Cigarette smoke is the primary cause of chronic obstructive pulmonary disease (COPD), an incurable condition characterized by irreversible airflow obstruction and alveolar destruction driven by chronic inflammation of the lungs and airways. The inflammatory response caused by cigarette smoke is typified by the recruitment of innate and adaptive immune cells to the lung. Paradoxically, many of these immune cells are functionally impaired by smoke. Notable among these are lung macrophages, which have reduced ability to clear apoptotic lung epithelial cells and neutrophils by efferocytosis when exposed to cigarette smoke. Lung macrophages may express the aryl hydrocarbon receptor (AhR), a receptor/transcription factor highly expressed in barrier organs including the lungs. The AhR protects against the damaging effects of cigarette smoke by attenuating pulmonary neutrophilia via an unknown mechanism. We used our preclinical cigarette smoke models, mutant AhR mice and techniques such as flow cytometry, Western blot and reverse transcription quantitative polymerase chain reaction (RT-qPCR) to show that the AhR promotes the resolution of cigarette smoke-induced inflammation in mice via enhanced efferocytosis. Moreover, the ability of macrophages to engulf apoptotic neutrophils in the lungs is due to a non-genomic AhR pathway that involves signaling through the IL-10/JAK/STAT pathway. Finally, we show that the non-toxic endogenous AhR ligand FICZ promotes macrophage uptake of neutrophils. Taken together, these results support the importance of AhR activity in mediating its anti-inflammatory functions in response to cigarette smoke. Further investigation of the precise mechanisms by which the AhR exerts its anti-inflammatory function may open the possibility for therapeutic agents to treat chronic inflammatory diseases.

Cigarette smokers have a greater risk for developing chronic obstructive pulmonary disease (COPD) and lung cancer because of smoking-induced lung injury. This injury is mitigated by smoking cessation, but former smokers are still more likely to develop these diseases than never smokers. This indicates that the lungs only partially recover with cessation, but what underlies this at a transcriptional level is unclear. To better understand the molecular impact of smoking cessation, we performed a meta-analysis of publicly available lung single cell RNA-sequencing (scRNA-seq) data from three studies, comprised of cells from 9 never, 5 active, and 7 former smokers. Genes in important cellular pathways were dysregulated in active smoker cells, including collagen genes in alveolar type 1 epithelial cells (AT1s), cytoskeleton genes in general capillary cells, and antigen presentation genes in macrophages. Cessation reduced antigen presentation gene dysregulation in some macrophages and fully resolved pathway dysregulation in AT1s. Cessation also had its own effects; former smoker capillary aerocytes had dysregulation of angiogenesis and cell junction genes despite having no dysregulated pathways in active smokers. Taken together, our findings shed light on how smoking permanently damages the lungs and heightens lung disease susceptibility in former smokers.

As a barrier organ, the lungs are continuously exposed to environmental stimuli and rely on molecular sensors to detect and respond to these external cues. One such sensor is the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor traditionally recognized for its role in xenobiotic metabolism. However, this function is a relatively recent evolutionary adaptation of the AhR. Beyond its detoxification role, AhR has been implicated in developmental and homeostatic processes, as evidenced by defects in AhR-deficient mice. Despite this, the contribution of endogenous AhR activity to pulmonary homeostasis remains poorly defined. To address this gap, we leveraged high-resolution proteomics from lung tissue, bronchoalveolar lavage fluid (BALF), and extracellular vesicles (EVs) of AhR-expressing and AhR-knockout mice to define the role of AhR in shaping the pulmonary proteomic landscape. Our analysis reveals that each compartment exhibits a distinct proteomic signature and that AhR plays a central role dictating that composition within each compartment. Notably, AhR regulates proteins involved in epithelial barrier function, alveolar macrophage lipid handling, and the selective packaging of bioactive cargo into EVs. These findings identify AhR as a key regulator of structural and immune balance in the lung and uncover new functions of this receptor in maintaining pulmonary homeostasis. Furthermore, our work highlights the potential of EVs as sensitive indicators of AhR activity and offers new insight into therapeutic strategies targeting AhR signaling in respiratory disease.

As cannabis use becomes increasingly mainstream for both recreational and medicinal purposes, scientific evaluation of its health effects has not kept pace with legalization and market expansion. This gap echoes historical missteps seen in tobacco regulation, where decades passed before sufficient mechanistic and epidemiologic data on health effects prompted policy action. To avoid repeating such delays in action, this paper advocates for the integration of New Approach Methodologies (NAMs) in cannabis toxicology research, particularly for inhaled products; these tools prioritize human relevance, mechanistic insight, and reduction of animal testing. We highlight three key domains of innovation: (1) cell models and organoids, including those from induced pluripotent stem cells (iPSCs), which provide insight into tissue-specific toxicity; and (3) computational toxicology platforms such as quantitative structure - activity relationship (QSAR) and physiologically-based pharmacokinetic (PBPK) modeling, which support high-throughput, mechanism-based risk assessment. Together, these tools offer a robust framework for evaluating the diverse and complex constituents of cannabis products, enabling proactive risk assessment and regulation for cannabis-based products.

Cannabis vaping is increasingly consumed as an alternative to cannabis smoke, yet its health consequences remain poorly defined. Vaporization may reduce the formation of combustion-related toxicants, but emerging data suggest that cannabis vaping still elicits deleterious effects in respiratory epithelial cells. While the actions of cannabis are commonly attributed to cannabinoid receptors, other molecular targets such as the aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, may play a role. In this study, we used wild-type (A549WT) and AhR-knockout (A549AHRKO) human alveolar epithelial cells cultured at the air–liquid interface (ALI) to investigate whether AhR mediates the response to cannabis vapor. Cells were exposed using the expoCube to achieve THC levels of 2 μg. Under basal conditions, AhR supported cell proliferation and regulated long non-coding RNAs (lncRNA) associated with cell cycle progression. Cannabis vapor exposure increased cell growth in an AhR-dependent manner. Transcriptomic profiling revealed that both protein-coding genes and lncRNA affiliated with cell cycle were upregulated in A549WT but not in A549AHRKO cells, indicating AhR is required to promote the transcriptional response to cannabis vapor. The AhR also modulated alternative splicing events in response to cannabis vapor, with increased splicing alterations observed in A549WT cells compared to A549AHRKO cells, signifying the AhR coordinates distinct post-transcriptional responses to cannabis vapor exposure. These results suggest a potential link between cannabis vapor exposure and oncogenic signaling, highlighting AhR as a molecular sentinel that shapes epithelial cell response to cannabis vapor through coordinated transcriptional and post-transcriptional regulation.

Since its legalization, approximately 27% of Canadians aged 16 and older report using cannabis, coinciding with widespread adoption of vaping amongst youth as an alternative to smoking, largely driven by the perception that it is a safer alternative to smoking. This shift is reshaping adolescent substance use. Although inhalation remains the primary method of cannabis consumption, vaping is steadily replacing smoking. Cannabis vaping involves inhaling an aerosol from heated cannabis products, available in both distillate and dry flower cartridges. However, we have previously demonstrated that cannabis vapor still contains carcinogens, teratogens, and respiratory toxicants that can interfere with cellular metabolism. While tobacco smoking is known to disrupt cellular metabolism, the effects of cannabis vapor on lung cell metabolism remain underexplored. Cellular metabolism disruptions are often overlooked as contributors to pulmonary pathophysiology. Disruptions in lung epithelial cell metabolism can impair tissue repair, compromise barrier integrity, and are associated with pulmonary diseases such as lung cancer and fibrosis. To investigate the metabolic effects of cannabis vapor, we exposed immortalized human lung epithelial cells to high-THC cannabis vapor and performed targeted metabolomic analysis via LC/MS. We found significant changes in metabolites related to lipid and membrane metabolism, with increased O-phosphorylethanolamine suggesting enhanced membrane turnover. We found elevated levels of oxidized glutathione, indicative of oxidative stress, and observed changes in energy production and nucleotide pathways suggesting potential impairments in cellular repair and function. Cannabis vapor exposure also increased levels of 5-hydroxymethylcytosine (5hmC), a marker of active DNA demethylation, suggesting the induction of epigenetic changes. Taken together, these findings indicate that cannabis vapor disrupts pulmonary epithelial cellular metabolism, possibly inducing epigenetic changes. These results underscore the need for more research on the respiratory effects of cannabis vaping and challenge its perception as a risk-free alternative to smoking.

Cannabis sativa (marijuana) is used by millions of people around the world. C. sativa produces hundreds of secondary metabolites including cannabinoids, flavones and terpenes. Terpenes are a broad class of organic compounds that give cannabis and other plants its aroma. Previous studies have demonstrated that terpenes may exert anti-inflammatory properties on immune cells. However, it is not known whether terpenes derived from cannabis alone or in combination with the cannabinoid ∆9-THC impacts the function of alveolar macrophages, a specialized pulmonary innate immune cell that is important in host defense against pathogens. Therefore, we investigated the immunomodulatory properties of two commercially-available cannabis terpene mixtures on the function of MH-S cells, a murine alveolar macrophage cell line. MH-S cells were exposed to terpene mixtures at sublethal doses and to the bacterial product lipopolysaccharide (LPS). We measured inflammatory cytokine levels using qRT-PCR and multiplex ELISA, as well as phagocytosis of opsonized IgG-coated beads or mCherry-expressing Escherichia coli via flow cytometry. Neither terpene mixture affected inflammatory cytokine production by MH-S cells in response to LPS. Terpenes increased MH-S cell uptake of opsonized beads but had no effect on phagocytosis of E. coli. Addition of ∆9-THC to terpenes did not potentiate cytotoxicity nor phagocytosis. These results suggest that terpenes from cannabis have minimal impact on the function of alveolar macrophages.

The increasing shift from cannabis smoking to cannabis vaping is largely driven by the perception that vaping to form an aerosol represents a safer alternative to smoking and is a form of consumption appealing to youth. Herein, we compared the chemical composition and receptor-mediated activity of cannabis smoke extract (CaSE) to cannabis vaping extract (CaVE) along with the biological response in human bronchial epithelial cells. Chemical analysis using HPLC and GC/MS revealed that cannabis vaping aerosol contained fewer toxicants than smoke; CaSE and CaVE contained teratogens, carcinogens, and respiratory toxicants. A bioluminescence resonance energy transfer (BRET)-based biosensor detected the receptor-mediated activity of the extracts, primarily driven by Δ9-THC concentration. RNA- sequencing showed both CaSE and CaVE induced similar transcriptional responses, significantly upregulating genes within pathways related to inflammation, cancer, and cellular stress. This was paralleled by downregulation of pathways related to lipid synthesis and metabolism similarly from both CaSE and CaVE. Targeted metabolomics revealed significant changes in metabolites involved in lipid and membrane metabolism, energy production, nucleotide/DNA/RNA pathways, and oxidative stress response, suggesting potential impairment of lung epithelial cell repair and function. Additionally, the upregulation of 5-hydroxymethylcytosine (5hmC) indicates epigenetic changes potentially contributing to inflammation, oxidative stress, and an increased risk of cancer. These findings challenge the notion that cannabis vaping is risk-free, highlighting an urgent need for comprehensive research into its respiratory health effects. This comparison of cannabis consumption methods offers insights that could inform public health policies and raise consumer awareness regarding the potential risks of inhaling cannabis aerosol.

As legalization of cannabis increases worldwide, vaping cannabis is gaining popularity due to the belief that it is less harmful than smoking cannabis. However, the safety of cannabis vaping remains untested. To address this, we developed a physiologically relevant method for in vitro assessment of cannabis vapor on alveolar epithelial cell cultures. We compared the transcriptional response in three in vitro models of cannabis vapor exposure using A549 epithelial cells in submerged culture, pseudo-air liquid interface (ALI) culture, and ALI culture coupled with the expoCube™ advanced exposure system. Baseline gene expression in ALI-maintained A549 cells showed higher expression of type 2 alveolar epithelial (AEC2) genes related to surfactant production, ion movement, and barrier integrity. Acute exposure to cannabis vapor significantly affected gene expression in AEC2 cells belonging to pathways related to cancer, oxidative stress, and the immune response without being associated with a DNA damage response. This study identifies potential risks of cannabis vaping and underscores the need for further exploration into its respiratory health implications. Graphical Abstract • Vaporizing cannabis is increasingly popular but remains largely untested. • We used three in vitro models to assess the effects of cannabis vapor on alveolar epithelial cells. • Cannabis vapor exposure alters pathways linked to cancer and metabolism, without causing DNA damage.

Cannabis is used by an estimated 192 million people around the world. Most people use cannabis through the inhalation of cannabis smoke, which contains combustion by-products that can negatively affect lung health. Knowledge of these risks has led to a growing interest in cannabis vaporizers, which heat the dry cannabis flower without burning. Vaporizing cannabis still releases cannabinoids for inhalation but heats the plant material at a lower temperature. There is currently no standardized in vitro model for assessing the effects of dry cannabis vapor. Therefore, we established a model for the exposure of lung cell cultures at an air-liquid interface (ALI), whereby cells are apically exposed to vaporized cannabis, thereby more accurately simulating lung epithelial cell physiology. This protocol ensures consistent and reproducible delivery of cannabis vapor to the cell surface, providing a reliable platform for investigating the cellular and molecular impacts of vaporized cannabis. This work is the first to standardize an in vitro cannabis vapor delivery method, which can serve as a benchmark for future preclinical cannabis research.

The advent of single-cell sequencing has revolutionized the study of cellular dynamics, providing unprecedented resolution into the molecular states and heterogeneity of individual cells. However, the rich potential of exon-level information and junction reads within single cells remains underutilized. Conventional gene-count methods overlook critical exon and junction data, limiting the quality of cell representation and downstream analyses such as subpopulation identification and alternative splicing detection. We introduce DOLPHIN, a deep learning method that integrates exon-level and junction read data, representing genes as graph structures. These graphs are processed by a variational graph autoencoder to improve cell embeddings. DOLPHIN not only demonstrates superior performance in cell clustering, biomarker discovery, and alternative splicing detection but also provides a distinct capability to detect subtle transcriptomic differences at the exon level that are often masked in gene-level analyses. By examining cellular dynamics with enhanced resolution, DOLPHIN provides new insights into disease mechanisms and potential therapeutic targets.

Cannabis use is prevalent worldwide, with smoking being the most common method of consumption. When smoking cannabis, users are exposed to both harmful combustion products and cannabinoids. The aryl hydrocarbon receptor (AhR), a transcription factor activated by both cannabinoids and combustion products, is known to regulate pulmonary responses to environmental insults. Therefore, we hypothesized that AhR activation would reduce susceptibility to the harmful effects of inhaled cannabis smoke. To investigate this hypothesis, Ahr+/- and Ahr-/- mice were exposed to air or cannabis smoke using a controlled puff regimen over a three-day period. In the first study to characterize the effects of cannabis smoke on lung tissue and the pulmonary secretome, we show that cannabis smoke activates AhR in lung tissue, leading to distinct immunological and proteomic responses across lung tissue, extracellular vesicles (EVs), and bronchoalveolar lavage fluid (BALF). AhR deficiency exacerbated neutrophilic inflammation, epithelial barrier disruption, and caused systemic cytokine elevation. Proteomic profiling revealed that AhR drives the activation of detoxification and metabolic pathways in lung tissue while suppressing cytoskeletal and adhesion proteins in response to cannabis smoke. In contrast, AhR loss shifted the proteomic response in EVs and BALF, altering coagulation, protease regulation, and metabolic stability. These findings demonstrate that AhR coordinates compartment-specific responses to cannabis smoke and plays a central role in preserving lung homeostasis and restraining inflammatory injury following cannabis exposure. These findings highlight not only the detrimental effects of cannabis smoke on lung health but also the pivotal role of the AhR as a key regulator of the pulmonary response to cannabis smoke exposure.

Despite their growing popularity, cannabis vape products remain understudied. Cannabis vape cartridges are used with battery-powered devices that aerosolize cannabis flower extracts containing high concentrations of cannabinoids such as THC. These types of products are commonly known as cannabis distillates. The potency of these products presents challenges in establishing effective dosing for preclinical studies. Currently, there are no established, standardized preclinical models for testing the safety and efficacy of these products in ways analogous to human use patterns. Thus, the in vivo cannabis distillate exposure regime required to achieve physiologically relevant doses in comparison to what is achieved in humans remains undetermined. To address this gap, a standardized preclinical murine model for inhalation of vaporized cannabis distillates has been developed using a computer-controlled delivery system. This protocol details procedures to administer cannabis vape distillates using a regimented puff topography to mice by a nose-only exposure tower. Methods to monitor mouse behavioral outcomes post-exposure and the utilization of a semi-quantitative ELISA to confirm THC delivery into the systemic circulation are also provided. This protocol will allow for the investigation of the pulmonary and systemic responses to cannabis vape distillate products by researchers interested in exploring the impact of cannabis vaping using real-world delivery protocols, thereby providing an opportunity for rigorous safety and therapeutic evaluation.

The advent of single-cell sequencing has revolutionized the study of cellular dynamics, providing unprecedented resolution into the molecular states and heterogeneity of individual cells. However, the rich potential of exon-level information and junction reads within single cells remains underutilized. Conventional gene-count methods overlook critical exon and junction data, limiting the quality of cell representation and downstream analyses such as subpopulation identification and alternative splicing detection. We introduce DOLPHIN, a deep learning method that integrates exon-level and junction read data, representing genes as graph structures. These graphs are processed by a variational graph autoencoder to improve cell embeddings. DOLPHIN not only demonstrates superior performance in cell clustering, biomarker discovery, and alternative splicing detection but also provides a distinct capability to detect subtle transcriptomic differences at the exon level that are often masked in gene-level analyses. By examining cellular dynamics with enhanced resolution, DOLPHIN provides new insights into disease mechanisms and potential therapeutic targets.

Single-cell RNA sequencing (scRNA-seq) provides high-resolution insights into cellular heterogeneity but remains costly, restricting its use to small cohorts that often lack comprehensive clinical data, reducing translational relevance. In contrast, bulk RNA sequencing is scalable and cost-effective but obscures critical single-cell insights. We introduce SIDISH, a neural network framework that integrates the granularity of scRNA-seq with the scalability of bulk RNA-seq. Using a variational autoencoder, deep Cox regression, and transfer learning, SIDISH identifies high-risk cell populations while enabling robust clinical predictions from large-cohort data. Its in silico perturbation module identifies therapeutic targets by simulating interventions that reduce high-risk cells associated with adverse outcomes. SIDISH also generalizes to spatial transcriptomics, identifying high-risk cells and mapping them within their native tissue microenvironment. Applied across diverse diseases, SIDISH establishes the link between cellular dynamics and clinical phenotypes, facilitating biomarker discovery and precision medicine. By unifying single-cell insights with large-scale clinical data, SIDISH advances computational tools for disease risk assessment and therapeutic prioritization, offering an integrative and scalable approach to precision medicine.

Rationale: Idiopathic pulmonary fibrosis (IPF) is the most common and severe interstitial lung disease (ILD), carrying a median survival of 3 years after diagnosis. In IPF, dysregulation of signaling pathways involved in extracellular matrix (ECM) production, such as transforming growth factor-β (TGF-β) signaling, lead to persistent activation of lung fibroblasts and results in the overproduction of ECM proteins such as collagen. We hypothesized that the aryl hydrocarbon receptor (AhR), a transcription factor originally characterized as a mediator of xenobiotic metabolism, is mechanistically involved in the development of fibrosis. To address this hypothesis, we evaluated the role of the AhR in fibroblast-mediated fibrotic outcomes in in-vitro and in-vivo models of pulmonary fibrosis. Methods: Primary mouse lung fibroblasts (MLFs) from AhR wild-type (Ahr+/+) and AhR knock-out (Ahr-/-) mice were treated with TGF-β (5 ng/mL) for 48 hours with or without the AhR inhibitor CH223191 (10µM). Collagen 1A1 (Col1a1) and α-smooth muscle actin (α-SMA) protein were measured via immunoblot. We performed proteomic analysis of cell lysate from Ahr+/+ and Ahr-/- MLFs treated with or without TGF-β via liquid chromatography-mass spectrometry (LC/MS). As a complement, the AhR was also knocked down in primary human lung fibroblasts (HLFs) using AhR-targeting siRNA. TGF-β-stimulated Col1a1 production was then evaluated via immunoblot. To evaluate AhR-dependency of pulmonary fibrosis in-vivo, Ahr+/+ and Ahr-/- mice were treated with bleomycin (2 mg/kg) via oropharyngeal administration and lung collagen deposition was evaluated 21 days later via Masson's Trichrome and quantified with hydroxyproline assay. Results: Col1a1 protein is reduced in Ahr-/- MLFs compared to Ahr+/+ controls both at baseline and in response to TGF-β. CH223191 attenuated TGF-β-stimulated Col1a1 production by Ahr+/+ MLFs. HLFs treated with AhR-targeting siRNA have reduced Col1a1 production compared to cells treated with siRNA controls. Proteomic analysis revealed reduced levels of proteins involved in collagen biosynthesis (Serpinh1, Plod3, Mia3, Colgalt1, Crtap, Ph31), actin polymerization (Cotl1, Arap1, Stmn1, Swap70, Flna), and mechanotransduction (Thy1, Rhog, Itga5, Itgb5) in AhR-deficient cells. Furthermore, Ahr-/- mice appear to be resistant to bleomycin-induced pulmonary fibrosis compared to Ahr+/+ controls. Conclusions: Our findings suggest that the AhR is essential for the development of the fibrotic phenotype in in-vitro and in-vivo models of pulmonary fibrosis. Future work will determine whether AhR antagonism can reverse the fibrotic phenotype, aiming to establish a novel therapeutic approach for combating fibrosis.