Phospholipid regulation of innate immunity and respiratory viral infection
2019; Elsevier BV; Volume: 294; Issue: 12 Linguagem: Inglês
10.1074/jbc.aw118.003229
ISSN1083-351X
AutoresDennis R. Voelker, Mari Numata,
Tópico(s)Neuroscience of respiration and sleep
ResumoToll-like receptors (TLRs) coupled to intracellular signaling cascades function as central elements of innate immunity that control transcription of numerous pro-inflammatory genes. Two minor anionic phospholipids present in the pulmonary surfactant complex, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI), antagonize the cognate ligand activation of TLRs 2 and 4. The lipids block recognition of activating ligands by the TLRs, either directly or via the TLR4 coreceptors CD14 and MD2. Antagonism of TLR activation results in inhibition of the initiating step of the pro-inflammatory signaling pathways. Evidence for this mechanism of action comes from direct binding studies between CD14 and MD2 with POPG and PI. Additional evidence for this mechanism of antagonism also comes from monitoring the reduction of protein phosphorylation events that characterize the intracellular signaling by activated TLRs. The pathogenesis of respiratory syncytial virus (RSV) and influenza A virus (IAV) have been linked to TLR4 activation, and we examined the action of POPG and PI as potential antagonists of the pathology of these viruses. Surprisingly, POPG and PI dramatically curtail infection, in addition to inhibiting inflammatory sequelae associated with RSV and IAV infections. The mechanism of action by the lipids is disruption of virus particle binding to host cell plasma membrane receptors, required for viral uptake. The antagonism of activation of TLRs and virus binding to the alveolar epithelium by resident constituents of the pulmonary surfactant system suggests that POPG and PI function in homeostasis, to prevent inflammatory processes that result in reductions in gas exchange within the alveolar compartment. Toll-like receptors (TLRs) coupled to intracellular signaling cascades function as central elements of innate immunity that control transcription of numerous pro-inflammatory genes. Two minor anionic phospholipids present in the pulmonary surfactant complex, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and phosphatidylinositol (PI), antagonize the cognate ligand activation of TLRs 2 and 4. The lipids block recognition of activating ligands by the TLRs, either directly or via the TLR4 coreceptors CD14 and MD2. Antagonism of TLR activation results in inhibition of the initiating step of the pro-inflammatory signaling pathways. Evidence for this mechanism of action comes from direct binding studies between CD14 and MD2 with POPG and PI. Additional evidence for this mechanism of antagonism also comes from monitoring the reduction of protein phosphorylation events that characterize the intracellular signaling by activated TLRs. The pathogenesis of respiratory syncytial virus (RSV) and influenza A virus (IAV) have been linked to TLR4 activation, and we examined the action of POPG and PI as potential antagonists of the pathology of these viruses. Surprisingly, POPG and PI dramatically curtail infection, in addition to inhibiting inflammatory sequelae associated with RSV and IAV infections. The mechanism of action by the lipids is disruption of virus particle binding to host cell plasma membrane receptors, required for viral uptake. The antagonism of activation of TLRs and virus binding to the alveolar epithelium by resident constituents of the pulmonary surfactant system suggests that POPG and PI function in homeostasis, to prevent inflammatory processes that result in reductions in gas exchange within the alveolar compartment. The lungs are uniquely situated at the interface between the external and internal environment, and in the alveolar compartment, where gas exchange occurs, a tissue layer of only 0.6 μm separates ambient air from the vascular compartment (1Hsia C.C. Hyde D.M. Weibel E.R. Lung structure and the intrinsic challenges of gas exchange.Compr. Physiol. 2016; 6 (27065169): 827-89510.1002/cphy.c150028Crossref PubMed Scopus (103) Google Scholar). Lung epithelia are continually exposed to pro-inflammatory airborne irritants, microbes, and microparticulates in the ∼104 liters of air inspired each day. Despite this daily onslaught of pro-inflammatory stimuli, the lungs remain relatively nonresponsive to such insults, in healthy individuals. This hyporesponsive state is conferred by a variety of molecular systems that suppress inflammatory signaling cascades until their engagement is essential. Upon activation of inflammatory processes, as occurs with active bacterial or viral infections, there is a rapid infiltration of neutrophils and monocytes into the tissue, release of numerous inflammatory mediators, increased vascular permeability, and reduced efficiency of O2/CO2 exchange within the alveolar compartment. Fundamentally, the lungs exhibit a high threshold for engaging inflammatory processes, until their activation is essential, and this homeostatic balance prevents unwanted inflammation and reduced gas exchange in response to routine exposure to environmental stimuli, such as ambient levels of airborne LPS, 3The abbreviations used are: LPSlipopolysaccharideTLRToll-like receptorDPPCdipalmitoylphosphatidylcholinePGphosphatidylglycerolPIphosphatidylinositolPOPGpalmitoyl-oleoyl-phosphatidylglycerolPOPCpalmitoyl-oleoyl-phosphatidylcholineTNFtumor necrosis factorMAPmitogen-activated proteinERKextracellular signal-regulated kinaseJNKc-Jun N-terminal kinaseRSVrespiratory syncytial virusIAVinfluenza A viruspfuplaque-forming unit. and other microbial products. Elucidation of the molecular mechanisms by which the lungs suppress inflammation is critical not only for understanding the function and homeostasis of the organ, but also because it presents opportunities to amplify, or complement, such intrinsic processes for therapeutic benefit in inflammatory disease states. lipopolysaccharide Toll-like receptor dipalmitoylphosphatidylcholine phosphatidylglycerol phosphatidylinositol palmitoyl-oleoyl-phosphatidylglycerol palmitoyl-oleoyl-phosphatidylcholine tumor necrosis factor mitogen-activated protein extracellular signal-regulated kinase c-Jun N-terminal kinase respiratory syncytial virus influenza A virus plaque-forming unit. Pulmonary surfactant is a secreted, extracellular complex of lipids and proteins, which lines the alveolar compartment at the external air/tissue interface, and also plays an important role in regulating inflammatory processes within the lung (2Numata M. Kandasamy P. Nagashima Y. Posey J. Hartshorn K. Woodland D. Voelker D.R. Phosphatidylglycerol suppresses influenza A virus infection.Am. J. Respir. Cell Mol. Biol. 2012; 46 (22052877): 479-48710.1165/rcmb.2011-0194OCCrossref PubMed Scopus (61) Google Scholar, 3Ariki S. Nishitani C. Kuroki Y. Diverse functions of pulmonary collectins in host defense of the lung.J. Biomed. Biotechnol. 2012; 2012 (22675254)53207110.1155/2012/532071Crossref PubMed Scopus (44) Google Scholar4Wright J.R. Immunoregulatory functions of surfactant proteins.Nat. Rev. Immunol. 2005; 5 (15630429): 58-6810.1038/nri1528Crossref PubMed Scopus (773) Google Scholar). The surfactant-associated proteins (SP-A, SP-B, SP-C, and SP-D) (5Kuronuma K. Mitsuzawa H. Takeda K. Nishitani C. Chan E.D. Kuroki Y. Nakamura M. Voelker D.R. Anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and U937 cells by binding the lipopolysaccharide-interacting proteins CD14 and MD-2.J. Biol. Chem. 2009; 284 (19584052): 25488-2550010.1074/jbc.M109.040832Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) constitute ∼10% of the complex. SP-A and SP-D are collectins (collagen domain containing lectins) located primarily within the alveolar hypophase, the thin layer of fluid that resides above the apical surface of the alveolar epithelium. SP-A and SP-D can regulate inflammatory processes by acting as opsonins, and by direct interaction with pathogens and Toll-like receptors (TLRs) (3Ariki S. Nishitani C. Kuroki Y. Diverse functions of pulmonary collectins in host defense of the lung.J. Biomed. Biotechnol. 2012; 2012 (22675254)53207110.1155/2012/532071Crossref PubMed Scopus (44) Google Scholar, 6Sano H. Sohma H. Muta T. Nomura S. Voelker D.R. Kuroki Y. Pulmonary surfactant protein A modulates the cellular response to smooth and rough lipopolysaccharides by interaction with CD14.J. Immunol. 1999; 163 (10384140): 387-395PubMed Google Scholar7Sano H. Chiba H. 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SP-B and SP-C are extraordinarily hydrophobic proteins that play important roles in regulating the transformation of lipids from their intracellular storage form within lipid-rich lamellar bodies in alveolar type 2 cells to their extracellular interfacial form at the external air/tissue interface (10Wert S.E. Whitsett J.A. Nogee L.M. Genetic disorders of surfactant dysfunction.Pediatr. Dev. Pathol. 2009; 12 (19220077): 253-27410.2350/09-01-0586.1Crossref PubMed Scopus (191) Google Scholar). Phospholipids are the major constituents of pulmonary surfactant, with phosphatidylcholine being the most abundant lipid class and dipalmitoylphosphatidylcholine (DPPC) being the most abundant molecular species (11Batenburg J.J. Haagsman H.P. The lipids of pulmonary surfactant: Dynamics and interactions with proteins.Prog. Lipid Res. 1998; 37 (10193527): 235-27610.1016/S0163-7827(98)00011-3Crossref PubMed Scopus (93) Google Scholar). DPPC plays a major role in respiratory mechanics by reducing surface tension at the air/tissue interface of the alveolar compartment (12Veldhuizen E.J. Haagsman H.P. Role of pulmonary surfactant components in surface film formation and dynamics.Biochim. Biophys. Acta. 2000; 1467 (11030586): 255-27010.1016/S0005-2736(00)00256-XCrossref PubMed Scopus (199) Google Scholar). Absence of DPPC leads to alveolar collapse and greatly increases the effort required for breathing and oxygen exchange. Indeed, prior to the development of artificial pulmonary surfactants, newborn respiratory distress, which results when parturition precedes full lung maturity, was the leading cause of mortality in neonates in developed countries (13Nkadi P.O. Merritt T.A. Pillers D.A. An overview of pulmonary surfactant in the neonate: Genetics, metabolism, and the role of surfactant in health and disease.Mol. Genet. Metab. 2009; 97 (19299177): 95-10110.1016/j.ymgme.2009.01.015Crossref PubMed Scopus (130) Google Scholar). The anionic phospholipids phosphatidylglycerol (PG) and phosphatidylinositol (13Nkadi P.O. Merritt T.A. Pillers D.A. An overview of pulmonary surfactant in the neonate: Genetics, metabolism, and the role of surfactant in health and disease.Mol. Genet. Metab. 2009; 97 (19299177): 95-10110.1016/j.ymgme.2009.01.015Crossref PubMed Scopus (130) Google Scholar) are minor constituents (∼2–10% of total phospholipid) of the pulmonary surfactant complex, but their concentration within the alveolar hypophase is extraordinarily high (∼5–10 mg/ml for PG and ∼2 mg/ml for PI) and greatly exceeds that for any other tissue or mucosal surface (11Batenburg J.J. Haagsman H.P. The lipids of pulmonary surfactant: Dynamics and interactions with proteins.Prog. Lipid Res. 1998; 37 (10193527): 235-27610.1016/S0163-7827(98)00011-3Crossref PubMed Scopus (93) Google Scholar). For decades the functions of PG and PI within the alveolar compartment remained enigmatic, but over the last several years elucidation of the actions of these lipids as regulators of innate immune processes has emerged (2Numata M. Kandasamy P. Nagashima Y. Posey J. Hartshorn K. Woodland D. Voelker D.R. Phosphatidylglycerol suppresses influenza A virus infection.Am. J. Respir. Cell Mol. Biol. 2012; 46 (22052877): 479-48710.1165/rcmb.2011-0194OCCrossref PubMed Scopus (61) Google Scholar). In our earliest studies on this topic, we serendipitously discovered that liposomes composed of POPG were potent suppressors of LPS-induced activation of the innate immune receptor TLR4 (5Kuronuma K. Mitsuzawa H. Takeda K. Nishitani C. Chan E.D. Kuroki Y. Nakamura M. Voelker D.R. Anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and U937 cells by binding the lipopolysaccharide-interacting proteins CD14 and MD-2.J. Biol. Chem. 2009; 284 (19584052): 25488-2550010.1074/jbc.M109.040832Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In contrast to the activity of POPG, a control phospholipid harboring the same fatty acids but a different polar head group, palmitoyl-oleoyl-phosphatidylcholine (POPC), was ineffective as a regulator of innate immunity (5Kuronuma K. Mitsuzawa H. Takeda K. Nishitani C. Chan E.D. Kuroki Y. Nakamura M. Voelker D.R. Anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and U937 cells by binding the lipopolysaccharide-interacting proteins CD14 and MD-2.J. Biol. Chem. 2009; 284 (19584052): 25488-2550010.1074/jbc.M109.040832Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In a typical experiment either primary human alveolar macrophages, or human macrophage cell lines, are treated with LPS, and this elicits the production and secretion of pro-inflammatory mediators, such as TNFα, which are detected by ELISA (Fig. 1). TNFα is one of a large spectrum of pro-inflammatory molecules produced and released by cells in response to TLR activation (14Kawai T. Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.Immunity. 2011; 34 (21616434): 637-65010.1016/j.immuni.2011.05.006Abstract Full Text Full Text PDF PubMed Scopus (2604) Google Scholar). The intracellular signaling events for TLR4 are outlined in Fig. 1; are well-known; and involve (in approximately sequential order) a pathogen-derived molecule binding to receptor, dimerization and a conformational change in the intracellular domain of the receptor, receptor interaction with MyD88, activation of IRAKs 1, 2, and 4, activation of TRAF6, phosphorylation, (inactivation) of IκBα, release of NF-κB from complex with IκBα, activation and nuclear import of NF-κB, and finally, induced transcription of genes regulated by NF-κB, such as TNFα (14Kawai T. Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.Immunity. 2011; 34 (21616434): 637-65010.1016/j.immuni.2011.05.006Abstract Full Text Full Text PDF PubMed Scopus (2604) Google Scholar). In parallel with the activation of NF-κB and induction of gene expression, MAP kinases (ERK, p38, and JNK) are also phosphorylated and lead to the activation of transcription factor AP1. The activation of TLR4 by LPS depends upon three LPS interacting proteins: LPS-binding protein (LBP), cluster of differentiation 14 (CD14) (15Haynes L.M. Moore D.D. Kurt-Jones E.A. Finberg R.W. Anderson L.J. Tripp R.A. Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.J. Virol. 2001; 75 (11602714): 10730-1073710.1128/JVI.75.22.10730-10737.2001Crossref PubMed Scopus (404) Google Scholar), and lymphocyte antigen 96 (MD2) (16Park B.S. Lee J.O. Recognition of lipopolysaccharide pattern by TLR4 complexes.Exp. Mol. Med. 2013; 45 (24310172): e6610.1038/emm.2013.97Crossref PubMed Scopus (625) Google Scholar). LBP is a plasma protein that facilitates transport of LPS through aqueous compartments. CD14 is a PI-glycan–anchored peripheral plasma membrane protein that binds LPS. MD2, which also binds LPS, is a peripheral plasma membrane protein tethered to TLR4. A generally accepted model is that LBP transfers LPS to CD14, which subsequently transfers LPS to MD2. A multimolecular complex of CD14-LPS-MD2-TLR4 is required to induce the dimerization and conformational change to TLR4 that initiates the intracellular signaling cascade (14Kawai T. Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.Immunity. 2011; 34 (21616434): 637-65010.1016/j.immuni.2011.05.006Abstract Full Text Full Text PDF PubMed Scopus (2604) Google Scholar). When we probed the plasma membrane signaling pathway from LPS-activated TLR4, we discovered that all the downstream signaling events we interrogated (phosphorylation of p38, JNK, ERK, and IκBα), expression of TNFα and MAP kinase phosphatase 1 (MKP1), and release of arachidonic acid were disrupted by POPG inhibition (5Kuronuma K. Mitsuzawa H. Takeda K. Nishitani C. Chan E.D. Kuroki Y. Nakamura M. Voelker D.R. Anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and U937 cells by binding the lipopolysaccharide-interacting proteins CD14 and MD-2.J. Biol. Chem. 2009; 284 (19584052): 25488-2550010.1074/jbc.M109.040832Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 17Kandasamy P. Numata M. Zemski Berry K. Fickes R. Leslie C.C. Murphy R.C. Voelker D.R. Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit Toll-like receptor 2 and 4 signaling.J. Lipid Res. 2016; 57 (27095543): 993-100510.1194/jlr.M065201Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). This result suggested that POPG acts far upstream in the TLR4 signaling pathway perhaps at the level of LPS recognition by CD14 and MD2. To examine this process further, we investigated the direct binding of CD14 and MD2 to POPG and discovered that both proteins bind to the lipid with high affinity (5Kuronuma K. Mitsuzawa H. Takeda K. Nishitani C. Chan E.D. Kuroki Y. Nakamura M. Voelker D.R. Anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and U937 cells by binding the lipopolysaccharide-interacting proteins CD14 and MD-2.J. Biol. Chem. 2009; 284 (19584052): 25488-2550010.1074/jbc.M109.040832Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) as shown in Fig. 2, C and D. Moreover, monoclonal antibodies specific for the LPS-binding site of CD14 significantly inhibit interaction of the protein with both POPG and PI (5Kuronuma K. Mitsuzawa H. Takeda K. Nishitani C. Chan E.D. Kuroki Y. Nakamura M. Voelker D.R. Anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and U937 cells by binding the lipopolysaccharide-interacting proteins CD14 and MD-2.J. Biol. Chem. 2009; 284 (19584052): 25488-2550010.1074/jbc.M109.040832Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Binding studies also revealed that fluid phase POPG liposomes readily blocked the recognition of solid phase LPS by CD14 (5Kuronuma K. Mitsuzawa H. Takeda K. Nishitani C. Chan E.D. Kuroki Y. Nakamura M. Voelker D.R. Anionic pulmonary surfactant phospholipids inhibit inflammatory responses from alveolar macrophages and U937 cells by binding the lipopolysaccharide-interacting proteins CD14 and MD-2.J. Biol. Chem. 2009; 284 (19584052): 25488-2550010.1074/jbc.M109.040832Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Collectively, these data all provide evidence for POPG acting as a competitive ligand for the LPS-binding sites on CD14 and MD2. We also investigated the actions of POPG upon TLR2-dependent inflammatory pathways. TLR2 forms heterodimers with either TLR1 or TLR6 to recognize a variety of bacterial lipopeptides (14Kawai T. Akira S. Toll-like receptors and their crosstalk with other innate receptors in infection and immunity.Immunity. 2011; 34 (21616434): 637-65010.1016/j.immuni.2011.05.006Abstract Full Text Full Text PDF PubMed Scopus (2604) Google Scholar). The synthetic ligand Pam3Cys activates TLR2/1 complexes, and the synthetic ligand MALP-2 activates TLR2/6 complexes; both heterodimeric forms use essentially the same intracellular signaling cascades described above in Fig. 1 for TLR4 to induce inflammatory mediator production (17Kandasamy P. Numata M. Zemski Berry K. Fickes R. Leslie C.C. Murphy R.C. Voelker D.R. Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit Toll-like receptor 2 and 4 signaling.J. Lipid Res. 2016; 57 (27095543): 993-100510.1194/jlr.M065201Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 18Kandasamy P. Zarini S. Chan E.D. Leslie C.C. Murphy R.C. Voelker D.R. Pulmonary surfactant phosphatidylglycerol inhibits Mycoplasma pneumoniae-stimulated eicosanoid production from human and mouse macrophages.J. Biol. Chem. 2011; 286 (21205826): 7841-785310.1074/jbc.M110.170241Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In our studies of TLR2 activation and its antagonism, we utilized mouse and human macrophages and monitored the expression and secretion of TNFα, the expression of cyclooxygenase 2, and the release of arachidonic acid, which serves as a marker for downstream eicosanoid synthesis (e.g. prostaglandins D and E and thromboxanes) (16Park B.S. Lee J.O. Recognition of lipopolysaccharide pattern by TLR4 complexes.Exp. Mol. Med. 2013; 45 (24310172): e6610.1038/emm.2013.97Crossref PubMed Scopus (625) Google Scholar, 17Kandasamy P. Numata M. Zemski Berry K. Fickes R. Leslie C.C. Murphy R.C. Voelker D.R. Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit Toll-like receptor 2 and 4 signaling.J. Lipid Res. 2016; 57 (27095543): 993-100510.1194/jlr.M065201Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). POPG potently inhibited TLR2 activation and the linked downstream phosphorylation of p38, ERKs, and IκBα, induced expression of COX2, and arachidonic acid release. POPG was significantly more effective than dipalmitoyl-PG as an antagonist of TLR2 activation, indicating that the fatty acid composition of the phospholipid is an important element of lipid structure related to antagonistic activity (17Kandasamy P. Numata M. Zemski Berry K. Fickes R. Leslie C.C. Murphy R.C. Voelker D.R. Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit Toll-like receptor 2 and 4 signaling.J. Lipid Res. 2016; 57 (27095543): 993-100510.1194/jlr.M065201Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Additional data in Fig. 2B reveal that dimyristoyl PG and PI are nearly as potent as POPG. In an additional line of investigation we examined the effects of manipulating the structure of the glycerol moiety of the POPG head group, upon its activity as an antagonist of TLR2 and TLR4 (17Kandasamy P. Numata M. Zemski Berry K. Fickes R. Leslie C.C. Murphy R.C. Voelker D.R. Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit Toll-like receptor 2 and 4 signaling.J. Lipid Res. 2016; 57 (27095543): 993-100510.1194/jlr.M065201Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). All the analogs we synthesized harbored the same fatty acids (palmitate and oleate) as found in POPG. We synthesized 12 compounds that altered the head group glycerol moiety, which consists of three carbons and three hydroxyl groups, by changing 1) the number of aliphatic carbons from 0 to 5, 2) the number of hydroxyl substituents from 1 to 3, 3) the position of the hydroxyl substitutions, and 4) the branching of the head group aliphatic chain. We also synthesized an analog in which the head group sn-2-hydroxyl moiety was substituted with an amino group (17Kandasamy P. Numata M. Zemski Berry K. Fickes R. Leslie C.C. Murphy R.C. Voelker D.R. Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit Toll-like receptor 2 and 4 signaling.J. Lipid Res. 2016; 57 (27095543): 993-100510.1194/jlr.M065201Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). For lipid antagonism of TLR2 activated by a MALP-2 ligand, POPG consistently had the lowest IC50, and elimination of hydroxyl groups from the head group aliphatic chain greatly reduced the efficacy of the lipids (17Kandasamy P. Numata M. Zemski Berry K. Fickes R. Leslie C.C. Murphy R.C. Voelker D.R. Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit Toll-like receptor 2 and 4 signaling.J. Lipid Res. 2016; 57 (27095543): 993-100510.1194/jlr.M065201Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). For lipid antagonism of TLR4 activated by LPS, nearly all the analogs were as effective as POPG with the exception of those with 3- or 4-carbon aliphatic chains and lacking hydroxyl substituents. Two additional structural features of analogs, which ablated lipid antagonism of both TLR2 and TLR4 activation, were complete deletion of the phospholipid head group distal to the phosphodiester (which creates phosphatidate) and introduction of an amino group in lieu of a hydroxyl group in the head group aliphatic chain. Comparison of IC50 data for POPG and analog inhibition of TLR2 and TLR4 also demonstrated that inhibition of TLR2 requires about 10 times the lipid concentration required for antagonism of TLR4 (17Kandasamy P. Numata M. Zemski Berry K. Fickes R. Leslie C.C. Murphy R.C. Voelker D.R. Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit Toll-like receptor 2 and 4 signaling.J. Lipid Res. 2016; 57 (27095543): 993-100510.1194/jlr.M065201Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Such discrepancies in dose-response curves make it plausible to selectively inhibit TLR4 and spare TLR2 in vivo, by simply altering antagonist concentration. The inhibitory activity of POPG upon TLR4 activation and signaling prompted us to investigate whether the lipid could also disrupt unexpected inflammatory processes dependent on this receptor that were revealed by genetic studies in mice. Surprisingly, one of these processes was the in vivo inflammatory response to respiratory syncytial virus (RSV) (15Haynes L.M. Moore D.D. Kurt-Jones E.A. Finberg R.W. Anderson L.J. Tripp R.A. Involvement of toll-like receptor 4 in innate immunity to respiratory syncytial virus.J. Virol. 2001; 75 (11602714): 10730-1073710.1128/JVI.75.22.10730-10737.2001Crossref PubMed Scopus (404) Google Scholar, 19Imai Y. Kuba K. Neely G.G. Yaghubian-Malhami R. Perkmann T. van Loo G. 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RSV-elicited lung inflammation was an attractive target to interrogate for lipid antagonism for several reasons, including 1) nearly all children are infected with the virus before age 2, and it is the primary cause of newborn hospitalizations in the United States; 2) worldwide, the early life mortality because of RSV is very high in underdeveloped countries, and rivals that attributable to malaria (21Griffiths C. Drews S.J. Marchant D.J. Respiratory syncytial virus: Infection, detection, and new options for prevention and treatment.Clin. Microbiol. Rev. 2017; 30 (27903593): 277-31910.1128/CMR.00010-16Crossref PubMed Scopus (280) Google Scholar); 3) there is no vaccine for the virus, and durable immunity does not develop following childhood infection (22Graham B.S. Vaccine development for respiratory syncytial virus.Curr. Opin. Virol. 2017; 23 (28525878): 107-11210.1016/j.coviro.2017.03.012Crossref PubMed Scopus (105) Google Scholar); 4) the virus has been implicated as a significant cause of exacerbations of chronic lung diseases in adults, especially those with asthma and chronic obstructive pulmonary disease (COPD) (22Graham B.S. Vaccine development for respiratory syncytial virus.Curr. Opin. Virol. 2017; 23 (28525878): 107-11210.1016/j.coviro.2017.03.012Crossref PubMed Scopus (105) Google Scholar). Our initial studies with in vitro RSV infection using primary human bronchial epithelial cells and bronchial cell lines demonstrated the virus elicited robust production of the inflammatory mediators IL-6 and IL-8, and this process was markedly inhibited by POPG and PI, but not by the control lipid POPC (23Numata M. Chu H.W. Dakhama A. Voelker D.R. Pulmonary surfactant phosphatidylglycerol inhibits respiratory syncytial virus-induced inflammation and infection.Proc. Natl. Acad. Sci. 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