Characterization of phospholipid-modified lung surfactant in vitro and in a neonatal ARDS model reveals anti-inflammatory potential and surfactant lipidome signatures.

A strong inflammatory immune response drives the lung pathology in neonatal acute respiratory distress syndrome (nARDS). Anti-inflammatory therapy is therefore a promising strategy for improved treatment of nARDS. We demonstrate a new function of the anionic phospholipids POPG, DOPG, and PIP2 as inhibitors of IL-1ß release by LPS and ATP-induced inflammasome activation in human monocyte-derived and lung macrophages. Curosurf® surfactant was enriched with POPG, DOPG, PIP2 and the head-group derivative IP3, biophysically characterized and applicability was evaluated in a piglet model of nARDS. The composition of pulmonary surfactant from piglets was determined by shotgun lipidomics screens. After 72 h of nARDS, levels of POPG, DOPG, and PIP2 were enhanced in the respective treatment groups. Otherwise, we did not observe changes of individual lipid species in any of the groups. Surfactant proteins were not affected, with the exception of the IP3 treated group. Our data show that POPG, DOPG, and PIP2 are potent inhibitors of inflammasome activation; their enrichment in a surfactant preparation did not induce any negative effects on lipid profile and reduced biophysical function in vitro was mainly observed for PIP2. These results encourage to rethink the current strategies of improving surfactant preparations by inclusion of anionic lipids as potent anti-inflammatory immune regulators.

Novel therapeutic roles for surfactant-inositols and -phosphatidylglycerols in a neonatal piglet ARDS model: a translational study.

The biological and immune-protective properties of surfactant-derived phospholipids and phospholipid subfractions in the context of neonatal inflammatory lung disease are widely unknown. Using a porcine neonatal triple-hit acute respiratory distress syndrome (ARDS) model (repeated airway lavage, overventilation, and LPS instillation into airways), we assessed whether the supplementation of surfactant (S; poractant alfa) with inositol derivatives [inositol 1,2,6-trisphosphate (IP3) or phosphatidylinositol 3,5-bisphosphate (PIP2)] or phosphatidylglycerol subfractions [16:0/18:1-palmitoyloleoyl-phosphatidylglycerol (POPG) or 18:1/18:1-dioleoyl-phosphatidylglycerol (DOPG)] would result in improved clinical parameters and sought to characterize changes in key inflammatory pathways behind these improvements. Within 72 h of mechanical ventilation, the oxygenation index (S+IP3, S+PIP2, and S+POPG), the ventilation efficiency index (S+IP3 and S+POPG), the compliance (S+IP3 and S+POPG) and resistance (S+POPG) of the respiratory system, and the extravascular lung water index (S+IP3 and S+POPG) significantly improved compared with S treatment alone. The inositol derivatives (mainly S+IP3) exerted their actions by suppressing acid sphingomyelinase activity and dependent ceramide production, linked with the suppression of the inflammasome nucleotide-binding domain, leucine-rich repeat-containing protein-3 (NLRP3)-apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)-caspase-1 complex, and the profibrotic response represented by the cytokines transforming growth factor-ß1 and IFN-γ, matrix metalloproteinase (MMP)-1/8, and elastin. In addition, IκB kinase activity was significantly reduced. S+POPG and S+DOPG treatment inhibited polymorphonuclear leukocyte activity (MMP-8 and myeloperoxidase) and the production of interleukin-6, maintained alveolar-capillary barrier functions, and reduced alveolar epithelial cell apoptosis, all of which resulted in reduced pulmonary edema. S+DOPG also limited the profibrotic response. We conclude that highly concentrated inositol derivatives and phosphatidylglycerol subfractions in surfactant preparations mitigate key inflammatory pathways in inflammatory lung disease and that their clinical application may be of interest for future treatment of the acute exudative phase of neonatal ARDS.

Lipopolysaccharide-binding protein is bound and internalized by host cells and colocalizes with LPS in the cytoplasm: Implications for a role of LBP in intracellular LPS-signaling.

The lipopolysaccharide-binding protein (LBP) is critically involved in innate immune responses to Gram-negative infections. We show here that human peripheral blood-derived monocytes, but not lymphocytes, stain positive for endogenous LBP on the cell surface. Studies on human macrophages demonstrate LBP binding at normal serum concentrations of 1-10 µg/ml. Binding was increased in a concentration-dependent manner by lipopolysaccharide (LPS). Fluorescence quenching experiments and confocal microscopy revealed constitutive and LPS-induced internalization of LBP by macrophages. Experiments with macrophages and HEK293 cell lines showed that binding and uptake of LBP do not depend on the LPS receptors CD14 and TLR4/MD-2. Fractionation of Triton X-100 solubilized cytoplasmic membranes revealed that LBP was primarily localized in non-raft domains under resting conditions. Cellular LPS stimulation elevated LBP levels and induced enrichment in fractions marking the transition between non-raft and raft domains. LBP was found to colocalize with LPS at the cytoplasmic membrane and in intracellular compartments of macrophages. In macrophages stimulated with LPS and ATP for inflammasome activation, LBP was observed in close vicinity to activated caspases. Furthermore, LBP conferred IL-1ß production by LPS in the absence of ATP. These data establish that LBP serves not only as an extracellular LPS shuttle but in addition facilitates intracellular transport of LPS. This observation adds a new function to this central immune regulator of LPS biology and raises the possibility for a role of LBP in the delivery of LPS to TLR4-independent intracellular receptors.