Anti-inflammatory and anti-viral actions of anionic pulmonary surfactant phospholipids

Abstract

Pulmonary surfactant is a mixture of lipids and proteins, consisting of 90% phospholipid, and 10% protein by weight, found predominantly in pulmonary alveoli of vertebrate lungs. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), are present within the alveoli at very high concentrations, and exert anti-inflammatory effects by regulating multiple Toll like receptors (TLR2/1, TLR4, and TLR2/6) by antagonizing cognate ligand-dependent activation. POPG also attenuates LPS-induced lung injury in vivo. In addition, these lipids bind directly to RSV and influenza A viruses (IAVs) and block interaction between host cells and virions, and thereby prevent viral replication in vitro. POPG and PI also inhibit RSV and IAV infection in vivo, in mice and ferrets. The lipids markedly inhibit SARS-CoV-2 infection in vitro. These findings suggest that both POPG and PI have strong potential to be applied as both prophylaxis and post-infection treatments for problematic respiratory viral infections.

Keywords: Antiviral; Innate immunity; Pulmonary surfactant phospholipids; Respiratory viruses; Toll-like receptors (TLRs).

Fig. 1.

Constituents of Pulmonary Surfactant. The surfactant complex consists mainly of phospholipids (~90%) and to a lesser extent proteins (~10%). The proteins consist of the surfactant proteins (SP-A, SP-B, SP-C and SP-D) with SP-B and SP-C being extraordinarily hydrophobic, and SP-A and SP-D being hydrophilic and highly oligomeric. The serum proteins found in the complex, consist of immunoglobulins (IgA and IgG) and albumin. Uteroglobin (CCL10) is also found associated with the complex. The lipids consist mostly of the phospholipid classes, Phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylinositol (PI) and the molecular species within these classes are shown in the inset tables. Minor-Trace amounts of sphingomyelin, PE, glycosphingolipids, triacylglycerols and cholesterol are also present. In Fig. 1, the major molecular species of PC is the disaturated 16:0/16:0 species, responsible for the surface tension lowering properties of the complex. Also, from the graphic in Fig. 1, the major molecular species of PG is the 16:0/18:1 species. As shown in the Fig. 1 graphic, the 18:1/18:1 molecular species of PI predominates [9].

Fig. 2.

Phosphatidylglycerol (PG) and phosphatidylinositol (PI) share structural similarity. The Schematic structures for PG and PI are shown, with emphasis placed upon the polar headgroups and their similarities. PtdO is the standard abbreviation for the phosphatidate moiety (containing a glycerolphosphate backbone to which is esterified two fatty acids) found in both the PG and PI classes [54] [55] [56].

Fig. 3.

Schematic depiction of mechanism of POPG and PI disruption of TLR signaling. As decoy ligands, POPG and PI prevent recognition of cognate ligands (e.g., LPS) by the TLR4 co-receptors CD14 and MD2. Similarly, POPG and PI disrupt the recognition of Pam3Cys and MALP2 by TLR2/1 and TLR2/6 complexes, respectively (adapted figure from ref. [57]).

Fig. 4.

POPG and PI prevent multiple TLR activations by cognate ligands. A) Arachidonic acid (AA) release from freshly isolated human alveolar macrophages can be used as a downstream indicator of TLR2/1, TLR4, and TLR2/6 activation. Cells prelabeled with [³H]-AA, release the AA in response to TLR activation by Pam3Cys for TLR2/1, LPS for TLR4 and MALP2 for TLR2/6. The release of [³H]-AA is inhibited by treatment with POPG liposomes, which disrupt early events in transmembrane signaling [16]. B-E) Beas2B cells were challenged with TLR agonists, B) Pam3Cysy for TLR2/1, C) Poly I:C for TLR3, D) Flagellin for TLR5, and E) MALP2 for TLR2/6, in either the absence, or presence of POPG, PI or POPC. IL-8 production was measured by ELISA at 48 h after stimulation with agonists [63].

Fig. 5.

POPG inhibits RSV infection and pulmonary injury in mice. Mice were challenged with 10⁷ pfu of RSV by intranasal inoculation in either the absence, or presence of anionic phospholipids. Panel A) shows histopathology scores in each group, and panel B) shows histopathological images of the lungs with each condition, consisting of sham treated group (CONL), RSV group (RSV), RSV challenge supplemented with POPG (RSV + POPG), and with treatment of POPG alone (POPG). Data are from ref. [14].

Fig. 6.

POPG and PI bind RSV with high affinity. Solid phase phospholipids were made by applying phospholipids (POPG, PI and POPC) in ethanol to microtiter wells, followed by air drying. Various amounts of RSV were added to the solid phases and incubated at 37°C for 60 min. Viral binding was detected with a polyclonal anti-RSV antibody [16]. The figure is adapted from ref. [16].

Fig. 7.

POPG and PI inhibit RSV infection as prophylaxis in mice. Balb/c mice were infected with RSV (1 × 10⁷ pfu/mouse) as described in Fig. 5. Panel A) shows viral burden in the lung at day 5 post infection. Virus alone (RSV), Virus + POPG simultaneous treatment (RPG), and Virus+ prophylaxis treatment with POPG (pRPG). POPG was administered to mice 45mins prior viral challenge. Panel B) shows viral burden in the lung in each group, virus alone (RSV), Virus + PI simultaneous treatment (RPI), and virus + prophylaxis treatment with PI (pRPI). Mice were treated with PI 2 h before viral challenge, The left lungs of infected animals were excised and homogenized and the viral titers of fresh homogenates were determined by plaque assay. Data are shown as, AVG ± SD, § indicates p < 0.01, §§ indicates p < 0.001. Statistical analysis by single Anova and unpaired t-test), respectively. Data are from refs [15,16].

Fig. 8.

POPG inhibits H3N2-influenza A attachment to epithelial cells. A) Solid phases of phospholipids were prepared in microtiter wells and hydrated, and subsequently defined amounts of virions were added and incubated for 1 h at 37° C. Virus attachment to phospholipids was quantified by immunodetection using HRP-conjugated polyclonal anti-IAV. B) The attachment of H3N2-IAV to monolayers of MDCK cells was quantified by western blotting, using Image J analysis. C) Virus attachment to cell monolayers in the absence and presence of phospholipids (POPG, POPC) was quantified by western blotting. Data are from ref. [107].

Fig. 9.

Binding studies demonstrate high affinity interactions between pH1N1-influenza A virions and phospholipids. Phospholipid solid phases were prepared in microtiter wells and binding interactions between pH1N1-virion and phospholipids were quantified by immunodetection (A₄₅₀). Figure is adapted from ref. [92].

Fig. 10.

POPG and PI inhibit pH1N1-influenza A viral burdens and consequent lung damage in mice. Panel A) Viral loads were determined by plaque assays and POPG treatment reduced viral burden. Data are shown as AVG ± SD, §§ represents p < 0.001. Panel B) Histology of lung sections in the absence (CONL) and presence (pH1N1) of virus, or virus + PG (pH1N1 + PG). The histopathology scores are shown in the lower left insets in the micrographs. Panel C) shows viral loads by plaque assay and antagonism by PI. Data are shown as AVG ± SD, §§ represents p < 0.001. Panel D) shows histology of lung sections for uninfected (CONL) and pH1N1 infected mice (pH1N1) and mice treated with PI alone; or virus + PI (pH1N1 + PI). The histopathology scores are shown in the lower left hand insets in the micrographs. Data are from ref. [92].

Fig. 11.

POPG and PI prevent cytopathic effect by SARS-CoV-2 and inhibit viral loads of SARS-CoV-2 in VeroE6 cells. Panel A) Hematoxylin-Eosin staining of VeroE6 cell monolayers of untreated (CONL), or SARS-CoV-2 infected cultures (SARS-CoV-2) that were also pretreated with lipids (+PC, +POPG, +PI) 30 mins prior to the time of infection. The white bar indicates 50 μm. Viral infection causes cell lysis, cell hypertrophy and hypochromic staining. Panel B) shows quantification of viral loads by plaque assays, with POPG reducing number by ~60%, and PI reducing numbers by ~90%.