The AhR Is a Critical Regulator of the Pulmonary Response to Cannabis Smoke

Abstract

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.

Keywords: aryl hydrocarbon receptor; cannabis; extracellular vesicles; pulmonary system.

FIGURE 1

Moderate cannabis smoke exposure activates pulmonary AhR. (A) Serum levels of THC‐COOH after acute cannabis smoke exposure in Ahr ^+/− and Ahr ^−/− mice shown as box plots indicating the mean, minimum, and maximum values. Relative levels of Cyp1a1 (B) and Cyp1b1 (C) mRNA in lung tissue after air or cannabis smoke exposure in Ahr ^+/− and Ahr ^−/− mice. Data represent mean ± SEM, with individual points indicating biological replicates. Statistical significance is shown as *p < 0.05, **p < 0.01.

FIGURE 2

Cannabis smoke alters innate immune cell populations in the lung, airspaces, and blood. (A) Schematic representation of immune cell populations assessed in the lung, airspaces, and blood. (B, C) Pulmonary monocytes and neutrophils, including total counts and percentages of CD45⁺ cells. (D) Representative images of BALF cells from Ahr ^+/− and Ahr ^−/− mice after air or cannabis smoke exposure. Scale bars represent 100 μm. (E–H) Differential cell counts in BALF, including total BALF cells (E), macrophages (F), epithelial cells (G), and neutrophils (H), with total counts and percentages shown. (I–K) Innate immune cell populations in blood, including neutrophils (I), eosinophils (J), and monocytes (K). Data represent mean ± SEM, with individual points indicating biological replicates. Statistical significance is denoted as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 3

Cannabis smoke induces cytokine expression in BALF and serum. Cytokine concentrations were measured in BALF (A–D) and serum (E–H) from Ahr ^+/− and Ahr ^−/− mice following exposure to air or cannabis smoke. (A) Tissue damage‐related cytokines VEGF and LIF. (B) Pleiotropic cytokine IL‐6. (C) Eosinophil‐related cytokines eotaxin and IL‐5. (D) T‐cell‐related cytokines IL‐2 and RANTES. (E) Systemic IL‐6 levels in serum. (F) Serum eosinophil‐related cytokine eotaxin. (G) Serum monocyte‐related cytokine MIG. (H) Serum T‐cell‐related cytokines IL‐17, RANTES, and IL‐2. Data are presented as mean ± SEM, with individual points representing biological replicates. Statistical significance is indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

FIGURE 4

Characterization of pulmonary compartments for proteomic analysis. (A) Schematic representation of the experimental workflow used to define and isolate lung tissue, EVs, and BALF for proteomic analysis. (B–D) Characterization of EVs including (B) TEM images of EVs from Ahr ^+/− and Ahr ^−/− mice following exposure to air or cannabis smoke. Scale bar represents 200 nm. (C) NTA of EVs, showing particle size distribution across 0–1000 nm. (D) Area under the curve (AUC) analysis of total EV concentration in BALF. Data represent mean ± SEM, with individual points indicating biological replicates.

FIGURE 5

Overlapping and unique upregulated DEPs and pathways across lung compartments in response to cannabis smoke. Venn diagram of upregulated DEPs (A) and pathways (B) in Ahr ^+/− mice exposed to cannabis smoke versus air. (C) Network analysis of upregulated pathways in Ahr ^+/−, with each node representing biological processes enriched in lung (green), EVs (orange), or BALF (purple). Venn diagram of upregulated DEPs (D) and pathways (E) in Ahr ^−/− mice exposed to cannabis smoke versus air. (F) Network analysis of upregulated pathways in Ahr ^−/−, with each node representing biological processes enriched in lung (green), EVs (orange), or BALF (purple).

FIGURE 6

Overlapping and unique downregulated DEPs and pathways across lung compartments in response to cannabis smoke. Venn diagram of downregulated DEPs (A) and pathways (B) in Ahr ^+/− mice exposed to cannabis smoke versus air. (C) Network analysis of upregulated pathways in Ahr ^+/− mice, with each node representing biological processes enriched in lung (green), EVs (orange), or BALF (purple). Venn diagram of downregulated DEPs (D) and pathways (E) in Ahr ^−/− mice exposed to cannabis smoke versus air. (F) Network analysis of upregulated pathways in Ahr ^−/−, with each node representing biological processes enriched in lung (green), EVs (orange), or BALF (purple).

FIGURE 7

Pathway analysis of DEPs in lung tissue of Ahr ^+/− and Ahr ^−/− mice exposed to cannabis smoke versus air. (A) Venn diagram showing the overlap of upregulated DEPs for Ahr ^+/− and Ahr ^−/− lung tissue after cannabis smoke exposure versus air. (B) Top 15 upregulated pathways induced by cannabis smoke exposure versus air in Ahr ^+/− and Ahr ^−/− mice. (C) Network clustering analysis of upregulated pathways. (D–E) Heatmaps of top 15 proteins in each cluster including detoxification (D) and nucleotide metabolism (E). (F) Venn diagram of downregulated DEPs for Ahr ^+/− and Ahr ^−/− lung tissue after cannabis smoke exposure versus air. (G) Top 15 downregulated pathways in response to cannabis smoke exposure in Ahr ^+/− and Ahr ^−/− mice. (H) Network analysis of downregulated pathways. (I, J) Heatmaps of top 15 proteins in the cytoskeletal organization (I) and cell adhesion (J) clusters. Heatmaps represent z‐scores of protein expression across conditions.

FIGURE 8

Pathway analysis of DEPs in EVs of Ahr ^+/− and Ahr ^−/− mice exposed to cannabis smoke versus air. (A) Venn diagram showing the overlap of upregulated DEPs for Ahr ^+/− and Ahr ^−/− EVs after cannabis smoke exposure versus air. (B) Top 15 upregulated pathways in Ahr ^+/− and Ahr ^−/− EVs in response to cannabis smoke exposure. (C) Network analysis of upregulated pathways. (D) Heatmap of top 15 coagulation‐related proteins. (E) Venn diagram of downregulated DEPs for Ahr ^+/− and Ahr ^−/− EVs after cannabis smoke exposure versus air. (F) Top 15 downregulated pathways in response to cannabis smoke exposure. (G) Network analysis of downregulated pathways, highlighting clusters related to protein metabolism, tRNA activation, and glutathione metabolism. (H–J) Heatmaps of top 15 proteins in the protein metabolism (H), tRNA activation (I), and glutathione metabolism (J) clusters, showing expression changes in response to cannabis smoke exposure. Heatmaps represent z‐scores of protein expression across conditions.

FIGURE 9

Pathway analysis of DEPs in BALF of Ahr ^+/− and Ahr ^−/− mice exposed to cannabis smoke versus air. (A) Venn diagram showing the overlap of upregulated DEPs for Ahr ^+/− and Ahr ^−/− BALF after cannabis smoke exposure versus air. (B) Top 15 upregulated pathways in Ahr ^+/− and Ahr ^−/− BALF in response to cannabis smoke exposure. (C) Network analysis of upregulated pathways. (D, E) Heatmaps of top 15 proteins in each cluster including coagulation (D) and protease regulation (E). (F) Venn diagram of downregulated DEPs for Ahr ^+/− and Ahr ^−/− BALF after cannabis smoke exposure versus air. (G) Top 15 downregulated pathways in response to cannabis smoke exposure. (H) Network analysis of downregulated pathways. (I–K) Heatmaps of top 15 proteins in the aldehyde metabolism (I), carbohydrate metabolism (J), and nucleotide metabolism (K) pathway clusters. Heatmaps represent z‐scores of protein expression across conditions.