Up-regulation of immunomodulatory effects of mouse bone-marrow derived mesenchymal stem cells by tetrahydrocannabinol pre-treatment involving cannabinoid receptor CB2

Abstract

Chronic pain is commonly and closely correlated with inflammation. Both cannabinoid signaling and mesenchymal stem cells (MSCs) have been demonstrated to reduce inflammatory pain. Although cannabinoid signaling is essential for mesenchymal stem cell survival and differentiation, little is known about its role in modulatory effect of MSCs on inflammation and pain sensitivity. Here we showed that mouse bone-marrow derived MSCs (BM-MSCs) expressed both cannabinoid receptor type 1 and 2 (CB1 and CB2). CB2 expression level in BM-MSCs increased with their maturation. In addition, we found that tetrahydrocannabinol (THC) activated CB2 receptor and ERK signaling, consequently enhancing the modulation of MSCs on inflammation-associated cytokine release from lipopolysaccharides-stimulated microglia. Consistent with in vitro data, THC pretreatment enhanced the immunomodulatory effects of BM-MSC on thermal hyperalgesia and mechanical allodynia in chronic constriction injury model, by decreasing the release of pro-inflammation cytokines. Our study revealed the crucial role of THC in promoting the immunomodulatory effects of MSCs and proposed a new strategy to alleviate pain based on stem cells therapy.

Keywords: Immune response; Immunity; Immunology and Microbiology Section; cannabinoid receptor; inflammation; mesenchymal stem cells; pain; tetrahydrocannabinol.

Figure 1. Phenotypic characterization of mouse BM-MSCs

a. Cultured BM-MSCs after initial seeding for 5 days. Scale bar, 100 μm. b.-f. Flow cytometry analysis on BM-MSCs shows the majority of cells are CD44⁺, Sca-1⁺, CD34⁻, CD14⁻ and CD45⁻, which are characteristic phenotypes of mouse BM-MSCs. g. BM-MSCs was capable of differentiating into osteogenic and adipogenic lineages. Cells were stained by Alizarin red staining for osteogenic differentiation and Oil red staining for adipogenic differentiation. Scale bar, 500 μm.

Figure 2. Expression of CB1 and CB2 receptors in mouse BM-MSCs

a.-b. RT-PCR analysis of the mRNA levels of CB1 and CB2 receptors in MSCs from passage 0 (P0) to passage 7 (P7). Gene GAPDH was used as control. c. Western blot analysis of CB1 and CB2 protein expression in MSCs from P0 to P7. GAPDH was used as control. d.-e. Relative CB1 and CB2 protein expression in the cells normalized to those of P0. Data were presented as mean ± SEM. *p < 0.05 and **p < 0.01 versus P0 group.

Figure 3. THC treatment affects viability, proliferation and immunomodulatory effects of BM-MSCs

a., b. THC treatment for 24 h at low concentration (0.5, 1 or 2 μM) had no effect on the viability and proliferation of BM-MSCs, while THC at higher concentrations (5 or 10 μM) significantly decreased cell viability and proliferation. Cell viability was measured by MTT assay. Proliferation was examined by CCK-8 assay. **p < 0.01 versus control (no THC treatment). c. Experimental diagram for MSC-CM collection and the following culture of primary microglia. Pre-treatment with 1 μM THC for 24 h significantly improved the immunomodulatory properties of BM-MSCs in primary microglia, as suggested by the lowest release of inflammatory cytokines in microglia when stimulated by LPS (100 ng/Ml, 24 h), including TNF-α d. IL-1β e. IL-6 f., IL-8 g. and IL-10 h. Data were presented as mean ± SEM. **p < 0.01 versus control group (the first column in d-h), #p < 0.05 and ##p < 0.01 versus LPS group (the second column in d-h).

Figure 4. Activation of CB2 receptors by THC stimulates IL-10 release and the ERK pathway in BM-MSCs

THC treatment (1 μM, 24 h) induced significant elevation of the anti-inflammatory IL-10 release a., intracellular IL-10 protein expression b., c., and stimulated p-ERK1/2 expression d., e. The release of IL-10 from BM-MSCs was measured by ELISA. The protein expressions of intracellular IL-10 and p-ERK1/2 were analyzed by western blot analysis and normalized to GAPDH. Meanwhile, THC treatment significantly caused CB2 receptor expression in BM-MSCs f., g. CB2 antagonist, AM 630, greatly blocked the THC-induced elevation of IL-10 release, intracellular IL-10 expression, p-ERK1/2 expression and CB2 expression, while AM251 (CB1 antagonist) failed to. Data were presented as mean ± SEM. *p < 0.05 and **p < 0.01 versus control group, ##p < 0.01 versus THC group.

Figure 5. THC-pre-treated BM-MSCs significantly enhances the effects of BM-MSCs on the thermal hyperalgesia

a. and mechanical allodynia b. in neuropathic mice (CCI model). BM-MSCs were pre-treated with 1 μM THC for 24 h, then administrated by intravenously injection at day 0 (indicated by the arrow). Thermal hyperalgesia was measured by Plantar test and mechanical allodynia was measured by Dynamic Plantar Aesthesiometer. Data were presented as mean ± SEM. **p < 0.01 versus CCI+veh group, ##p < 0.01 versus CCI+MSC (THC pre-treated) group.

Figure 6. TNF-α (a and e), IL-1β (b and f), IL-6 (c and g) and IL-10 (d and h) mRNA expression and protein content in ipsilateral sciatic nerve of experimental mice 7 days after MSC administration

The cytokine mRNA levels were determined by RT-PCR with GAPDH as a control and were normalized to those of sham+veh group. Cytokine protein contents were measured by ELISA and normalized to sample total protein. Data were presented as mean ± SEM. **p < 0.01 versus sham+veh group, ##p < 0.01 versus CCI+veh group, +p < 0.05 and ++p < 0.01 versus CCI+MSC group.

Figure 7. THC pre-treatment has no impact on the percentage of GFP-labeled BM-MSCs in the sciatic nerve 24 h after administration

Green indicated the GFP-labeled BM-MSCs, while blue indicated the nucleus. Data were presented as mean ± SEM. N.S indicates no significance.