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

Figures & data

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 h 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, ~ 1 h 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 h 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 h 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 h (NA; not analyzed at 24-h timepoint). Results are representative from two separate experiments. * p < 0.05 as compared to LPS/Oil at each respective timepoint.

Table 1.  Serum levels of CBD.

	Sal/CBD 6 h	LPS/CBD 6 h	Sal/CBD 24 h	LPS/CBD 24 h
CBD (ng/ml)	40.9 ± 4.3 (3)	40.2 ± 12.6 (7)	NQ (3)	1.6 ± 0.8* (5)
Low spike recovery (%)	76.0 ± 30.0 (2)	124.8 ± 23.0 (5)	NA	169.4 ± 28.0 (2)
High spike recovery (%)	88.0 ± 8.2 (3)	97.0 ± 9.3 (7)	NA	95.0 ± 5.8 (4)

Blood was collected at necropsy and serum was isolated. Serum was extracted with methanol (1 part serum:10 parts methanol), centrifuged, and supernatants were analyzed by LC/MS/MS.

Serum was pre-spiked with either 31.5 ng/ml (low) or 315 ng/ml (high) CBD concentrations prior to extraction.

Values shown are mean ± SE. Number in parentheses indicates number analyzed. Of 11 serum samples taken from mice that did not receive CBD, nine were NQ and the average CBD concentration from two animals was 2.4 (± 1.2) ng/ml.

NQ, not quantifiable; NA, not applicable.

* p < 0.01 vs both 6 h groups.