Differential metabolic pathways underlie THC- and CBD-mediated inhibition of B-cell activation in both young and aged mice

Abstract

Objective: B lymphocytes play a crucial role in immunity but also contribute to the pathogenesis of various diseases. Cannabis plants produce numerous biologically active compounds, including cannabinoids. The two most studied phytocannabinoids are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD). These cannabinoids exert diverse and potent biological effects primarily through the endocannabinoid system (ECS), which also plays a key role in mature B-cell function. Both the immune system and the ECS undergo age-related changes that lead to a clinically significant decline in function.

Methods: This study compares the effects of THC and CBD on B-cell activity in young and aged mice. Murine B lymphocytes were activated using lipopolysaccharide (LPS) and interleukin-4 (IL-4), and the impact of cannabinoid treatments was assessed in terms of cell phenotype, proliferation, antibody secretion, tumor necrosis factor-alpha (TNFα) secretion, extracellular signal-regulated kinase (ERK) phosphorylation, and the cellular metabolome.

Results: Both THC and CBD exhibited dose-dependent inhibitory effects on B-cell activation in young and aged mice. However, we show here, for the first time, that the treatments induce distinct metabolic profiles. Although some metabolites, such as glucose-6-phosphate, pentose phosphate pathway (PPP) and nucleotide metabolites, were reduced by both cannabinoids, THC selectively reduced the levels of a distinct set of amino acids, while only CBD increased the levels of Citrulline and Allantoin. Additionally, the effects of THC and CBD differed between young and aged B cells, suggesting that age-related changes in the ECS may influence cannabinoid sensitivity.

Conclusions: These findings provide insights into the distinct mechanisms by which THC and CBD regulate immune activation and may open the door for investigating the mechanisms behind cannabinoids effects on the immune system. They also highlight the need for further research into phytocannabinoid-based therapies, particularly in age-specific contexts. Given the immunoregulatory properties of cannabinoids, especially CBD, tailored therapeutic strategies may enhance their clinical applications.

Keywords: B lymphocytes; PDL 1; aged; cannabidiol (CBD); cannabinoid; immune activation; metabolome; Δ9-tetrahydrocannabinol (THC).

Figure 1

The effect of CBD and THC on the phenotype of activated spleen derived B-cells. Splenocytes were obtained from C57bl/6 female mice and activated for 24h with LPS+IL4, in the presence of cannabinoid treatments. Cell surface expression levels of CD69 (A, n=6 per group) and PDL1 (B, n=6 per group) on B220 positive cells were determined by flow cytometry. For each mouse, the results are calculated as % of activated sample’s GM. Results expressed as mean +SEM. p value as compare to activated control cells *<0.05; **<0.01; ***<0.001. NA, non-activated; ACT, activated cells; NA, non-activated cells; THC, D9 tetrahydrocannabinol; CBD, cannabidiol.

Figure 2

The effect of CBD and THC on activated B-cell function. Isolate B-cells were activated with LPS+IL4, in the presence of cannabinoid treatments (5µg/ml). (A) TNF alpha levels in the culture medium after 24h activation, n=6 per group. (B) Proliferation of CFSE-stained cells after 96h activation, n=6. (C) IgG levels in the culture medium after 72h activation, n=6 per group. (D) ERK, p-ERK and GAPDH levels were detected by western blot analysis of Isolated B-cells after 4h incubation with IL4 followed by 30 minutes activation with LPS, n=4 per group. Results are expressed as mean +SEM. p value as compare to activated control cells *<0.05; **<0.01; ***<0.001. NA, non-activated; ACT, activated cells; NA, non-activated cells; THC, D9 tetrahydrocannabinol; CBD, cannabidiol; TNFa, Tumor necrosis factor alpha; IgG, Immunoglobulin G; pERK, phosphorylated ERK; T-ERK, total ERK. n.s., not significant.

Figure 3

Alteration of WBC populations due to reduced lymphocyte counts and elevated granulocyte counts, in old female mice. Blood samples were collected from young (2–3 month old) and aged (18–20 month old) C57BL/6 mice. (A) Complete blood counts (CBC). (B) Cell surface expression levels of PDL1 (n=12 per group) and PD1 (n=6 per group) on B-Cells (B220+), CD4 or CD8 positive cells were determined by flow cytometry. Results are expressed as mean +SEM. p value young as compare to old cells **<0.01; ***<0.001.

Figure 4

B lymphocytes from old mice are responsive to cannabinoid treatments. Splenocytes were obtained from young and aged C57BL/6 mice. For A and B cells were activated for 24h with LPS+IL4, in the presence of cannabinoid treatments (5µg/ml). Cell surface expression levels of CD69 (A, n=12 per group) and PDL1 (B, n=8 per group) on B220 positive cells were determined by flow cytometry. C, Isolated B-cells were activated with LPS+IL4 for 24h, in the presence of cannabinoid treatments. TNF alpha levels in the culture medium were detected by ELISA, n=6 per group. D. splenocytes were activated for 72h. IgG levels in the culture medium were measured by ELISA, n=7 per group. Results are expressed as mean +SEM. Results in the right side of the figure are calculated as % of activated sample’s GM, for each mouse. Results expressed as mean +SEM. Relative p value as compare to activated control cells **<0.01; ***<0.001. p value THC compare to CBD treated cells #,<0.05; ##,<0.01; NA-non-activated ACT–activated cells, NA, non-activated cells; THC, D9 tetrahydrocannabinol; CBD, cannabidiol; TNFa, Tumor necrosis factor alpha; IgG, Immunoglobulin G. n.s., not significant.

Figure 5

THC and CBD treated activated B lymphocytes show different metabolite composition. LCMS metabolomics analysis of isolated B-cells, activated with LPS+IL4, in the presence of cannabinoid (5µg/ml), n=7 per group, including 4 young and 3 aged mice. Mean of each treatment is marked. p value as compare to activated control cells *<0.05; **<0.01; ***<0.001. ACT, activated cells; THC-D9, tetrahydrocannabinol; CBD, cannabidiol.