Phosphatidylinositol inhibits respiratory syncytial virus infection

Cell culture, viruses, and reagents

HEp2 cells and BEAS2B cells were obtained from American Type Tissue Culture Collection and cultured as previously described (11). The human RSV A2 strain (VR-1540) was also obtained from American Tissue Culture Collection. Virus stocks were propagated in HEp2 cell monolayers using DMEM/Ham’s F12 Medium (GIBCO) plus 5% bovine growth serum (Hyclone) and purified using methods previously described (11, 19, 20). Viral titers and growth were measured using quantitative plaque assays (21). The TLR5 agonist, flagellin, was purchased from Alexis. Agarose was obtained from MP Biomedicals LLC.

Phospholipids

All phospholipids used in these studies were obtained from Avanti Polar Lipids. The biological source of PI was soybean. The molecular species distribution of the PI used in the experiments was determined by MS and consisted of the following molecular ions (mol%): 34:2 (58.4%), 36:2 (14.9%), 36:4 (9.5%), 36:3 (7.8%), 34:3 (6.5%), and 36:5 (1.8%). MS and MS/MS experiments were performed on an AB Sciex QTRAP. An autosampler was used to directly infuse the sample into the mass spectrometer utilizing electrospray ionization in negative ion mode. A methanol-acetonitrile-water (60:20:20, v/v/v) solvent system was used, and soybean PI was diluted to 20 ng/μl. MS/MS experiments utilized a collision energy = 50 eV. The relative intensities of the molecular ion peaks were used to estimate the mol fraction of molecular species within the mass range of 300–900.

In vitro viral infection and phospholipid treatments

BEAS2B cells were seeded at 1.0 × 10⁵/well and grown in 24-well plates for 48 h before the addition of virus. To compare the potency of PI and POPG as antagonists of RSV-elicited inflammatory responses, the cells were challenged with virus at a multiplicity of infection (MOI) of 0.5 in either the absence or presence of 200 μg/ml phospholipids. Phospholipids used in these and all other experiments were bath sonicated until they produced a clear solution, and sterilized by filtration prior to addition to cell cultures. At 48 h after infection, interleukin-8 (IL-8) secretion into the medium was determined by ELISA (11). The phospholipid 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) was used as a negative control lipid (11, 12).

To rule out nonspecific effects of lipids on the ability of BEAS2B cells to respond to proinflammatory stimuli, the cells were challenged with the TLR 5 agonist flagellin (10 ng/ml) in either the absence or presence of 200 μg/ml POPG, POPC, or PI. After 48 h, the culture supernatants were harvested, and IL-8 production was measured by ELISA. Additional control experiments were performed to determine the effects of 200 μg/ml phospholipids on BEAS2B protein synthesis, by monitoring the incorporation of ³H leucine into macromolecular pools precipitable by 5% trichloroacetic acid (14)

In additional experiments, we determined the activity of POPG, PI, POPC, and 1,2-dipalmitoyl-phosphatidylcholine (DPPC) as inhibitors of in vitro RSV infection assessed by viral plaque assay. HEp2 cell monolayers (5 × 10⁶ cells/35 mm well) were infected with serial dilutions of virus in the presence or absence of varying concentrations of phospholipid (0–200 μg/ml). After 2 h of viral adsorption, the infecting inoculum and lipids were removed from the wells, which were overlayed with medium containing 0.3% agarose. After 6 days of further incubation, the cultures were fixed and stained, and the viral plaques were quantified (12, 21).

RSV binding to solid-phase phospholipids and HEp2 cells

RSV binding to phospholipids was measured using phospholipid solid phases in microtiter wells, as previously described (11, 13–15). Phospholipids in 100% ethanol were added in 50 μl aliquots (1.5 nmol lipid), to wells in flat-bottomed microtiter plates (Corning). The ethanol was removed under a stream of warm air, and the wells were hydrated and blocked with solutions of 20 mM Tris pH 7.4, 100 mM NaCl, 10 mM CaCl₂, containing 3% BSA, and then incubated with varying concentrations of RSV (11, 13). The binding reactions were conducted at 37°C for 2 h. Subsequently, the wells were washed three times with the binding buffer. Specific virus binding was detected with HRP-conjugated anti-human RSV antibody (1:500) (AbD Serotec) by ELISA, using orthophenylenediamine substrate and measuring absorbance at 450 nm (11, 13).

RSV binding to epithelial cell plasma membrane was detected using monolayers of HEp2 cells (2.5 × 10⁵/well in 24-well plates) incubated with varying concentrations of RSV (MOI = 0–10) at 18°C (to minimize endocytosis) for 2 h. The incubations with virus were performed in either the absence or presence of 200 μg/ml phospholipids [POPG, PI, POPC, phosphatidylserine (PS), or phosphatidic acid (PA)]. Following the binding reaction, the monolayers were washed with PBS at 0°C, three times, to remove unattached viruses. The cultures were next harvested by scraping in SDS-PAGE loading buffer and briefly sonicated, and denatured at 95°C, and aliquots were electrophoresed on 8–20% polyacrylamide gels. After electrophoresis, the gels were processed for immunoblotting, and viral proteins were detected with HRP-conjugated anti-human RSV antibody (1:500) using enhanced chemiluminescence (22). The viral G-protein band was chosen for sample comparison, and films were scanned and then analyzed using the National Institutes of Health Image J analysis program (22).

We also conducted phospholipid interaction experiments with RSV to test for the potential viricidal activity of phospholipids. High titer preparations of RSV (1 × 10⁸/ml) were incubated either without or with 200 μg/ml PI or POPC for 1 h at 37°C. Following this incubation, the preparations were diluted 10⁵- to 10⁶-fold and then adsorbed to monolayers of HEp2 cells for 1 h and finally overlayed with 0.3% agarose. After 5 days, the wells were stained, and viral plaques were quantified and compared among the different conditions.

RSV infection in vivo and determination of the PI prophylaxis efficacy window

All experiments involving animals were reviewed and approved by the National Jewish Health Institutional Animal Care and Use Committee. Female BALB/c mice at 6 weeks of age were obtained from Jackson Laboratory. Mice were anesthetized with intraperitoneal Avertin (0.25 g/kg) (11) and either sham infected or infected with RSV [10⁷ plaque forming units (pfu)] in either the absence or presence of 300 μg of PI, or treated with PI alone. The total volume of all intranasal inoculations was kept constant at 50 μl with 25 μl introduced into each nostril.

In a separate series of studies, mice were pretreated with PI to evaluate the activity of this lipid in preventing infection. To determine the efficacy window for PI prophylaxis, mice were anesthetized by isoflurane inhalation and inoculated with PI (600 μg/mouse) at various time points prior to a second round of anesthesia and inoculation with RSV. The prophylaxis studies also included a control group that received PI at the same time as the RSV. For all studies in mice, the experiments were ended at 5 days after infection (the usual peak of infectious virus number) with euthanasia by CO₂ asphyxiation. Sample recovery from mice involved lavaging the lungs with 1 ml of saline and dissection and removal of the lungs for tissue processing. The left lungs were harvested and homogenized and processed for RSV plaque assays. The right lungs were used for histopathology scoring (11, 23) by a pathologist who was blinded to the experimental protocol. The lavage material was used to determine total cell infiltrates and quantify different cell populations. The lavage fluid was centrifuged at 200 g × 5 min to remove cells, and the supernatant was used to determine IFN-γ levels (11).

PI turnover in mice

To determine the turnover of instilled PI in vivo, anesthetized mice were inoculated with 150 μg of the lipid in 50 μl of PBS. Subsequently, the mice were euthanized with CO₂, and five serial lavages (1 ml each) were performed with buffer (HBSS containing 0.5 mM EGTA and 4.4 mM sodium bicarbonate) at 0, 30, 60, 180, and 360 min after inoculation. Cells were removed by centrifugation at 200 g × 5 min, and the supernatant fluid was processed for lipid extraction as previously described (24). Phospholipid content was determined by measurement of lipid phosphate (25). Phospholipid classes were analyzed by TLC and phosphate determination as previously described (25, 26).

PI inhibits IL-8 production induced by RSV in human epithelial cells

POPG is a minor anionic phospholipid constituent of pulmonary surfactant that has previously been shown to inhibit virus-dependent inflammatory cytokine production (11, 22, 27). In these experiments, we investigated whether PI, another minor anionic phospholipid present in pulmonary surfactant, has similar activity. Initially, we compared PI and POPG as antagonists of RSV-elicited IL-8 production from BEAS2B epithelial cells (11). The cells were pretreated with lipids (200 μg/ml) for 1 h and then infected with RSV (MOI = 0.5), and IL-8 secretion into the culture supernatant was measured at 48 h after infection. As shown in Fig. 1A, both POPG and PI inhibit IL-8 production in a dose-dependent manner. The IC₅₀ value for POPG was 11.4 ± 1.7 (μg/ml), and that of PI was 21.9 ± 5.6 (μg/ml). The control lipid POPC did not have a significant effect on IL-8 production. To exclude any toxic effects of POPG and PI, BEAS2B cells were stimulated with the 10 ng/ml of the TLR5 agonist flagellin, and the response was quantified (11). Neither POPG (200 μg/ml) nor PI (200 μg/ml) significantly inhibited IL-8 production induced by flagellin (Fig. 1B). As an additional control to evaluate lipid toxicity, cultures were incubated with ³H-leucine for 48 h, and the trichloroacetic acid precipitable protein-associated radioactivity was quantified in the absence and presence of the lipid treatment. As shown in Fig. 1C, neither lipid affected protein synthesis measured by ³H-leucine incorporation into macromolecules (11). These data demonstrate that PI disrupts the inflammatory response of BEAS2B cells by a process that does not involve general suppression of TLR signaling, or gross alteration of cellular homeostasis.

PI inhibits RSV infection in vitro

In the next series of experiments, we tested the activity of PI as an inhibitor of RSV infection. The antiviral activity of PI was quantified by infecting HEp2 cells for 2 h in either the absence or presence of the indicated concentrations of PI. Subsequently, the viral inoculum and lipids were removed from the cultures, and the cell monolayers were overlayed with agarose. After 5 days, the appearance of plaques was quantified. In Fig. 1D, the data demonstrate that both PI and POPG potently reduce viral plaque formation in a concentration-dependent manner. The activity of PI is comparable to that of POPG, which was previously shown to potently inhibit RSV infection and replication (11). At concentrations of 200 μg/ml, PI reduces viral infection in vitro by a factor >10³. These data demonstrate that PI, like POPG, strongly suppresses RSV infection in vitro. We also performed additional experiments examining 200 μg/ml DPPC as a potential inhibitor of viral infection because this phospholipid is the most abundant in pulmonary surfactant. The findings from two independent experiments demonstrated that DPPC did not have any significant inhibitory activity upon RSV plaque formation (virus alone = 3 × 10⁷ pfu/ml, virus + DPPC = 3.3 × 10⁷ pfu/ml, virus + POPG = 3 × 10³ pfu/ml).

PI binds to RSV with high affinity and blocks viral attachment to epithelial cells

We next sought to elucidate the mechanism by which PI inhibits RSV infection. Previous studies with POPG demonstrated this lipid disrupts RSV infection by direct, high-affinity interactions with the virus that block attachment to epithelial cell plasma membranes (11). We performed solid-phase binding studies to test for direct interactions between RSV and PI, and the results are shown in Fig. 2. RSV bound to solid-phase PI with high affinity and in a saturable reaction, yielding half-maximal binding at a viral titer of 0.25 × 10⁷ pfu/ml. The interaction of the virus with PI was similar to that for POPG. In contrast, the binding of RSV to solid-phase POPC was low affinity and nonspecific.

In additional experiments, we examined the activity of PI as an antagonist of RSV attachment to monolayers of HEp2 cells, using Western blotting to detect viral antigens. The experiments were performed at 18°C to block internalization of the virus. For comparison, we also quantified the inhibition of virus binding by other anionic phospholipids including POPG, PA, and PS, and the zwitterionic lipid, POPC. Representative Western blots are shown in Fig. 3A, C, and quantitative data are shown in Fig. 3B, D. The results show that RSV binds to HEp2 cells in a concentration-dependent reaction, and PI effectively disrupts virus attachment to epithelial cells; whereas PA, PS, and POPC do not significantly alter viral attachment. The activity of PI as an antagonist of viral attachment to cells is comparable to that observed for POPG. The data also reveal that the inhibition of RSV binding to cells can be overcome as the viral titer is increased, consistent with PI and POPG exerting a competitive inhibition of the process. From these data, we conclude that specific structural aspects of POPG and PI confer binding affinity and specificity that is not duplicated by either the anionic character, or hydrophobic constituents of other negatively charged lipids, or the hydrophobic constituents of zwitterionic phospholipids (12, 13).

PI does not act as a viricidal agent

The data in Fig. 3 demonstrate high-affinity interactions between RSV and PI that disrupt viral attachment to epithelial cells but do not eliminate viricidal effects as an additional mechanism of lipid action. We designed experiments to test whether PI could compromise the integrity of RSV and if this could also contribute to the mode of action of the lipid. To examine the inhibitory effect of PI against RSV in more detail, we tested for the reversibility of PI binding to RSV and potential viricidal effects of the phospholipid. High titer preparations of RSV (1 × 10⁸/ml) were incubated with either PI or POPC (200 μg/ml) at 37°C for 1 h. Subsequently, the solutions were diluted 10⁵-fold, and aliquots were analyzed by plaque assay. As shown in Fig. 4, there were no significant differences in the number of recovered plaques with the lipid treatments. The data in Figs. 3 and 4 clearly establish that the principal mode of action of PI as an antagonist of RSV infection is by inhibition of viral attachment to cell surfaces, and not compromise of virus integrity.

PI suppresses RSV infection in vivo

We next conducted experiments to evaluate the effectiveness of PI as an antagonist of RSV infection in vivo. Mice were inoculated with RSV (1 × 10⁷ pfu/mouse) in either the absence or presence of PI (300 μg/mouse), and the progress of viral infection was evaluated after 5 days by quantifying inflammatory cell infiltrates recovered in lavage, IFN-γ production, and the viral burden of RSV within the lung (11, 13). As shown in Fig. 5A, total cell numbers present in bronchoalveolar lavage fluid (BALF) increased ∼25-fold with RSV infection, but simultaneous treatment with RSV plus PI reduced the cell numbers to those of sham-infected animals (CONL = 2.3 ± 0.1 × 10⁴ cells/ml, RSV = 58 ± 2.0 × 10⁴ cells/ml, RSV + PI = 3.0 ± 0.1 × 10⁴ cells/ml, PI alone = 2.5 ± 0.2 × 10⁴ cells/ml). The major virus-elicited, inflammatory cell populations in the BALF were neutrophils and lymphocytes (Fig. 5B), and the presence of both of these cell types was markedly diminished by the PI treatment. High levels of IFN-γ (6.5 ± 0.5 ng/ml) are produced in response to active RSV infections as shown in Fig. 5C, but PI suppression of viral infection and inflammation resulted in no detectable IFN-γ in the BALF (11). We examined the RSV burden present in the left lungs of mice by plaque assay, as previously described (11, 21). The results shown in Fig. 5D demonstrate that PI reduced viral titers by a factor of ∼30. As a control condition, in these infection experiments we also inoculated mice with PI alone, and the results in Fig. 5A–C demonstrate that this treatment was without a significant effect on any of the measured parameters.

We also examined the effects of RSV and PI treatment of the mice by quantitative histopathology using light microscopy, which provides a means for comparing tissue damage and inflammation among the different treatment groups. The data in Fig. 5E demonstrate that PI significantly reduces the histological criteria for pulmonary inflammation and infection. Figure 5F shows representative micrographs of right lung sections for the different experimental conditions applied to the mice. The results show that RSV elicits inflammatory responses and damage to the bronchial and alveolar epithelia that are markedly curtailed by PI treatment. Collectively, the data in Fig. 5 demonstrate that in mice, PI significantly suppresses the effects of RSV infection on the lung.

Supplemental PI has a relatively long turnover time in mouse lung

Recently, we reported that supplemental POPG has a relatively short turnover time (t_1/2 ∼45 min) in the mouse lung, and this corresponds to a short prophylaxis window for the use of this phospholipid (13). We performed similar turnover studies with PI in mice using a 150 μg (172 nmol) intranasal inoculum. Following administration of PI, groups of mice were euthanized and lavaged after 0, 30, 60, 180, and 360 min, and the amount of PI recovered in the cell-free BALF was measured, using TLC and lipid phosphate determination (13, 25). The data presented in Fig. 6A show that the supplemental PI can be readily recovered and detected in the BALF immediately after the administration. Chemical measurement of PI by phosphate analysis in Fig. 6B demonstrates that the PI turns over relatively slowly with ∼50% of the lipid recoverable at 0 time remaining after 6 h. Figure 6C reveals that the pool of introduced PI recoverable from lavage exceeds that of the endogenous pool by ∼25-fold immediately after instillation and ∼15-fold at 6 h after instillation. Despite these large changes in BALF lipid composition, the levels of PI used in these experiments did not produce any overt signs of distress in the mice, and the histopathology from the experiments described in Fig. 5 also does not show any signs of adverse effects. A plot of the time-dependent loss of PI from the BALF is shown in Fig. 6D and demonstrates an initial rapid turnover followed by slower turnover. The markedly slower turnover phase suggests that PI can persist in the extracellular compartment for extended periods of time.

PI provides significantly longer prophylaxis against RSV than POPG

The slower turnover rate of PI compared with that previously reported for POPG (13, 22) prompted us to test whether PI would provide improved protection from RSV infection in mice. In preliminary studies (data not shown), we treated mice with 600 μg of PI at either 6 h or 3 h before RSV infection. The 6 h pretreatment was ineffective, but the 3 h pretreatment reduced viral plaque numbers recovered from the left lungs by ∼50% (data not shown). We next performed in vivo studies using a 600 μg treatment of mice with PI at 2 h prior to infection with RSV, and the results of these experiments are shown in Fig. 7. The RSV infection elicited an expected 14-fold increase in cellular infiltrates recoverable in lavage, which was almost completely suppressed by either simultaneous treatment or pretreatment with PI (Fig. 7A). Likewise, the PI pretreatment reduced the neutrophil and lymphocyte populations to near control levels (Fig. 7B). The data in Fig. 7C demonstrate that the effects of pretreatment with PI upon RSV-elicited IFN-γ levels (75% reduction compared with viral infection alone) were not as pronounced as that observed with simultaneous PI treatment but still demonstrate significant suppression of viral infection. The PI pretreatment reduced the viral burden in the lungs by 85% (Fig. 7D) as compared with the 97% suppression effected by simultaneous treatment with PI. Thus PI prophylaxis provides significant benefit for protecting against RSV infection. Compared with previous prophylaxis studies using POPG, PI more than doubles the efficacy window for protection against the virus.