Serum Lipoproteins Are Critical for Pulmonary Innate Defense against Staphylococcus aureus Quorum Sensing Free

Hyperlipidemia is a clinical syndrome with high circulating levels of cholesterol, triglycerides, and the lipoprotein particles that carry them, including very low–density lipoproteins (VLDLs) and low-density lipoproteins (LDLs) (1). Hyperlipidemia is a risk factor for cardiovascular disease (2, 3), yet serum lipoproteins also contribute to host innate defense against infection (4, 5). Interestingly, hyperlipidemia results in impaired intrapulmonary host immunity (6), suggesting that maintenance of normal circulating cholesterol and lipoprotein levels is crucial for optimal host innate defense in the lung. Although severe hypolipidemia, which often accompanies the acute-phase response after surgery or trauma (reviewed in Refs. 7–9), has been associated with bacterial pneumonia (10–12), a critical gap in knowledge remains regarding the impact of extremely low serum lipoprotein levels on host innate defense in the lung.

Staphylococcus aureus, and methicillin-resistant S. aureus in particular, accounts for 20–40% of hospital-acquired pneumonia cases in the United States, as well as a growing number of cases of community-acquired pneumonia (13–15). Invasive pulmonary infection caused by S. aureus requires the expression of virulence factors controlled by the accessory gene regulator (agr) operon (16–19), which encodes a two-component quorum-sensing (QS) system for bacterial communication and coordinated gene expression (reviewed in Refs. 20, 21). QS is facilitated by secretion of a cyclic autoinducing peptide (AIP), which binds to and activates its cognate surface receptor AgrC. This in turn leads to expression of >200 virulence factors (22), many of which are pretranscriptionally and posttranscriptionally regulated by a small RNA molecule, called RNAIII, produced by transcription from the agr P3 promoter (20, 21). Importantly, apolipoprotein B (apoB), the sole protein component of LDL lipoprotein particles, and not other serum apoproteins or associated lipids, binds and sequesters AIP, thereby inhibiting agr-signaling and limiting pathogenesis during S. aureus skin infection (23–25). However, the impact of apoB deficiency and hypolipidemia on host innate defense in the lung, and against S. aureus pneumonia in particular, has not been investigated. We hypothesized that serum lipoproteins would contribute to pulmonary host defense against S. aureus QS and agr-mediated inflammation.

In this study, we used a sublethal model of S. aureus pneumonia to demonstrate that severe hypolipidemia impairs the early host innate defense response to lung infection. Specifically, lipoprotein deficiency impairs host control of S. aureus QS in the lung, resulting in agr-dependent increases in pulmonary proinflammatory cytokine production and neutrophil influx. Furthermore, apoB inhibits agr-dependent inflammatory cytokine expression by human alveolar epithelial cells, supporting a role for apoB in limiting QS-dependent virulence and inflammation during human lung infection. Given that serum lipoproteins also limit inflammation via sequestration of lipoteichoic acid (LTA) and LPS (26–28), these studies may have broad implications for hypolipidemia in increased susceptibility to posttrauma pneumonia caused by both Gram-positive and -negative pathogens.

USA300 LAC and its isogenic agr deletion mutant (USA300 LACΔagr) were provided by Dr. Frank DeLeo (Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT) and Dr. Michael Otto (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD), LACΔhla was provided by Dr. Juliane Bubeck-Wardenburg (Department of Microbiology, University of Chicago, Chicago, IL), and AH1677 agr::P3-yfp (USA300 LACyfp) was provided by Dr. Alex Horswill (Department of Microbiology, Carver College of Medicine, University of Iowa). Bacteria were grown in trypticase soy broth (TSB), and early exponential phase frozen stocks were prepared as previously described (29). CFUs of frozen stocks were determined by plating of serial dilutions on blood agar (BD Biosciences, Franklin Lakes, NJ).

Animal work was carried out at the American Association for the Accreditation of Laboratory Animal Care–accredited Animal Research Facility of the University of New Mexico Health Sciences Center in accordance with recommendations in the Eighth Edition of The Guide for the Care and Use of Laboratory Animals and the U.S. Animal Welfare Act. The protocol was approved by the Institutional Animal Care and Use Committee of the University of New Mexico. Eight- to 12-wk-old male mice [C57BL/6, Pcsk9^−/− on the B6 × 129 background, and B6 × 129 wild-type (WT)] were purchased from Jackson Laboratories (Bar Harbor, ME). Mice receiving 4-aminopyrazole-(3,4-D)pyrimidine (4APP) treatment were injected i.p. with 100 μl of 5.15 mg/ml 4APP (Sigma-Aldrich, St. Louis, MO) prepared as previously described (23, 24), or buffer control at 48 and 24 h before infection, as well as at the time of infection. Reductions in serum cholesterol were determined using Infinity Cholesterol Liquid Stable Reagent (Thermo Scientific, Middletown, VA) according to the manufacturer’s directions.

The mouse model of S. aureus pneumonia was performed as previously described (17). In brief, mice were anesthetized by isoflurane inhalation and 30 μl sterile saline containing ∼ 4 × 10⁸ CFU S. aureus was administered intranasally, followed by an additional 30 μl saline alone. Mice were weighed at the time of infection and again before sacrifice. At 6 h postinfection, mice were sacrificed by CO₂ asphyxiation, and blood and tissues were collected. For CFU determinations, right lungs were collected in bead-beating tubes containing 2.3-mm Zirconia/Silica beads (BioSpec Products, Bartlesville, OK) in 1 ml HBSS⁻ (Life Technologies, Grand Island, NY) with 0.2% human serum albumin (Sigma-Aldrich), and lung tissue was disrupted for 1 min using a Mini-Bead Beater-24 (Biospec). Homogenates were diluted 1:10 in PBS with 0.1% Triton X-100, sonicated, and serial dilutions plated on blood agar (BD Biosciences, Franklin Lakes, NJ). For agr::P3-YFP promoter activation assays, bacteria from diluted homogenates were pelleted by centrifugation and fixed for 10 min with 1% paraformaldehyde containing 25 mM CaCl₂. Promoter activation was determined by measuring mean channel fluorescence by flow cytometry (Accuri C6; BD Accuri Cytometers, Ann Arbor, MI). For cytokine and Western blot analyses, lung homogenates were clarified by centrifugation at 12,500 × g and supernatant was stored at −80°C until use as described later. For transcription analyses, left lungs were harvested, immediately placed into 1.8 ml RNAlater (Qiagen, Valencia, CA), and frozen at −80°C.

For A549 assays, bacteria were cultured in TSB (5 ml with 10:1 air/volume ratio) for 6 h at 37°C with shaking and with or without 100 nM human LDL. Bacteria were then pelleted by centrifugation, and supernatants filter sterilized by passage through 0.2-μm filters, aliquoted, and frozen at −80°C until use. A549 human alveolar epithelial cells were grown to 80–90% confluence in 12-well plates in DMEM supplemented with 10% heat-inactivated FBS and penicillin/streptomycin. Twenty-four hours before use, cell culture medium was changed to DMEM with 10% human lipoprotein-deficient serum with penicillin/streptomycin. Sterile bacterial supernatants were thawed, diluted to working concentrations in TSB, and added to A549 cells at a 1:10 dilution in cell culture media. Postculture human LDL was added to respective supernatants at 100 nM concentration before addition to A549 cells. Cells were incubated at 37°C for 4 h. At 4 h postexposure, cell culture supernatants were collected and frozen at −80°C for future analysis. To collect RNA, we immediately lysed cells with Qiagen RLT buffer with 1% 2-ME and QIAshredder spin columns (Qiagen). RNA was processed using an RNeasy Mini kit, according to the manufacturer’s Animal Cell Spin protocol (Qiagen).

Cytokines were measured in cell culture supernatant or clarified supernatant from mouse lung homogenates using custom-designed Milliplex Cytokine Magnetic kits according to the manufacturer’s specifications (Millipore, Billerica, MA). Cell culture supernatants were pooled from identically treated triplicates. Myeloperoxidase (MPO) was also measured in clarified supernatants using the Mouse Myeloperoxidase DuoSet ELISA (R&D Systems, Minneapolis, MN) according to manufacturer’s directions.

Clarified lung homogenate supernatants were quick thawed (37°C), and protein concentrations were determined by A₂₈₀ absorption (Nanodrop 100 Spectrophotometer; Thermo Fisher Scientific, Wilmington, DE). For apoB Western blots, equivalent amounts of total protein were separated by SDS-PAGE on 3–8% Tris-Acetate gels (Novex Life Technologies, Grand Island, NY) before transfer to polyvinylidene fluoride membranes. Membranes were blocked with 5% nonfat milk in TBS (20 mM Tris pH 7.5, 150 mM NaCl) containing 0.1% TBST, then probed with rabbit anti-apoB Ab (Abcam, Cambridge, MA). Unbound Ab was removed by triplicate 5-min washes with TBST, and membranes were developed using goat anti-rabbit IgG-alkaline phosphatase–conjugated secondary Ab (KPL, Gaithersburg, MD) and 1-Step NBT/5-bromo-4-chloro-3-indolyl-phosphate (Thermo Scientific).

For Western blot analysis of secreted Hla in bacterial supernatants applied to A549 cells (see earlier), equivalent amounts of total protein were separated by SDS-PAGE on 4–12% Bolt gels (Novex Life Technologies, Grand Island, NY) before transfer to polyvinylidene fluoride membranes and overnight blocking. Membranes were probed with mouse anti-Hla mAb (Integrated BioTherapeutics, Gaithersburg, MD), followed by goat anti-rabbit IgG poly-HRP (Thermo Fisher Scientific). Blots were developed using Thermo Pierce SuperSignal West Pico Chemiluminescent substrate (Thermo Fisher Scientific). All blots were imaged using a ProteinSimple FluorChem R instrument and quantified with AlphaView software (Protein Simple, Santa Clara, CA).

Sterile bacterial culture supernatants prepared as described earlier and used for A549 cytokine induction assays were tested for hemolytic activity as previously described (30). In brief, a 4% solution of rabbit RBCs in PBS was incubated at 37°C for 1 h with 2-fold serial dilutions of sterile-filtered bacterial supernatant. Lysis was determined by absorbance at 450 nm of the resulting RBC supernatant. Data were analyzed by nonlinear regression and are shown as the HA₅₀, which equals 1/the log of the dilution needed for 50% complete lysis.

For bacterial RNA isolation, bronchial alveolar lavage fluid (BALF) was collected as described later but in a 10-fold volume of RNAlater (Qiagen) and frozen at −80°C for later processing. After thawing, bacteria were pelleted by centrifugation, RNAlater was aspirated, and pellets were incubated with 100 μl Tris-EDTA buffer, with 20 μl/ml proteinase K (Qiagen) and 20 μg/ml lysostaphin for 10 min. Thawed lungs were removed from RNAlater and RNA extracted using QIAzol according to manufacturer’s directions (Qiagen). RNA isolation from cell culture was performed as described above. An RNeasy Mini kit (Qiagen) was used for RNA purification and cDNA was generated using a high-capacity cDNA RT kit with RNase inhibitor together with random hexamer primers (Applied Biosystems, Foster City, CA). TaqMan Gene Expression master mix (Applied Biosystems) and an ABI7000 Real-Timer PCR system were used for quantitative PCR. Prime Time Predesigned qPCR primers and probes (Integrated DNA Technologies, Coralville, IA) were used for transcriptional analyses of mouse hprt, il-1b, mip-2, and il-6, and for human hrpt and il-8. Primers and probes for quantification of S. aureus 16S, RNAIII, and lukS were previously described (31). Gene expression was quantified using SDS RQ Manager Version 1.2.2 software (Applied Biosystems) relative to mouse or human hprt, and S. aureus 16S, as appropriate.

BALF was collected from sacrificed mice by intratracheal lavage, using three sequential washes (700 μl each) with ice-cold Dulbecco’s PBS (2.67 mM KCl, 1.47 mM KH₂PO₄, 138 mM NaCl, 8.1 mM Na₂HPO₄) (Corning, Corning, NY). Total cell count and live/dead counts were determined by trypan blue staining using a TC 20 Automated Cell Counter System (Bio-Rad, Hercules, CA). For immunostaining, cells were centrifuged at 800 × g for 3 min; then pelleted cells were resuspended in cold PBS with 0.5% BSA and 0.075% sodium azide. Cells were blocked for 30 min with 2% BSA, followed by a 1-h, 4°C incubation with anti-mouse Ly6G (neutrophils) (BioXCell, West Lebanon, NH) conjugated to Alexa Fluor 488 (Protein labeling kit; Molecular Probes, Eugene, OR), anti-mouse CD11c-PE (alveolar macrophages) (Biolegend, San Diego, CA), or isotype controls. Cells were washed with PBS before analysis by flow cytometry (Accuri C6; BD Accuri Cytometers).

All data were analyzed using GraphPad Prism Version 5.04 for Windows (GraphPad Software, San Diego, CA). Parametric data were analyzed using the two-tailed Student t test or ANOVA with either Dunnett’s post hoc test or Bonferroni’s multiple comparison test, and are displayed as the mean ± SEM. Nonparametric data were analyzed by the Mann–Whitney U test and displayed as the median plus 5th–95th percentiles.

Because lipoproteins are detectable in lungs shortly after intranasal infection with S. aureus (32), and apoB on LDL binds S. aureus AIP and inhibits agr-activation (23–25), we predicted that decreased circulating lipoprotein levels would result in impaired control of S. aureus QS in the lung. To address this, we compared S. aureus agr-activation in the lungs of mice with reduced serum apoB levels versus controls. Given that APOB deletion is embryonic lethal (33), we used pharmacological treatment with 4APP, which inhibits lipoprotein secretion from the liver (34), and Pcsk9^−/− mice, which have reduced circulating LDL levels because of overexpression of the LDL receptor (35), as models of apoB deficiency. Both models have an ∼50% reduction in apoB measured in the serum (Fig. 1A, 1B), consistent with those of trauma patients at risk for infection (9). Using a sublethal challenge model (17), we intranasally infected mice with a reporter strain of the agr⁺ clinical isolate LAC, which expresses YFP under the control of the agr::P3 promoter (36). We measured agr-activation at 6 h postinfection, at which time agr activation is detectable in the lung, as are differences in lung pathogenesis and bacterial burden between mice infected with agr⁺ versus Δagr isolates (16, 37). As expected, agr::P3 promoter activation was significantly increased in the lungs of both 4APP-treated and Pcsk9^−/− mice compared with controls (Fig. 2A, 2D). In addition, bacteria in BALF collected from LAC-infected, 4APP-treated mice showed increased transcription of agr-regulated RNAIII and lukS-PV, the latter encoding the Panton-Valentine leukocidin (Supplemental Fig. 1). Weight loss, used as a measure of morbidity, over the course of the 6-h infection was slight, with median weight loss ranging from ∼0.45 to 1.12 g and from ∼1.5 to 4.8% of total body weight (Fig. 2B, 2E, Supplemental Fig. 2). However, 4APP-treated and Pcsk9^−/− mice infected with LAC, but not with an isogenic agr mutant (LACΔagr), showed increased weight loss, compared with vehicle-treated and WT controls, respectively, suggesting increased virulence in the lipoprotein-deficient mice caused by an inability to control agr-signaling. Importantly, differences in QS and weight loss at this time point were not the result of increased bacterial burden, as the number of CFUs in the lungs of lipoprotein-deficient mice did not differ from that of controls (Fig. 2C, 2F). Therefore, these results suggest that serum lipoproteins contribute to host control of S. aureus QS-dependent virulence in the lungs, and that this contribution is independent of host control of bacterial burden.

Increased agr-signaling in the lungs of S. aureus–infected, lipoprotein-deficient mice (Fig. 2A, 2D) suggested that these mice would have decreased apoB present in the lungs compared with lipoprotein-sufficient controls. As suggested, LAC-infected Pcsk9^−/− mice showed significantly decreased apoB in the lungs at 6 h postinfection compared with WT controls (Fig. 3A, 3B). Similarly, compared with vehicle-treated mice, BALF from mice treated with 4APP and infected with LAC also showed significant reductions in apoB at the 6-h time point (Fig. 3C, 3D). In contrast, differences in apoB levels in BALF from mice infected with LACΔagr did not reach statistical significance. Together, these data suggest that the presence of apoB in the lung during S. aureus infection, and thus its ability to control agr-signaling in the lung, is dependent upon circulating lipoprotein concentrations.

S. aureus stimulates the release of a variety of cytokines and chemokines in the lungs of infected mice, including cytokines produced in response to agr-regulated virulence factors (17, 18, 32, 38–41). In particular, expression of the agr-regulated virulence factor α-hemolysin (Hla), a pore-forming toxin, induces pulmonary release of the proinflammatory cytokine IL-1β (38, 39), suggesting there would be an agr- and Hla-dependent increase in IL-1β in the lungs of lipoprotein-deficient mice compared with controls. As predicted, 4APP-treated mice infected with LAC, but not with LACΔagr or LACΔhla, had significantly increased pulmonary IL-1β levels at 6 h postinfection compared with vehicle-treated mice (Fig. 4A), consistent with increased morbidity (weight loss) relative to vehicle controls only with LAC infection (Fig. 2B, 4B). Also, as seen for LAC and LACΔagr infected mice, bacterial burden did not differ between 4APP- and vehicle-treated mice infected with LACΔhla (Fig. 4C). Interestingly, IL-1β expression was reduced in lipoprotein-deficient mice infected with LACΔagr (Fig. 4A), although the mechanism driving this response remains unclear. Therefore, although Hla expression in lung homogenate of LAC-infected mice was below the limit of detection at 6 h postinfection (Western blot data not shown), these results suggest that lipoproteins inhibit agr-dependent Hla production and associated virulence in the lungs early during S. aureus infection.

In addition to inhibiting agr-signaling, apoB on LDL also binds LTA from S. aureus and limits LTA-induced expression of IL-6 (28). To distinguish between agr-dependent and -independent effects of lipoprotein deficiency on pulmonary inflammatory cytokine production, we measured lung cytokine levels in WT and Pcsk9^−/− mice intranasally infected with LAC or LACΔagr. In addition to IL-1β and IL-6, we measured production of MIP-2, a neutrophil chemotactic protein, which has also been shown to be driven by agr-regulated Hla (42). At 6 h postinfection, IL-1β and MIP-2 levels were significantly increased in the lungs of Pcsk9^−/− mice infected with LAC, but not with LACΔagr (Fig. 5A, 5B), consistent with impaired host control of QS in the lungs of lipoprotein-deficient mice. In contrast, compared with WT mice, IL-6 was increased in the lungs of both LAC- and LACΔagr-infected Pcsk9^−/− mice (Fig. 5C), further supporting a role for apoB in limiting LTA-induced expression of this inflammatory cytokine (28). Therefore, these data demonstrate that apoB limits S. aureus–induced inflammatory cytokine production in the lung in both an agr-dependent and an agr-independent manner.

Appropriate recruitment of neutrophils during S. aureus infection is critical for bacterial clearance (43); however, excessive neutrophil recruitment can result in severe injury to sensitive lung tissues (reviewed in Refs. 44, 45). Given that MIP-2 levels were increased in the lungs of lipoprotein-deficient mice infected with LAC, but not with LACΔagr, we compared MPO levels, a surrogate marker of neutrophil influx, in the lungs of LAC-infected Pcsk9^−/− mice versus controls. MPO levels were significantly higher in the lungs of lipoprotein-deficient mice (Fig. 6A), and this paralleled an increase in Ly6G⁺ cells in BALF from 4APP- versus vehicle-treated mice also infected with LAC (Fig. 6B). In contrast, neutrophil influx and MPO levels did not differ between 4APP- and vehicle-treated mice infected with LACΔagr (Fig. 6B) or LACΔhla (Supplemental Fig. 3), respectively. Furthermore, although macrophage levels overall were increased in BALF from LAC-infected versus LACΔagr-infected mice (Fig. 6C), macrophage presence was independent of circulating lipoprotein levels. Together, these data indicate that lipoproteins are important for early host control of agr-dependent neutrophil influx during S. aureus lung infection.

Murine MIP-2 is a functional homolog of human IL-8, and Hla strongly induces IL-8 release by human alveolar epithelial cells at toxin concentrations below those causing cell lysis (46). To demonstrate that lipoproteins protect against early inflammatory cytokine production in the lung via inhibition of agr activation, rather than by direct effects on lung cells or sequestration of agr-regulated virulence factors (28, 47, 48), we measured IL-8 expression by human alveolar epithelial cells exposed to sterile bacterial supernatant from LAC cultured in the presence versus the absence of LDL. Consistent with previous reports (23, 24), LDL limited accumulation of Hla in LAC culture supernatant (Fig. 7A), and thus hemolytic activity (Fig. 7B), whereas LDL incubated with sterile supernatant postculture and, either used immediately or after a 1-h coincubation, did not inhibit hemolysis. Using A549 cells as a model for human alveolar epithelial cells, LAC supernatant induced il-8 transcription in a dose-dependent manner (Fig. 7C). Furthermore, compared with IL-8 release after exposure to LAC supernatant, IL-8 production by A549 cells was significantly reduced when exposed to supernatant from LAC cultured in the presence of LDL (Fig. 7D). This reduction in IL-8 was not due to direct effects of LDL on the A549 cells because LDL incubated with cells for 1 h before the addition of LAC supernatant did not inhibit IL-8 production. Also, although there was a small reduction in IL-8 levels when LDL was coincubated with LAC supernatant for 1 h before addition to the A549 cells, this reduction did not reach statistical significance. This suggests that the impact of LDL on IL-8 expression in these assays is mainly independent of sequestration of virulence factors or pathogen-associated molecular patterns within the supernatant (28, 47, 48). Importantly, IL-8 production did not significantly differ between cells exposed to supernatant from LAC cultured with LDL and supernatant from cultures of LACΔagr or LACΔhla. Therefore, these results support a mechanism whereby apoB limits S. aureus QS in the lung and protects the host against excessive inflammatory cytokine production in response to agr-regulated virulence factors.

Considerable attention has been given to the role of hyperlipidemia in cardiovascular disease, such that the terms dyslipidemia and hyperlipidemia are often used synonymously (49). This illustrates the limited consideration given to the potential health consequences of the other dyslipidemia, severe hypolipidemia and lipoprotein deficiency, often experienced posttrauma and by other critically ill patients (7–9). Because hypolipidemia has been clinically associated with bacterial pneumonia (10–12), we sought to determine the impact of extremely low circulating lipoprotein levels on host innate defense in the lung. Using a sublethal mouse model of S. aureus pneumonia, we demonstrate that lipoprotein deficiency impairs early pulmonary innate defense against bacterial pathogenesis. Specifically, apoB is present in the lung early postinfection and its levels decrease significantly with lipoprotein deficiency. This decrease coincides with impaired host control of S. aureus agr-signaling, as well as increased agr-dependent morbidity and inflammation, in alignment with the role of apoB in controlling S. aureus QS-dependent virulence in the skin (23–25). In addition, lipoprotein deficiency results in an agr-independent increase in pulmonary IL-6 expression, consistent with the ability of apoB to bind LTA and limit LTA-mediated inflammation (28). Given that hyperlipidemia also impairs lung innate defense mechanisms (6), these results strongly suggest that maintenance of normal serum cholesterol and lipoprotein levels is necessary for optimal host innate defense in the lung.

The contribution of host lipoproteins to pulmonary innate immunity is not surprising considering their previously described host defense contributions, together with their demonstrated uptake by lung capillary endothelium (50) and carriage on infiltrating leukocytes (51). For example, circulating lipoproteins, including LDL and high-density lipoprotein (HDL), nonspecifically bind and sequester LPS, thus limiting endotoxin-induced toxicity and lethality (26, 27, 52). In addition, apoB from LDL and apolipoproteins A1 and A2 from HDL bind soluble LTA and inhibit LTA-mediated cytokine release from both human and murine cells (28). The most pronounced inhibition comes from LDL, which dose-dependently inhibits LTA-mediated IL-6 expression by human PBMCs. In support of this finding, in this study we show that lipoprotein deficiency results in significantly increased IL-6 levels in the lungs of S. aureus–infected mice independent of agr status. Although in vivo validation of the role of lipoproteins in controlling LTA-mediated inflammation in the lung will require comparison of lipoprotein-sufficient versus -deficient mice after pulmonary administration of LTA or infection with LTA-deficient S. aureus mutants (53, 54), our data and that of others suggest that hypolipidemia may broadly impact posttrauma pneumonia susceptibility to both Gram-positive and -negative pathogens.

Along with sequestration of AIP, lipoproteins have other roles in host defense against S. aureus pathogenesis. Specifically, LDL has been reported to bind and partially inactivate Hla (48), whereas the phenol soluble modulins (22, 55, 56), which attract and lyse neutrophils, are also bound by lipoproteins, in particular by HDL (47). In this regard, the data reported in this article indicate that LDL limits IL-8 production by human alveolar epithelial cells by inhibiting expression and secretion of agr-regulated virulence factors. This is supported by our findings that: 1) A549 cells produce IL-8 when exposed to LAC sterile supernatant, but not when exposed to sterile supernatant from LACΔagr or LACΔhla, 2) that inclusion of LDL during LAC culture limits both Hla accumulation in the supernatant (23, 24) and the ability of the resulting supernatant to induce IL-8 release by A549 cells, and 3) that the addition of LDL directly to LAC supernatant does not inhibit IL-8 release by A549 cells. Although these data suggest that LDL primarily functions by prevention of virulence factor expression in this system, lipoproteins may also directly inhibit virulence factor function in the lung. In this respect, pulmonary administration of LAC supernatant, purified Hla, or phenol soluble modulins to serum lipoprotein-sufficient versus -deficient mice would further clarify this issue. Regardless, our findings and those of others (6, 26–28, 47–49) strongly suggest that lipoproteins likely contribute in a variety of both direct and indirect means to pulmonary host innate defense.

In addition to the negative effects of lipoprotein deficiency on pulmonary innate immunity reported in this study, severe hypolipidemia may also impair the innate defense function of lung surfactant (reviewed in Ref. 57). Lung surfactant is composed of a mixture of lipids, phospholipids, and proteins, and maintenance of the appropriate ratios of these components is required for functional surfactant self-assembly (58–60). Of particular importance to surfactant-mediated host innate defense are the surfactant-associated proteins, SP-A and SP-D, which act as opsonins to promote bacterial clearance (61–65). Because lipoproteins contribute to the regulation of surfactant cholesterol metabolism (66), it is unclear whether severe hypolipidemia results in impaired surfactant assembly and negatively impacts bacterial clearance by altering the distribution or otherwise impairing the antibacterial functions of SP-A and SP-D. Although we saw no evidence of impaired pulmonary bacterial clearance in lipoprotein-deficient mice at the early postinfection time point investigated in this study, the potential impact of severe hypolipidemia on pulmonary bacterial clearance warrants further investigation.

By focusing on early control of S. aureus QS in the lung, we have demonstrated one mechanism by which severe hypolipidemia impairs pulmonary host innate defense. Although a significant gap in knowledge remains regarding the full impact of hypolipidemia on infection susceptibility, disease progression, and survival during pneumonia caused by S. aureus and other microbial pathogens, the work reported in this study regarding hypolipidemia, together with the work of others focused on hyperlipidemia (6), represent important advances in understanding the impact of both extremes of dyslipidemia on pulmonary host innate defense. Furthermore, these studies point to the potential clinical impact of severe hypolipidemia on pulmonary innate defense in critically ill patients.

We thank Drs. Pavan Muttil and Jon Femling for critical review of the manuscript and Dr. Hattie Gresham for many helpful discussions.

The authors have no financial conflicts of interest.