Cannabidiol (CBD) enhances lipopolysaccharide (LPS)-induced pulmonary inflammation in C57BL/6 mice

Abstract

Cannabidiol (CBD) is a plant-derived cannabinoid that has been predominantly characterized as anti-inflammatory. However, it is clear that immune effects of cannabinoids can vary with cannabinoid concentration, or type or magnitude of immune stimulus. The present studies demonstrate that oral administration of CBD enhanced lipopolysaccharide (LPS)-induced pulmonary inflammation in C57BL/6 mice. The enhanced inflammatory cell infiltrate as observed in bronchoalveolar lavage fluid (BALF) was comprised mainly of neutrophils, with some monocytes. Concomitantly, CBD enhanced pro-inflammatory cytokine mRNA production, including tumor necrosis factor-α (Tnfa), interleukins (IL)-5 and -23 (Il6, Il23), and granulocyte colony stimulating factor (Gcsf). These results demonstrate that the CBD-mediated enhancement of LPS-induced pulmonary inflammation is mediated at the level of transcription of a variety of pro-inflammatory genes. The significance of these studies is that CBD is part of a therapeutic currently in use for spasticity and pain in multiple sclerosis patients, and therefore it is important to further understand mechanisms by which CBD alters immune function.

Figure 1. Characterization of LPS-induced pulmonary inflammation

LPS or saline (Sal) was instilled intranasally and inflammatory cell infiltration in the BALF was assessed. Values shown are mean cells/ml ± SE (≥ 6 animals/group). (A) Neutrophil and (B) monocyte counts over time in response to 50 μg LPS/mouse. (C) Neutrophil and (D) monocyte counts in response to various doses of LPS at 6 and 24 hr post-instillation. NA, naive (untreated); Sal, 2 h post-Sal. *p < 0.05 as compared to Sal.

Figure 2. Effect of CBD on LPS-induced pulmonary inflammatory cell infiltration

Mice were administered CBD (75 mg/kg/day) in corn oil by oral gavage for 3 days. On the third day, approximately 1 hr before the last dose of CBD, mice received either Sal or LPS (10 μg/mouse) intranasally. Values shown are mean cells/ml ± SE (≥ 6 animals/group). Results are representative of two separate experiments. *p < 0.05 as compared to LPS/Oil.

Figure 3. Effect of CBD on LPS-induced acute pulmonary centriacinar inflammation

Mice were treated as outlined in the legend for Figure 2. Left lung lobes (6 hr post LPS) were fixed and sections were stained with NIMP-R14 anti-neutrophil antibody and counter-stained with hematoxylin. (A) Sal/Oil; (B) LPS/Oil; (C) Sal/CBD; (D) LPS/CBD. Arrows depict positive staining for neutrophils in the centriacinar region of the lung. a, alveoli; ad, alveolar duct; tb, terminal bronchiole. Neutrophilic inflammation is present in B and D, with enhanced neutrophilic influx in the proximal alveolar ducts and associated alveoli in D compared to B.

Figure 4. Effect of CBD on type II epithelial cell proliferation

Mice were treated as outlined in the legend for Figure 2. Left lung lobes (24 hr post-LPS) were fixed and sections were stained with hematoxylin and eosin. (A) Sal/CBD; (B) LPS/CBD. Dashed arrow depicts neutrophils; arrow depicts type II epithelial cell proliferation. a, alveoli; ad, alveolar duct.

Figure 5. Effect of CBD on LPS-induced pro-inflammatory cytokines

Mice were treated as outlined in the legend for Figure 2. Total RNA was isolated from right lung lobes and QRT-PCR was performed. Bars represent fold-change relative to naive (NA) group at 6 hr (NA; not analyzed at 24-hr timepoint). Results are representative from two separate experiments. *p < 0.05 as compared to LPS/Oil at each respective timepoint.