Neutrophil extracellular traps are present in the airways of ENaC-overexpressing mice with cystic fibrosis-like lung disease

Abstract

Background Neutrophils are key components of the exacerbated inflammation and tissue damage in cystic fibrosis (CF) airways. Neutrophil extracellular traps (NETs) trap and kill extracellular pathogens. While NETs are abundant in the airways of CF patients and have been hypothesized to contribute to lung damage in CF, the in vivo role of NETs remains controversial, partially due to lack of appropriate animal models. The goal of this study was to detect NETs and to further characterize neutrophil-mediated inflammation in the airways of mice overexpressing the epithelial sodium channel (βENaC-Tg mice on C57BL/6 background) in their lung with CF-like airway disease, in the absence of any apparent bacterial infections. Methods Histology scoring of lung tissues, flow cytometry, multiplex ELISA, immunohistochemistry and immunofluorescence were used to characterize NETs and the airway environment in uninfected, βENaC-Tg mice at 6 and 8 weeks of age, the most chronic time points so far studied in this model. Results Excessive neutrophilic infiltration characterized the lungs of uninfected, βENaC-Tg mice at 6 and 8 weeks of age. The bronchoalveolar lavage fluid (BALF) of βENaC-Tg mice contains increased levels of CF-associated cytokines and chemokines: KC, MIP-1α/β, MCP-1, G-CSF, IL-5, and IL-6. The BALF of βENaC-Tg mice contain MPO-DNA complexes, indicative of the presence of NETs. Immunofluorescence and flow cytometry of BALF neutrophils and lung tissues demonstrated increased histone citrullination, a NET-specific marker, in βENaC-Tg mice. Conclusions NETs are detected in the airways of βENaC-Tg mice, in the absence of bacterial infections. These data demonstrate the usefulness of the βENaC-Tg mouse to serve as a model for studying the role of NETs in chronic CF airway inflammation.

Myeloperoxidase and Other Markers of Neutrophil Activation Associate With Malaria and Malaria/HIV Coinfection in the Human Placenta

“…NETs in tissue samples have similarly been shown as extracellular DNA colocalized with neutrophil-derived proteins ( 49 ). NET remnants such as DNA and neutrophil-derived protein complexes (MPO-DNA; neutrophil elastase (NE)-DNA) and citrullinated histones can be determined by ELISA in fluid samples ( 50 , 51 ) or detected by flow cytometry ( 52 , 53 ) as indicators of NETosis.…”

Noscapine Acts as a Protease Inhibitor of In Vitro Elastase-Induced Collagen Deposition in Equine Endometrium

“…Hepatic [ 47 ], cardiac [ 48 ] and renal [ 49 ] fibrosis was also linked to NETs persistence. Recently, NETs were found in the lungs of a cystic fibrosis mouse model in the absence of bacterial infection [ 50 ].…”

RGS10 Reduces Lethal Influenza Infection and Associated Lung Inflammation in Mice

“…MPO-DNA complexes were detected in the BALF of infected WT and Rgs10-/- mice following a published protocol ( 26 , 27 ) adapted to murine samples ( 28 ). BALFs were diluted 1:25 in PBS and were subsequently added to a high-binding 96-well ELISA microplate pretreated overnight at 4 °C with capture anti-MPO (1:200 RD Systems, Cat#: AF3667).…”

“…Because of the abundant neutrophil infiltrates in infected airways of Rgs10-/-mice, we explored whether soluble markers of NETs are also increased in the lungs of PR8-infected Rgs10-/-mice. Cell-free BALF supernatants were collected from WT and Rgs10-/-mice at 0 and 5 dpi and the presence of NET-specific MPO-DNA complexes was assessed as previously (26)(27)(28). While there was no significant difference in the BALF MPO-DNA complexes level between WT and Rgs10-/-mice at 0 dpi, we found that there was a significant, 4-fold increase in the absorbance values in the BALFs of infected Rgs10-/-mice compared to their WT littermate controls (Figure 6A), indicating the enhanced release of NETs in infected airways of these animals.…”