Highly lipophilic drugs such as the phytocannabinoids CBD and THC are good candidates for intestinal lymphatic transport28. In light of this, we have recently shown that the systemic bioavailability of CBD and THC can be significantly enhanced when administered orally in conditions facilitating intestinal lymphatic transport, specifically co-administration with dietary lipids18. The results of the current study indicate that the intestinal lymphatic transport of CBD and THC in rats was, indeed, enhanced following oral co-administration of lipids as denoted by the dramatic increase in the concentrations recovered in MLN (Fig. 1a,b). More importantly, the biodistribution of CBD and THC to lymphoid tissues in the intestinal lymphatic system (MLN) was substantially higher than the distribution to the largest lymphatic tissue in the central compartment, the spleen (Fig. 2a). It is also important to note the extremely high concentration of cannabinoids recovered in intestinal lymph fluid compared with plasma (Fig. 2b). Similar trends were previously reported for other lipophilic compounds, dexanabinol and PRS-211,220, when orally administered with LCT to rats19. Therefore, given our findings, we suggest that oral administration with dietary lipids has the potential to be an approach suitable for targeting delivery of CBD and THC to the intestinal lymphatic system. This targeting approach, as we have shown in our previous work, has the potential to increase the concentrations in plasma as well18.

The intestinal lymphatic system is the major host of immune cells. It has been proposed that the lymphatic system is an attractive target for immunomodulators whereby drugs can achieve high local concentrations and avoid systemic dilution28,29. This concept is supported by the fact that immune cells within the lymphatic system move more slowly and experience lower shear stress relative to those within the circulation30. In this study, the effect of CBD and THC on the proliferation of immune cells isolated from lymphoid tissues in the intestinal lymphatic system and spleen of rats was assessed. Both cannabinoids significantly inhibited the proliferation of mitogen-stimulated immune cells from MLN and spleen at concentrations that are highly unlikely to be achieved in rat plasma, but can be easily obtained in the intestinal lymphatic system after co-administration of lipids (Fig. 3). In addition, the results suggest that CBD has more potent anti-proliferative effect than THC (Fig. 3). Moreover, in this study, CBD and THC attenuated the expression of TNF-α and IFN-γ, key pro-inflammatory cytokines in the pathogenesis of autoimmune diseases31, by CD3+ T cells isolated from MLN and spleen (Fig. 4). In line with the anti-proliferative effect, cannabinoids also suppressed the frequency of inflammatory cytokines-expressing T cells at micromolar concentrations, which are achievable in the intestinal lymphatic system, but not in general circulation. To note, the reduced frequency of cytokines-expressing T cells can be potentially due to a decrease in the proliferation of cells. The immunosuppressive effects of CBD and THC on murine immune cells in the same concentration range were also reported elsewhere12,32. Intestinal lymphatic transport could be a potential mechanism of previously reported therapeutic effects of CBD and THC in murine models of autoimmune diseases in which the cannabinoids were administered orally with substantial amounts of lipids3,10.

To assess the immunomodulatory effect of cannabinoids on human cells, proliferation assays were performed on PBMCs isolated from venous blood of human volunteers. Our results demonstrate that CBD and THC, at relatively high concentrations, inhibit the proliferation of mitogen-stimulated PBMCs isolated from healthy volunteers (Fig. 5a,e). These results are consistent with previous reports13. Importantly, these concentrations would not be achievable in systemic blood circulation even if high oral doses of cannabinoids were consumed. Consroe et al.33 reported that the maximum plasma concentration recovered following repeated oral administration of 10 mg/kg/day CBD (in a small-amount lipid-based formulation) in humans was 25 ng/mL. This is considerably lower than the 5 μg/mL concentration required to significantly inhibit PBMCs proliferation in this study (Fig. 5a). In contrast, high doses of THC are limited by the psychotropic side effects reported for this drug34. Nevertheless, given the results obtained in this study, it is likely that oral administration of CBD and THC in humans in conditions facilitating intestinal lymphatic transport (with lipid dose in the range of 10 g35) can result in significant increases in intestinal lymphatic transport similar to what has been obtained for rats in this study. This is also supported by the fact that CBD and THC showed comparable association values with human and rat CM18. It should be noted that CM association in the enterocytes is a prerequisite for the intestinal lymphatic delivery of lipophilic compounds when orally administered with lipids18,19,36,37. Importantly, in the current study, CM-associated cannabinoids were still able to inhibit the proliferation of PBMCs isolated from healthy volunteers, showing similar effects to cannabinoids in solution (Fig. 5b,f). Therefore, our results suggest that the fact that cannabinoids are delivered to the intestinal lymphatic system in a CM-associated form would not reduce the immunomodulatory effects. A possible explanation for this maintained effect in CM-associated form is that lymphocytes possess lipoprotein lipase enzyme activity, which enables them to utilise fatty acids and triglycerides from CM38 and therefore to be exposed to cannabinoids.

In addition, our results indicate that PBMCs isolated from MS patients are more sensitive to the immunomodulatory effects of cannabinoids compared to PBMCs isolated from healthy volunteers (Fig. 5c,g). This could be related to the up-regulation of cannabinoid CB 2 receptors in the immune cells of MS patients39. These receptors are thought to be involved in the immunosuppressive effect of cannabinoids32,40. Interestingly, some other reports suggested that cannabinoids exert their immunosuppressive effect by CB 2 -independent mechanisms as well41. Another aspect demonstrated in this study is that cannabinoids have comparable anti-proliferative effects on PMBC isolated either from blood of cancer patients under chemotherapy regimens or from healthy volunteers (Fig. 5d,h). However, cancer patients on chemotherapy usually have low or low-borderline blood lymphocyte counts42, which was also the case in this study (Table 2). Therefore, there could be a potential effect of further immunosuppression in some people, such as cancer patients, when cannabinoids are administered in conditions that facilitate intestinal lymphatic transport.

It is widely accepted that TNF-α, IFN-γ, and IL-2 produced by T H 1 cells are actively involved in the pathogenesis of many autoimmune diseases43. Recently, T H 17 cells (which produce IL-17A) have emerged as a major factor in the pathogenesis of autoimmune diseases, as well as the contribution of GM-CSF to drive the inflammatory effects of T H 1744 and T H 145,46 cells. In this study, CBD and THC induced a profound decrease in the frequency of IL-2 and GM-CSF expressing T cells separated from healthy volunteers. This is consistent with the demonstration of a link between IL-2 production and GM-CSF induction47. CBD showed higher immunosuppressive effect than THC as manifested by the effect on TNF-α, IFN-γ, and IL-17A expressing T cells (Fig. 6a,d). For PBMCs isolated from MS patients, similar to the anti-proliferative effect, cannabinoids displayed more potent suppression of cytokine expression compared to cells from healthy volunteers (Fig. 6b,e). Yet, these effects were only observed at micromolar concentrations, consistent with previous reports13,48. Collectively, the effects of cannabinoids on lymphocyte proliferation and the frequency of cytokines-producing T cells explored in the current study suggest that targeting lipophilic cannabinoids to the intestinal lymphatic system for enhanced immunomodulatory effects in the treatment of autoimmune diseases could be a promising therapeutic approach. This approach could extend the therapeutic value of cannabinoids currently being used for symptomatic relief in MS patients25 to a disease modifying treatment, which could delay the progression of MS. Moreover, the results suggest that CBD has higher therapeutic effectiveness in autoimmune diseases compared with THC as it has more pronounced immunomodulatory effects, is devoid of psychotropic side effects, and is well tolerated in humans following acute and chronic intake of relatively high doses49.

On the other hand, adequate levels of the above-mentioned cytokines are important to maintain adaptive immune responses to fight infections50,51. In this study, a substantial decrease in the frequency of cytokines-expressing T cells has been demonstrated with cannabinoids in PBMCs isolated from cancer patients under chemotherapy regimen (Fig. 6c,f). This can potentially further deteriorate chemotherapy-induced immunosuppression in these patients. In addition, it has been reported that CBD and THC have inhibitory immunomodulatory effects on innate immune cells, particularly macrophages and natural killer cells (NK)52. It is well recognised that some cancer patients self-medicate and consume cannabis or cannabis-based medicinal formulations orally to alleviate nausea and vomiting associated with chemotherapy26. Given the results of this study, in this patients group, oral administration of immunosuppressive drugs such as cannabinoids in conditions facilitating intestinal lymphatic transport requires caution.

In summary, it has been demonstrated in this work that oral co-administration of cannabis or cannabis-based medicines with lipids results in extremely high levels of lipophilic cannabinoids in the intestinal lymphatic system and prominent immunomodulatory effects. Therefore, administering cannabinoids with a high-fat meal, as cannabis-containing food, or in lipid-based formulations has the potential to be a therapeutic approach to improve the treatment of MS, or indeed other autoimmune disorders. Whether cannabinoids as used in this study can also induce regulatory cytokines in addition to suppressing inflammatory ones as shown here remains to be determined. Further studies would be required to assess if both lymph and peripheral concentrations of cannabinoids have to be high for effective immunomodulation or only targeting to lymphatic system is sufficient. In addition, more work would have to be done to elucidate if cannabinoids could be used alone or as an adjuvant to other treatment. However, in immunocompromised patients, administration of cannabinoids in this way could potentially deepen the immunosuppressive effects. Further studies will be needed to evaluate the clinical significance of these effects.