Compatible solutes prevent lung inflammation and loss of CFTR induced by combustion-derived nanoparticles in human and rodent experimental systems.
Description
The compatible solute ectoine is known to attenuate inflammatory effects in the airways after exposure to combustion-derived nanoparticles. Proinflammatory signaling in epithelial cells as well as antiapoptotic mechanisms in neutrophilic granulocytes, both triggered by particles, are reduced by this substance. Here we investigated the preventive potential on airway inflammation of additional substances originating from the ectoine metabolism, NADA and 5-hydroxyectoine, in a mouse model and in human neutrophilic granulocytes. Furthermore, effects of these substances on the loss of CFTR, as an additional pathogenic endpoint of nanoparticle exposure was investigated. All three substances exhibited beneficial effects at the level of inflammatory cells in lung lavages from exposed mice. The loss of CFTR in lung tissue of exposed mice was mitigated by the substances. In primary human neutrophils and in neutrophilic differentiated HL-60 cells the reduction of apoptosis rates after particle exposure was effectively abolished. The loss of CFTR from cytoplasmic membrane in neutrophilic cells was also counteracted by the substances. The data identify both NADA and 5-hydroxyectoine as additional substances for molecular prevention of airway effects of environmental particles. Furthermore, the reduction of CFTR loss might be a relevant finding for patients suffering from impaired function of this ion channel.
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Steps to reproduce
Carbon nanoparticles (carbon black, Printex 90) were purchased from Degussa, Germany. Carbon nanoparticle suspension was made in PBS by sonication for 15 min, working concentration was 33 µg/ml. Compatible solutes ectoin, hydroxyectoin and NADA were provided by bitop AG (Dortmund, Germany). Solutes were dissolved in PBS, working concentration of each solute was 1mM. Human peripheral blood neutrophils were isolated from venous blood by discontinuous Percoll density gradient. The human myeloid leukemia HL-60 cell line (wild type CCL-240) was from ATCC. Differentiation into a neutrophil-like state was induced by incubation of HL-60 cells with 1 µM all-trans retinoic acid (ATRA, Sigma) and 1% DMSO (Roth) for 5 days. Apoptosis in human neutrophils and HL-60 cells was estimated according to Nicoletti protocol by direct DNA staining in propidium iodide (Sigma) hypotonic solution and flow cytometry (as %hypodiploidy or %sub-G1). Cell surface and intracellular CFTR staining in HL-60 cells were carried out using a fluorescently conjugated human antibody CFTR-APC (#NBP2-54509 Novus Biologicals) and flow cytometry. Intracellular CFTR staining was validated using a monoclonal unconjugated CFTR antibody (#ab660 Cystic Fibrosis Foundation Therapeutics – CFTR Antibody Distribution program and flow cytometry. For animal experiments female C57BL/6JRj mice (from Janvier Labs, Le Genet St. Isle, France) were subjected to pharyngeal aspiration of particle suspensions or mock controls (PBS) in the absence or presence of compatible solutes. 12h after exposure immune cell identification in broncho-alveolar lavage samples, as parameter of neutrophil lung inflammation, was carried out using specific fluorescently conjugated antibodies GR-1-PE (#108407 BioLegend) and CD11c-APC (#117309 BioLegend) and flow cytometry analysis. The percentages of CD11c+ cells representing monocytes/macrophages, GR-1+ cells representing neutrophils, and CD11c- GR-1- cells representing lymphocytes were estimated by analysis of the respective dot plot (quadrant statistic). Additionally, CFTR expression in murine lung homogenates was analyzed using CFTR antibody (#sc-376683 Santa Cruz) and Western blot technique.
Institutions
- Leibniz-Institut fur umweltmedizinische Forschung an der Heinrich-Heine-Universitat Dusseldorf gGmbH