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.

Figure 1.

Palmitoyl-oleoyl-phosphatidylglycerol (POPG) attenuates H3N2–influenza A virus (IAV)–induced IL-8 production by bronchial epithelial cells. IL-8 production by cells from a human bronchial epithelial cell line (Beas2B) was determined by ELISA after a 48-hour H3N2-IAV challenge in either the absence or presence of 200 μg/ml of POPG or palmitoyl-oleoyl-phosphatidylcholine (POPC). The cells were either sham treated (UN) or challenged with virus at a multiplicity of infection (MOI) of 2. Values shown are means (±SE) for three independent experiments; *P < 0.05. UN, uninfected.

Figure 2.

POPG prevents the cytopathic effects of H3N2 upon cultured Madin-Darby canine kidney (MDCK) cells. Monolayers of MDCK cells were either uninfected (CONL) or treated with H3N2-IAV at an MOI of 1 (IAV), in either the absence or presence of POPG (200 μg/ml or 1,000 μg/ml) or POPC (200 μg/ml), as indicated. After 36 hours, the cultures were examined by light microscopy.

Figure 3.

POPG suppresses neuraminidase (NA) and M1 protein expression elicited by H3N2-IAV infection of MDCK cells. (A) Monolayers of MDCK cells were either uninfected (UN) or infected with H3N2-IAV at MOIs of 0.5 or 1 for 2 hours in either the absence or presence of POPG (200 μg/ml or 1,000 μg/ml) or POPC (200 μg/ml). After 36 hours, the wells were harvested and analyzed for the expression of NA and M1 (M-P) by SDS-PAGE and immunoblotting. Control experiments also included incubation with POPG (1,000 μg/ml) and POPC (200 μg/ml) in the absence of viral infection. (B) Quantification of NA and M1 protein (MP) expression from three independent experiments. Values shown are means (±SE). *P < 0.05, upon comparison of virally infected cells without phosphatidylglycerol (PG) addition to those with PG addition.

Figure 4.

POPG inhibits hemagglutinin (HA) messenger RNA (mRNA) expression in H3N2-IAV–infected MDCK cells. (A) Monolayers of MDCK cells were either uninfected (UN) or infected at MOIs of 0.5, or 1 for 2 hours in either the absence or presence of POPG (200 μg/ml or 1,000 μg/ml) or POPC (200 μg/ml). After 24 hours, the wells were harvested and processed for RNA extraction and subjected to quantitative RT-PCR and gel electrophoresis. (B) The results from three independent experiments are shown. Values are means (±SE). *P < 0.02; **P < 0.001.

Figure 5.

POPG binds IAV with high affinity and inhibits cell surface binding of H3N2-IAV. (A) Aliquots of 1.25 nmol phospholipid (POPG or POPC) were adsorbed onto microtiter wells, and the indicated concentrations of H3N2-IAV were added and incubated at 37°C for 2 hours. The viral binding was detected by ELISA and quantified by A490. (B) Solid-phase lipids were prepared as in (A), and the binding of H1N1-PR8-IAV was performed at 37°C for 2 hours. The viral binding was detected by ELISA and quantified by A450. (C) Monolayers of MDCK cells, at 19°C, were challenged with H3N2-IAV at MOIs of 0–10, as indicated, in either the absence or presence of POPG (200 μg/ml or 1,000 μg/ml) or POPC (200 μg/ml). The cultures were harvested and processed for SDS-PAGE and immunoblotting. (D) Quantification of three immunoblotting experiments performed as described for (C). Values shown in (A), (B), and (D) are means (±SE) for three independent experiments. p.f.u., plaque-forming units.

Figure 6.

The POPG effect upon IAV infection is reversible and dependent upon the timing of lipid and virus addition. (A) Monolayers of MDCK cells were infected with IAV at an MOI of 1 at 8°C for 4 hours, and then treated with POPG (200 μg/ml or 1,000 μg/ml) or POPC (1,000 μg/ml) at 8°C for 4 hours. The cultures were shifted to 37°C and the infections were allowed to proceed for 36 hours. The contents of each tissue culture well were recovered and processed for immunoblotting with polyclonal goat anti-IAV and rabbit anti–β-actin antibodies. Immunoblots from a representative experiment are shown. (B and C) Quantification of MP and NA expression, normalized to β-actin expression, from three experiments is summarized. (D) Aliquots of H3N2-IAV (10⁸ pfu/ml) were incubated for 1 hour at 37°C in either the absence or presence of 1,000 μg/ml POPG. Subsequently, the viral aliquots were diluted 10³-fold in either the absence (Reversal) or presence of POPG (POPG) and used to infect monolayers of MDCK cells at an MOI of 0.05. At 7 hours after the initiation of infection, the cultures were washed with cold PBS and the monolayers were fixed with paraformaldehyde, permeabilized, and stained for the presence of viral antigens with polyclonal goat anti-IAV antibody and rabbit anti-goat Alexa 548 antibody. Fluorescent foci were scored using a Zeiss 200-M microscope (Zeiss, Oberkochen, Germany) and Slidebook software (Leeds Precision Instruments, Minneapolis, MN) at 10× magnification. (E) Cells were either uninfected (UN), treated with 1,000 μg/ml POPG alone (POPG), or challenged with virus (+ H3N2) in either the absence or presence of POPG, as indicated. POPG was added either 0.5 or 0 hours before (designated by minus sign), or 1 or 2 hours after (designated by plus sign) viral addition. In one group of 0.5-hour lipid treatment before viral addition, the POPG was washed out (WO) of the culture well before viral infection. In all other groups the POPG level was maintained until cell harvest. In each case, viral adsorption was conducted for 1 hour at 37°C, followed by removal of unbound virus. Culture wells were harvested and the cells present in both the supernatant and the adherent monolayer were counted and stained for viability using 0.02% trypan blue. Viral infection at an MOI of 0.05 occurred at 0 hours. Values shown in bar graphs are means (±SE) for three independent experiments. *P < 0.05; ^§P < 0.001.

Figure 7.

POPG inhibits H1N1-PR8-IAV infection and inflammation in vivo and suppresses the histopathology elicited by the virus. BALB/c mice were infected with 80 pfu H1N1-PR8 (H1N1) by intranasal inoculation in either the absence or presence of 3 mg of POPG, as indicated. Sham (CONL) and lipid-only treatments (POPG) were also performed. After 5 days of infection, the animals were killed. (A) Amount of virus present in the left lung was quantified using plaque assays. *P < 0.01. (B) Lavage fluid collected from animals was used to quantify total cells recovered from the bronchoalveolar compartment. *P < 0.02. (C) Cytospin preparations were used to quantify the percentage of lymphocytes (Lymph.) and neutrophils (Neut.) present in the lavage fluid. *P < 0.01, ^§§P < 0.001. (D) The production of IFN-γ was measured in cell-free lavage fluid by ELISA. *P < 0.01. Each group contains four to six animals per individual experiment. (E) Paraffin sections (4 μm) were stained with hematoxylin and eosin, analyzed by light microscopy, and assigned a histopathology score. *P < 0.05. (F) Representative micrographs from the experiment. Values shown in A–E are means (±SE) for three independent experiments. Scale bar, 200 μm.