Cannabinoids Reduce Inflammation but Inhibit Lymphocyte Recovery in Murine Models of Bone Marrow Transplantation

Abstract

Cannabinoids, the biologically active constituents of Cannabis, have potent neuronal and immunological effects. However, the basic and medical research dedicated to medical cannabis and cannabinoids is limited. The influence of these treatments on hematologic reconstitution and on the development of graft versus host disease (GVHD) after bone marrow transplantation (BMT) is largely unknown. In this research, we compared the influence of D9 tetrahydrocannabinol (THC) and cannabidiol (CBD) on lymphocyte activation in vitro and in murine BMT models. Our in vitro results demonstrate that these treatments decrease activated lymphocyte proliferation and affect cytokine secretion. We also discovered that CBD and THC utilize different receptors to mediate these effects. In vivo, in a syngeneic transplantation model, we demonstrate that all treatments inhibit lymphocyte reconstitution and show the inhibitory role of the cannabinoid receptor type 2 (CB2) on lymphocyte recovery. Although pure cannabinoids exhibited a superior effect in vitro, in an allogeneic (C57BL/6 to BALB/c) BMT mouse model, THC-high and CBD-high cannabis extracts treatment reduced the severity of GVHD and improved survival significantly better than the pure cannabinoids. Our results highlights the complexity of using cannabinoids-based treatments and the need for additional comparative scientific results.

Keywords: D9 tetrahydrocannabinol; bone marrow transplantation; cannabidiol; cannabinoid receptor 2; cannabis; graft versus host disease; hematopoiesis; immune; lymphocyte; phytocannabinoids.

Figure 1

The influence of pure CBD/THC and cannabis extracts on lymphocyte activation. Proliferation of CFSE-stained, CD3-activated splenocytes from C57BL/6 (A,C) and Balb/c (B) mice was analyzed on day 4 after activation using flow cytometry analysis. (A) Summary of six independent experiments. When Comparing all treatments to control, the differences are significant starting from 3 µg/mL. The differences between THC/CBD and THC BDS/CBD BDS are significant starting from 5 µg/mL. (B) Summary of three independent experiments. When Comparing all treatments to control, THC to CBD and THC BDS to CBD BDS, significant differences were observed starting from 5 µg/mL. (C) Flow cytometry analysis of activated C57BL/6 splenocytes stained with anti-CD4 and anti-CD8 antibodies. Data is summarized from six independent experiments for pure cannabinoids and four independent experiments for BDS. Results are expressed as mean + SEM. p Value as compared to the activated control cells *, <0.05; **, <0.001; ***, <0.0001. (D) C57BL/6 splenocytes were activated for 4 h, stained with anti-CD69 antibodies and analyzed using flow cytometry. Data is summarized from three independent experiments. The differences of all treatments as compared to control are significant at 15 µg/mL, act: activated splenocytes, THC: D9 tetrahydrocannabinol, CBD: cannabidiol, BDS: Botanical Drug Substance.

Figure 1

The influence of pure CBD/THC and cannabis extracts on lymphocyte activation. Proliferation of CFSE-stained, CD3-activated splenocytes from C57BL/6 (A,C) and Balb/c (B) mice was analyzed on day 4 after activation using flow cytometry analysis. (A) Summary of six independent experiments. When Comparing all treatments to control, the differences are significant starting from 3 µg/mL. The differences between THC/CBD and THC BDS/CBD BDS are significant starting from 5 µg/mL. (B) Summary of three independent experiments. When Comparing all treatments to control, THC to CBD and THC BDS to CBD BDS, significant differences were observed starting from 5 µg/mL. (C) Flow cytometry analysis of activated C57BL/6 splenocytes stained with anti-CD4 and anti-CD8 antibodies. Data is summarized from six independent experiments for pure cannabinoids and four independent experiments for BDS. Results are expressed as mean + SEM. p Value as compared to the activated control cells *, <0.05; **, <0.001; ***, <0.0001. (D) C57BL/6 splenocytes were activated for 4 h, stained with anti-CD69 antibodies and analyzed using flow cytometry. Data is summarized from three independent experiments. The differences of all treatments as compared to control are significant at 15 µg/mL, act: activated splenocytes, THC: D9 tetrahydrocannabinol, CBD: cannabidiol, BDS: Botanical Drug Substance.

Figure 2

The influence of pure CBD/THC and cannabis extracts on cytokine secretion. Quantification of IL-17a (A), IL-10 (B), TNFα (C), and IL-5 (D) secretion from C57bl/6 splenocytes activated for 4 days which were treated with cannabinoids/cannabis, was performed using enzyme-linked immunosorbent assay on culture medium of activated cells. Data are summarized for five independent experiments for CBD BDS and six independent experiments for the other treatments. Results are expressed as mean + SEM. p Value as compared to activated control cells *, <0.05; **, <0.001; ***, <0.0001, act: activated splenocytes, THC: D9 tetrahydrocannabinol, CBD: cannabidiol, BDS: Botanical Drug Substance.

Figure 3

Receptors involved in THC and CBD’s effect on lymphocyte proliferation. (A) Proliferation of CFSE-stained, 4 days CD3-activated, splenocytes from CB2 knockout mice was analyzed using flow cytometry. Summary of four independent experiments. The differences of CBD, THC BDS and CBD BDS as compared to control are significant starting from 3 µg/mL. The differences of THC as compared to control are significant starting from 10 µg/mL. The differences of THC when compared to CBD is significant starting from 3 µg/mL. (B) The influence of PPARγ antagonist, GW9662, on CBD’s effect on lymphocyte activation. Proliferation of CFSE-stained, CD3-activated murine splenocytes was analyzed using flow cytometry analysis. Summary of eight independent experiments. p Value—samples were compared to act spl + CBD (right) or act spl (left). (C) Real time PCR analysis for the expression of cyp1a1 in activated splenocytes treated with THC or CBD. Summary of four independent experiments. Results are expressed as mean + SEM. p Value *, <0.05; **, <0.001;, act spl: activated splenocytes, THC: D9 tetrahydrocannabinol, CBD: cannabidiol, BDS: Botanical Drug Substance.

Figure 4

Cannabis/Cannabinoids administration to syngeneic BMT model. (A) Recipient C57BL/6 mice received lethal whole-body irradiation and were reconstituted with 8 × 10⁶ donor C57BL/6 bone marrow cells. Cannabis/cannabinoids were administered IP every other day, for 2 weeks from the day of transplantation. Blood samples for CBC were obtained once a week. Average lymphocyte counts in pure cannabinoid-treated groups (B) and BDS-treated groups (C) are presented. Average counts at different time points (left) and day 21 after transplantation counts (right). (D) Average platelet counts in pure cannabinoid-treated groups (left) and BDS-treated groups (right), day 14 after transplantation. Data are summarized from four independent experiments (lymphocytes) and three independent experiments (platelets). p Value *, <0.05; **, <0.001; ***, <0.0001. % of normal-% of the mean cell concentrations (cells/µL) in healthy C57BL/6 mice, non act: non-activated, act spl: activated splenocytes, THC: D9 tetrahydrocannabinol, CBD: cannabidiol, BDS: Botanical Drug Substance, ns: not significant.

Figure 5

The role of CB2 in lymphocyte recovery. (A) Recipient C57BL/6 mice underwent syngeneic BMT. CB2 reverse agonist, SR144528, was administered IP once a day for 1 week from the day of transplantation. Blood samples were obtained once a week. Average lymphocyte counts. (B) Syngeneic BMT from CB2 KO donor mice to C57BL/6 WT mice. Average counts at different time points (left) and day 21 after transplantation (right). (C) Syngeneic BMT from C57BL/6 WT donor mice to CB2 KO mice. Average counts at different time points (left) and day 21 after transplantation (right). Data are summarized from three independent experiments. p Value ***, <0.0001. % of normal-% of the mean cell concentrations (cells/µL) in healthy C57BL/6 mice. WT: wild type, CB2KO: CB2 knock out.

Figure 6

Cannabis/Cannabinoids administration for GVHD prophylaxis. (A) Recipient BALB/c mice received lethal whole-body irradiation and were reconstituted with 8 × 10⁶ donor C57BL/6 bone marrow cells and 2 × 10⁶ spleen cells. Cannabis/cannabinoids were administered IP every other day, for 2 weeks from the day of transplantation. The clinical condition of the mice was evaluated for up to 67 days after transplantation. (B) Survival curve. Differences between control and THC BDS as well as CBD BDS is significant. The difference between THC and THC BDS is also significant. Data are summarized from two independent experiments, six mice/group in each experiment. (C) Average GVHD score (Days 15–26). Differences between THC BDS/CBD BDS to the control group are significant. The same control group is shown in the left and right graph.