Diesel particulate matter induces dose-dependent cytotoxicity, multi-faceted inflammation, MAPK activation, and oxidative stress in RAW 264.7 macrophages: an integrated transcriptomic and protein–protein interaction network analysis
Beyond classical inflammation, these integrated findings reveal coordinated network-level perturbations consistent with ferroptosis vulnerability, genome instability, paradoxical immune dysregulation, and potential macrophage identity loss consistent with ferroptosis vulnerability, genome instability, paradoxical immune dysregulation, and potential macrophage identity loss.
Abstract
Diesel particulate matter (DPM), a Group 1 carcinogen classified by the International Agency for Research on Cancer, is a major component of traffic-related air pollution. Although macrophages are central to the response to inhaled particles, the integrated network-level cellular response to DPM remains incompletely characterized. A combined three analytical methods was applied to RAW 264.7 macrophages exposed to DPM (NIST SRM2975) for 24 h: (i) dose–response phenotyping across twelve CCK-8 concentrations (0–750 μg/mL) and multi-modal characterization (Western blot, RT-qPCR, Griess assay, DCFH-DA ROS imaging, TNF-α ELISA) at sub-cytotoxic to near-IC50 doses; (ii) 3′ mRNA-sequencing at 100 μg/mL with edgeR-based differential expression (|fold change|≥ 1.5) and Gene Ontology, KEGG, and Reactome enrichment; and (iii) STRING-based protein–protein interaction (PPI) network analysis with Markov Cluster (MCL) modular clustering in Cytoscape. DPM reduced viability dose-dependently (IC50 ≈ 135–140 μg/mL) and induced concerted activation of iNOS, COX-2, MAPK phosphorylation, pro-inflammatory cytokines (TNF-α, IL-1β), antioxidant enzymes (SOD2, HO-1, Catalase), nitrite, ROS, and secreted TNF-α. Transcriptomics identified 750 differentially expressed genes (331 up; 419 down), with enrichment in cytokine production, NF-κB/MAPK signaling, inflammasome, and oxidative stress. PPI analysis resolved ten up- and ten down-regulated functional modules, with key hubs Ccl2, Gpx4, Ifit3/Stat1, Dna2/Rad52, and Csf1r. Beyond classical inflammation, these integrated findings reveal coordinated network-level perturbations consistent with ferroptosis vulnerability, genome instability, paradoxical immune dysregulation, and potential macrophage identity loss — hypothesis-generating axes for future mechanistic validation.
Ambient fine particulate matter (PM2.5) is a chemically complex mixture whose health impacts are not fully captured by particle mass. Here, we developed an interpretable chemotranscriptomic framework to attribute PM2.5-induced molecular perturbations to toxicity-relevant components. PM2.5 collected from urban roadside and coastal environments was separated into whole, extractable, and unextractable fractions, characterized by LC/GC × GC-HRMS-based nontarget analysis and inductively coupled plasma mass spectrometry (ICP-MS), and evaluated using cytotoxicity testing and transcriptomic profiling in human bronchial epithelial cells. Urban PM2.5 exhibited greater cytotoxic potency per unit mass than coastal PM2.5, with extractable fractions accounting for most cytotoxic and pathway-level responses. Transcriptomics revealed distinct site-specific modes of action: urban PM2.5 preferentially induced oxidative stress, xenobiotic metabolism, and cell cycle suppression, consistent with acute, nonapoptotic injury, whereas coastal PM2.5 elicited weaker cytotoxicity but stronger interferon-mediated immune and apoptosis-related signaling. Integrating chemical abundance with pathway activity using random forest regression, SHAP interpretation, and mechanistic corroboration reduced 5,033 detected features to 444 pathway-linked candidate drivers. Fewer than 5% of features explained ∼95% of cumulative model contribution. Standard-confirmed contributors included plasticizer-related compounds, aromatic and heteroaromatic combustion products, and copper for urban PM2.5 and secondary/aged organics and nickel for coastal PM2.5. These findings support mechanism-informed prioritization of hazardous PM2.5 components beyond mass-based assessment.
Shihao Wang, Xinyu Li, Yong Han et al.· Environmental Science and Te...· 0 citations
Background Airborne pollutants comprise biological agents such as lipopolysaccharide (LPS) and engineered nanomaterials, including carbon nanotubes (CNTs), which are increasingly prevalent in industrial and consumer applications. Although the pulmonary toxicity of high-dose CNT exposure is established, the inflammatory consequences of low-dose, non-cytotoxic CNT exposure, particularly in combination with other aerosols, remain poorly understood. Methods We investigated cytotoxicity and proinflammatory responses to multi-walled CNTs in three-dimensional A549 human epithelial spheroids, compared with A549 monolayers and MM6 monocytes. Cells were exposed to two CNTs differing in diameter (CNT1: 7–15 nm; CNT2: 110–170 nm), alone or combined with LPS. Cell viability was assessed by WST-1 assay and F-actin junction staining, while IL-8 and IL-6 production was quantified by ELISA and qPCR. Results A549 spheroids were more susceptible to CNT-induced cytotoxicity than monolayers, with CNT1 inducing greater cytotoxicity than CNT2. At non-cytotoxic concentrations, CNTs alone did not elicit cytokine release; however, co-exposure with LPS significantly enhanced cytokine secretion in spheroids, particularly in the presence of CNT2. This synergistic inflammatory response was not observed in MM6 monocytes. Conclusion Our study demonstrates that non-cytotoxic CNTs can potentiate LPS-driven inflammation in A549 epithelial spheroids, suggesting potential respiratory risks from combined exposure to environmental endotoxins and nanomaterials, even at low doses.
Jian Liu, Li Zeng, W. Abate et al.· Frontiers in Immunology· 0 citations
Combustion of composite solid propellants generates complex aerosols containing metallic nanoparticles (NPs) and acidic gases, raising concerns about inhalation toxicity. Here, we investigated the effects of alumina (Al₂O₃) NPs, alone or combined with hydrochloric acid (HCl), in a human alveolar-capillary barrier (ACB) model exposed at the air-liquid interface. The model consisted of hAELVi alveolar epithelial cells co-cultured with HPMEC-ST1.6R pulmonary microvascular endothelial cells and exposed using a Vitrocell Cloud system under acute or repeated scenarios. Deposited Al₂O₃ NPs doses corresponding to low-to-high alveolar exposure scenarios were 0.007, 1.35 ± 0.1 and 2.3 ± 0.2µg/cm², with or without HCl at 1.37mM. Barrier integrity, viability, inflammatory and remodeling mediators, oxidative stress and DNA damage were assessed 24h after the final exposure. The co-culture formed a tight functional barrier with high transepithelial electrical resistance, low permeability and organized junctional protein expression. Acute exposure to the highest Al₂O₃ NPs dose induced a compartment-specific inflammation-related response, based on the biomarkers investigated, with increased IL-1α, IL-8, IL-1β, CCL2, DKK1 and angiogenin, without cytotoxicity or barrier disruption. Repeated exposure shifted the response toward moderate cytotoxicity at the highest dose, with viability decreasing to 77% for Al₂O₃ NPs alone and 66% for Al₂O₃ NPs with HCl, while permeability remained unchanged. No significant oxidative stress or γ-H2AX-associated DNA damage was detected. HCl co-exposure did not enhance Al₂O₃ NPs toxicity. However, because HCl was administered in dissolved form prior to nebulization, the contribution of potential gas-particle interactions occurring in combustion plumes could not be evaluated and the results should not be interpreted as evidence for or against synergistic effects arising from gas-particle interactions occurring in combustion plumes. These findings show that exposure pattern critically shapes alveolar-capillary responses, supporting human air-liquid interface co-culture models for mechanistic inhalation toxicology.
M. Cherrière, S. De Araujo, M. Oger et al.· Toxicology· 0 citations
Several studies have been conducted on human exposure to ultrafine particulate matter (UFPM), Black carbon (BC), and polystyrene nanoplastics (PS-NPs). However, it remains unclear whether different chemical types of environmental nanoparticles induce a similar mitochondrial stress response or a unique particle-specific response. In the present study, we examined the molecular mechanisms underlying nanoparticle-induced mitochondrial stress response and immunotoxicity using human peripheral blood mononuclear cells exposed to UFPM, BC, and PS-NPs under similar experimental conditions. Oxidative stress, mitochondrial adaptation, respiratory chain integrity, mitochondrial integrated stress response, inflammatory signaling, and systems-level interactions between molecules were analyzed through the evaluation of the expression of NRF2, HIF-1α, PGC-1α, TFAM, OMA1, DELE1, mitochondrial ND1, Complex I-V, NF-κB, TNF-α, and NLRP3 and the use of principal component analysis, hierarchical clustering, and correlation networks. All three nanoparticles caused oxidative stress and mitochondrial dysfunction with different kinetics and mechanisms. UFPM mostly induced an acute antioxidant response and mitochondrial adaptation; BC led to chronic mitochondrial dysfunction, chronic activation of the OMA1-DELE1-mediated mitochondrial ISR pathway, and inflammation; while PS-NPs induced low but chronic mitochondrial adaptation along with mitochondrial biogenesis and stress responses. Our systems-level analysis showed that oxidative stress, mitochondrial adaptation, mitochondrial ISR, and inflammation represent a highly connected molecular network regardless of the physicochemical nature of the nanoparticles, with the OMA1- DELE1 axis being a key regulatory node connecting mitochondrial stress response and inflammation. Overall, we have found that mitochondrial stress response is a common mechanism underlying the toxicity of chemically different nanoparticles and have also revealed particle-specific stress-response dynamics responsible for the degree and persistence of cellular damage. The current work presents novel insights into the molecular mechanisms of nanoparticle-induced immunotoxicity and suggests OMA1, DELE1, NRF2, PGC-1α, TFAM, ND1, and Complex I-V as potential biomarkers.
P. K. Mishra, Apoorva Chouksey, Aneha K. Rajan et al.· bioRxiv· 0 citations
Air pollutants, including vehicle emissions and photochemical smog such as ozone, have become a significant health concern in most industrialized nations. Inhaling ozone has repeatedly been linked to increased lung inflammation, immune responses, and cancer development, ultimately speeding up lung cancer metastasis and leading to higher death rates. However, the direct connection between ozone exposure and tumor cell migration has not yet been definitively established. This study aims to investigate the effects of ozone exposure on cancer spread and its underlying mechanisms, focusing on the hypoxia-inducible factor 1α (HIF-1α)/C-X-C motif chemokine receptor 4 (CXCR4) pathway, which influences cancer cell migration and epithelial-mesenchymal transition (EMT). Using B16F10 melanoma and LL2 Lewis lung carcinoma cells, we examined how 1 ppm ozone exposure affects reactive oxygen species (ROS) production and its subsequent impact on the HIF-1α/CXCR4 pathway. Our results show a significant increase in HIF-1α/CXCR4 expression and EMT-related proteins after ozone exposure. Additionally, the use of arbutin, a known ROS scavenger, significantly reduced HIF-1α/CXCR4 levels and cell migration, confirming the role of ROS in ozone-induced metastasis. In summary, our research suggests that ozone promotes cancer spread by activating the HIF-1α/CXCR4 signaling pathway, highlighting an environmental factor that could facilitate cancer progression.
Che-Hsin Lee, Chun-Sheng Chuang, P. Chiu et al.· American Journal of Cancer R...· 0 citations
Microplastics (MPs) pollution represents a pressing global environmental challenge, with studies increasingly highlighting their associated health risks. Although MPs have been detected in human lung tissues, the majority of existing research has concentrated on their physicochemical characteristics, environmental distribution and pulmonary health risks. Consequently, our understanding of the specific biological targets and effective intervention strategies against these risks remains limited. To identify therapeutic targets, we screened for pulmonary differential metabolites between normal mice and mice exposed to airborne MPs, derived from dust fall of 10 cities in China. Proteomics results showed adenosine 5'-monophosphate-activated protein kinase (AMPK) signalling pathway was one of critical targets. Through molecular docking and molecular dynamics stimulation, honokiol (HNK) was selected as therapeutic drug to regulate AMPK. In vitro results demonstrated that HNK significantly ameliorated autophagy inhibition in RAW264.7 cell, and alleviated mitochondrial dysfunction in BEAS-2B cell. Drug mechanism research revealed that HNK activated autophagy via the AMPK/mammalian target of rapamycin (AMPK/mTOR) pathway, and promoted mitophagy through the AMPK/E3 ubiquitin protein ligase parkin (AMPK/Parkin) pathway, thereby restoring mitochondrial function. Further targeted energy metabolomics analysis illustrated that HNK regulated the guanosine triphosphate to guanosine diphosphate (GTP/GDP) ratio, adenosine triphosphate (ATP) production, and nucleotide metabolism. These functions accelerated the restoration of autophagic flux, mitophagy reactivation and DNA repair. In conclusion, HNK effectively alleviates airborne MPs-induced autophagy inhibition, mitochondrial dysfunction and energy metabolism disorder via AMPK signalling, providing a promising intervention strategy for pulmonary injury caused by airborne MPs.
A new machine-learning framework aims to improve the success rate of computational protein design while moving away from results that reproduce sequences found in nature.
