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Cannabidiol Modulates the Immunophenotype and Inhibits the Activation of the Inflammasome in Human Gingival Mesenchymal Stem Cells

View Article: PubMed Central - PubMed

ABSTRACT

Human Gingival Mesenchymal Stem Cells (hGMSCs) are multipotential cells that can expand and differentiate in culture under specific and standardized conditions. In the present study, we have investigated whether in vitro pre-treatment of hGMSCs with Cannabidiol (CBD) can influence their expression profile, improving the therapeutic potential of this cell culture. Following CBD treatment (5 μM) for 24 h, gene expression analysis through Next Generation Sequencing (NGS) has revealed several genes differentially expressed between CBD-treated hGMSCs (CBD-hGMSCs) and control cells (CTR-hGMSCs) that were linked to inflammation and apoptosis. In particular, we have demonstrated that CBD treatment in hGMSCs prevented the activation of the NALP3-inflammasome pathway by suppressing the levels of NALP3, CASP1, and IL18, and in parallel, inhibited apoptosis, as demonstrated by the suppression of Bax. CBD treatment was also able to modulate the expression of the well-known mesenchymal stem cell markers (CD13, CD29, CD73, CD44, CD90, and CD166), and other surface antigens. Specifically, CBD led to the downregulation of genes codifying for antigens involved in the activation of the immune system (CD109, CD151, CD40, CD46, CD59, CD68, CD81, CD82, CD99), while it led to the upregulation of those implicated in the inhibition of the immune responses (CD47, CD55, CD276). In conclusion, the present study will provide a new simple and reproducible method for preconditioning hGMSCs with CBD, before transplantation, as an interesting strategy for improving the hGMSCs molecular phenotype, reducing the risk of immune or inflammatory reactions in the host, and in parallel, for increasing their survival and thus, their long-term therapeutic efficacy.

No MeSH data available.


Immunocytochemical staining for IL18, NALP3 and CASP1. CBD-hGMSCs showed a negative staining for IL-18, NALP3, and CASP1 compared to CTR-hGMSCs. Instead, hGMSCs-SR141716A and hGMSCs-AM630 showed a significant positive staining for IL-18, NALP3, and CASP1 compared to CBD-hGMSCs. The graph represented the densytometric quantitative analysis. For IL-18 CTR-hGMSCs vs. CBD-hGMSCs ***p < 0.001; SR141716A-hGMSCs vs. AM630-hGMSCs ****p < 0.0001. For NALP3 CTR-hGMSCs vs. CBD-hGMSCs **p < 0.01; SR141716A-hGMSCs vs. AM630-hGMSCs **p < 0.01. For CASP-1 CTR-hGMSCs vs. CBD-hGMSCs ***p < 0.001; SR141716A-hGMSCs vs. AM630-hGMSCs ***p < 0.001.
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Figure 2: Immunocytochemical staining for IL18, NALP3 and CASP1. CBD-hGMSCs showed a negative staining for IL-18, NALP3, and CASP1 compared to CTR-hGMSCs. Instead, hGMSCs-SR141716A and hGMSCs-AM630 showed a significant positive staining for IL-18, NALP3, and CASP1 compared to CBD-hGMSCs. The graph represented the densytometric quantitative analysis. For IL-18 CTR-hGMSCs vs. CBD-hGMSCs ***p < 0.001; SR141716A-hGMSCs vs. AM630-hGMSCs ****p < 0.0001. For NALP3 CTR-hGMSCs vs. CBD-hGMSCs **p < 0.01; SR141716A-hGMSCs vs. AM630-hGMSCs **p < 0.01. For CASP-1 CTR-hGMSCs vs. CBD-hGMSCs ***p < 0.001; SR141716A-hGMSCs vs. AM630-hGMSCs ***p < 0.001.

Mentions: The NALP3-inflammasome has been correlated with several inflammatory conditions and increasing evidences have indicated that the inhibition of the inflammasome could be a promising strategy for treating inflammatory disorders and also for preventing allograft-rejections in cell-based therapies (Foley, 2013; Coll et al., 2015; Shah et al., 2015). Our gene expression data have revealed a downregulation of several genes codifying for key players of the NALP3-inflammasome in CBD-hGMSCs. In order to confirm the ability of CBD to modulate the NALP3-inflammasome, we have performed immunocytochemistry and western blot analyses. Achieved immune-localization data have shown that NALP3, IL-18, and CASP1 were completely negative in CBD-hGMSCs, in a significant manner compared to CTR-hGMSCs (Figure 2). A representative negative control of immunocytochemistry is showed in Supplementary Material. The expression levels of CASP1 were also investigated by Western blotting and normalized on pro-CASP1, showing a reduced CASP1 expression in CBD-hGMSCs compared to CTR-hGMSCs (Figure 3).


Cannabidiol Modulates the Immunophenotype and Inhibits the Activation of the Inflammasome in Human Gingival Mesenchymal Stem Cells
Immunocytochemical staining for IL18, NALP3 and CASP1. CBD-hGMSCs showed a negative staining for IL-18, NALP3, and CASP1 compared to CTR-hGMSCs. Instead, hGMSCs-SR141716A and hGMSCs-AM630 showed a significant positive staining for IL-18, NALP3, and CASP1 compared to CBD-hGMSCs. The graph represented the densytometric quantitative analysis. For IL-18 CTR-hGMSCs vs. CBD-hGMSCs ***p < 0.001; SR141716A-hGMSCs vs. AM630-hGMSCs ****p < 0.0001. For NALP3 CTR-hGMSCs vs. CBD-hGMSCs **p < 0.01; SR141716A-hGMSCs vs. AM630-hGMSCs **p < 0.01. For CASP-1 CTR-hGMSCs vs. CBD-hGMSCs ***p < 0.001; SR141716A-hGMSCs vs. AM630-hGMSCs ***p < 0.001.
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Figure 2: Immunocytochemical staining for IL18, NALP3 and CASP1. CBD-hGMSCs showed a negative staining for IL-18, NALP3, and CASP1 compared to CTR-hGMSCs. Instead, hGMSCs-SR141716A and hGMSCs-AM630 showed a significant positive staining for IL-18, NALP3, and CASP1 compared to CBD-hGMSCs. The graph represented the densytometric quantitative analysis. For IL-18 CTR-hGMSCs vs. CBD-hGMSCs ***p < 0.001; SR141716A-hGMSCs vs. AM630-hGMSCs ****p < 0.0001. For NALP3 CTR-hGMSCs vs. CBD-hGMSCs **p < 0.01; SR141716A-hGMSCs vs. AM630-hGMSCs **p < 0.01. For CASP-1 CTR-hGMSCs vs. CBD-hGMSCs ***p < 0.001; SR141716A-hGMSCs vs. AM630-hGMSCs ***p < 0.001.
Mentions: The NALP3-inflammasome has been correlated with several inflammatory conditions and increasing evidences have indicated that the inhibition of the inflammasome could be a promising strategy for treating inflammatory disorders and also for preventing allograft-rejections in cell-based therapies (Foley, 2013; Coll et al., 2015; Shah et al., 2015). Our gene expression data have revealed a downregulation of several genes codifying for key players of the NALP3-inflammasome in CBD-hGMSCs. In order to confirm the ability of CBD to modulate the NALP3-inflammasome, we have performed immunocytochemistry and western blot analyses. Achieved immune-localization data have shown that NALP3, IL-18, and CASP1 were completely negative in CBD-hGMSCs, in a significant manner compared to CTR-hGMSCs (Figure 2). A representative negative control of immunocytochemistry is showed in Supplementary Material. The expression levels of CASP1 were also investigated by Western blotting and normalized on pro-CASP1, showing a reduced CASP1 expression in CBD-hGMSCs compared to CTR-hGMSCs (Figure 3).

View Article: PubMed Central - PubMed

ABSTRACT

Human Gingival Mesenchymal Stem Cells (hGMSCs) are multipotential cells that can expand and differentiate in culture under specific and standardized conditions. In the present study, we have investigated whether in vitro pre-treatment of hGMSCs with Cannabidiol (CBD) can influence their expression profile, improving the therapeutic potential of this cell culture. Following CBD treatment (5 &mu;M) for 24 h, gene expression analysis through Next Generation Sequencing (NGS) has revealed several genes differentially expressed between CBD-treated hGMSCs (CBD-hGMSCs) and control cells (CTR-hGMSCs) that were linked to inflammation and apoptosis. In particular, we have demonstrated that CBD treatment in hGMSCs prevented the activation of the NALP3-inflammasome pathway by suppressing the levels of NALP3, CASP1, and IL18, and in parallel, inhibited apoptosis, as demonstrated by the suppression of Bax. CBD treatment was also able to modulate the expression of the well-known mesenchymal stem cell markers (CD13, CD29, CD73, CD44, CD90, and CD166), and other surface antigens. Specifically, CBD led to the downregulation of genes codifying for antigens involved in the activation of the immune system (CD109, CD151, CD40, CD46, CD59, CD68, CD81, CD82, CD99), while it led to the upregulation of those implicated in the inhibition of the immune responses (CD47, CD55, CD276). In conclusion, the present study will provide a new simple and reproducible method for preconditioning hGMSCs with CBD, before transplantation, as an interesting strategy for improving the hGMSCs molecular phenotype, reducing the risk of immune or inflammatory reactions in the host, and in parallel, for increasing their survival and thus, their long-term therapeutic efficacy.

No MeSH data available.