Mechanical ventilation, a critical life support in respiratory failure, can paradoxically cause ventilator-induced lung injury (VILI). While the mechanisms remain incompletely understood, neutrophil infiltration is central to the inflammatory process. Neutrophils employ various mechanisms to contribute to lung injury, including protease secretion (e.g., elastase), reactive oxygen species production, and release of pro-inflammatory cytokines. A recent focus has been on neutrophil extracellular traps (NETs), structures composed of DNA and granular proteins released by neutrophils. While NETs serve a crucial antimicrobial function, their release can also cause tissue damage. This study investigates the hypothesis that NET formation (NETosis) contributes to VILI.

Previous research has linked neutrophil accumulation in the lungs during injury (infection or aspiration) to worsening lung damage. Recent studies have highlighted the role of NETs in various pathologies. NETs, which trap and kill pathogens, have been implicated in tissue damage and autoimmune diseases. Their presence has been observed in transfusion-related acute lung injury (TRALI), and targeting NET components showed protective effects in a mouse model of TRALI. NETs have also been detected in human allergic asthma and mouse models of pneumonia. However, their role in VILI was unclear. NETosis, the process of NET formation, is a cell death pathway distinct from apoptosis or necrosis and involves reactive oxygen species-dependent and -independent pathways. Several known inducers of NETosis, including the cytokines IL-8 and IL-1β, and the damage-associated molecular pattern molecule HMGB1, are elevated in mechanical ventilation models, suggesting a potential link between NETs and VILI. This study aimed to clarify this link.

This study employed a two-hit mouse model to investigate NET formation in VILI. The model involved intratracheal lipopolysaccharide (LPS) instillation, followed by high tidal volume mechanical ventilation. Mice were randomized into groups receiving either LPS or phosphate-buffered saline (PBS) pretreatment, and either high tidal volume (HV+) or protective low tidal volume (LV) ventilation. The effects of DNase I treatment (to degrade extracellular DNA) and blockade of HMGB1 (with glycyrrhizin) or IL-1β (with anakinra) were also examined. Several parameters were assessed, including: * **Lung mechanics:** Static and dynamic compliance were measured using pressure-volume loops and perturbation methods. * **Bronchoalveolar lavage fluid (BALF) analysis:** BALF was collected for the quantification of protein, DNA (using Quant-iT Picogreen®), and cytokines (using Milliplex). * **NET markers:** Western blot analysis was used to quantify citrullinated histone-3 (Cit-H3), a key marker of NETosis, and myeloperoxidase in BALF. Immunofluorescence staining was performed to visualize Cit-H3 and DNA colocalization in lung tissue sections. * **Blood gas analysis:** Arterial blood gas measurements were taken to assess oxygenation and ventilation. * **Cell counts:** BALF cell counts (neutrophils, macrophages) were performed after differential staining. * **Morphometric analysis:** Hematoxylin and eosin staining and morphometric analysis of lung tissue sections were conducted to evaluate lung injury. Statistical analysis included Student's t-test, Mann-Whitney U test, two-way ANOVA, ANOVA on ranks, and Fisher's exact test, as appropriate.

The key findings of the study are: 1. **LPS alone induced neutrophil recruitment but not significant NET formation.** The combination of LPS pretreatment and high tidal volume ventilation was required for significant induction of NET markers (Cit-H3 and DNA) in BALF. 2. **High tidal volume ventilation significantly increased BALF DNA and Cit-H3 levels in LPS-treated mice.** This increase was accompanied by elevated levels of HMGB1 and IL-1β. 3. **Intratracheal DNase treatment effectively reduced BALF DNA and Cit-H3 levels.** DNase treatment improved lung mechanics (static compliance) without significantly affecting other measures of lung injury (BALF protein, blood gases). 4. **Blockade of HMGB1 or IL-1β did not prevent NETosis or protect against lung injury.** This suggests that other pathways might be involved in NETosis induction in this model. 5. Significant increases in several inflammatory cytokines (IL-1β, MCP-1, IL-6, and MIP-2) were observed only in the group receiving both LPS and high tidal volume ventilation. 6. Other cytokines (TNFα, KC, G-CSF) increased primarily in response to LPS and not to mechanical ventilation, suggesting a role in neutrophil recruitment but not necessarily NETosis initiation under the experimental conditions. 7. Morphometric analysis revealed no differences in tissue damage between vehicle and DNase-treated groups. 8. Although DNase treatment decreased levels of NET markers and improved lung compliance, it failed to improve other measures of lung injury, including oxygenation, BALF protein, and inflammation.

This study demonstrates that NETosis is induced in a two-hit model of VILI. The combination of LPS and high tidal volume ventilation was critical for NET formation, suggesting that a pre-existing inflammatory environment, along with the mechanical stress, is necessary to trigger this response. While DNase treatment effectively reduced NETs and improved lung compliance, other aspects of lung injury remained unaffected. This contrasts with findings in TRALI models, where targeting NETs offered broader protection. The lack of efficacy of HMGB1 and IL-1β blockade suggests that other mechanisms are implicated in NETosis in this model. The results indicate that NETs may primarily affect lung mechanics, rather than playing a dominant role in the overall VILI inflammatory response, particularly when existing inflammation is already present.

This research establishes a two-hit LPS/VILI model to study NETs in airways, demonstrating NET formation only during high tidal volume ventilation. Elevated BALF HMGB1 and IL-1β correlate with NET formation. DNase treatment successfully eliminated NETs and improved compliance but did not prevent airway inflammation, highlighting a potential, but limited, role for NETs in VILI pathogenesis. Future research should explore other signaling molecules or pathways that induce NET formation and determine whether strategies targeting these alternative pathways might mitigate VILI.

The study used a mouse model, which may not fully replicate human VILI. The LPS pretreatment may introduce confounding variables affecting the interpretation of results. The time frame of the study (6 hours) might not capture the full extent of long-term effects of NETs on lung injury. The use of a single DNase dose limits the conclusions about its therapeutic potential. The study did not analyze the exact mechanism of how mechanical ventilation triggers NET formation.