Dyslipidemia Induces Opposing Effects on Intrapulmonary and Extrapulmonary Host Defense through Divergent TLR Response Phenotypes¹

Reagents

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

Mice and diets

Female mice (7–14 weeks old, weighing 18–22 g) were used in all experiments. C57BL/6 and ldlr null (backcrossed 10 generations onto C57BL/6) mice were from Jackson Laboratories (Bar Harbor, ME). Myd88 and Tlr4 null mice backcrossed >8 generations onto C57BL/6 (21, 22) were provided by Shizuo Akira. All experiments were performed in accordance with the Animal Welfare Act and the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals after review by the Animal Care and Use Committee of the National Institute of Environmental Health Sciences. Mice were placed for 2–5 weeks on either a high-cholesterol diet (HCD)(15.8% fat, 1.25% cholesterol, 0.5% cholate [Harlan Teklad TD.90221]) or a normal diet (ND), which was identical except for omission of cocoa butter, cholesterol, and cholate (TD.95138). In other studies, mice fed TD.95138 were compared to those fed a matching control diet supplemented with 0.5% sodium cholate (TD.98244), or were fed a cholate-free Western diet (TD.88137, 4 weeks).

In vivo exposures

Exposure to aerosolized LPS (300 μg/ml, 20 min) was as previously described (23). KC (0.5 μg in 60 μl), IL-17 (3 μg in 60 μl), and K. pneumoniae (1500 or 2000 CFU in 60 μl) were delivered intratracheally by oropharyngeal aspiration during isoflurane anesthesia. In other experiments, K. pneumoniae was injected i.v. (7 × 10⁴ CFU) into the retro-orbital venous plexus or i.p. (1×10⁶ CFU), or LPS (2 mg/kg) was injected i.p. PBS (1×, pH 7.4), or native or oxidized LDL (200 μg) were delivered i.v. into the retro-orbital venous plexus 2 hrs before and 2 hrs following exposure to aerosolized LPS.

Bronchoalveolar Lavage Fluid (BALF) Collection and Analysis

BALF was collected immediately following sacrifice, and total leukocyte and differential counts were performed, as previously described (23). Total protein was quantified by the method of Bradford.

In vitro chemotaxis

Bone marrow-isolated PMNs(24) (1x10⁶, 0.1 mL) were seeded into the upper chamber of a transwell system (polycarbonate membrane, 3.0 μm pore)(Corning, Corning, NY). Media (0.4 mL) with or without MIP-2 (5 ng/ml) or KC (25 ng/ml)(Peprotech) was added to the lower chamber. Cells were counted in the lower chamber in triplicate after 1 hr (37°C).

Bactericidal assays

Lung, spleen, and liver were excised after sacrifice, homogenized in PBS, and serial dilutions plated on tryptic soy agar for bacterial quantification, as described (23). Blood was collected from the inferior vena cava and plated after serial dilution. Intracellular killing capacity of PMNs against i.p.-injected K. pneumoniae was quantified as reported (25).

Synthesis of fPEG₁₀₀₀₀-chol

To PEG₁₀₀₀₀- bis(amine)(1 g, 0.1 mmol) in dichloromethane (DCM)(75 mL) was added triethylamine (TEA)(41 μL, 0.3 mmol). Cholesteryl chloroformate (54 mg, 0.12 mmol) dissolved in DCM (5 mL) was then added dropwise, and stirred (RT, overnight). TLC analysis indicated formation of dicholesteryl and monocholesteryl products. The reaction mixture was diluted with DCM (425 mL), washed with 5% potassium hydrogen sulfate, brine, and 5% sodium bicarbonate, dried over sodium sulfate, and evaporated to dryness. Column chromatography using a gradient of methanol (0–20%) in DCM afforded monocholesteryl PEG (Chol-PEG₁₀₀₀₀-NH₂) as a white solid (574 mg, 55%), confirmed by MALDI-MS and ¹H NMR. To Chol-PEG₁₀₀₀₀-NH₂ (133 mg, 0.012 mmol) in 0.1 M sodium bicarbonate (13 mL) was added FITC isomer I and the reaction mixture was stirred (72 hrs), after which all Chol-PEG₁₀₀₀₀-NH₂ was consumed by HPLC analysis. The reaction mixture was concentrated (Millipore Centriprep, YM-3) over eight rounds after which HPLC analysis of the retentate showed only one peak. This was lyophilized, loaded onto a PD-10 column, and eluted with water. The desired MW fractions from the PD-10 column were pooled and lyophilized to give the final product as an orange solid (108 mg, 78%), which was confirmed by HPLC, MALDI-MS, and ¹H NMR analysis.

Neutrophil and macrophage harvests and culture

Mature murine bone marrow PMNs were isolated from mouse femurs and tibias by discontinuous Percoll gradient centrifugation, as previously described (24). This preparation yields cell populations that are >95% PMNs (data not shown). Peritoneal exudate macrophages were harvested by peritoneal lavage 96 hours after i.p. injection of Brewer’s thioglycollate (2 ml, 4% solution). Alveolar macrophages were harvested by BAL of resting, unexposed animals (macrophages represent >98% of airspace cell pellet). Both macrophage types were plated in DMEM supplemented with 10% FBS, penicillin, and streptomycin. RAW 264.7 cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 100 μg/ml streptomycin, and 100 U/ml penicillin under a humidified 5% CO₂ atmosphere at 37ºC. For in vitro cell exposures, highly purified E. coli 0111:B4 LPS (List Biological, Campbell, CA, product #201) and endotoxin-free PAM3CSK4 (Invivogen, San Diego, CA) were used.

Western blotting

Plated macrophages were lysed in 1x Laemmli/20 mM DTT. Protein was resolved by 10% SDS-PAGE, transferred to nitrocellulose (Bio-Rad), and probed with primary antibodies. Rabbit anti-PO₄-p38 (1:1000), rabbit anti-p38 (1:1000), rabbit anti-PO₄-JNK, and rabbit anti-JNK were from Santa Cruz (Santa Cruz, CA). Membranes were then washed in Tween Tris Buffered Saline (TTBS), and exposed for 60 minutes to a 1:5000 dilution of species-specific, HRP-conjugated secondary antibody (GE Healthcare) in 5% milk/TTBS. Following further washes, signal was detected with ECL™ Western Blot Detection Reagents (GE Healthcare), followed by film exposure (GE Healthcare).

NF-κB activity assay

Activation of p65 NF-κB in the nuclear fraction of lung homogenates (Nuclear Extract Kit, Active Motif) was quantified by ELISA (p65 TransAM™ kit, Active Motif, Carlsbad, CA) after normalizing nuclear protein input (Bradford assay).

Cytokine analysis

Cytokines were quantified by multiplex assay (Bio-Plex, Bio-Rad, Hercules, CA). LIX was quantified by ELISA (ELISA Tech, Aurora, 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), TNFα (3.0 pg/ml), KC (2.7 pg/ml), IL-6 (1.0 pg/ml), LIX (8.0 pg/ml).

Peripheral blood leukocyte typing and enumeration

The blood samples were analyzed using the HEMAVET 1700 hematology analyzer (Drew Scientific, Inc.). Manual WBC differential counts were reported and smear estimates were used to confirm values.

Serum lipoprotein cholesterol quantification

Plasma lipoprotein separation was achieved by size exclusion chromatography (FPLC). Cholesterol concentrations were measured by gas chromatography or enzymatically, as previously described (26).

Intracellular bacterial killing assay

A previously reported protocol was followed (25). Mice received 2.5 ml of 4% thioglycollate i.p., followed 4 hrs later by 1x10⁸ CFUs of K. pneumoniae i.p. Intraperitoneal leukocytes were collected 30 minutes later by lavage (5 ml HBSS containing 100 μg/ml gentamicin), and then washed. Cells (1x10⁶) were then incubated (37ºC) 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 APC), -CD11c (PE-Cy5), and isotype control antibodies were from Biolegend. Anti-CD14 (PE) and –TLR4 (PE) were from Ebioscience. Cells were stained and fixed with 2% paraformaldehyde/PBS. For fPEG-chol staining, cells were fixed (2% paraformaldehyde), washed, and then exposed to fPEG-chol (10 μg/ml, 10 min, RT). Flow cytometry was performed using an LSR II (BD Biosciences) and analyzed using FlowJo (Tree Star, Inc., Ashland, OR) and FCS Express (De Novo Software, Los Angeles, CA) software.

Statistical analysis

Analysis was performed using GraphPad Prism statistical software (San Diego, CA). Data are represented as mean ± SEM. Two-tailed student’s t test was applied for comparisons of two groups, and ANOVA for analyses of three or more groups. Survival was evaluated by log rank test. For all tests, p<0.05 was considered significant.

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

In order to investigate the effects of dyslipidemia on responses in the airspace, we used a well-established model of high cholesterol diet (HCD)-induced dyslipidemia in wild type (C57BL/6) mice (27, 28). The effects of dyslipidemia upon airspace:blood compartmental levels of cholesterol, leukocytes, and cytokines in the steady state were first defined. As expected, compared to normal diet (ND)-fed mice, HCD-fed mice developed significantly elevated serum total cholesterol, with increases in the LDL and very low density lipoprotein fractions and a decrease in HDL cholesterol (Fig. S1A). There was no difference in bronchoalveolar lavage fluid (BALF) cholesterol between mice fed the two diets (Fig. S1B), suggesting that cholesterol levels are regulated by distinct mechanisms in the airspace and serum. By contrast, peritoneal lavage fluid cholesterol was increased in HCD-fed mice (Fig. S1C), indicating that extravascular compartments differ in regulation of cholesterol levels during dyslipidemia.

Whereas no significant difference was noted between ND- and HCD-fed mice in steady state BAL total leukocyte count/differential (data not shown), HCD-fed mice displayed increased circulating PMNs and monocytes (Table I). Dyslipidemia is reported to induce endovascular inflammation through TLR4 and its intracellular adaptor, Myeloid Differentiation Primary Response Protein 88 (MyD88)(29, 30). Nonetheless, we found that HCD induction of circulating PMNs and monocytes were both MyD88-independent, as they occurred equivalently in wild type (wt) and Myd88 null mice (Table I). Whereas the circulating lymphocyte count was unchanged by HCD in wt mice, it was significantly increased by HCD in Myd88 null mice (Table I), suggesting that MyD88-dependent signals may suppress HCD-induced lymphocytosis. HCD did not impact peripheral blood hemoglobin, erythrocyte count, or platelet count (data not shown), suggesting a selective effect upon hematopoietic lineages. IL-17 reportedly regulates granulopoiesis through its target gene G-CSF (31, 32), though we are unaware of any prior report of IL-17 regulation during dyslipidemia. We found that HCD modestly upregulated IL-17 and its target gene macrophage inflammatory protein-2 (MIP-2) in the serum (Fig. 1A–B), though did not upregulate G-CSF, nor TNFα (Fig. 1C–D). HCD-associated induction of IL-17 and MIP-2 were noted to occur in a MyD88- and TLR4-dependent fashion, as animals deficient in these genes, unlike wt animals, had no HCD induction of IL-17 and blunted induction of MIP-2 (Figure 1A–B). Unlike the serum compartment, there was no induction by HCD of basal BALF levels of MIP-2 (data not shown).

Taken together, these findings indicate that: 1) cholesterol is regulated differentially between serum and airspace during dyslipidemia, yielding divergent lipid environments; 2) dyslipidemia impacts steady state compartmental leukocyte and cytokine levels differentially between airspace and serum; and 3) TLR4/MyD88 regulates dyslipidemia-associated induction of serum cytokines but not expansion of neutrophils and monocytes in the bloodstream.

Dyslipidemia attenuates induced trafficking of PMNs to the inflamed airspace

In order to determine the effect of dyslipidemia upon induced trafficking of leukocytes to the airspace, we next exposed mice to aerosolized LPS, using an established model (23). Despite their relative circulating neutrophilia compared to ND-fed mice, HCD-fed mice had significantly reduced influx of total leukocytes (WBCs) into the BALF following LPS inhalation (Fig. 2A). The reduction in WBCs was accounted for by a reduction in BALF PMNs (Fig. 2A), as BALF macrophage and lymphocyte numbers were not different between mice on the two diets (data not shown). The reduction of LPS-induced airspace PMNs was observed following as little as 2 weeks of HCD feeding (data not shown). By contrast, there was no difference in LPS-induced accumulation of airspace PMNs between mice on ND and mice on ND supplemented with cholate (Fig. 2B), indicating that the cholate component of HCD, previously reported to regulate pro-inflammatory genes in the liver via Farnesoid X Receptor (33), does not itself modulate the pulmonary innate immune response. Moreover, low density lipoprotein receptor (ldlr) null mice on a (cholate-free) Western diet, an alternate commonly used mouse model of dyslipidemia, also had significantly fewer total leukocytes/PMNs recruited to the airspace after LPS than Western diet-fed wt mice (Fig. 2C). Oxidized LDL (oxLDL), like native LDL (nLDL), is increased in the serum by HCD and mediates effects of dyslipidemia upon vascular inflammation and innate immunity (3, 4, 6). Consistent with possible roles for nLDL and oxLDL in our model, naïve (chow-fed) wild type mice subjected to pharmacologic dysplipidemia by intravenous (i.v.) injection of either oxLDL or nLDL had lesser WBC influx into the airspace after inhaled LPS than did mice treated with i.v. PBS (vehicle)(Fig. 2D). Taken together, these data indicate that dyslipidemia, modeled by several independent approaches, dysregulates systemic vs. pulmonary compartmentalization of PMNs during an inflammatory stimulus.

Dyslipidemia modulates airspace cytokine induction by inhaled LPS

Circulating PMNs migrate to the inflamed or infected airspace in response to locally expressed chemokines. In pursuit of mechanisms 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 begin to accumulate in the airspace at ~4–6 hrs post-LPS in this model (data not shown)(23), we assayed BALF 2 hrs post-LPS as a temporally relevant time point largely reflecting cytokine production by lung-resident cells. As shown in Fig. 3, BALF levels of MIP-2 and keratinocyte-derived chemokine (KC), two CXC chemokines that play critical roles in PMN recruitment to the LPS-exposed lung (23), were lower in HCD than in ND mice, whereas expression of the chemokine LIX (CXCL5) was equivalent between diets. TNFα, a cytokine important in PMN recruitment to lung, was also lower in HCD mice, whereas BALF IL-6, a negative regulator of LPS-induced PMN recruitment to the lung (34), was modestly higher in HCD mice. Serum KC, MIP-2, and IL-6 were equivalent between ND- and HCD-fed mice 2 hrs post-LPS inhalation (data not shown). Taken together, these findings suggest that airspace cyto-/chemokine expression is differentially modulated during dyslipidemia. Reductions in BALF TNFα and in airspace-serum chemokine gradients may possibly contribute to the observed reduction in LPS-induced airspace neutrophilia, as proposed in other models (35).

LPS induction of KC and MIP-2 in the lung is driven by the transcription factor nuclear factor (NF)-κB in lung structural cells (36). Notably, we found that DNA binding of p65 NF-κB in lung parenchymal nuclear isolates 2 hours post-LPS was indeed lower in HCD-fed than in ND-fed mice (Fig. 3F), suggesting that dyslipidemia may modulate airspace cytokine production through effects upon NF-κB activation.

Dyslipidemia impairs PMN migration

Given the relatively modest reductions in LPS-induced BALF chemokines in dyslipidemic animals, we next investigated whether dyslipidemia also modifies PMN migration itself. In order to model PMN migration in response to airspace chemokines in vivo, we first instilled KC directly into the airspace (23) of ND- and HCD-fed mice. HCD-fed mice indeed had reduced airspace recruitment of PMNs in response to intratracheal (i.t.) KC (Fig. 4A). In order to more directly examine effects of dyslipidemia on PMN migratory function, we next isolated mature PMNs from the bone marrow of ND- and HCD-fed mice, and quantified their chemotaxis in vitro. Consistent with the in vivo model, PMNs isolated from HCD mice had impaired chemotaxis to KC, as well as MIP-2 (Fig. 4B). Cell surface expression of CXCR2 was decreased in circulating PMNs from HCD- as compared to ND-fed mice (Fig. 4C). By contrast, CD18 cell surface expression was equivalent in PMNs between the two diets (data not shown). Thus, in addition to reduction of chemokine expression by lung-resident cells, the dyslipidemic environment also confers a chemotactic defect upon circulating PMNs that may reflect, at least in part, downregulated cell surface expression of chemokine receptors.

Dyslipidemia attenuates airspace neutrophilia and cytokines in response to K. pneumoniae

Given the observed deficits in PMN migration and in recruitment of PMNs to the LPS-exposed lung, we predicted that PMN migration to the lung in response to intact Gram-negative bacteria would also be deficient during dyslipidemia. Indeed, compared to ND-fed, HCD-fed mice had fewer PMNs in the airspace following i.t. inoculation with K. pneumoniae (Fig. 5A). As with LPS, in response to K. pneumoniae, HCD-fed animals had markedly reduced BALF MIP-2 (Fig. 5B), a chemokine of established importance to PMN recruitment to the K. pneumoniae-infected lung.(37) There was, however, no significant alteration in BALF IL-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, and G-CSF in the K. pneumoniae-infected lung (38). Given the varying effects of HCD upon MIP-2, LIX, KC, and G-CSF expression in the lung, we questioned whether dyslipidemic animals might have an underlying change in pulmonary IL-17 expression or activity. HCD-fed mice had significantly increased BALF IL-17 24 hours after i.t. K. pneumoniae (Fig. 5F). BALF concentrations of MIP-2, LIX, KC, and G-CSF were equivalent between ND- and HCD-fed animals 2 hours following i.t. delivery of recombinant IL-17 (data not shown), suggesting that IL-17 responsiveness is not modified in lung-resident cells during dyslipidemia. Collectively, these findings suggest that reduced PMN recruitment to the airspace in response to i.t. K. pneumoniae during dyslipidemia reflects both impaired PMN chemotaxis and reduced airspace expression of chemokines, and that the latter occurs at least in part through an IL-17-independent mechanism.

Dyslipidemia exerts opposing effects on intra- and extrapulmonary antibacterial host defense

Airspace-recruited PMNs play a critical role in clearance of pulmonary infection with extracellular pathogens, such as K. pneumoniae. Thus, we predicted that intrapulmonary host defense against K. pneumoniae would be impaired in HCD-fed animals. Indeed, compared to ND-, HCD-fed animals had significantly increased K. pneumoniae CFUs in lung homogenates following i.t. inoculation (Fig. 6A), indicating impaired intrapulmonary host defense. As IL-17 induction in the infected lung parallels K. pneumoniae burden (38), increased microbial load may account for the increased BALF IL-17 in HCD-fed mice, and, conversely, highlights the significance of the BALF MIP-2 deficit in these animals (Fig. 5B).

Dissemination of bacteria from the infected lung to the bloodstream and from there to peripheral organs generally tracks in parallel with intrapulmonary bacterial burden (23). Nonetheless, we found that HCD-fed animals had markedly reduced K. pneumoniae CFUs in spleen and liver following i.t. infection (Fig. 6B–C). This suggested the two possibilities that either: 1) the integrity of the alveolocapillary barrier was enhanced during dyslipidemia, thus reducing bacterial dissemination despite increased bacteria in the airspace; or 2) extrapulmonary (i.e., bloodstream) killing of K. pneumoniae might be paradoxically enhanced during dyslipidemia. Arguing against the first possibility, BALF protein concentration, an indicator of pulmonary microvascular integrity, was equivalent between ND and HCD animals following pneumonia induction, and higher in HCD animals following LPS inhalation (Fig. S2). As PMNs, the major cellular effector of host defense against Klebsiella, were increased in the blood of HCD-fed mice (Table I), we favored the second possibility. Indeed, confirming enhanced clearance of bacteremia in HCD- as compared to ND-fed animals, blood and splenic CFUs were both significantly reduced in HCD-fed animals following direct i.v. inoculation with K. pneumoniae (Fig. 6D–E). Perhaps reflecting counterbalancing effects of dyslipidemia upon host defense within vs. outside of the lung, survival following i.t. inoculation with K. pneumoniae was 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 TLR responses

We next explored mechanisms in addition to neutrophilia and cytokine induction (Table I, Fig. 1) that might underlie the enhanced systemic host defense response during dyslipidemia. Consistent with dyslipidemia priming for a more robust systemic response to infection, HCD-fed mice had markedly enhanced (~20-fold greater than ND-fed mice) serum TNFα and IL-1β (~3-fold greater) but not MIP-2 following i.p. LPS (Fig. 7A). Suggesting a contribution from dyslipidemia-primed extrapulmonary macrophages to this enhanced systemic response, cultured peritoneal exudate macrophages (PEMs) from HCD-fed mice also had enhanced TNFα induction (Fig. 7B), and enhanced activation of p38, JNK, and NF-κB (Fig. 7C) following in vitro treatment with either LPS or the TLR2 ligand PAM3CSK4. By contrast, alveolar macrophages harvested from unexposed ND- and HCD-fed mice, while not strictly analogous to elicited peritoneal macrophages nonetheless informative of the airway macrophage response, produced equivalent amounts of TNFα protein in response to LPS ex vivo (Fig. 7D).

Cholesterol-loading of the macrophage plasma membrane in vitro enhances cellular responses to TLR2 and TLR4 ligands through expansion of lipid raft microdomains (39, 40). However, it is unknown whether a similar phenomenon occurs to macrophages in vivo during hypercholesterolemia. To investigate this, we synthesized an analog (Fig. S3) of a compound that has previously been used as a raft probe, fluorescein ester of polyethylene glycol cholesteryl ether (fPEG-chol)(41–43). fPEG-chol is a cholesterol derivative that intercalates into the exofacial leaflet of the plasma membrane in raft microdomains in parallel to membrane cholesterol content (41, 43). F4/80⁺ peritoneal exudate cells harvested from HCD-fed animals bearing increased peritoneal fluid cholesterol (Fig. S1), indeed had greater fPEG-chol signal by flow cytometry than their counterparts from ND-fed animals (Fig. 7E, Fig. S4A), consistent with increased lipid rafts. On the other hand, alveolar macrophages from HCD- and ND-fed animals, harvested from equivalent BALF cholesterol environments (Fig. S1B), had equivalent rafts as assessed by fPEG-chol staining (Fig. 7F). F4/80⁺ peritoneal cells from HCD-fed animals were equivalent to their ND counterparts in cell-surface expression of CD14 (Fig. 7G, Fig. S4B), but, notably, had significantly greater expression of TLR4 (Fig. 7H, Fig. S4C). In support of the premise that extracellular cholesterol may be incorporated into membrane rafts and enhance TLR4 responses, RAW 264.7 macrophages loaded with cholesterol in vitro using cyclodextrin-cholesterol complex (17, 40) had increased fPEG-chol signal and produced increased levels of TNFα in response to LPS (Fig. S5). Consistent with previous reports, cholesterol loading was itself sufficient to induce TNFα in the macrophage (Fig. S5)(17, 40).

Dyslipidemia enhances PMN influx into and host defense within the peritoneum

In order to address whether dyslipidemia globally impairs PMN transvascular migration into all peripheral loci, or, alternatively, whether it 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-associated PMN chemotactic defect, i.p. LPS (Fig. 8A) and i.p. K. pneumoniae (Fig. 8B) both induced greater intraperitoneal PMN accumulation in HCD- than ND-fed mice. HCD-fed mice also had increased peritoneal lavage fluid levels of TNFα 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 effectively from their peritonea than their ND-fed counterparts (Fig. 8G); in parallel, HCD mice had lesser dissemination of peritoneal K. pneumoniae to the spleen (Fig. 8H). In order to determine whether dyslipidemia modulates intrinsinc PMN bactericidal capacity in vivo, we pre-elicited PMNs into the peritoneum with thioglycollate, exposed the elicited PMNs in vivo to K. pneumoniae by i.p. injection, and then harvested the PMNs for further ex vivo incubation, as previously described (25). Quantification of intracellular K. pneumoniae CFUs revealed similar time-dependent killing curves between PMNs from ND- and HCD-fed mice (Fig. S6), indicating no diet-induced modification of intracellular bactericidal function.