Discussion
This study marks the first application of organ-on-a-chip and organoid technologies to create a human-derived, more physiologically relevant, and functionally diverse model for fire-related smoke inhalation acute lung injury (SI-ALI). Through the integration of advanced methodologies, deeper insights into the pathophysiological mechanisms of fire-related SI-ALI have been achieved, along with the identification of potential therapeutic agents. This research provides a novel and robust platform for advancing both the study and treatment of fire-related SI-ALI.
Previous research on lung injury from fire-related smoke inhalation primarily used wood combustion smoke, which posed limitations due to the lack of control over combustion processes and unclear smoke concentrations, often relying on subjective estimates, thus compromising model stability[19]. Additionally, the composition of natural wood differs significantly from modern construction materials, making traditional data inadequate for contemporary fire-related SI-ALI studies[20]. By controlling material proportions and combustion processes, a system was developed to simulate modern non-metallic material combustion accurately (Figure 2B, Supplementary Figure 2C and Supplementary Text 1). Fourier-transform infrared spectroscopy confirmed the system’s stability, providing a foundation for a reproducible SI-ALI model.
To create a more physiologically relevant SI-ALI model, organ-on-a-chip and organoid technologies were integrated. However, traditional lung chip models had limitations[21], such as complex perfusion pumps prone to bubble formation and contamination, as well as limited cell seeding capacity. To address these issues, an optimized lung chip model was developed with a gravity-driven, pump-less system to reduce contamination risks and improve practicality[22]. The expanded cell seeding area allowed for three parallel experiments within a single chip (details in the methodology section), increasing efficiency and enabling advanced molecular research. The chip design included a non-overlapping area between the vascular and alveolar layers, allowing real-time observation of capillary leakage and other pathological processes (Figures 1B and 2A).
A co-culture system of human alveolar epithelial and endothelial cells was established on the chip, simulating the alveolar-capillary barrier (Figure 3F). Over time, increased transepithelial resistance indicated tight junction formation (Supplementary Figure 1B). Confocal microscopy confirmed the bilayer structure, demonstrating successful barrier formation (Supplementary Figures 1C-D). This model allowed for the direct introduction of smoke into the alveolar chamber, simulating fire-related lung damage (Figure 2C). The chip technology bypassed the need for gases to dissolve in an aqueous solution, providing a more accurate replication of smoke damage to the alveolar epithelium and allowing for comprehensive analysis of intercellular interactions during injury[23].
The chip’s response to prolonged toxic smoke exposure revealed oxidative stress and apoptosis, consistent with clinical findings[24-26]. Electron microscopy showed significant subcellular abnormalities, such as dense mitochondria and altered endoplasmic reticulum structures. Increased macrophage adhesion and chemotaxis were observed, aligning with clinical immune cell infiltration findings. The non-overlapping chip areas enabled real-time observation of alveolar-capillary leakage, challenging to replicate in traditional experiments. ELISA analysis indicated a substantial release of inflammatory cytokines following smoke exposure, independent of immune cell presence (Figures 3 and 4).
The advanced chip design allowed for separate extraction of endothelial and epithelial cells for molecular analysis. Despite direct smoke stimulation of epithelial cells, qPCR analysis showed a similar response in endothelial cells, with upregulated inflammation and apoptosis-related genes, highlighting intercellular signaling during smoke exposure. This interaction, overlooked in previous single-cell models, warrants further investigation in fire-related SI-ALI (Figure 4).
To further elucidate the mechanisms of fire-related SI-ALI, a proteomic analysis was conducted to identify differentially expressed proteins (DEPs) post-smoke exposure in the chip model. KEGG enrichment analysis indicated these DEPs are involved in critical pathways such as cysteine and methionine metabolism, amino acid biosynthesis, mineral absorption, carbon metabolism, lysosomal function, and spliceosomal activity (Figure 5B and Supplementary Text 2). Notably, cysteine and methionine metabolism, linked to sepsis-related lung damage, and amino acid biosynthesis, associated with ferroptosis and drug efficacy in sepsis-induced lung injury, were emphasized[27, 28]. The role of mineral absorption, pertinent to injuries caused by particulate inhalation (e.g., graphene, asbestos), aligns with the smoke’s solid particle composition[29, 30]. The enrichment of PSAT1 in carbon metabolism suggests novel research avenues for SI-ALI[31]. Autophagy’s role, especially in lysosomal function, is highlighted in sepsis, COPD, and COVID-19 contexts[32-34], while the underexplored spliceosome’s involvement is supported by its association with phosgene-induced lung injury[35], reinforcing the relevance of the identified pathways.
Gene Ontology Biological Process (GOBP) analysis revealed pathways including lymphocyte aggregation, CAMKK-AMPK signaling, mitochondrial protein processing, DNA methylation maintenance, and L-serine biosynthesis. Lymphocyte aggregation, linked to the inflammatory cascade in acute lung injury[36, 37]and CAMKK-AMPK signaling, associated with smoking-related lung cancer[38], were observed. Mitochondrial damage, linked to ferroptosis[39], aligns with the damage seen in smoke-exposed cells (Figure 3E), consistent with proteomic findings. The maintenance of DNA methylation is crucial for normal lung function, with its disruption noted in COPD[40]. Serine’s role in nucleotide synthesis and antioxidant defense was noted[41, 42], while the hypermethylation of CpG islands, particularly the IFN-γ promoter via the PI3K-Akt-DNMT3b pathway, was linked to lung inflammation[43]. The depalmitoylation pathway remains underexplored in lung injury, but our analysis suggests its relevance, highlighting the complex pathology mirrored in clinical presentations.
To compare in vivo and chip models, a mouse model of fire-related SI-ALI was established. Histopathological analysis revealed significant lung damage (Supplementary Figure 2A and 2B), consistent with oxidative stress and cellular damage findings, aligning with the results of previous research[44, 45] and the chip model. Proteomic analysis showed more DEPs in the animal model, likely due to tissue complexity. However, both models shared enrichment in carbon metabolism and spliceosomal pathways, with common pathways including SRP-dependent co-translational protein targeting and small GTPase-mediated signaling, associated with metabolism, ROS, and apoptosis. The observed differences are attributed to species-specific variations but underscore the models’ collective reflection of SI-ALI pathology, providing insights into underlying mechanisms (Supplementary Text 3).
Subsequent focus was on molecules with significant fold-change in the chip model, specifically DOCK2, TST, NIT1, RAP1GDS1, and KRR1. These molecules were investigated for endothelial and epithelial cell responses to smoke exposure (Figure 5D and Figure 6A). TST, involved in enhancing mitochondrial function and reducing ROS[46], and NIT1, responsible for hydrolyzing deaminated glutathione, have been underexplored in lung injury[47]. RAP1GDS1, linked to lung carcinogenesis[48, 49], and DOCK2, with anti-inflammatory properties, suggest potential as SI-ALI markers and therapeutic targets[50, 51]. These findings highlight critical molecules relevant to fire-related SI-ALI, warranting further investigation.
In the combined proteomic analysis of animal and chip models, only COMT consistently showed an upward expression trend, observed at both transcriptional and translational levels in endothelial and epithelial cells (Figure 5C-D and Figure 6A). COMT, involved in catecholamine metabolism, may reduce oxidative stress from polycyclic aromatic hydrocarbons (PAHs) in A549 cells[52]and regulate lung water and sodium storage[53]. However, its role in smoke-induced acute lung injury (SI-ALI) remains largely unexplored. Our findings suggest that COMT could be crucial in the pathology of fire-related SI-ALI.
To identify potential therapies, a computational analysis of COMT’s structure was performed[54]. Screening against the FDA-approved drug library revealed compounds like Ractopamine HCl, Bimatoprost, and Fenoterol Hydrobromide (Figure 6B and Supplementary Text 4). These drugs, targeting 5-HT, adrenergic, and prostaglandin receptors, are mainly used for glaucoma and respiratory conditions, yet their relevance to lung injury is unexamined. Our study indicates they may offer therapeutic potential in fire-related SI-ALI, although further validation is needed.
The chip model’s drug screening ability was further explored for SI-ALI treatment development. Severe oxidative stress and inflammation following smoke exposure were consistent with clinical observations. To address these, intravenous vitamin C and nebulized budesonide were tested, significantly reducing oxidative stress and inflammation (Figure 7).
Vitamin C is known for its antioxidant and immune-modulating effects, with high doses shown to mitigate organ damage in sepsis[55], suggesting it may benefit SI-ALI treatment. However, the absence of clinical trials leaves no established guidelines. Glucocorticoids, despite their anti-inflammatory properties, pose risks like electrolyte imbalances and immunosuppression[56]. Budesonide, when nebulized, may minimize systemic side effects, offering potential for fire-related SI-ALI[57], though clinical evidence remains limited.
This study indicates that the combined therapy holds promise, providing a foundation for clinical use while underscoring the need for further research. The chip model’s advanced features enabled evaluation of different administration routes, which traditional models struggle to achieve. This reinforces the chip’s value in preclinical drug testing and new treatment development for SI-ALI.
Given the superior tissue specificity of human-derived lung organoids compared to cell lines[58], ethical approval was obtained to culture lung organoids (Figure 8A-C). A lung organoid chip was then developed, and smoke exposure produced similar damage responses to those seen in cell line-derived chips (Figure 8D), confirming model stability. Analysis of key molecules in the organoid model showed transcriptional trends consistent with previous findings, except for KRR1 (Figure 8E). The therapeutic effects of vitamin C and budesonide were validated, showing mitigation of smoke-induced inflammatory gene expression. These results further corroborate our findings using the organoid chip model.