Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14+ Monocytes

PLOS ONE, Dec 2019

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. Conclusion 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.

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Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14+ Monocytes

January Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14+ Monocytes Kirsten Glaser 0 1 2 Markus Fehrholz 0 1 2 Tore Curstedt 0 1 2 Steffen Kunzmann 0 1 2 Christian P. Speer 0 1 2 0 1 University Children ́s Hospital, University of Würzburg , Würzburg, Germany , 2 Department of Molecular Medicine and Surgery, Karolinska Institutet at Karolinska University Hospital , Stockholm , Sweden 1 Competing Interests: CP Speer has a consultancy agreement with Chiesi Farmaceutici S.p.A. (Parma, Italy). This project was supported by Chiesi Farmaceutici S.p.A. , supplying CHF5633, its synthetic components and Poractant alfa 2 Editor: Umberto Simeoni, Centre Hospitalier Universitaire Vaudois , FRANCE - OPEN ACCESS Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors gratefully acknowledge the generous financial support of this project by Georg Bierich, Duesseldorf, Germany. 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 Results Highly purified adult CD14+ cells, either native or simultaneously stimulated with LPS, were exposed to CHF5633, its components, or poractant alfa (Curosurf1). 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. 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 (Curosurf1). This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials. 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). LPSinduced IL-8 and IL-10 mRNA and protein expression were unaffected by CHF5633. For all cytokines, the observed CHF5633 effects paralleled a Curosurf1-induced modulation of cytokine response. TLR2 and TLR4 mRNA and protein expression were not affected by CHF5633 and Curosurf1, neither in native nor in LPS-stimulated adult monocytes. Conclusion 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. Introduction Surfactant replacement therapy using exogenous surfactant preparations derived from bovine or porcine lungs has significantly improved the outcome in respiratory distress syndrome (RDS) in preterm infants and has become the standard of care for the prevention and treatment of RDS [ 1–8 ]. Pulmonary surfactant prevents alveolar collapse and improves lung compliance by significant reduction of surface tension at low lung volumes or minimal alveolar size–both conditions particulary prone to collapse according to Laplace´s tidal equations [ 9– 11 ]. It promotes gas exchange, thus, allowing for rapid de-escalation of ventilation strategy and the use of lower concentrations of oxygen [12]. A number of animal-derived surfactants are available, isolated mainly from porcine or bovine lungs [ 6,8 ]. These preparations containing variable amounts of lipids, mainly dipalmitoylphosphatidylcholine (DPPC), and residual hydrophobic surfactant proteins (SP)-B and SP-C, are very effective, but supplies are limited. Development and introduction of new generation synthetic surfactants is a promising approach to further improve surfactant replacement therapy and to widen indications [ 8,13– 17 ]. In contrast to natural surfactants, synthetic surfactants may have standardized composition, increased resistance against inactivatio (...truncated)


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Kirsten Glaser, Markus Fehrholz, Tore Curstedt, Steffen Kunzmann, Christian P. Speer. Effects of the New Generation Synthetic Reconstituted Surfactant CHF5633 on Pro- and Anti-Inflammatory Cytokine Expression in Native and LPS-Stimulated Adult CD14+ Monocytes, PLOS ONE, 2016, Volume 11, Issue 1, DOI: 10.1371/journal.pone.0146898