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PhenotypesDefense through Divergent TLR ResponseIntrapulmonary and Extrapulmonary Host Dyslipidemia Induces Opposing Effects on

FesslerMartha D. Wilson, Lawrence L. Rudel and Michael B.Smoak, Haitao Li, Gary L. Griffiths, Benjamin T. Suratt, Jennifer H. Madenspacher, David W. Draper, Kathleen A.

http://www.jimmunol.org/content/185/3/1660doi: 10.4049/jimmunol.0903501June 2010;

2010; 185:1660-1669; Prepublished online 25J Immunol 

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The Journal of Immunology

Dyslipidemia Induces Opposing Effects on Intrapulmonaryand Extrapulmonary Host Defense through Divergent TLRResponse Phenotypes

Jennifer H. Madenspacher,* David W. Draper,* Kathleen A. Smoak,* Haitao Li,†

Gary L. Griffiths,† Benjamin T. Suratt,‡ Martha D. Wilson,x Lawrence L. Rudel,x and

Michael B. Fessler*

Dyslipidemia influences innate immune responses in the bloodstream, but whether and how pulmonary innate immunity is sensitive

to circulating lipoproteins is largely unknown. To define whether dyslipidemia impacts responses to bacteria in the airspace and, if

so, whether differently from its effects in other tissues, airspace, bloodstream, and i.p. responses to LPS and Klebsiella pneumoniae

were investigated using murine models of dyslipidemia. Dyslipidemia reduced neutrophil (PMN) recruitment to the airspace in

response to LPS and K. pneumoniae by impairing both chemokine induction in the airspace and PMN chemotaxis, thereby

compromising pulmonary bacterial clearance. Paradoxically, bacteria were cleared more effectively from the bloodstream during

dyslipidemia. This enhanced systemic response was due, at least in part, to basal circulating neutrophilia and basal TLR4/MyD88-

dependent serum cytokine induction and enhanced serum cytokine responses to systemically administered TLR ligands. Dysli-

pidemia did not globally impair PMN transvascular trafficking to, and host defense within all loci, because neutrophilia, cytokine

induction, and bacterial clearance were enhanced within the infected peritoneum. Peritoneal macrophages from dyslipidemic

animals were primed for more robust TLR responses, reflecting increased lipid rafts and increased TLR4 expression, whereas

macrophages from the airspace, in which cholesterol was maintained constant during dyslipidemia, had normal responses and

rafts. Dyslipidemia thus imparts opposing effects upon intra- and extrapulmonary host defense by inducing tissue-divergent TLR

response phenotypes and dysregulating airspace/blood compartmental levels of PMNs and cytokines. We propose that the airspace

is a “privileged” site, thereby uniquely sensitive to dyslipidemia. The Journal of Immunology, 2010, 185: 1660–1669.

Serum lipoproteins, such as low-density lipoprotein (LDL),exert complex effects upon intravascular innate immunity.Lipoproteins neutralize bacterial LPS (1). Oxidation of

LDL, which may occur during dyslipidemia and infection (2, 3),also generates lipoprotein species with activity upon TLR4, theLPS receptor. Stimulation of TLR4 by minimally oxidized LDL(oxLDL) (4) is proposed to underlie TLR4-dependent vascularinflammation induced by dyslipidemia (5). In contrast, more com-pletely oxLDL inhibits TLR responses (3, 6), attenuating antimi-crobial functions in dendritic cells during dyslipidemia (3). Perhaps

reflecting the complexity of cross-talk between lipoproteins andinnate immunity, varying effects of dyslipidemia upon host defensehave also been reported. Clearance of some pathogens (e.g., Leish-mania) is compromised during dyslipidemia (3, 7, 8), whereas forothers (e.g., Klebsiella pneumoniae), both enhanced and impairedhost defense are reported (9, 10). Most reports have used models ofbloodstream infection, and several are complicated by the use ofanimals deficient in apolipoprotein E, itself a direct regulator ofinflammation and LPS (1, 11). Given the high prevalence of dysli-pidemia in humans, an improved understanding of the effects ofdyslipidemia on host defense at the initial extravascular sites ofpathogen encounter may bear important implications.Pulmonary host defense is critically important as acute lower

respiratory tract infections impose the greatest burden of mor-bidity and mortality of all infections in the United States (12).Serum lipoproteins are taken up by pulmonary endothelium (13)and modify the function of pulmonary cells in vitro (14–16).Moreover, recent studies of ATP binding cassette transporter G1null mice indicate that cholesterol trafficking and inflammationmay be coupled uniquely within the lung (17, 18). Despite this,there have been very few reports of the effect of dyslipidemia onpulmonary innate immunity; indeed, regulation of airspace lipidsduring dyslipidemia and the effects of dyslipidemia upon lungbiology in general are both poorly understood. The lung may, infact, have unique challenges, given its anatomical positioningimmediately downstream of return of lipoprotein-rich first-passthoracic duct lymph and portal blood and the sensitivity of sur-factant function to cholesterol (19). Moreover, as recruitment ofneutrophils (PMNs) to the airspace entails several microvascularevents distinct from those in other tissues (20), PMN-dependent

*Laboratory of Respiratory Biology, National Institute of Environmental HealthSciences, Research Triangle Park, NC 27709; †Imaging Probe Development Center,National Heart, Lung, and Blood Institute, National Institutes of Health, Rockville,MD 20850; ‡Vermont Lung Center, University of Vermont College of Medicine,Burlington, VT 05405; and xSection on Lipid Sciences, Department of Pathology,Wake Forest University Health Sciences, Winston-Salem, NC 27157

Received for publication October 26, 2009. Accepted for publication May 22, 2010.

This work was supported in part by the Intramural Research Program of the NationalInstitutes of Health, National Institute of Environmental Health Sciences and byNational Institutes of Health Grants R01 HL84200 and HL-49373.

Address correspondence and reprint requests to Dr. Michael B. Fessler, National In-stitute of Environmental Health Sciences, 111 T. W. Alexander Drive, P.O. Box 12233,MD D2-01, Research Triangle Park, NC 27709. E-mail address: fesslerm@niehs.nih.gov

The online version of this article contains supplemental material.

Abbreviations used in this paper: BAL, bronchoalveolar lavage; BALF, bronchoalveolarlavage fluid; Chol-PEG10000-NH2, monocholesteryl polyethylene glycol; DCM,dichloromethane; fPEG-chol, fluorescein ester of polyethylene glycol cholesterylether; HCD, high-cholesterol diet; i.t., intratracheal(ly); KC, keratinocyte-derived che-mokine; LDL, low-density lipoprotein; LIX, lipopolysaccharide-induced CXC chemo-kine; LXR, liver X receptor; MFI, mean fluorescence intensity; ND, normal diet; nLDL,native low-density lipoprotein; oxLDL, oxidized low-density lipoprotein; PEG, poly-ethylene glycol; PEM, peritoneal exudatemacrophage; PMN, neutrophil;WT,wild-type.

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intra-alveolar host defense may have distinct sensitivity to circulat-ing lipoprotein levels.We hypothesized that dyslipidemia would modify pulmonary

innate immunity. In this paper, we report that dyslipidemia attenu-ates PMN trafficking to the airspace triggered by both LPS andK. pneumoniae, compromising pulmonary clearance of the latter.The reduced influx of PMNs stems from deficient induction of air-space chemokines associated with reduced pulmonary NF-kBactivation as well as impaired PMN chemotaxis, indicating thatdyslipidemia impacts airway responses by modulating both lung-resident and circulating cells. Paradoxically, bacteria are clearedmuch more effectively from the bloodstream during dyslipidemia,reflecting basal circulating neutrophilia and activation of systemicinnate immunity as well as hypersensitivity to TLR ligands in theserum. Dyslipidemia does not globally impair PMN egress into, andTLR responses within, all peripheral loci, because PMN influx andTLR responses are indeed enhancedwithin the infected peritoneum.Lipid rafts and cell surface TLR4 are increased in peritoneal macro-phages during dyslipidemia, whereas rafts are unchanged in alveo-lar macrophages. Dyslipidemia thus imparts opposing effects uponintra- and extrapulmonary host defense by dysregulating compart-mental TLR responses and airspace/blood compartmentalizationof PMNsand cytokines.Wepropose that the airspacemaybe a “priv-ileged” site, thereby uniquely sensitive to dyslipidemia.

Materials and MethodsReagents

Escherichia coli 0111:B4 LPS, penicillin, streptomycin, and methyl-b-cyclodextrin-cholesterol were from Sigma-Aldrich (St. Louis, MO).For the latter, concentrations were based on cyclodextrin formula weight.K. pneumoniae 43816 (serotype 2), DMEM, FBS, and RAW 264.7 cellswere from American Type Culture Collection (Manassas, VA). Kerati-nocyte-derived chemokine (KC), MIP-2, and IL-17 were from R&DSystems (Minneapolis, MN).

Mice and diets

Female mice (7–14 wk old, weighing 18–22 g) were used in all experiments.C57BL/6 and LDL receptor (ldlr) null (backcrossed 10 generations ontoC57BL/6) mice were from The Jackson Laboratory (Bar Harbor, ME).Myd88 and Tlr4 null mice backcrossed less than eight generations ontoC57BL/6 (21, 22) were provided by S. Akira (Osaka University, Osaka, Ja-pan). All experiments were performed in accordance with the Animal Wel-fare Act and the U.S. Public Health Service Policy on Humane Care and Useof Laboratory Animals after review by the Animal Care and Use Committeeof theNational Institute ofEnvironmentalHealth Sciences.Micewere placedfor 2–5 wk on either a high-cholesterol diet (HCD) (15.8% fat, 1.25% cho-lesterol, and 0.5% cholate [TD.90221; Harlan Laboratories, Indianapolis,IN]) or a normal diet (ND), which was identical, except for omission ofcocoa butter, cholesterol, and cholate (TD.95138). In other studies, micefed TD.95138 were compared with those fed a matching control diet supple-mented with 0.5% sodium cholate (TD.98244) or were fed a cholate-freeWestern diet (TD.88137, 4 wk).

In vivo exposures

Exposure to aerosolized LPS (300 mg/ml, 20 min) was as described pre-viously (23). KC (0.5 mg in 60 ml), IL-17 (3 mg in 60 ml), and K. pneumo-niae (1500 or 2000 CFU in 60 ml) were delivered intratracheally (i.t.) byoropharyngeal aspiration during isoflurane anesthesia. In other experi-ments, K. pneumoniae was injected i.v. (7 3 104 CFU) into the retro-orbital venous plexus or i.p. (13 106 CFU), or LPS (2 mg/kg) was injectedi.p. PBS (13, pH 7.4), or native LDL (nLDL) or oxLDL (200 mg) wasdelivered i.v. into the retro-orbital venous plexus 2 h before and 2 hfollowing exposure to aerosolized LPS.

Bronchoalveolar lavage fluid collection and analysis

Bronchoalveolar lavage fluid (BALF) was collected immediately followingsacrifice, and total leukocyte and differential counts were performed, asdescribed previously (23). Total protein was quantified by the method ofBradford.

In vitro chemotaxis

Bone marrow-isolated PMNs (24) (1 3 106, 0.1 ml) were seeded into theupper chamber of a Transwell system (polycarbonate membrane, 3.0 mmpore) (Corning, Corning, NY). Medium (0.4 ml) with or without MIP-2(5 ng/ml) or KC (25 ng/ml) (PeproTech, Rocky Hill, NJ) was added to thelower chamber. Cells were counted in the lower chamber in triplicate after1 h (37˚C).

Bactericidal assays

Lung, spleen, and liver were excised after sacrifice and homogenized inPBS, and serial dilutions were plated on tryptic soy agar for bacterialquantification, as described previously (23). Blood was collected from theinferior vena cava and plated after serial dilution. Intracellular killingcapacity of PMNs against i.p.-injected K. pneumoniae was quantified asreported previously (25).

Synthesis of fPEG10000-chol

To polyethylene glycol (PEG)10,000-bis(amine) (1 g, 0.1 mmol) indichloromethane (DCM) (75 ml) was added triethylamine (41 ml, 0.3mmol). Cholesteryl chloroformate (54 mg, 0.12 mmol) dissolved inDCM (5 ml) was then added dropwise and stirred (room temperature,overnight). TLC analysis indicated formation of dicholesteryl and mono-cholesteryl products. The reaction mixture was diluted with DCM (425ml), washed with 5% potassium hydrogen sulfate, brine, and 5% sodiumbicarbonate, dried over sodium sulfate, and evaporated to dryness. Col-umn chromatography using a gradient of methanol (0–20%) in DCMafforded monocholesteryl PEG (Chol-PEG10000-NH2) as a white solid(574 mg, 55%), confirmed by MALDI-mass spectroscopy and 1H NMR.To Chol-PEG10000-NH2 (133 mg, 0.012 mmol) in 0.1 M sodiumbicarbonate (13 ml) was added FITC isomer I, and the reaction mixturewas stirred (72 h), after which all Chol-PEG10000-NH2 was consumed byHPLC analysis. The reaction mixture was concentrated (MilliporeCentriprep, YM-3) over eight rounds, after which HPLC analysis of theretentate showed only one peak. This was lyophilized, loaded ontoa PD-10 column, and eluted with water. The desired m.w. fractions fromthe PD-10 column were pooled and lyophilized to give the final product asan orange solid (108 mg, 78%), which was confirmed by HPLC, MALDI-mass spectroscopy, and 1H NMR analysis.

PMN and macrophage harvests and culture

Mature murine bone marrow PMNs were isolated from mouse femurs andtibias by discontinuous Percoll gradient centrifugation, as described pre-viously (24). This preparation yields cell populations that are.95% PMNs(data not shown). Peritoneal exudate macrophages (PEMs) were harvestedby peritoneal lavage 96 h after i.p. injection of Brewer’s thioglycollate(2 ml, 4% solution). Alveolar macrophages were harvested by BAL of re-sting, unexposed animals (macrophages represent .98% of airspace cellpellet). Both macrophage types were plated in DMEM supplemented with10% FBS, penicillin, and streptomycin. RAW 264.7 cells were culturedin DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 mg/mlstreptomycin, and 100 U/ml penicillin under a humidified 5% CO2 atmo-sphere at 37˚C. For in vitro cell exposures, highly purified E. coli 0111:B4LPS (product number 201; List Biological, Campbell, CA) and endotoxin-free PAM3CSK4 (InvivoGen, San Diego, CA) were used.

Western blotting

Plated macrophages were lysed in 13 Laemmli/20 mM DTT. Protein wasresolved by 10% SDS-PAGE, transferred to nitrocellulose (Bio-Rad, Her-cules, CA), and probed with primary Abs. Rabbit anti–PO4-p38 (1/1000),rabbit anti-p38 (1/1000), rabbit anti–PO4-JNK, and rabbit anti-JNK werefrom Santa Cruz Biotechnology (Santa Cruz, CA). Membranes were thenwashed in Tween 20 and Tris-buffered saline and exposed for 60 min toa 1/5000 dilution of species-specific, HRP-conjugated secondary Ab (GEHealthcare, Piscataway, NJ) in 5% milk/Tween 20 and Tris-buffered saline.Following further washes, the signal was detected with ECLWestern BlotDetection Reagents (GE Healthcare), followed by film exposure (GEHealthcare).

NF-kB activity assay

Activation of p65 NF-kB in the nuclear fraction of lung homogenates(Nuclear Extract Kit, Active Motif, Carlsbad, CA) was quantified byELISA (p65 TransAM kit; Active Motif) after normalizing nuclearprotein input (Bradford assay).

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Cytokine analysis

Cytokines were quantified by multiplex assay (Bio-Plex; Bio-Rad). LPS-induced CXC chemokine (LIX) was quantified by ELISA (ELISATech, Au-rora, CO). The lower limits of detection were as follows: IL-17 (2.0 pg/ml),MIP-2 (2.0 pg/ml), G-CSF (2.8 pg/ml), TNF-a (3.0 pg/ml), KC (2.7 pg/ml),IL-6 (1.0 pg/ml), and LIX (8.0 pg/ml).

Peripheral blood leukocyte typing and enumeration

The blood samples were analyzed using the HEMAVET 1700 hematologyanalyzer (Drew Scientific, Waterbury, CT). Manual WBC differential countswere reported, and smear estimates were used to confirm values.

Serum lipoprotein cholesterol quantification

Plasma lipoprotein separation was achieved by size-exclusion chromatog-raphy (fast protein liquid chromatography). Cholesterol concentrationswere measured by gas chromatography or enzymatically, as described pre-viously (26).

Intracellular bacterial killing assay

A previously reported protocol was followed (25). Mice received 2.5 ml 4%thioglycollate i.p., followed 4 h later by 1 3 108 CFU K. pneumoniae i.p.Intraperitoneal leukocytes were collected 30 min later by lavage (5 mlHBSS containing 100 mg/ml gentamicin) and then washed. Cells (13 106)were then incubated (37˚C) for varying durations, followed by lysis (0.1%Triton X-100) and CFU quantification by plating of serial dilutions.

Flow cytometry

Anti-CXCR2 (PerCP/Cy5.5), -Gr-1 (FITC), -F4/80 (PE or allophycocyanin),-CD11c (PE-Cy5), and isotype control Abs were from BioLegend (SanDiego, CA). Anti-CD14 (PE) and -TLR4 (PE) were from eBioscience(San Diego, CA). Cells were stained and fixed with 2% paraformalde-hyde/PBS. For fPEG10000-chol staining, cells were fixed (2% paraformalde-hyde), washed, and then exposed to fPEG10000-chol (10 mg/ml, 10 min,room temperature). Flow cytometry was performed using an LSR II (BDBiosciences, San Jose, CA) and analyzed using FlowJo (Tree Star, Ashland,OR) and FCS Express (De Novo Software, Los Angeles, CA) software.

Statistical analysis

Analysis was performed using GraphPad Prism statistical software (SanDiego, CA). Data are represented as mean 6 SEM. Two-tailed Student’s ttest was applied for comparisons of two groups and ANOVA for analysesof three or more groups. Survival was evaluated by log-rank test. For alltests, p , 0.05 was considered significant.

ResultsDyslipidemia induces circulating neutrophilia and TLR4/MyD88-dependent serum cytokines

To investigate the effects of dyslipidemia on responses in theairspace, we used a well-established model of HCD-induced dys-lipidemia in wild-type (WT) (C57BL/6) mice (27, 28). The effectsof dyslipidemia upon airspace/blood compartmental levels of cho-lesterol, leukocytes, and cytokines in the steady state were firstdefined. As expected, compared with ND-fed mice, HCD-fed micedeveloped significantly elevated serum total cholesterol, with in-creases in the LDL and very LDL fractions and a decrease in high-

density lipoprotein cholesterol (Supplemental Fig. 1A). There wasno difference in BALF cholesterol between mice fed the two diets(Supplemental Fig. 1B), suggesting that cholesterol levels are reg-ulated by distinct mechanisms in the airspace and serum. Bycontrast, peritoneal lavage fluid cholesterol was increased inHCD-fed mice (Supplemental Fig. 1C), indicating that extravas-cular compartments differ in regulation of cholesterol levels dur-ing dyslipidemia.Whereas no significant difference was noted between ND- and

HCD-fed mice in steady-state BAL total leukocyte count/differen-tial (data not shown), HCD-fed mice displayed increased circulat-ing PMNs and monocytes (Table I). Dyslipidemia is reported toinduce endovascular inflammation through TLR4 and its intracel-lular adaptor, MyD88 (29, 30). Nonetheless, we found that HCDinduction of circulating PMNs and monocytes were both MyD88independent, because they occurred equivalently in WTandMyd88null mice (Table I). Whereas the circulating lymphocyte count wasunchanged by HCD in WT mice, it was significantly increased byHCD in Myd88 null mice (Table I), suggesting that MyD88-dependent signals may suppress HCD-induced lymphocytosis.HCD did not impact peripheral blood hemoglobin, erythrocytecount, or platelet count (data not shown), suggesting a selectiveeffect upon hematopoietic lineages. IL-17 reportedly regulates gran-ulopoiesis through its target gene G-CSF (31, 32), although we areunaware of any prior report of IL-17 regulation during dyslipide-mia. We found that HCD modestly upregulated IL-17 and its targetgene MIP-2 in the serum (Fig. 1A, 1B); although, HCD did notupregulate G-CSF or TNF-a (Fig. 1C, 1D). HCD-associated induc-tion of IL-17 and MIP-2 were noted to occur in a MyD88- andTLR4-dependent fashion, as animals deficient in these genes, unlikeWT animals, had no HCD induction of IL-17 and blunted inductionof MIP-2 (Fig. 1A, 1B). Unlike the serum compartment, there wasno induction by HCD of basal BALF levels of MIP-2 (data notshown).Taken together, these findings indicate that 1) cholesterol is

regulated differentially between serum and airspace during dys-lipidemia, yielding divergent lipid environments; 2) dyslipidemiaimpacts steady-state compartmental leukocyte and cytokine levelsdifferentially between airspace and serum; and 3) TLR4/MyD88regulates dyslipidemia-associated induction of serum cytokinesbut not expansion of PMNs and monocytes in the bloodstream.

Dyslipidemia attenuates induced trafficking of PMNs to theinflamed airspace

To determine the effect of dyslipidemia upon induced trafficking ofleukocytes to the airspace, we next exposed mice to aerosolizedLPS, using an established model (23). Despite their relative cir-culating neutrophilia compared with ND-fed mice, HCD-fed micehad significantly reduced influx of total leukocytes (WBCs) intothe BALF following LPS inhalation (Fig. 2A). The reduction inWBCs was accounted for by a reduction in BALF PMNs (Fig.2A), because BALF macrophage and lymphocyte numbers were

Table I. Blood leukocyte count and differential in Myd88+/+ and Myd882/2 mice fed ND or HCD

Cell Count (3103/ml)

Myd88+/+ Myd882/2

ND (n = 13) HCD (n = 13) ND (n = 10) HCD (n = 8)

WBCs 2.35 6 0.71 2.68 6 1.03 1.95 6 0.46 3.34 6 0.79*PMNs 0.24 6 0.1 0.39 6 0.24† 0.21 6 0.12 0.45 6 0.24†

Monocytes 0.09 6 0.05 0.14 6 0.06‡ 0.08 6 0.03 0.12 6 0.04†

Lymphocytes 1.93 6 0.63 2.05 6 0.7 1.49 6 0.43 2.67 6 0.64*Eosinophils 0.03 6 0.02 0.05 6 0.06 0.04 6 0.03 0.04 6 0.04

Values are mean 6 SD.pp , 0.001; †p , 0.05; ‡p = 0.05 for HCD versus ND comparison of cell type count within genotype.

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not different between mice on the two diets (data not shown). Thereduction of LPS-induced airspace PMNs was observed followingas little as 2 wk of HCD feeding (data not shown). By contrast,there was no difference in LPS-induced accumulation of airspacePMNs between mice on ND and mice on ND supplemented withcholate (Fig. 2B), indicating that the cholate component of HCD,previously reported to regulate proinflammatory genes in the livervia farnesoid X receptor (33), does not itself modulate the pulmo-nary innate immune response. Moreover, ldlr null mice on a (cho-late-free) Western diet, an alternate commonly used mouse modelof dyslipidemia, also had significantly fewer total leukocy-tes/PMNs recruited to the airspace after LPS than Western diet-fed WT mice (Fig. 2C). oxLDL, like nLDL, is increased in theserum by HCD and mediates the effects of dyslipidemia uponvascular inflammation and innate immunity (3, 4, 6). Consistentwith possible roles for nLDL and oxLDL in our model, naive(chow-fed) WT mice subjected to pharmacologic dysplipidemiaby i.v. injection of either oxLDL or nLDL had lesser WBC influxinto the airspace after inhaled LPS than did mice treated with i.v.PBS (vehicle) (Fig. 2D). Taken together, these data indicate thatdyslipidemia,modeled by several independent approaches, dysregu-lates systemic versus pulmonary compartmentalization of PMNsduring an inflammatory stimulus.

Dyslipidemia modulates airspace cytokine induction byinhaled LPS

Circulating PMNs migrate to the inflamed or infected airspace inresponse to locally expressed chemokines. In pursuit of mecha-nisms underlying the reduced recruitment of PMNs to the LPS-exposed lung during dyslipidemia, we thus first measured LPS-induced cyto-/chemokine expression in BALF. As PMNs beginto accumulate in the airspace at ∼4–6 h post-LPS in this model(data not shown) (23), we assayed BALF 2 h post-LPS as a tem-porally relevant time point largely reflecting cytokine productionby lung-resident cells. As shown in Fig. 3, BALF levels ofMIP-2 and KC, two CXC chemokines that play critical roles inPMN recruitment to the LPS-exposed lung (23), were lower inHCD than in ND mice, whereas expression of the chemokineLIX (CXCL5) was equivalent between diets. TNF-a, a cytokineimportant in PMN recruitment to lung, was also lower in HCDmice, whereas BALF IL-6, a negative regulator of LPS-inducedPMN recruitment to the lung (34), was modestly higher in

HCD mice. Serum KC, MIP-2, and IL-6 were equivalent be-tween ND- and HCD-fed mice 2 h post-LPS inhalation (datanot shown). Taken together, these findings suggest that airspacecyto-/chemokine expression is differentially modulated duringdyslipidemia. Reductions in BALF TNF-a and in airspace-serum chemokine gradients may possibly contribute to the ob-served reduction in LPS-induced airspace neutrophilia, as pro-posed in other models (35).LPS induction of KC and MIP-2 in the lung is driven by the tran-

scription factor NF-kB in lung structural cells (36). Notably, wefound that DNA binding of p65 NF-kB in lung parenchymalnuclear isolates 2 h post-LPS was indeed lower in HCD-fed thanin ND-fed mice (Fig. 3F), suggesting that dyslipidemia may mod-ulate airspace cytokine production through effects upon NF-kBactivation.

Dyslipidemia impairs PMN migration

Given the relatively modest reductions in LPS-induced BALF che-mokines in dyslipidemic animals, we next investigated whetherdyslipidemia also modifies PMN migration itself. To modelPMNmigration in response to airspace chemokines in vivo, we firstinstilled KC directly into the airspace (23) of ND- and HCD-fedmice. HCD-fed mice indeed had reduced airspace recruitment ofPMNs in response to i.t. KC (Fig. 4A). To more directly examineeffects of dyslipidemia on PMN migratory function, we next isolated

FIGURE 1. Dyslipidemia induces serum cytokines through TLR4 and

MyD88. WT,Myd882/2, and Tlr42/2 mice were fed either ND or HCD for

5 wk, following which serum cytokines (A, IL-17; B, MIP-2; C, G-CSF; D,

TNF-a) were quantified by Bioplex assay (n = 5/genotype/diet). pp , 0.01

compared with WT/ND; †p , 0.05 compared with WT/HCD.

FIGURE 2. Dietary, genetic, and pharmacologic dyslipidemia attenu-

ate PMN recruitment to the LPS-exposed murine airspace. A, Leukocyte

recruitment to LPS-exposed airway. BAL total leukocytes (WBCs) and

PMNs were enumerated at various time points following exposure of ND-

and HCD-fed C57BL/6 mice to aerosolized LPS (n = 10–13/diet/time

point). pp , 0.01. B, BAL WBCs were enumerated 24 h following aero-

solized LPS in mice fed ND or ND supplemented with sodium cholate

(n = 10/diet). C, BALWBCs were enumerated 24 h following aerosolized

LPS in ldlr+/+ and ldlr2/2 mice fed Western diet (n = 10/genotype). pp ,0.01. D, Mice were pretreated i.v. with equal volumes of 13 PBS, or

200 mg nLDL or oxLDL 2 h before exposure to aerosolized LPS, and

then BAL WBCs were enumerated 8 h postexposure (n = 10/treatment).

pp = 0.01; ppp , 0.01.

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mature PMNs from the bone marrow of ND- and HCD-fed miceand quantified their chemotaxis in vitro. Consistent with thein vivo model, PMNs isolated from HCD mice had impaired che-motaxis to KC as well as MIP-2 (Fig. 4B). Cell surface expressionof CXCR2 was decreased in circulating PMNs from HCD-fed ascompared with ND-fed mice (Fig. 4C). By contrast, CD18 cellsurface expression was equivalent in PMNs between the two diets(data not shown). Thus, in addition to reduction of chemokineexpression by lung-resident cells, the dyslipidemic environmentalso confers a chemotactic defect upon circulating PMNs that mayreflect, at least in part, downregulated cell surface expression ofchemokine receptors.

Dyslipidemia attenuates airspace neutrophilia and cytokines inresponse to K. pneumoniae

Given the observed deficits in PMNmigration and in recruitment ofPMNs to the LPS-exposed lung, we predicted that PMN migrationto the lung in response to intact Gram-negative bacteria would alsobe deficient during dyslipidemia. Indeed, compared with ND-fedmice, HCD-fed mice had fewer PMNs in the airspace followingi.t. inoculation with K. pneumoniae (Fig. 5A). As with LPS, inresponse to K. pneumoniae, HCD-fed animals had markedly re-duced BALF MIP-2 (Fig. 5B), a chemokine of establishedimportance to PMN recruitment to the K. pneumoniae-infectedlung (37). There was, however, no significant alteration in BALFIL-6 (Fig. 5C), LIX (Fig. 5D), or KC (data not shown) and a ∼2-fold increase in G-CSF in HCD-fed mice (Fig. 5E).IL-17 plays a critical role in secondary induction of PMN-

attracting chemokines and cytokines including MIP-2, LIX, andG-CSF in the K. pneumoniae-infected lung (38). Given the

varying effects of HCD upon MIP-2, LIX, KC, and G-CSF ex-pression in the lung, we questioned whether dyslipidemic animalsmight have an underlying change in pulmonary IL-17 expressionor activity. HCD-fed mice had significantly increased BALF IL-1724 h after i.t. K. pneumoniae (Fig. 5F). BALF concentrations ofMIP-2, LIX, KC, and G-CSF were equivalent between ND- andHCD-fed animals 2 h following i.t. delivery of rIL-17 (data notshown), suggesting that IL-17 responsiveness is not modified inlung-resident cells during dyslipidemia. Collectively, these find-ings suggest that reduced PMN recruitment to the airspace in re-sponse to i.t. K. pneumoniae during dyslipidemia reflects bothimpaired PMN chemotaxis and reduced airspace expression ofchemokines and that the latter occurs at least in part through anIL-17–independent mechanism.

Dyslipidemia exerts opposing effects on intra- andextrapulmonary antibacterial host defense

Airspace-recruited PMNs play a critical role in the clearance of pul-monary infection with extracellular pathogens, such as K. pneumo-niae. Thus, we predicted that intrapulmonary host defense againstK. pneumoniaewould be impaired inHCD-fed animals. Indeed, com-pared with ND-fed animals, HCD-fed animals had significantly in-creased K. pneumoniae CFUs in lung homogenates following i.t.inoculation (Fig. 6A), indicating impaired intrapulmonary host de-fense.As IL-17 induction in the infected lungparallelsK.pneumoniaeburden (38), increased microbial load may account for the increasedBALF IL-17 in HCD-fed mice and, conversely, highlights the signif-icance of the BALF MIP-2 deficit in these animals (Fig. 5B).Dissemination of bacteria from the infected lung to the blood-

stream and from there to peripheral organs generally tracks in parallelwith intrapulmonary bacterial burden (23). Nonetheless, we foundthat HCD-fed animals had markedly reduced K. pneumoniae CFUsin spleen and liver following i.t. infection (Fig. 6B, 6C). This sug-gested the two possibilities that either: 1) the integrity of the alveo-locapillary barrier was enhanced during dyslipidemia, thus reducing

FIGURE 4. PMN migration is impaired during dyslipidemia. A, BAL

PMNs were enumerated 4 h following i.t. delivery of 0.5mg KC by aspiration

to ND- and HCD-fed mice (n = 10/diet). pp, 0.05. B, In vitro chemotaxis to

MIP-2 and KC of bone marrow-derived PMNs isolated from ND- and HCD-

fed mice was quantified by modified Boyden chamber assay (n = 9/diet/che-

mokine). pp , 0.01; ppp , 0.001. C, Surface expression of CXCR2 on

circulatingPMNs fromND-andHCD-fedmicewas quantifiedbyflowcytom-

etry. PMNs were gated as Gr-1hi. Isotype control (gray) and CXCR2 in HCD

(blue trace) and ND (black trace) Gr-1hi cells are shown at center; CXCR2

is quantified as MFI at right, representative of two independent experiments

(n = 3/diet). pp, 0.01. MFI, mean fluorescence intensity.FIGURE 3. LPS-induced airspace cyto-/chemokines are modulated dur-

ing dyslipidemia. A–E, ND- and HCD-fed mice were exposed to aerosol-

ized LPS. BALF cytokines, including TNF-a (A), MIP-2 (B), KC (C), LIX

(D), and IL-6 (E) were quantified by ELISA 2 h postexposure (n = 13/diet).

pp, 0.05; ppp, 0.01. F, NF-kB p65 DNA binding was quantified in lung

parenchymal nuclear isolates from ND- and HCD-fed mice at baseline and

at 2 h post-LPS inhalation (n = 4–7/diet/time point). pp , 0.05.

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bacterial dissemination despite increased bacteria in the airspace; or2) extrapulmonary (i.e., bloodstream) killing of K. pneumoniaemight be paradoxically enhanced during dyslipidemia. Arguingagainst the first possibility, BALF protein concentration, an indi-cator of pulmonary microvascular integrity, was equivalent be-tween ND and HCD animals following pneumonia inductionand higher in HCD animals following LPS inhalation (Supple-mental Fig. 2). As PMNs, the major cellular effector of host de-fense against Klebsiella, were increased in the blood of HCD-fedmice (Table I), we favored the second possibility. Indeed, confirm-ing enhanced clearance of bacteremia in HCD—as compared withND-fed animals, blood and splenic CFUs were both significantlyreduced in HCD-fed animals following direct i.v. inoculation withK. pneumoniae (Fig. 6D, 6E). Perhaps reflecting counterbalancingeffects of dyslipidemia upon host defense within versus outside ofthe lung, survival following i.t. inoculation with K. pneumoniaewas equivalent between ND- and HCD-fed animals (Fig. 6F).Although the final survival rate was modestly higher in HCD-fed animals, this difference was not statistically significant.

Dyslipidemia primes for more robust extrapulmonary TLRresponses

We next explored mechanisms in addition to neutrophilia and cy-tokine induction (Fig. 1, Table I) that might underlie the enhancedsystemic host defense response during dyslipidemia. Consistentwith dyslipidemia priming for a more robust systemic response toinfection, HCD-fed mice had markedly enhanced (∼20-fold greaterthan ND-fed mice) serum TNF-a and IL-1b (∼3-fold greater) butnot MIP-2 following i.p. LPS (Fig. 7A). Suggesting a contributionfrom dyslipidemia-primed extrapulmonary macrophages to this

enhanced systemic response, cultured PEMs from HCD-fed micealso had enhanced TNF-a induction (Fig. 7B) and enhancedactivation of p38, JNK, and NF-kB (Fig. 7C) following in vitrotreatment with either LPS or the TLR2 ligand PAM3CSK4. Bycontrast, alveolar macrophages harvested from unexposed ND-and HCD-fed mice, although not strictly analogous to elicitedperitoneal macrophages nonetheless informative of the airwaymacrophage response, produced equivalent amounts of TNF-aprotein in response to LPS ex vivo (Fig. 7D).Cholesterol loading of the macrophage plasma membrane

in vitro enhances cellular responses to TLR2 and TLR4 ligandsthrough expansion of lipid raft microdomains (39, 40). However, itis unknown whether a similar phenomenon occurs to macrophagesin vivo during hypercholesterolemia. To investigate this, we syn-thesized an analog (Supplemental Fig. 3) of a compound that haspreviously been used as a raft probe, fPEG-chol (41–43). fPEG-chol is a cholesterol derivative that intercalates into the exofacialleaflet of the plasma membrane in raft microdomains in parallel tomembrane cholesterol content (41, 43). F4/80+ peritoneal exudatecells harvested from HCD-fed animals bearing increased perito-neal fluid cholesterol (Supplemental Fig. 1) indeed had greaterfPEG-chol signal by flow cytometry than their counterparts fromND-fed animals (Fig. 7E, Supplemental Fig. 4A), consistent withincreased lipid rafts. In contrast, alveolar macrophages fromHCD- and ND-fed animals, harvested from equivalent BALF cho-lesterol environments (Supplemental Fig. 1B), had equivalent raftsas assessed by fPEG-chol staining (Fig. 7F). F4/80+ peritoneal

FIGURE 6. Dyslipidemia confers opposing effects upon antibacterial

host defense in the airspace and bloodstream. A–C, Bacterial CFUs in lung

(A), spleen (B), and liver (C) homogenates were quantified 24 and 48 h

following i.t. delivery of 2000 CFU K. pneumoniae to HD- and HCD-fed

mice (n = 15/diet/time point). pp, 0.05. D and E, Bacterial CFUs in blood

(D) or spleen (E, 4 h) were quantified at time points following i.v. injection

of 7 3 104 CFU K. pneumoniae into ND- and HCD-fed mice (n = 10/diet/

time point). ppp , 0.01. F, Survival was monitored in ND- and HCD-fed

mice following i.t. delivery of 1500 CFU K. pneumoniae (n = 30/diet, three

independent experiments). p = NS by log-rank test.

FIGURE 5. Dyslipidemia attenuates PMN recruitment to airspace and

modifies airspace cytokine expression in response to i.t. K. pneumoniae. A,

BAL PMNs were quantified at time points following i.t. delivery of 2000

CFU K. pneumoniae by aspiration to ND- and HCD-fed mice (n = 10/diet/

time point). pp , 0.01. B–F, BALF cytokines, including MIP-2 (B), IL-6

(C), LIX (D), G-CSF (E), and IL-17 (F) were quantified 24 h following i.t.

delivery of K. pneumoniae (n = 10/diet). pp , 0.05; †p = 0.05.

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cells from HCD-fed animals were equivalent to their ND counter-parts in cell surface expression of CD14 (Fig. 7G, SupplementalFig. 4B) but, notably, had significantly greater expression of TLR4(Fig. 7H, Supplemental Fig. 4C). In support of the premise thatextracellular cholesterol may be incorporated into membrane raftsand enhance TLR4 responses, RAW 264.7 macrophages loadedwith cholesterol in vitro using cyclodextrin-cholesterol complex(17, 40) had increased fPEG-chol signal and produced increasedlevels of TNF-a in response to LPS (Supplemental Fig. 5).Consistent with previous reports, cholesterol loading was itselfsufficient to induce TNF-a in the macrophage (SupplementalFig. 5) (17, 40).

Dyslipidemia enhances PMN influx into and host defensewithin the peritoneum

To address whether dyslipidemia globally impairs PMN trans-vascular migration into all peripheral loci or, alternatively, whetherit exerts effects more specific to PMN accumulation in the airspace,two models of neutrophilic peritonitis were next investigated.Contrary to responses in the lung, and despite the HCD-associatedPMN chemotactic defect, i.p. LPS (Fig. 8A) and i.p. K. pneumo-niae (Fig. 8B) both induced greater i.p. PMN accumulation inHCD- than ND-fed mice. HCD-fed mice also had increased peri-toneal lavage fluid levels of TNF-a and MIP-2 following both i.p.LPS and K. pneumoniae (Fig. 8C–F). Also contrary to the lung,HCD-fed mice cleared i.p.-injected K. pneumoniae more effec-tively from their peritonea than their ND-fed counterparts (Fig.8G); in parallel, HCD mice had lesser dissemination of peritonealK. pneumoniae to the spleen (Fig. 8H). To determine whetherdyslipidemia modulates intrinsic PMN bactericidal capacityin vivo, we pre-elicited PMNs into the peritoneum with thiogly-collate, exposed the elicited PMNs in vivo to K. pneumoniae byi.p. injection, and then harvested the PMNs for further ex vivoincubation, as described previously (25). Quantification of intra-cellular K. pneumoniae CFUs revealed similar time-dependentkilling curves between PMNs from ND- and HCD-fed mice (Sup-plemental Fig. 6), indicating no diet-induced modification of in-tracellular bactericidal function.

DiscussionSerum lipoproteins influence innate immunity, but whether and howinnate immune responses in the airspace are sensitive to dyslipidemia

FIGURE 7. Dyslipidemia primes for more robust extrapulmonary TLR

responses. A, Serum cytokines were quantified in ND- and HCD-fed mice

2 h following i.p. injection of 2 mg/kg E. coli LPS (n = 6/diet). pp , 0.05;

ppp , 0.0001. B, TNF-a release into media by PEMs harvested from ND-

and HCD-fed mice was quantified by ELISA 2 h after ex vivo treatment

with LPS (10 ng/ml) or PAM3CSK4 (PAM, 10 ng/ml). Data are represen-

tative of four independent experiments. pp , 0.01. C, Cultured PEMs

harvested from ND- or HCD-fed mice were exposed as shown, following

which whole-cell lysates were immunoblotted for the indicated targets.

Findings are representative of three independent experiments. Hatched line

demarcates juxtaposition of different sections of the same gel. D, Alveolar

macrophages (∼3 3 104, .98% purity) collected from unchallenged

ND- and HCD-fed mice were plated in DMEM/10% FBS and exposed

to 10 ng/ml E. coli 0111:B4 LPS. TNF-a protein release into the media

16 h postexposure was quantified by ELISA (n = 8/diet). E, Lipid rafts

were quantified by flow cytometry in F4/80+ peritoneal exudate cells from

ND- and HCD-fed mice after in vitro treatment with fPEG-chol, a fluoro-

scein-labeled raft probe (representative of two experiments involving n =

7–9 animals/diet). pp , 0.001. F, Lipid rafts were quantified by flow

cytometry of fPEG-chol in CD11c+ alveolar macrophages (n = 5/diet).

G, Cell surface CD14 was quantified by flow cytometry in F4/80+ perito-

neal exudate cells (n = 4–5/diet). H, Cell surface TLR4 was quantified by

flow cytometry in F4/80+ peritoneal exudate cells (left panel), and percent-

age of F4/80+ cells expressing TLR4 was determined (right panel) (n = 4–

5/diet). pp , 0.01. MFI, mean fluorescence intensity.

FIGURE 8. PMN recruitment to and host defense responses within the

peritoneal cavity are enhanced during dyslipidemia. A and B, PMNs were

quantified in peritoneal lavage fluid 4 h after i.p. injection of 2mg/kg LPS (n =

5/diet,A) and 1 h after i.p. injection of 13 106 CFUK. pneumoniae (n = 9–10/

diet,B). pp, 0.05.C–F, TNF-a proteinwas quantified byELISA inperitoneal

lavage fluid 2 or 2.5 h after i.p. injection of LPS (C) or K. pneumoniae (E),

respectively; parallel measurements were made of peritoneal fluid MIP-2 (D,

F) (n = 4–10/diet/injectable). pp, 0.05; †p = 0.08. G and H, Bacterial CFUs

were quantified in peritoneal lavage fluid (G) and in splenic homogenate (H)

2.5 h after i.p. injection of 13 106 CFU K. pneumoniae (n = 12–19/diet over

three independent experiments). pp, 0.05.

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has, to a large extent, not been described. In this paper, we report thatdyslipidemia induceswidely contrasting effects upon innate immunityand host defense within versus outside of the lung. Airspace fluid, asevaluated in the form of BALF, maintains cholesterol levels underconditions inducing adoublingof serumcholesterol.This suggests thatthe airspacemaybe aprivilegedcompartment duringdyslipidemia andconfirms that intra-alveolar and circulating immune cells are exposedto divergent environments during dyslipidemia. Dyslipidemia primessystemic innate immune responses by 1) inducing basal serum cyto-kines through TLR4/MyD88; 2) enhancing LPS induction of serumcytokines invivo andmacrophage cytokines ex vivo; and 3) expandingcirculating PMNs andmonocytes; together, 4) enhancing bloodstreamkilling of bacteria. In contrast, dyslipidemia attenuates intrapulmonaryinnate immunity in the form of reduced: 1) LPS-stimulated airspacecytokines; 2)migration of PMNs into the airspace in response to intra-alveolarLPS,KC, andbacteria; and3) bacterial killingwithin the lung.Enhanced bloodstream killing and deficient pulmonary killing of K.pneumoniae are unified by the dysregulation of blood/airspace PMNcompartmentalization that occurs during dyslipidemia. This, in turn,appears to be due, at least in part, to a PMN migration defect. Ofinterest, dyslipidemic animals with pulmonary bacterial overgrowthnevertheless clear bacteria that have egressed from lung to blood andperipheral sites more effectively than their ND-fed counterparts. Op-posite to the airspace, PMNmigration into the peritoneum in responseto both LPS and K. pneumoniae, and i.p. clearance of the latter, isenhanced in the dyslipidemic state. Taken together, these findings in-dicate that, despite impairing chemotaxis, dyslipidemia does not im-pose a global defect upon transvascular migration of the PMN into allinflammatory loci. Rather, dyslipidemia selectively impairs host de-fense in the airspace.Our findings that HCD modulates LPS-induced BALF cytokines

and lung parenchymal NF-kB suggest that lung-resident cells areindeed sensitive to dyslipidemia and divergently so compared withextrapulmonary LPS-responsive cells (Figs. 3, 7, 8). Cholesterol-loaded macrophages have been detected in lung tissue of hyper-cholesterolemic rabbits, although the interstitial versus alveolarlocalization of these cells is uncertain (44). Airspace fluid appearsto be relatively sheltered from changes in circulating cholesterol,as the lipid environments of the serum and of the alveolus, towhich alveolar macrophages and alveolar epithelium are exposed,diverge during dyslipidemia (Supplemental Fig. 1). Complicatingthe matter, dyslipidemic serum contains an array of mediatorswith conflicting activities upon innate immunity. Serum-lung com-partmentalization of these mediators has not been determined; inaddition, it is not known whether alveolocapillary disruption com-promises mediators’ compartmentalization during lung injury. Werecently reported that pharmacologic activation of liver X receptor(LXR), a cell sensor of oxysterols expressed in both alveolarmacrophages and alveolar epithelium, reduces LPS induction ofTNF-a and MIP-2 in BALF and airspace recruitment of PMNs inmice (23). As dyslipidemia and oxLDL stimulate LXR, it is pos-sible that the inhibitory effects of dyslipidemia on airspace cyto-kine expression in the current study may reflect an LXR-dependent counterregulatory response in alveolar cells.Given the complex milieu during dyslipidemia, it is unlikely that

a single mediator is sufficient for the HCD phenotype. ElevatedLDL is unlikely by itself to explain our findings. Although ldlr nullmice and nLDL-infused WT mice have a similar pulmonary LPSphenotype to that of HCD-fed mice (Fig. 2), neutralization of LPSby excess serum LDL in ldlr null mice is thought to account fortheir reported dampened induction of serum cytokines followingi.p. LPS (10). Also unlike the HCD phenotype, ldlr null mice arereported to have normal, not enhanced, clearance of i.v. K. pneumo-niae from blood, liver, and spleen (10). Interestingly, contrary to the

in vivo ldlr null i.p. LPS phenotype, ldlr null murine macrophages(10) and mutant ldlrmonocytes from familial hypercholesterolemiapatients (45), like HCD peritoneal macrophages in the currentstudy, are hyperresponsive to LPS in vitro. This suggests their in-trinsic modification in vivo by a mediator not requiring interactionwith the LDL receptor. OxLDL is induced in serum by HCD (3)and taken up by scavenger receptors, but several papers and ourown findings (data not shown) indicate that oxLDL inhibits LPSinduction of cytokines in macrophages (6). Thus, although circu-lating oxLDL also recapitulates some features of our model, re-ducing PMN influx into the LPS-exposed airspace (Fig. 2D), it doesnot appear sufficient to mediate the full phenotype.Cellular cholesterol loading per se also exerts complex effects

upon innate immune function. Increasing plasmalemmal-free cho-lesterol through either genetic manipulation or in vitro cholesterolloading reportedly enhances TLR2 and TLR4 activation throughexpanding lipid raft mass (39, 40). We provide the first paper, toour knowledge, that macrophage lipid rafts are increased in vivoduring hypercholesterolemia and that there is an associated in-crease in cell surface TLR4. These findings raise the interestingpossibility that cellular raft cholesterol/mass, and with it, TLR4expression, may also vary with lipid status in human subjects andact as a determinant of cellular inflammatory phenotype. Althoughwe found significant differences between alveolar and peritonealelicited macrophages from the HCD condition, caution is war-ranted in drawing direct comparisons between these two substan-tially different cell types. Thus, future studies are required to morecarefully characterize the phenotype of different macrophage pop-ulations during the dyslipidemic state.In our study, unchallenged HCD-fed mice displayed evidence of

basal systemic inflammation in the form of circulating neutrophiliaand TLR4-/MyD88-dependent induction of serumMIP-2 and IL-17,although it should be noted that the serum cytokine induction wasmodest and of uncertain biological impact. Of interest, in humans,dyslipidemia, sometimes in the context of obesity and/or “metabolicsyndrome” is also associated with peripheral leukocytosis and in-creased serum cytokines (46). Obesity was not a factor in the phe-notype in our study, because HCD-fed animals had modestly lowerbody weight than ND-fed animals did (data not shown). We areunaware of any investigations of alveolar inflammation in dyslipi-demic humans, although we recently reported an inverse relationshipbetween serum cholesterol and asthma, an inflammatory airwaysdisease, in a national survey (47). Thus, the findings of the currentstudy may well have relevance to human disease.Other mouse models may provide insight into the pathogenesis

in our dyslipidemia model. Interestingly, circulating neutrophilia,increased plasma IL-17, and impaired PMN transvascularmigrationare hallmarks of murine models of adhesion molecule deficiency(i.e., CD18, E/P/L-selectin knockouts) (31, 32). Indeed, granulo-poiesis and trafficking of PMNs to tissues are homeostaticallylinked insofar as successful PMN trafficking to tissues includingthe lung curbs their induction of IL-17/G-CSF (32). Contrary to thedeficit in PMN migration to the peritoneum reported in adhesionmolecule-deficient models, we found that HCD feeding was asso-ciated with enhanced inducible migration of PMNs into the peri-toneum. We also found PMN surface expression of CD18 to beunchanged by HCD (data not shown).We speculate that the basal systemic TLR4-/MyD88-dependent

inflammation induced by dyslipidemia may contribute causally tothe deficit in inducible PMN transvascular migration into the air-space. Reminiscent of our findings, PMN recruitment into the air-space in response to i.t. LPS is attenuated during endotoxemiabecause of impaired PMN chemotaxis to MIP-2 (48). Systemicadministration of bacterial lipoprotein, a MyD88-dependent

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stimulus, also impairs PMN migration to peripheral inflammatoryloci through downregulation of chemokine receptors includingCXCR2 (49). Long-term exposure to circulating CXC chemo-kines, a feature of our HCD model (Fig. 1), was also previouslyreported in the context of genetic overexpression to impair PMNtransvascular emigration through CXCR2 desensitization (50).Taken together, these studies suggest that dyslipidemia-inducedsystemic inflammation has the potential to produce several fea-tures of the PMN compartmentalization phenotype in the currentstudy through effects upon PMN migration. Despite the manyfeatures of PMN transvascular emigration that are unique to thelung (e.g., transcapillary instead of transvenular transit, absence ofrolling) (20), we are unaware of other reported models in whichPMN migration into airspace and peritoneum are oppositely reg-ulated. One important feature of dyslipidemia that may contributeto this lung-selective phenotype is the parallel, discordant effect ofdyslipidemia upon local intra-alveolar versus i.p. induction ofcytokines that act to attract the circulating PMN.The lung has not been widely conceived of as an organ sensitive

to systemic dyslipidemia. We report that dyslipidemia regulatesinnate immunity and host defense discordantly between lung andblood/peritoneum and connect this to dysregulated compartmentallevels of PMNs and cytokines, as well as to a tissue-divergent LPSresponse phenotype that occurs during dyslipidemia. The “unique”relationship between the airspace and serum lipids likely arisesfrom unique features of lung biology, including the requirementfor maintenance of alveolar surfactant lipid, and the unique andincompletely understood cellular/molecular features of the trans-capillary passage of PMNs from blood to interstitium to alveolus.Future studies will be required to differentiate the roles of variousdyslipidemic serum mediators in lung pathophysiology and to in-vestigate their therapeutic potential for manipulation of pulmo-nary innate immunity.

AcknowledgmentsWe thank Laura M. DeGraff for technical assistance with i.v. injection, San-

dra Ward for peripheral leukocyte counting and typing, Dr. Shizuo Akira for

generously providing Myd88 and Tlr4 null mice, and Drs. Donald Cook

and Stavros Garantziotis for manuscript review.

DisclosuresL.L.R. has received research funding from Amgen and GlaxoSmithKline

in excess of $5000 within the past 5 years.

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