title: Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14+ Monocytes.
abstract: BACKGROUND
  Surfactant replacement therapy is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome. New generation synthetic surfactants represent a promising alternative to animal-derived surfactants. CHF5633, a new generation reconstituted synthetic surfactant containing SP-B and SP-C analogs and two synthetic phospholipids has demonstrated biophysical effectiveness in vitro and in vivo. While several surfactant preparations have previously been ascribed immunomodulatory capacities, in vitro data on immunomodulation by CHF5633 are limited, so far. Our study aimed to investigate pro- and anti-inflammatory effects of CHF5633 on native and LPS-stimulated human adult monocytes.
  
  
  METHODS
  Highly purified adult CD14+ cells, either native or simultaneously stimulated with LPS, were exposed to CHF5633, its components, or poractant alfa (Curosurf®). Subsequent expression of TNF-α, IL-1β, IL-8 and IL-10 mRNA was quantified by real-time quantitative PCR, corresponding intracellular cytokine synthesis was analyzed by flow cytometry. Potential effects on TLR2 and TLR4 mRNA and protein expression were monitored by qPCR and flow cytometry.
  
  
  RESULTS
  Neither CHF5633 nor any of its components induced inflammation or apoptosis in native adult CD14+ monocytes. Moreover, LPS-induced pro-inflammatory responses were not aggravated by simultaneous exposure of monocytes to CHF5633 or its components. In LPS-stimulated monocytes, exposure to CHF5633 led to a significant decrease in TNF-α mRNA (0.57 ± 0.23-fold, p = 0.043 at 4h; 0.56 ± 0.27-fold, p = 0.042 at 14h). Reduction of LPS-induced IL-1β mRNA expression was not significant (0.73 ± 0.16, p = 0.17 at 4h). LPS-induced IL-8 and IL-10 mRNA and protein expression were unaffected by CHF5633. For all cytokines, the observed CHF5633 effects paralleled a Curosurf®-induced modulation of cytokine response. TLR2 and TLR4 mRNA and protein expression were not affected by CHF5633 and Curosurf®, neither in native nor in LPS-stimulated adult monocytes.
  
  
  CONCLUSIONS
  The new generation reconstituted synthetic surfactant CHF5633 was tested for potential immunomodulation on native and LPS-activated adult human monocytes. Our data confirm that CHF5633 does not exert unintended pro-inflammatory effects in both settings. On the contrary, CHF5633 significantly suppressed TNF-α mRNA expression in LPS-stimulated adult monocytes, indicating potential anti-inflammatory effects.
authors.affiliation: University Children´s Hospital, University of Würzburg, Würzburg, Germany.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Glaser
authors.affiliation: University Children´s Hospital, University of Würzburg, Würzburg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Fehrholz
authors.affiliation: Department of Molecular Medicine and Surgery, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden.
authors.forename: Tore
authors.initials: T
authors.lastname: Curstedt
authors.affiliation: University Children´s Hospital, University of Würzburg, Würzburg, Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Kunzmann
authors.affiliation: University Children´s Hospital, University of Würzburg, Würzburg, Germany.
authors.forename: Christian P
authors.initials: CP
authors.lastname: Speer
mesh_terms: D000328:Adult; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000633:toxicity*; D008297:Male; D009000:Monocytes / Q000378:metabolism*; D015846:Monokines / Q000096:biosynthesis*; D010446:Peptide Fragments / Q000494:pharmacology*; D010713:Phosphatidylcholines / Q000494:pharmacology*; D037701:Pulmonary Surfactant-Associated Protein B / Q000494:pharmacology*; D037721:Pulmonary Surfactant-Associated Protein C / Q000494:pharmacology*; D051195:Toll-Like Receptor 2 / Q000096:biosynthesis; D051197:Toll-Like Receptor 4 / Q000096:biosynthesis
chemical_list: D000893:Anti-Inflammatory Agents; C000592408:CHF5633; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D015846:Monokines; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D037701:Pulmonary Surfactant-Associated Protein B; D037721:Pulmonary Surfactant-Associated Protein C; C495342:TLR2 protein, human; C495345:TLR4 protein, human; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4