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How Cigarette Smoke and Neutrophil Extracellular Traps Contribute to Chronic Airway Inflammation in COPD

/ by Lifeline® Cell Technology

Lung conditions such as chronic obstructive pulmonary disease (COPD), emphysema, and asthma affect millions of people worldwide. Exposure to air pollutants can damage the airways, promote inflammation, and contribute to the development or worsening of respiratory diseases. For people living with chronic lung conditions, repeated or prolonged exposure can further exacerbate airway inflammation and severely impact lung health.

For example, cigarette smoke (CS) is a major risk factor for COPD and is strongly linked to airway inflammation. While short-term exposure can trigger acute inflammatory responses, inflammation in COPD can persist even after an individual stops smoking. In recent years, research has implicated neutrophil extracellular traps (NETs) as important contributors to COPD-associated inflammation and disease severity. A recent study investigated how the DNA component of NETs contributes to persistent airway inflammation and look at whether targeting NETs and associated signaling pathways could represent a potential therapeutic strategy for COPD.

DNA of NETs Promotes NF-κB-Dependent Autoimmunity in COPD

In this study, Chen and colleagues investigated how the immunoregulatory effects of NETs-DNA on airway epithelial cells (AECs) contribute to long-term CS-induced COPD, which remain poorly understood. NETs are web-like structures composed of DNA, histones, and cytotoxic enzymes released by activated neutrophils in response to pathogens. However, excessive NETs formation (NETosis) can contribute to tissue damage and chronic inflammation through their effects on AECs and dendritic cells (DCs).

The researchers found that cigarette smoke extract (CSE) induced NETosis in human neutrophils in a dose- and time-dependent manner as quantified by immunofluorescence. Specifically, neutrophils derived from patients with COPD exhibited a greater capacity to release CSE-induced NETs, which contained high levels of oxidatively damaged mitochondrial DNA (mtDNA) and chromosomal DNA (chDNA), compared with neutrophils isolated from healthy individuals.

Next, they evaluated the effects of NETs on human airway cells, using primary hAECs obtained from Lifeline® Cell Technology maintained in BronchiaLife™ Medium. These cells were treated with 6, 12, or 24 μg/mL of NETs for 48 or 72 hours. CSE-induced NETs stimulated hAEC proliferation and increased the production of NF-κB-dependent cytokines (CXCL5, IL-8, TNF-α, and IL-1β), as well as type I interferons (IFNs), including IFN-β1 and IL-12. NETs also promoted the maturation of hDCs.

Given the high levels of damaged mtDNA and chDNA in CSE-induced NETs, the researchers next investigated how NETs-DNA is detected by hAECs. They focused on two DNA-sensing pathways, cyclic GMP-AMP synthase (cGAS), and Toll-like receptor 9 (TLR9), which can activate downstream inflammatory signaling in response to aberrant or extracellular DNA. siRNA silencing of both cGAS and TLR9 reduced NETs-mediated hAECs proliferation  and inhibited NETs-induced NF-κB (P65 and P50) activation and the production of NF-κB-dependent cytokines, suggesting that NETs-DNA modulate airway inflammation through cGAS/TLR9 pathways.

The researchers followed up with in vivo knockout mouse experiments to confirm these findings. Compared with CS-exposed control mice, cGAS knockout mice exhibited reduced production of NF-κB-dependent cytokines, decreased airway inflammation and NET infiltration, and improved lung function, while type I IFN production was not significantly affected. Similar findings were observed in TLR9 knockout mice, suggesting that both DNA-sensing pathways participate in NET-mediated inflammatory responses.

To further investigate the contribution of NETosis to airway inflammation, the researchers treated a long-term CS-induced COPD mouse model with mitoTEMPO, an inhibitor of mitochondrial reactive oxygen species (mtROS) that suppresses CSE-induced NETosis. Inhibition of NETosis reduced airway inflammation, NET infiltration, and the production of NF-κB-dependent cytokines (but not type I IFNs). The researchers also found that degradation of NETs-DNA using DNase I reduced NET infiltration and emphysema-related changes in the COPD mouse model. Together, these in vivo findings show that inhibition of NETosis, blocking NETs-DNA sensing, or degrading NETs-DNA can reduce several features of CSE-induced airway inflammation.

Finally, the researchers examined bronchoalveolar lavage fluid (BALF) from COPD patients. NET-associated components, including myeloperoxidase (MPO) and neutrophil elastase activity, were elevated in COPD smokers and correlated with disease severity and NF-κB-dependent cytokine levels, but not with type I IFN levels. In particular, MPO levels correlated with IL-1β and CXCL8, further linking NETosis with NF-κB-dependent inflammatory signaling in COPD.

Taken together, the results from this study suggest that DNA components within CSE-induced NETs promote NF-κB-dependent autoimmunity through cGAS/TLR9 signaling in long-term CS-induced COPD. The study also suggests that targeting the sensing-blockage/inhibition/degradation of NETs-DNA could be potential areas for therapeutic intervention to treat COPD.

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