The effect of cannabis-derived terpenes on alveolar macrophage function

Abstract

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.

Keywords: cannabis; inflammation; macrophages; phagocytosis; terpenes.

FIGURE 1

Terpene Mix A does not attenuate inflammatory cytokine production in response to LPS. MH-S cells were pretreated for 1 h with Terpene Mix A followed by the addition of LPS for 24 h before measurement of (A) mRNA and (B) protein for IL-1β, IL-6 and TNF. Results are expressed as the mean ± SEM of three independent experiments. Kruskall Wallis multiple comparisons test was applied in panel B due to a protein concentration of 0 for untreated condition; ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.

FIGURE 2

Terpene Mix B does not attenuate inflammatory cytokine production in response to LPS. MH-S cells were pretreated for 1 h with Terpene Mix B followed by the addition of LPS for 24 h before analysis of (A) mRNA and (B) protein for IL-1β, IL-6 and TNF. Results are expressed as the mean ± SEM of 3–4 independent experiments. Kruskall Wallis multiple comparisons test was applied in panel B due to a protein concentration of 0 for untreated samples; ns, not significant, *P < 0.05, ****P < 0.0001.

FIGURE 3

Terpene Mix B increases phagocytosis against opsonized beads. (A) Representative gating strategies for quantification of phagocytic cells using flow cytometry. (B) Terpene Mix B increased the percentage of phagocytic macrophages by approximately 30% when challenged with fluorescently labelled latex beads opsonized with IgG. (C) Neither terpene mixture significantly affected phagocytosis of mCherry-expressing Escherichia coli. Results are expressed as the mean ± SEM of 3–6 independent experiments. ns, not significant, *P < 0.05.

FIGURE 4

Terpenes do not potentiate the effects of Δ⁹-THC. Treatment of MH-S cells with terpenes in combination with Δ⁹-THC had no additive effect as measured by MTT assay (A) or Annexin-V PI staining assay (B). (C) Representative gating strategy for Annexin-V PI assay as measured by flow cytometry. (D) Terpene Mix A in combination with Δ⁹-THC did not affect phagocytosis of mCherry-expressing E. coli. Results are expressed as the mean ± SEM of 4–9 independent experiments; ns, not significant.