Alcohol and cannabis use alter pulmonary innate immunity

Abstract

Purpose: Cannabis use is increasing due to recent legislative changes. In addition, cannabis is often used in conjunction with alcohol. The airway epithelium is the first line of defense against infectious microbes. Toll-like receptors (TLR) recognize airborne microbes and initiate the inflammatory cytokine response. The mechanism by which cannabis use in conjunction with alcohol affects pulmonary innate immunity mediated by TLRs is unknown.

Methods: Samples and data from an existing cohort of individuals with alcohol use disorders (AUDs), along with samples from additional participants with cannabis use alone and with AUD were utilized. Subjects were categorized into the following groups: no alcohol use disorder (AUD) or cannabis use (control) (n = 46), AUD only (n = 29), cannabis use-only (n = 39), and AUD and cannabis use (n = 29). The participants underwent bronchoscopy with bronchoalveolar lavage (BAL) and airway epithelial brushings. We measured IL-6, IL-8, TNF⍺, and IL-10 levels in BAL fluid, and performed real-time PCR for TLR1-9 on the airway epithelial brushings.

Results: We found significant increases in TLR2 with AUD alone, cannabis use alone, and cannabis use with AUD, compared to control. TLR5 was increased in cannabis users compared to control, TLR6 was increased in cannabis users and cannabis users with AUD compared to control, TLR7 was increased in cannabis users compared to control, and TLR9 was increased in cannabis users compared to control. In terms of cytokine production, IL-6 was increased in cannabis users compared to control. IL-8 and IL-10 were increased in AUD only.

Conclusions: AUD and cannabis use have complex effects on pulmonary innate immunity that promote airway inflammation.

Keywords: Alcoholism; Cigarette smoke; Ethanol; IL-6; IL-8; Marijuana; TNF⍺; Toll-like receptors.

Figure 1:. TLR mRNA expression at baseline in those with AUDs and cannabis use.

Airway brushings were collected from subjects with no history of AUD or cannabis use, AUD, Cannabis use, or AUD and cannabis use. mRNA was extracted from the brushings and real time PCR for TLR1–9 was assayed. Each TLR was normalized to the housekeeping gene 18s ribosomal RNA. TLR8 was undetectable in all samples (Data not shown). Significant differences were seen in TLR2, TLR5, TLR6, TLR7 and TLR9.

Figure 2:. IL-6 is increased in the bronchial washings of cannabis users.

Bronchial washings were available from a subset of subjects (without AUD and without cannabis use (n=40), AUD only (n=26), Cannabis use only (n=32), AUD and cannabis use (n=27)) and we measured the cytokines IL-6, IL-8, TNFα and IL-10. There was a significant increase (p=0.02) in IL-6 in the cannabis users compared to control. There was also a significant increase in IL-8 in those with AUDs (p=0.03) and an increase in IL- 0 with those with AUDs (p=0.003).

Figure 3:. TLR5, TLR6 and TLR9 are increased in those that have used cannabis 11 years or longer

We tested whether TLR1–9 levels vary based on the years of cannabis use. The mean is represented by the bar graph ± SEM. Each individual data point is represented by the open circles. There was a statistically significant increase in TLR5, TLR6 and TLR9 in those with 11 years of cannabis use or longer compared to non-users.

Figure 4:. IL-6 is increased with longer duration of cannabis use.

We tested whether bronchial washing cytokine levels varied with the duration of cannabis use. Bronchial washings were not available for all subjects. The number available for each of the groups is as follows: 0 (n=66) 1–10 (n=24), 11–20 (n=23) 21–40 (n=12). Only IL-6 was increased compared to non-users. There is trend of increasing IL-6 with longer duration of cannabis use.