Finally, and because PLTP expression led here to an amplification, not a reduction, in the inflammatory response to triacyl lipid A when injected in mice, we turned toward the working hypothesis of a systemic rather than a local effect of PLTP.

At the systemic level, PLTP deficiency was associated in the present study with the formation of fewer HDL-triacyl lipid A complexes. Unlike total plasma LPS concentration, which increased in PLTP-deficient mice by comparison with WT mice (15), PLTP deficiency was associated in the present study with decreased total plasma concentration of triacyl lipid A. This probably reflects the slower clearance of HDL-triacyl lipid A complexes (formed in large amounts in PLTP-containing plasma) as compared with unbound triacyl lipid A in the plasma lipoprotein-free fraction. The slower clearance of triacyl lipid A in PLTP-expressing mice contrasted with earlier studies in which the PLTP-mediated formation of HDL-LPS complexes was found to accelerate the clearance of bacterial LPS aggregates (15).

Differences in the behavior of hexaacyl LPS compared with triacyl lipid A might actually relate to differences in their physicochemical properties and physical state in aqueous media. Because OM-174 contains only 3 acyl chains (against 6 usually present in natural lipid A) and a polar head with glucosamines and ionized phosphates, its critical micellar concentra-

<figure><img src="image_27.png" /></figure>

**Figure 7.** Determination of appropriate experimental conditions for the antitumoral effect of triacyl lipid A. B16-F10 melanoma cells were injected subcutaneously in WT mice, and were left for 3 d. Treatment was started at time 0 (arrow), and animals received either triacyl lipid A (8 mg/kg=continuous lines) or vehicle only (dotted lines) with 1-, 3-, or 5-d intervals. Tumor was monitored every 2 or 3 d by using a caliper rule, and tumor size (mm³) was calculated as length × width² × 0.5.
<figure><img src="image_28.png" /><figcaption>**Figure 8.** Effect of triacyl lipid A treatment on tumor onset in WT and PLTP<sup>-/-</sup> mice. B16-F10 melanoma cells were injected subcutaneously in WT (A, C) or PLTP<sup>-/-</sup> (B, D) mice, and were left for 3 d. Treatment was started at time 0 (arrow), and animals received either triacyl lipid A (8 mg/kg; squares) or vehicle only (circles) over a 20-d period with 5-d intervals. Presence of early tumors (A, B) was assessed by palpation, and tumor volumes exceeding 4 mm<sup>3</sup> (C, D) were assessed by using a caliper rule. \*P < 0.05 vs. untreated controls. Experiments were done twice with similar results.</figcaption></figure>

tion (CMC, i.e., the concentration above which it forms large, complex structures) is high (0.1 mM for triacyl lipid A vs. <0.1 µM for hexaacyl lipid A or LPS) (30–32). At the concentration used in the present study (leading to a value of ~40 µM for the highest circulating concentrations), triacyl lipid A behaves like highly soluble monomers and micelles (30, 33). Because of their small size (<10 nm), monomers and micelles are supposed to diffuse easily out of the bloodstream by passage through endothelial fenestrae that are wide enough (mean diameter, 60–100 nm) (34). This might explain the rapid clearance and the loss of function of triacyl lipid A when it associates poorly with plasma lipoproteins, as observed in PLTP<sup>-/-</sup> mice in the present study. In contrast, hydrophobic, hexaacyl bacterial LPS mainly form large lamellar vesicles and inverted micelles (diameter range, 30–340 nm) (33, 35, 36). These are highly active (37, 38), and a disaggregation step that leads to the association with lipoproteins is needed before hexaacyl LPS can be neutralized and eliminated (15, 39, 40). The present study now demonstrates that the lipoprotein-binding process of bacterial LPS (14, 15, 41, 42) actually applies to, and thus modulates, the behavior and properties of triacyl lipid A but with opposite consequences on the metabolic fate of these 2 compounds. It comes in support of the hypothesis that PLTP fits with and modulates the activity of the wide variety of LPS that are expressed by innumerable microorganisms.

In the quest for alternative therapeutic strategies to delay cancer progression, lipid A derivatives have been the subject of intense research, and triacyl lipid A is now undergoing pharmacological development. Given the low toxicity of triacyl lipid A, it can be administered in high enough doses to make it useful

as a therapeutical tool *in vivo*. OM-174 triacyl lipid A was found in earlier studies to cure colon cancer in a syngenic rat model (19) and to reduce the number of metastases induced by melanoma cells in C57BL/6 mice (2). In phase I trials in patients with solid cancer, OM-174 triacyl lipid A was found to be able to produce an appropriate TLR2/TLR4-dependent response, as assessed by increased production of TNFα, IL-8, and IL-10 (2). In contrast with observations in WT mice (20; present study), it is shown here that triacyl lipid A is ineffective in preventing B16 tumor growth in PLTP<sup>-/-</sup> mice. This suggests that when triacyl lipid A binds to extracellular lipoproteins *via* PLTP, its residence time in the bloodstream is extended, and its biological activity is thus enhanced. The lack of an antitumor effect of triacyl lipid A in PLTP<sup>-/-</sup> mice could not be explained by a blunted cytotoxic response of PLTP-deficient macrophages. This provides further support for systemic rather than locally produced PLTP accounting for the amplified anticancer response in WT mice.

In summary, the present study sheds light on the efficacy and bioavailability of lipid A derivatives. It provides support to the hypothesis that PLTP-mediated association with lipoproteins could be a promising means to prolong residence time and thus efficacy of triacyl lipid A treatment. Therefore, and besides having a potential role in modulating the systemic inflammatory response in critically ill patients (43), increasing circulating levels of PLTP appears here to be a promising strategy to enhance the antitumoral effect of triacyl lipid A in anticancer therapy.

[J]

The authors thank Philip Bastable for manuscript editing. This work was supported by grants from the Université de
<figure><img src="image_29.png" /></figure>

<figure><img src="image_30.png" /><figcaption>**Figure 9.** Effect of triacyl lipid A treatment on tumor growth in WT and PLTP<sup>-/-</sup> mice. WT (A) and PLTP<sup>-/-</sup> (B) mice were treated as described in legend to Fig. 7. Tumor growth was monitored every 2 d with a caliper rule, and tumor size was calculated as length × width<sup>2</sup> × 0.5. *P* < 0.05 vs. untreated controls. Experiments were done twice with similar results.</figcaption></figure>

Bourgogne, the Conseil Régional de Bourgogne, INSERM (Institut National de la Santé et de la Recherche Médicale), the ANR (Agence Nationale de la Recherche), and the Fondation pour la Recherche Médicale (FRM).

REFERENCES

1. Miller, S. I., Ernst, R. K., and Bader, M. W. (2005) LPS, TLR4 and infectious disease diversity. *Nat. Rev. Microbiol.* **3**, 36–46

2. Garay, R. P., Viens, P., Bauer, J., Normier, G., Bardou, M., Jeannin, J. F., and Chiavaroli, C. (2007) Cancer relapse under chemotherapy: why TLR2/4 receptor agonists can help. *Eur. J. Pharmacol.* **563**, 1–17

3. Casella, C. R., and Mitchell, T. C. (2008) Putting endotoxin to work for us: monophosphoryl lipid A as a safe and effective vaccine adjuvant. *Cell. Mol. Life Sci.* **65**, 3231–3240

4. Sprong, T., Netea, M. G., van der Ley, P., Verver-Jansen, T. J., Jacobs, L. E., Stalenhoef, A., van der Meer, J. W., and van Deuren, M. (2004) Human lipoproteins have divergent neutralizing effects on E. coli LPS, N. meningitidis LPS, and complete gram-negative bacteria. *J. Lipid Res.* **45**, 742–749

5. Kitchens, R. L., Thompson, P. A., Viriyakosol, S., O'Keefe, G. E., and Munford, R. S. (2001) Plasma CD14 decreases monocyte responses to LPS by transferring cell-bound LPS to plasma lipoproteins. *J. Clin. Invest.* **108**, 485–493

6. Brandenburg, K., Jürgens, G., Andrä, J., Lindner, B., Koch, M. H., Blume, A., and Garidel, P. (2002) Biophysical characterization

of the interaction of high-density lipoprotein (HDL) with endotoxins. *Eur. J. Biochem.* **269**, 5972–5981

7. Cai, L., Ji, A., de Beer, F. C., Tannock, L. R., and van der Westhuyzen, D. R. (2008) SR-BI protects against endotoxemia in mice through its roles in glucocorticoid production and hepatic clearance. *J. Clin. Invest.* **118**, 364–375

8. Van Lenten, B. J., Fogelman, A. M., Haberland, M. E., and Edwards, P. A. (1986) The role of lipoproteins and receptor-mediated endocytosis in the transport of bacterial lipopolysaccharide. *Proc. Natl. Acad. Sci. U. S. A.* **83**, 2704–2708

9. Van Lenten, B. J., Wagner, A. C., Nayak, D. P., Hama, S., Navab, M., and Fogelman, A. M. (2001) High-density lipoprotein loses its anti-inflammatory properties during acute influenza a infection. *Circulation* **103**, 2283–2288

10. Hyka, N., Dayer, J. M., Modoux, C., Kohno, T., Edwards, C. K. 3rd, Roux-Lombard, P., and Burger, D. (2001) Apolipoprotein A-I inhibits the production of interleukin-1beta and tumor necrosis factor-alpha by blocking contact-mediated activation of monocytes by T lymphocytes. *Blood* **97**, 2381–2389

11. Cockerill, G. W., Huehns, T. Y., Weerasinghe, A., Stocker, C., Lerch, P. G., Miller, N. E., and Haskard, D. O. (2001) Elevation of plasma high-density lipoprotein concentration reduces interleukin-1-induced expression of E-selectin in an in vivo model of acute inflammation. *Circulation* **103**, 108–112

12. Vreugdenhil, A. C., Snoek, A. M., van 't Veer, C., Greve, J. W., and Buurman, W. A. (2001) LPS-binding protein circulates in association with apoB-containing lipoproteins and enhances endotoxin-LDL/VLDL interaction. *J. Clin. Invest.* **107**, 225–234

13. Thompson, P. A., Tobias, P. S., Viriyakosol, S., Kirkland, T. N., and Kitchens, R. L. (2003) Lipopolysaccharide (LPS)-binding protein inhibits responses to cell-bound LPS. *J. Biol. Chem.* **278**, 28367–28371

14. Hailman, E., Albers, J. J., Wolfbauer, G., Tu, A. Y., and Wright, S. D. (1996) Neutralization and transfer of lipopolysaccharide by phospholipid transfer protein. *J. Biol. Chem.* **271**, 12172–12178

15. Gautier, T., Klein, A., Deckert, V., Desrumaux, C., Ogier, N., Sberna, A. L., Paul, C., LeGuern, N., Athias, A., Montange, T., Monier, S., Piard, F., Jiang, X. C., Masson, D., and Lagrost, L. (2008) Effect of plasma phospholipid transfer protein deficiency on lethal endotoxemia in mice. *J. Biol. Chem.* **283**, 18702–18710

16. Tzotzas, T., Desrumaux, C., and Lagrost, L. (2009) Plasma phospholipid transfer protein (PLTP): review of an emerging cardiometabolic risk factor. *Obes. Rev.* **10**, 403–411

17. Nishijima, M., Amano, F., Akamatsu, Y., Akagawa, K., Tokunaga, T., and Raetz, C. R. (1985) Macrophage activation by monosaccharide precursors of *Escherichia coli* lipid A. *Proc. Natl. Acad. Sci. U. S. A.* **82**, 282–286

18. Jeannin, J. F., Onier, N., Lagadec, P., von Jeney, N., Stütz, P., and Liehl, E. (1991) Antitumor effect of synthetic derivatives of lipid A in an experimental model of colon cancer in the rat. *Gastroenterology* **101**, 726–733

19. Onier, N., Hilpert, S., Arnould, L., Saint-Giorgio, V., Davies, J. G., and Jeannin, J. F. (1999) Cure of colon cancer metastasis in rats with the new lipid A OM 174: apoptosis of tumor cells and immunization of rats. *Clin. Exp. Metastasis* **17**, 299–306

20. D'Agostini, C., Pica, F., Febbraro, G., Grelli, S., Chiavaroli, C., and Garaci, E. (2005) Antitumour effect of OM-174 and cyclophosphamide on murine B16 melanoma in different experimental conditions. *Int. Immunopharmacol.* **5**, 1205–1212

21. Jiang, X. C., Bruce, C., Mar, J., Lin, M., Ji, Y., Francone, O. L., and Tall, A. R. (1999) Targeted mutation of plasma phospholipid transfer protein gene markedly reduces high-density lipoprotein levels. *J. Clin. Invest.* **103**, 907–914

22. Olsson, N. O., Leclerc, A., Jeannin, J. F., and Martin, F. (1982) A simple photometric microassay for the quantitative evaluation of macrophage-mediated cytotoxicity on adherent cancer cells. *Ann. Immunol. (Paris)* **133D**, 245–254

23. Sponar, B., Krasnik, L., Hryniewiecki, T., Gamian, A., and Larsson, L. (2003) Distribution of 3-hydroxy fatty acids in tissues after intraperitoneal injection of endotoxin. *Clin. Chem.* **49**, 1149–1153

24. Büchau, A. S., Morizane, S., Trowbridge, J., Schauber, J., Kotol, P., Bui, J. D., and Gallo, R. L. (2010) The host defense peptide cathelicidin is required for NK cell-mediated suppression of tumor growth. *J. Immunol.* **184**, 369–378
25. Buchwald, H. (1992) Cholesterol inhibition, cancer, and che-
motherapy. *Lancet* **339**, 1154–1156

26. Guyard-Dangremont, V., Desrumaux, C., Gambert, P., Lalle-
mant, C., and Lagrost, L. (1998) Phospholipid and cholesteryl
ester transfer activities in plasma from 14 vertebrate species:
relation to atherogenesis susceptibility. *Comp. Biochem. Physiol. B
Biochem. Mol. Biol.* **120**, 517–525

27. Lakomy, D., Rébé, C., Sberna, A. L., Masson, D., Gautier, T., Chevriaux, A., Raveneau, M., Ogier, N., Nguyen, A. T., Gambert, P., Grober, J., Bonnotte, B., Solary, E., and Lagrost, L. (2009) Liver X receptor-mediated induction of cholesteryl ester transfer protein expression is selectively impaired in inflammatory macrophages. *Arterioscler. Thromb. Vasc. Biol.* **29**, 1923–1929

28. Vikstedt, R., Ye, D., Metso, J., Hildebrand, R. B., Van Berkel, T. J., Ehnholm, C., Jauhiainen, M., and Van Eck, M. (2007) Macrophage phospholipid transfer protein contributes significantly to total plasma phospholipid transfer activity and its deficiency leads to diminished atherosclerotic lesion development. *Arterioscler. Thromb. Vasc. Biol.* **27**, 578–586

29. Klimp, A. H., de Vries, E. G., Scherphof, G. L., and Daemen, T. (2002) A potential role of macrophage activation in the treatment of cancer. *Crit. Rev. Oncol. Hematol.* **44**, 143–161

30. Brandenburg, K., Lindner, B., Schromm, A., Koch, M. H., Bauer, J., Merkli, A., Zbaeren, C., Davies, J. G., and Seydel, U. (2000) Physicochemical characteristics of triacyl lipid A partial structure OM-174 in relation to biological activity. *Eur. J. Biochem.* **267**, 3370–3377

31. Brandenburg, K., Mayer, H., Koch, M. H., Weckesser, J., Ri-
etschel, E. T., and Seydel, U. (1993) Influence of the supramo-
lecular structure of free lipid A on its biological activity. *Eur.
J. Biochem.* **218**, 555–563

32. Shnyra, A., Hultenby, K., Lindberg, A. A. (1993) Role of the physical state of Salmonella lipopolysaccharide in expression of biological and endotoxic properties. *Infect. Immun.* **61**, 5351–5360

33. Sasaki, H., and White, S. H. (2008) Aggregation behavior of an
ultra-pure lipopolysaccharide that stimulates TLR-4 receptors.
*Biophys. J.* **95**, 986–993

34. Salmon, A. H., Neal, C. R., and Harper, S. J. (2009) New aspects of glomerular filtration barrier structure and function: five

layers (at least) not three. *Curr. Opin. Nephrol. Hypertens.* **18**,
197–205

35. Koch, P. J., Frank, J., Schüler, J., Kahle, C., and Bradaczek, H. (1999) Thermodynamics and structural studies of the interaction of polymyxin B with deep rough mutant lipopolysaccharides. *J. Colloid Interface Sci.* **213**, 557–564

36. Bergstrand, A., Svanberg, C., Langton, M., and Nydén, M. (2006) Aggregation behavior and size of lipopolysaccharide from *Escherichia coli* O55:B5. *Colloids Surf. B Biointerfaces* **53**, 9–14

37. Mueller, M., Lindner, B., Kusumoto, S., Fukase, K., Schromm, A. B., and Seydel, U. (2004) Aggregates are the biologically active units of endotoxin. *J. Biol. Chem.* **279**, 26307–26313

38. Seong, S. Y., and Matzinger, P. (2004) Hydrophobicity, an
ancient damage-associated molecular pattern that initiates in-
nate immune responses. *Nat. Rev. Immunol.* **4**, 469–478

39. Ulevitch, R. J., Johnston, A. R., and Weinstein, D. B. (1981) New function for high density lipoproteins: isolation and characterization of a bacterial lipopolysaccharide-high density lipoprotein complex formed in rabbit plasma. *J. Clin. Invest.* **67**, 827–837

40. Kitchens, R. L., Wolfbauer, G., Albers, J. J., and Munford, R. S. (1999) Plasma lipoproteins promote the release of bacterial lipopolysaccharide from the monocyte cell surface. *J. Biol. Chem.* **274**, 34116–34122

41. Levels, J. H., Marquart, J. A., Abraham, P. R., van den Ende, A. E., Molhuizen, H. O., van Deventer, S. J., and Meijers, J. C. (2005) Lipopolysaccharide is transferred from high-density to low-density lipoproteins by lipopolysaccharide-binding protein and phospholipid transfer protein. *Infect. Immun.* **73**, 2321–2326

42. Vesy, C. J., Kitchens, R. L., Wolfbauer, G., Albers, J. J., and Munford, R. S. (2000) Lipopolysaccharide-binding protein and phospholipid transfer protein release lipopolysaccharides from gram-negative bacterial membranes. *Infect. Immun.* **68**, 2410–2417

43. Wendel, M., Paul, R., and Heller, A. R. (2007) Lipoproteins in inflammation and sepsis. II. Clinical aspects. *Intensive Care Med.* **33**, 25–35

Received for publication January 13, 2010.
Accepted for publication April 8, 2010.