MH-S cells were obtained from American Type Culture Collection (ATCC; Manassas, VA, United States) and cultured in RPMI (Roswell Park Memorial Institute) 1640 media (WISENT Inc, Saint-Jean Baptiste, Canada) containing 10% fetal bovine serum (FBS) (WISENT Inc), gentamicin (WISENT Inc), antibiotic-antimycotic (A/A; WISENT Inc) and 2-mercaptoethanol (Millipore Sigma, Burlington, MA, United States). Cells were treated with the following: Terpene Mix A (CRM40755; Sigma-Aldrich, St. Louis, United States), Terpene Mix B (CRM40937; Sigma-Aldrich) (Table 1), LPS (LPS 0111: B4; Sigma-Aldrich), the toll-like receptor 2 (TLR2) agonist Pam3CSK4 (tlrl-pms; InvivoGen, San Diego, United States), ∆⁹-THC (ISO60157; Cayman Chemical, Ann Arbor, United States) or the appropriate controls which included methanol (MeOH) or serum-free (SF) RPMI 1640. All cells were incubated in humidified chambers at 37°C and exposed to 21% O₂ and 5% CO₂.

MH-S cells were plated at 4.7 × 10⁴ cells/cm² in a sterile, flat-bottomed 96 well plate (Thermo Fisher Scientific) in RPMI 1640 containing 10% FBS. Forty-eight h later, cells were treated with SF-RPMI 1640, varying concentrations of Terpene Mix A and Terpene Mix B in SF-RPMI 1640, MeOH, PBS (positive control), and 3 μg/mL of ∆⁹-THC (Sigma Aldrich) with and without Terpene Mix A and B for 24 h. Then, a 5 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT; Sigma-Aldrich M-2128) in PBS was prepared and 10 μL of this solution was added to each well. Following a 4-h incubation period, the plates were centrifuged at 800 RPM for 5 min, the supernatant was discarded, and the contents of each well were resuspended in 200 µL of dimethyl sulfoxide (DMSO; Sigma Aldrich). Ten minutes later, plates were read on an iMark microplate reader (Bio-Rad Laboratories, Canada) using Microplate Manager Software Version 6 at 570 nm.

Cells were plated at 4 × 10⁴ cells/cm² in a 6-well plate (Thermo Fisher Scientific) and upon reaching 80% confluency, 24–48 h later, were pre-treated with 1 μg/mL of Terpene Mix A or Terpene Mix B. One hour later, 0.1 μg/mL LPS was added for 24 h. RNA was isolated using Aurum Total RNA Mini Kit (Bio-Rad Laboratories, Canada) in accordance with instructions from the manufacturer. RNA was quantified using Nanodrop 1000 Spectrophotometer Infinite M200 Pro (TECAN, Mannedorf, Switzerland). Reverse transcription was accomplished with iScript Reverse Transcription Supermix (Bio-Rad Laboratories, Canada) to a final concentration of 10 ng/mL. Primer sequences for the genes were: 18S (forward) CGG​AAA​ATA​GCC​TTC​GCC​ATC​AC, (reverse) ATC​ACT​CGC​TCC​ACC​TCA​TCC​T; Tnf (forward) CTA​TGT​CTC​AGC​CTC​TTC​TC, (reverse) GGG​AAC​TTC​TCA​TCC​CTT​T; Il1b (forward) GGA​CAT​GAG​CAC​CTT​CTT, (reverse) CCT​GTA​GTG​CAG​TTG​TCT​AA and Il6 (forward) CCAGAGTCCT TCAGAGAGATACA, (reverse) CCT​TCT​GTG​ACT​CCA​GCTTAT​C. qPCR was done by combining 1 μL of cDNA and 0.25 μL of forward and reverse primers with SsoFast Evagreen (Bio-Rad Laboratories). Amplification was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories). Thermal cycling was initiated at 95°C for 3 min followed by 39 cycles of denaturation at 95°C for 10 s and annealing at 55°C or 52°C for S18/il6 and il1b and tnf, respectively, for 5 s. RNA expression was calculated using the ΔΔCt method and results were expressed as the log₂ fold change normalized to housekeeping gene 18S.

Following treatments for 24 h, the cell supernatant was collected and a Mouse Cytokine Array Proinflammatory Focused 10-plex (MDF10) Assay was conducted (Eve Technologies, Alberta, Canada). Only the results are shown for IL-1β, IL-6 and TNF.

MH-S cells were plated in a 6-well plate (Thermo Fisher Scientific) and treated with SF-RPMI 1640, 100 ng/mL Pam3CSK4 (positive control), MeOH (vehicle) or 1 μg/mL of terpene mixes A or B; 24 h later, IgG-coated beads were added to each well at a final volume of 10 μL per well (dilution 1:100 in SF-RPMI 1640) and incubated for 4 h. Medium was then aspirated, cells were washed 2x with PBS and detached using 1 mL of Accutase Cell Detachment Solution (Innovative Cell Technologies, United States). Alveolar macrophages were then stained with Amcyan fixable viability dye (Thermo Fisher Scientific) for 30 min. Cells were acquired using a FACSCanto flow cytometer (BD Biosciences, Canada) and analyzed using FlowJo software version 10.

XL1 Gold E. coli containing the replicative plasmid pUC18-miniTn7T2.1::PA1/04/03-mCherry, which constitutively expresses mCherry fused to a synthetic Lac promoter (Zhao et al., 2013), was grown on LB agar plates (Thermo Fisher Scientific) containing 10 mg/mL gentamicin. The mCherry plasmid was isolated from these colonies using a plasmid miniprep kit (GeneJet Plasmid Miniprep Kit K0503, Thermo Scientific) according to the manufacturer’s instructions. The plasmid concentration was measured using a NanoDrop Microvolume Spectrophotometer (Thermo Fisher Scientific) and subsequently transformed into K12 E. coli via electroporation as previously described (Dower et al., 1988). Electroporated K12 E. coli was then plated on LB agar containing 10 mg/mL gentamicin for positive selection. Successful transformation of the mCherry plasmid was confirmed by measuring the fluorescence at an excitation wavelength of 587 nm and an emission wavelength at 620 nm as well as the optical density at 600 nm of 200 μL samples of cultures from five individual colonies grown in Miller LB broth (Thermo Fisher Scientific) at 37°C, shaken at a duration of 5 s and an amplitude of 1 mm once an hour for 18 h using the Infinite 200 pro plate reader (TECAN, Zürich, Switzerland). The colony presenting the highest fluorescence to optical density ratio was plated on LB agar containing 10 mg/mL gentamicin for 24 h before storage in LB with 15% v/v glycerol at −80°C.

MH-S cells were plated in a 6-well plate and treated with SF-RPMI 1640, MeOH (vehicle) and 1 μg/mL of terpene mixes A or B with or without 3 μg/mL of ∆⁹-THC. Twenty-four hours later, mCherry-expressing K12 E. coli was applied to the wells at an MOI of 50. To synchronize phagocytosis, plates were centrifuged at 700 RPM for 3 min before incubation at 37°C and 5% CO₂ for 75 min. After incubation, cells were washed three times with ice cold PBS to stop degradation in the phagolysosome and to wash away residual extracellular bacteria. Cells were then detached with 1 mL Accutase Cell Detachment Solution, stained with Amcyan fixable viability dye for 30 min, and fixed with 4% paraformaldehyde in PBS (Thermo Fisher Scientific) for 15 min. Cells were acquired using the LSRFortessa X-20 flow cytometer (BD Biosciences; Canada) and analyzed using FlowJo software version 10 (Supplemental Figure S1).

Cells were plated and treated as previously described. Twenty-four h after treatment, cells were detached with 1 mL Accutase Cell Detachment Solution and simultaneously stained with 5 μL APC-Annexin V and 3 μL PI for 15 min. Cells were then acquired using the FACS Canto flow cytometer (BD Biosciences, Canada) and data were analyzed using FlowJo software version 10 (Supplemental Figure S2).

Unless otherwise stated, statistical analysis was performed using a one-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test to assess differences between the untreated control group and any treatment groups. Statistical analyses were performed on GraphPad Prism 10 (version 10.0.3; GraphPad Software Inc, San Diego, CA, United States). Results are presented as mean ± standard error of the mean (SEM). Statistical significance was considered where the P value <0.05.

Some studies have suggested that terpenes found in cannabis and other plants are anti-inflammatory (Rufino et al., 2015; Gallily et al., 2018). Because most people consume cannabis via inhalation, we hypothesized that cannabis-derived terpenes would dampen the ability of alveolar macrophages to mount an inflammatory response. To be sure that any observed effect was not confounded by alterations in cellular viability, we first determined the maximal concentration of each terpene mixture that did not significantly reduce cell survival to be 1 μg/mL (Supplemental Figure S3); this concentration was used in all subsequent experiments. We then analyzed these cannabis-derived mixtures on inflammatory cytokine mRNA and protein expression of MH-S cells in response to LPS as previously described (Preteroti et al., 2023b). IL-1β, IL-6 and TNF were selected as our primary outcomes because of their potent transcriptional induction by LPS as well as their downregulation by ∆⁹-THC (Rossol et al., 2011). As expected, LPS significantly increased the mRNA and protein expression of IL-1β, IL-6 and TNF after 24 h (Figures 1, 2). Neither Terpene Mix A (Figure 1A) nor Terpene Mix B (Figure 2A) alone affected the transcription of Il1b, Il6, or Tnf. Neither Terpene Mix A (Figure 1A) or Terpene Mix B (Figure 2A) affected LPS-induced gene expression. Although LPS also significantly increased the release of IL-1β, IL-6, and TNF protein after 24 h, this was not attenuated by either terpene mixture (Figures 1B, 2B). Further, the protein levels of GM-CSF, IFNγ, IL-2, IL-4, IL-10, IL-12p70 and MCP-1 were also not affected by terpene treatment (data not shown). Thus, cannabis-derived terpenes have minimal impact on inflammatory mediator production in response to LPS in MH-S cells.

A primary function of alveolar macrophages is to phagocytose inhaled pathogens and foreign debris (Joshi et al., 2018). Hence, we analyzed the phagocytic capability of MH-S cells in response to treatment with cannabis-derived terpenes. First, we quantified the percentage of phagocytic cells by using flow cytometry to measure the uptake of latex beads opsonized with IgG and conjugated to a phycoerythrin (PE) fluorophore (Figure 3A). As a positive control, we used Pam3CSK4, a TLR2 agonist which has been previously described as a potent enhancer of phagocytosis against opsonized ligands (Sigola et al., 2016). Pam3CSK4 increased the percentage of MH-S cells that took up the IgG-conjugated beads by approximately 30% (Figure 3B). While Terpene Mix A did not significantly affect uptake of the fluorescent beads (Figure 3), Terpene Mix B significantly increased IgG internalization (Figure 3B). Thus, terpenes can selectively increase the ability of alveolar macrophages to phagocytose opsonized particles.

Macrophages can recognize and phagocytose multiple types of pathogens, and opsonized ligands are one type of particle that binds to the fragment crystallizable receptor gamma (FcGammaR) to stimulate internalization. Hence, we extended our analysis by using a common non-virulent laboratory strain of E. coli which is detected by scavenger receptors on macrophages. We transformed wild type K12 E. coli with a plasmid encoding for a constitutively expressed fluorescent mCherry protein to track association of the bacteria to MH-S cells (Supplemental Figure S4). Because we did not perform a gentamicin treatment following co-incubation of MH-S cells with bacteria, our assay did not exclude bacteria stuck to the outside of the MH-S cell membrane, and thus complete internalization could not be confirmed. However, pretreatment with either cannabis-derived terpene mixture A or B before incubation with these bacteria had no significant effect on the ability of MH-S cells to phagocytose E. coli (Figures 3A, C). Therefore, these results suggest that terpenes do not affect the ability of MH-S cells to phagocytose bacteria.

Our data above imply that cannabis-derived terpenes have minimal effect on alveolar macrophage function. However, a prevailing concept is that cannabis-derived terpenes may exert a synergistic effect with ∆⁹-THC, which is commonly referred to as the entourage effect (Russo, 2011). However, there exists very little evidence to suggest that terpenes in fact synergize with ∆⁹-THC to potentiate its effects, and no information exists for the entourage effect in alveolar macrophages. Therefore, we utilized ∆⁹-THC, together with the cannabis-derived terpene mixtures, and assessed cell viability and phagocytosis. We used concentrations of 3 μg/mL and 1 μg/mL ∆⁹-THC and Terpene Mix A or B (Supplemental Figure S3) (Preteroti et al., 2023b). When ∆⁹-THC was included with Terpene Mix A or B, there was no significant effect on cell viability when compared to each treatment individually as measured by MTT assay (Figure 4A). As a complement, we also performed the Annexin-V/PI staining, which allows the differentiation between healthy, apoptotic and necrotic cells (Miller, 2004). Here, there was no difference in apoptosis or necrosis between cells treated with ∆⁹-THC and/or cannabis-derived terpenes (Figures 4B, C). Finally, we measured the phagocytic capability of MH-S cells. Treatment with Terpene Mix A nor ∆⁹-THC alone significantly affected MH-S cell phagocytosis (Figure 4D). There was also no difference when Terpene Mix A was combined with ∆⁹-THC (Figure 4D). These results suggest that cannabis-derived terpenes do not enhance the effects of ∆⁹-THC in MH-S cells. Overall, these results show that terpenes have minimal impact on the function of alveolar macrophages, resident pulmonary cells that are important in protecting the lungs against inhaled pathogens.