Phosphatidylglycerol suppresses influenza A virus infection.

Abstract

Influenza A virus (IAV) is a worldwide public health problem causing 500,000 deaths each year. Palmitoyl-oleoyl-phosphatidylglycerol (POPG) is a minor component of pulmonary surfactant, which has recently been reported to exert potent regulatory functions upon the innate immune system. In this article, we demonstrate that POPG acts as a strong antiviral agent against IAV. POPG markedly attenuated IL-8 production and cell death induced by IAV in cultured human bronchial epithelial cells. The lipid also suppressed viral attachment to the plasma membrane and subsequent replication in Madin-Darby canine kidney cells. Two virus strains, H1N1-PR8-IAV and H3N2-IAV, bind to POPG with high affinity, but exhibit only low-affinity interactions with the structurally related lipid, palmitoyl-oleoyl-phosphatidylcholine. Intranasal inoculation of H1N1-PR8-IAV in mice, in the presence of POPG, markedly suppressed the development of inflammatory cell infiltrates, the induction of IFN-γ recovered in bronchoalveolar lavage, and viral titers recovered from the lungs after 5 days of infection. These findings identify supplementary POPG as a potentially important new approach for treatment of IAV infections.

Full text

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Phosphatidylglycerol Suppresses Influenza A Virus Infection

Mari Numataa, Pitchaimani Kandasamya, Yoji Nagashimab, Janelle Poseya, Kevan Hartshornc,
David Woodlandd and Dennis R. Voelkera
aDepartment of Medicine, Program in Cell Biology, National Jewish Health, Denver, CO 80206
bDepartment of Pathology, Yokohama City University School of Medicine, Yokohama, JAPAN
cDepartment of Medicine, Boston University School of Medicine, Boston, MA 02118
dTrudeau Institute, Saranac Lake, NY, 12983
Correspondence:
Dennis R. Voelker
Dept Med
National Jewish Health
1400 Jackson St
Denver, CO 80206

Email: voelkerd@njhealth.org
Phone: 303-398-1300
Fax: 303-398-1806

Grant Support:

This work was supported by NIH HL094629, HL 073907, CBDE-2009

Running Title:

Phosphatidylglycerol antagonism of Influenza A infection


This article has an online data supplement, which is accessible from this issue's table of content
online at www.atsjournals.org

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AJRCMB Articles in Press. Published on November 3, 2011 as doi:10.1165/rcmb.2011-0194OC
Copyright (C) 2011 by the American Thoracic Society.

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Abstract
Influenza A virus (IAV) is a worldwide public health problem causing 500,000 deaths each
year. Palmitoyl-oleoyl-phophatidylglycerol (POPG) is a minor component of pulmonary
surfactant, which has recently been reported to exert potent regulatory functions upon the innate
immune system. In this report we demonstrate that POPG acts as a strong anti-viral agent
against IAV. POPG markedly attenuated IL-8 production and cell death induced by IAV in
cultured human bronchial epithelial cells. The lipid also suppressed viral attachment to the
plasma membrane and subsequent replication in MDCK cells. Two virus strains, H1N1-PR8-
IAV and H3N2-IAV bind to POPG with high affinity but exhibit only low affinity interactions
with the structurally related lipid palmitoyl-oleoyl-phosphatidylcholine. Intranasal inoculation of
H1N1-PR8-IAV in mice, in the presence of POPG, markedly suppressed the development of
inflammatory cell infiltrates and the induction of IFN-γ recovered in bronchoalveolar lavage, and
viral titers recovered from the lungs after 5 days of infection. These findings identify
supplementary POPG as a potentially important new approach for treatment of IAV infections.

Key words: Antiviral, innate immunity, pulmonary surfactant









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INTRODUCTION
IAV is one of the most common viruses causing global health problems and life-threatening
infections, resulting in an estimated 500,000 deaths each year (1). In the US, 5-20% of the
population is infected annually, producing 200,000 hospitalizations and 36,000 deaths (1-3).
Patients with chronic pulmonary disease (e.g. chronic obstructive pulmonary disease, asthma) are
more susceptible to IAV infection and typically develop more severe symptoms requiring
hospitalization, or ICU admissions (4-6). In 2009-2010, the pandemic influenza A outbreak was
caused by a novel IAV of swine origin and the H1N1 subtype. This pandemic spread rapidly and
is illustrative of the problems of emergence of new strains (2, 7). Vaccination is the standard
strategy for prevention of influenza, but this effect varies and depends upon successful matching
of the vaccine antigen with the epidemic, or pandemic virus, and population compliance with
vaccination programs (1). Vaccine shortages for rapidly spreading pandemic viruses can also
limit population coverage and further intensify disease outbreaks and persistence.

Two classes of drugs currently available for treatments of influenza in non-immune individuals
are the ion channel inhibitors (e.g. amantadine, remantadine), and the neuraminidase inhibitors
(NAI) (e.g. Oseltamivir, Zanamivir, Peramivir) (1). The near complete loss of efficacy of the ion
channel inhibitors has led to heavy reliance upon NAIs (8), which are currently standard drugs of
choice for both prophylaxis and the early treatment of IAV infection. The frequency of NAI
resistant IAV is approximately 1% in adults, and 4-8 % in children (9). This resistance usually
develops from prior application, or prophylaxis treatment with NAIs. However, the CDC
recently reported that seasonal oseltamivir-resistant IAV has appeared independently of
oseltamivir use. The osteltamvir-resistant IAV is a more frequent and serious problem in children
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(10). The growing frequency of NAI-resistant IAV strains (9-11) highlights the importance of
developing new agents for the treatment of influenza infection with novel mechanisms of action.

Pulmonary surfactant is a lipid and protein complex that regulates biophysical properties of the
alveoli, and innate immune responses in the lung (12). It is well recognized that the hydrophilic
surfactant proteins (SP-A and SP-D) play multiple roles in regulating host defense. SP-A and SP-
D bind to a variety of bacteria, fungi, and viruses with high affinity, and regulate the innate
immune responses to these pathogens in the lung (12, 13). Surfactant proteins are minor
components of the surfactant complex accounting for approximately 10% of the material. The
major constituents of pulmonary surfactant are phospholipids, with phosphatidylcholines (PC) as
the dominant molecular class. Dipalmitoyl-PC is the most abundant lipid molecular species in
surfactant and is the lipid most responsible for the reduction of surface tension at the air-tissue
interface, within the alveolar compartment (13). Phosphatidylglycerol (PG) is also present in
surfactant and comprises approximately 10-mole % of the lipids. In humans, palmitoyl-oleoyl
PG (POPG) is the most abundant molecular species present within the PG class (14). The
concentration of phospholipids in the extracellular pulmonary surfactant present in the alveolar
hypophase, is estimated to be approximately 35 mg/ml (15). These extraordinarily high
extracellular phospholipid levels are not found in any other organ system. In addition, no other
organ has such high levels of PG, although trace levels of PG are found in numerous organs
where this lipid primarily functions at the subcellular level as a precursor to mitochondrial
cardiolipin. The functions of such high levels of extracellular PG within the lung have been
unclear, but recent studies now provide evidence that this lipid plays an important role in
regulating innate immunity and viral infection (16-21). We recently reported that POPG,
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suppresses LPS induced-inflammatory responses in vivo and in vitro through direct interactions
with CD14 and MD2 (16). Previous studies have also reported that PG antagonizes ligand
recognition by LPS binding protein (LBP), and CD14; and reduces LPS-induced inflammatory
responses (17-19). In addition to regulating cellular responses to LPS, CD14 has been
implicated in the innate immune response to respiratory syncytial virus (RSV) (22). This latter
connection prompted recent examination of the effects of POPG upon RSV induced
inflammation and infection (20). These studies produced the unanticipated finding that POPG
blocks RSV infection in vitro and in vivo by disrupting viral attachment to epithelial cell
surfaces. An additional unanticipated finding was that supplemental POPG administered
intranasally, markedly attenuated RSV infection in vivo, in mice (20). This unexpected anti-viral
activity of surfactant lipid led us to examine the effect of POPG as an IAV antagonist. The goals
of this study were to determine if POPG could, 1) suppress the inflammatory response and cell
death induced by IAV infection in epithelial cells in vitro, 2) inhibit viral attachment and
subsequent replication in epithelial cells, 3) directly interact with IAV, and 4) attenuate IAV
infection in vivo. Our findings demonstrate that supplemental POPG is a potent anti-viral agent
against IAV. These findings strongly suggest that POPG and related compounds play an
important role in pulmonary innate immunity and could be developed and used as a novel
therapy against IAV infection.

MATERIAL AND METHODS
Viruses, tissue culture, infection and surfactant lipid treatments.
Influenza A viruses, Philippines 82/H3N2 and H1N1/PR8, were prepared as previously
described (23-25). Madin-Darby Canine Kidney (MDCK) cells and Beas2B cells were obtained
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from ATCC (Manassas, Virginia, USA). Phospholipids were obtained from Avanti (Alabaster,
AL, USA), and uni-lamellar liposomes were prepared as previously reported (16, 20). To
examine the effects of phospholipids on H3N2-IAV infection, cells were pretreated for 1h with
POPG or POPC liposomes (20).

Viral protein expression.
MDCK cells were grown in 24-wells plates, and pretreated for 1h with phospholipids prior to
virus addition. Viruses were added to cells in the presence or absence of phospholipids, and the
total well lysates were subjected to immunoblotting after 36hrs using Goat polyclonal anti IAV
antibody (Millipore, Billerica, MA, USA) and β-actin (Cell Signaling Technology, USA).
Quantification of MP and NA protein expression was performed using NIH Image J1.34
software.

HA mRNA analysis by qRT-PCR.
MDCK cells were grown in 24-well plates, and H3N2-IAV adsorption was performed using a
multiplicity of infection (MOI) of 0.5-1.0 for 2 hr at 37°C. Immediately following the
adsorption, and at 24h, total well contents were processed for RNA extraction using a Qiagen
RN-easy kit (Quagenm, Maryland, USA). HA mRNA expression was quantified using a qRT-
PCR kit (Invitrogen, Camarillo, CA, USA).

Binding of Influenza A viruses to phopholipids and MDCK cells.
To examine the direct interactions between IAV and phospholipids, solid phase binding assays
were performed (20). Phospholipid coated wells were incubated for 2 hr at 37°C with varying
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concentrations of viruses. Viral attachment was detected with goat anti IAV antibody added with
3% BSA at 37°C. The bound viruses were quantified by absorbance at 450nm, or 490nm.
For cellular binding studies, MDCK cells were grown in 24-well plates and IAV was adsorbed to
the monolayers, for 2 hr at 19°C, either in the absence, or presence of phospholipids. At 19°C
endocytosis by MDCK cells is minimal, and this temperature allows viral binding to reach
equilibrium within 2 h. The cell monolayers were processed at 0 °C for subsequent analysis by
quantitative immunoblotting.

In vivo suppression of Influenza A infection.
Female BALB/c mice at 6 weeks old, were obtained from Jackson Laboratory (Bar Harbor,
Maine, USA). Mice were anesthetized with 0.25g/kg avertin introduced intraperitoneally (20).
Anesthetized mice were inoculated intranasally with a total volume of 50 µl of PBS in groups
consisting of sham infection, IAV infection (80pfu/mouse), IAV infection plus POPG, and POPG
alone. POPG liposomes were prepared in PBS (16, 20), and mice were inoculated with 3 mg of
the lipid premixed with the virus. On specific days, mice were euthanized by intraperitoneal
injection of 0.25 ml of Nembutal (10 mg/ml). Bronchoalveolar lavage fluid (BALF) was used for
differential cell quantification and IFN-γ analysis (20). Homogenates of the left lungs were used
for IAV plaque assays (26). The right lungs were processed for lung histopathology score (20,
27). Animal studies followed all prescribed guidelines and were approved by the Institutional
Animal Care and Use Committee.

RESULTS
POPG attenuates H3N2-IAV induced cytokine production in human bronchial epithelial
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cells.
We first examined the effects of POPG upon IL-8 production induced by H3N2-IAV in a human
bronchial epithelial cell line (BEAS2B). IL-8 is a typical early alarm cytokine released by
tissues to recruit neutrophils to sites of injury and infection. Cells were pretreated with POPG, in
the form of small unilamellar vesicles, for 1 hr, and challenged with H3N2-IAV at an MOI of
2/cell. As shown in Figure 1, H3N2-IAV induced a 6000-fold increase of IL-8 compared with
uninfected cells. POPG (200 µg/ml) treatment inhibited H3N2-IAV induced IL-8 production by
91%. A control lipid, palmitoyl-oleoyl-phosphatidylcholine (POPC), did not alter the virally
induced IL-8 production. POPG and POPC contain identical hydrophobic domains, but differ in
their hydrophilic domains, which contain phosphoglycerol and phosphocholine, respectively.
Treatment of BEAS 2B cells with either POPG or POPC, in the absence of virus had no effect
upon basal IL-8 production. From these experiments we conclude that POPG acts as a potent
inhibitor of the inflammatory response elicited by H3N2-IAV in cultured human epithelial cells.
These results also indicate that the polar portion of POPG plays a major role in dictating the
specificity of the lipid as an antagonist of H3N2-IAV induction of IL-8 production.
The concentrations of POPG used in these experiments are less than 10% of the PG levels found
in pulmonary surfactant, suggesting that in vivo resident PG pools may provide significant
protection from the virus. Previous studies have shown that the actions of POPG are not broadly
pleiotropic for inhibition of IL-8 production, since the lipid does not suppress the expression of
the cytokines induced by the TLR5 agonist, flagellin (20). Additional control experiments
demonstrate that POPG does not alter cellular protein synthesis, or growth. These findings
clearly demonstrate that POPG can significantly suppress H3N2-IAV induced inflammatory
cytokine production.
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POPG prevents cell death and suppresses IAV protein expression.
We next investigated the action of POPG upon the cytopathic effects of the virus against MDCK
cells, which are routinely used for plaque assays and in vitro propagation of many strains of IAV.
As shown in Figure 2, untreated MDCK cells form monolayers with a typical cobblestone
appearance. Infection of the cells with H3N2-IAV, at an MOI of 1, destroys the cell monolayer
after 36 hr. In contrast, treatment of the cells with virus in the presence of 1 mg/ml POPG
completely protects the cells from the cytopathic effects of IAV. At 200 µg/ml POPG also
provides significant protection of the monolayer from the lytic effects of the virus, although a
few cytopathic foci are evident. Treatment of the cultures with virus in the presence of 200 µg/ml
POPC fails to prevent cell death by IAV infection. From these data we conclude that POPG acts
early in the infectious cycle to protect cells from IAV.

To estimate phospholipid antagonism of virus propagation in MDCK cells, we examined the
effects of POPG upon the expression of M1 Protein (MP) and neuraminidase (NA), which were
measured following infection of the cells with H3N2-IAV for 36 hr. As shown in Figure 3A, at 2
hr after infection, using viral MOIs of either 0.5 or 1.0, neither MP, nor NA protein were
detectable; but after 36hr infection, MP and NA protein expression clearly increased and was
readily measurable. POPG treatment attenuated both MP and NA protein expression in a dose
dependent manner with 1 mg/ml being significantly more effective than 200 µg/ml; (Figure 3A,
3B). At 1mg/ml, POPG inhibited NA expression by 80% and MP expression by 75%. In contrast
to POPG, POPC was completely ineffective. In addition to the H3N2 strain, we also performed
experiments with the mouse adapted H1N1-PR8-IAV strain, because it is routinely used to
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perform in vivo studies with mice. As shown in Figure S1, infection of MDCK cells with H1N1-
PR8-IAV at an MOI of 0.5, for 36hr, in the presence of 1 mg/ml POPG or POPC; produced
results similar to that found for H3N2-IAV. The treatment with POPG at 1 mg/ml attenuated MP
expression by 70% whereas POPC at 1 mg/ml did not alter MP expression. From these results,
we conclude that POPG can suppress IAV protein expression from both H3N2-IAV and H1N1-
PR8 strains, and the effect is dependent upon phospholipid structure.

We also examined the mRNA expression for the hemagglutinin (HA) gene using quantitative RT-
PCR. The results from these experiments are shown in Figure 4. After 2 hr of viral adsorption,
RNA for HA was not detectable at MOIs of either 0.5, or 1.0. After 24 hr, a robust RT-PCR
signal was obtained from cells infected at an MOI of 0.5 (Figure 4A). POPG treatment
significantly attenuated the HA-mRNA signal, but POPC did not alter the HA-mRNA signal.
Quantitative analysis of the RT-PCR data in Figure 4B, showed that the inclusion of 200 µg/ml
POPG during infection suppressed HA gene expression by 75%, and 1mg/ml POPG suppressed
the expression by 88%. In contrast to the findings with POPG, the treatment with POPC was
ineffective. These data demonstrate that POPG suppresses IAV HA mRNA expression in MDCK
cells. Collectively, the experiments examining cytopathology, protein expression and mRNA
expression demonstrate that POPG disrupts the IAV infection process at an early stage, and
consequently prevents viral replication and cell death. These findings suggested that POPG
might directly interact with the virus and interfere with cell binding, and additional experiments
were conducted to test this idea.

POPG binds to influenza A virus with high affinity.
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To investigate the mechanism of the anti-viral effect of POPG, we examined the binding
interaction between IAV and the lipid. Figure 5A shows the direct binding of H3N2-IAV to a
POPG solid phase adsorbed to an ELISA plate. This binding interaction is virus concentration
dependent, high affinity and saturable. In contrast to the virus binding to POPG, the binding to
POPC has the characteristic of a low affinity, non-specific interaction. Figure 5B shows the
results of similar experiments using H1N1-PR8 IAV with POPG, and also demonstrates high
affinity, concentration dependent, and saturable binding. Compared with POPG, the lipid POPC
is a weak-binding ligand for H1N1-PR8 IAV and the interaction is non-saturable and non-
specific.
Additional experiments examined whether POPG could interrupt binding of H3N2-IAV to cell
surfaces. In these studies H3N2-IAV was adsorbed to MDCK cells at varying multiplicities of
infection for 2 hr at 19°C (to block endocytosis), in either the absence, or presence of POPG.
Following viral adsorption, cell monolayers were washed with PBS to remove unbound viruses,
and processed to detect attached viruses by immunoblotting for MP. The results presented in
Figure 5C demonstrate that MP detection increased with increasing H3N2-IAV titer. The
attachment of the virus to the cell surface was high affinity and saturable. The recovery of MP
was inhibited 75% by 200 µg/ml POPG, and inhibited 93% by 1mg/ml POPG, at viral MOIs as
high as 10/cell (Figure 5D). POPC failed to block the binding between MDCK cells and IAV.
These data provide clear evidence that POPG binds directly to IAV and disrupts viral adsorption
to epithelial cell surfaces, thereby suppressing infection (Figures 2, 3 and 4) and the
inflammatory response (Figure 1).

POPG antagonism of H3N2-IAV infection is reversible and dependent upon the timing of
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lipid addition.
The data presented in Figure 5 provide compelling evidence that POPG directly binds to IAV and
this interaction inhibits cell surface attachment of the virus. However, these findings do not
provide any information about the consequences of POPG binding to IAV upon the integrity of
the virus. In order to examine this issue in more detail and assess whether the lipid has direct
virucidal activity, we conducted two types of experiments. In the first line of experimentation,
H3N2-IAV (MOI of 1) was preadsorbed to MDCK cells in culture for 4hrs at 8C° (a temperature
that inhibits all viral endocytosis), and then the adsorbed virus was exposed to POPG at either
200 µg/ml, or 1000 µg/ml, for an additional 4hr at 8C°. As a control phospholipid, POPC was
added to separate MDCK monolayers also harboring preadsorbed virus. Following the
incubation with lipid, the cultures were washed and warmed to 37C° to allow the viral infection
to proceed. We reasoned that if the POPG acted as a virucidal agent to compromise the integrity
of the virus, the lipid treatment should reduce the subsequent progress of the infection. As
shown in Figure 6A, B and C, treatment of cell surface associated virus with POPG failed to
disrupt cell infection, as monitored by the production of the viral proteins NA and MP, at 36hrs
following the infection.

A second approach to examining whether POPG was directly acting as a virucidal agent, tested
the reversibility of the interaction of POPG with IAV. In these experiments H3N2-IAV (108
pfu/ml) was incubated with 1mg/ml POPG at 37C° for 1hr. Following the incubation, the virus
and lipid were diluted 103-fold in either the absence, or presence of 1 mg/ml POPG, and then
used to infect monolayers of MDCK cells. As a control for these manipulations, identical
aliquots of H3N2-IAV were incubated at 37C for 1hr in the complete absence of phospholipid.
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The infectivity of the IAV in these experiments was examined by quantifying fluorescent foci of
viruses formed on the MDCK monolayers 6 hrs after infection, detected by antibody. The results
presented in Figure 6D show that H3N2-IAV alone produced 1.01 ± 0.25 x 104 fluorescent foci
per well, and virus transiently exposed to 1mg/ml lipid and then diluted 103-fold produced 0.87 ±
0.23 x 104 foci per well, whereas virus exposed to a constant level of POPG produced 0.23 ±
0.04 x104 foci per well. These results demonstrate that the effects of POPG upon H3N2-IAV are
reversible. Together, the data in panels 6A-D provide strong evidence that POPG is not directly
virucidal.

An additional conclusion from the data in panels 6A-C is that POPG must act prior to viral
attachment to the cell surface, because virus already bound to cells is resistant to the antagonistic
effects of the lipid. To further examine this latter conclusion, we applied a viral challenge to
MDCK monolayers that were exposed to POPG for various periods before and after the addition
of virus. To quantify the effects of viral infections in these experiments we measured cell
viability at 36 hrs after adding viruses to the cultures. The results of these experiments are
presented in Figure 6E. The viability of uninfected cells, 98.7 + 0.24%, and cells treated with
POPG alone, 98.6 ± 0.3%, was equivalent. The addition of H3N2-IAV at an MOI of 0.5 reduced
cell viability to 45.2 ± 6.8%. Pretreatment of MDCK cells with POPG followed by washout of
the lipid prior to addition of virus, resulted in 44.7 ± 3.9% viability; whereas omission of the
lipid washout resulted in 79.3 ± 1.7% viability. Simultaneous addition of lipid and virus
provided significant protection to the monolayer and produced 70.8 ± 3.0% viability. The
addition of lipid 1hr after infection showed marginal protection and yielded 57.1 ± 2.5% viability.
When the lipid was added at 2hrs after infection, there was no significant protection of the
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cultures from the extent of cell death produced by virus alone. Collectively, these data show
that: 1) preincubation of cells with POPG followed by washout, prior to viral challenge, does not
block infection, 2) the anti-viral effects of POPG are nearly the same whether cells are pre-
incubated with lipid or simultaneously incubated with lipid at the time of viral challenge; and 3)
the protective effects of the lipid diminish rapidly with time following viral challenge. All of
these conclusions are consistent with the lipid acting prior to viral binding to the cell surface.
These in vitro properties of POPG suggested that the lipid might function as an effective anti-
viral agent in vivo, and further experiments were conducted to examine in vivo efficacy.


Intranasal administration of POPG suppresses IAV infection in mice.
We examined the potency of POPG as an anti IAV-agent using a mouse model of viral infection.
6 week old female BALB/c mice were inoculated intranasally, with the mouse adapted influenza
strain, H1N1-PR8-IAV (80 pfu/mouse) either in the absence, or presence of 3 mg of POPG. Five
days after infection, the animals were euthanized and the lungs were lavaged and harvested, and
analyzed for the effects of viral infection (26). The results presented in Figure 7A demonstrate
the POPG treatment clearly suppressed viral propagation in the lung by a factor 10 (IAV
infection = 6.21 ± 0.6 x 105 pfu, IAV + POPG = 0.6 ± 0.2 x 105 pfu). No plaques were obtained
from uninfected animals, or animals challenged with UV inactivated IAV, or animals treated with
POPG alone. Lavage from control mice produced a total cell number of 5.3 ± 1.6 x 104 cells/ml,
and H1N1-IAV increased the total cells recovered in lavage to 15.3 ± 1.5 x 104 cells/ml (Figure
7B). The POPG treatment reduced the total cell number in lavage, induced by virus by 50%. The
data presented in Figure 7C demonstrate that POPG significantly suppressed the proportional
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increase in lymphocytic and neutrophilic cellular infiltrates in lavage by 60%. IFN-γ production
was undetectable in lavage from control animals, and was 111.5 ± 24.7 pg/ml following virus
infection. The POPG treatment suppressed the virally induced IFN-γ response by 81% (Figure
7D).

Lung tissue from experimental animals was examined and assigned a histopathology score (20),
and the data are shown in Figure 7E. H1N1-IAV infected animals had a 3-fold higher
histopathology score than sham infected controls; and animals receiving virus plus POPG were
not significantly different from sham-infected animals. Representative micrographs are shown in
Figure 7E and reveal H1N1-IAV infection elicited a significant influx of inflammatory cells in
alveolar and peribronchial areas, and pneumonia. POPG treatment markedly attenuated these
virus-induced inflammatory changes, and the lipid treatment alone did not cause significant
histological changes. From the data shown in Figures 6 and 7, we conclude that POPG
suppresses H1N1-PR8 IAV infection and viral replication in vivo, and markedly reduces the
inflammatory responses to the virus. These findings strongly suggest that supplementary POPG
could be an important and novel approach for prevention and treatment of IAV infections.

DISCUSSION
In this report we provide strong evidence that supplementary POPG, the major molecular
species of PG present in pulmonary surfactant, potently suppresses the infection of epithelial
cells by IAV in vitro and in vivo. By interfering with the initial infective process, the lipid also
disrupts the release of inflammatory cytokines, such as IL-8, by the epithelium. The in vitro
doses of POPG capable of disrupting IAV infection are similar to in vitro doses of the lipid
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