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VDR ligands modulate peptide-specific recall responses from MOG-immunized mice. (a) Compound A inhibits the proliferation of MOG-specific splenocytes. Splenocytes from groups (n = 5) of MOG-immunized mice were harvested at day 28 and analyzed ex vivo for proliferative responses to MOG at the concentrations indicated. Cells were cultured in triplicate in 96-well plates for 60 hours, and proliferation was measured by 3H-thymidine incorporation during the final 8 hours of culture. Values represent the mean ± SE of triplicate for each peptide concentration. Ovalbumin (OVA) peptide was used as specificity control since the mice were immunized with MOG peptide. Vehicle group consisted of MOG immunized mice treated with vehicle (sesame seed oil). CFA group was mock immunized with CFA only (without MOG peptide) and was not treated with any ligands. (b) Effect of VDR ligands on cytokine elaboration in MOG-immunized animals. For dendritic cell IL-10 production, CD11c+ cells were purified on day 28 from splenocytes obtained from MOG-immunized animals. The effect of VDR ligands on IL-10 levels from splenic CD11c+ cultures stimulated for 24 hours with 100 ng/mL LPS is shown. VDR ligands decreased IFN-γ elaboration from splenocyte cultures stimulated for 48 hours with 80 μg/mL MOG peptide. IL-10 and IFN-γ protein levels were measured by ELISA.
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fig9: VDR ligands modulate peptide-specific recall responses from MOG-immunized mice. (a) Compound A inhibits the proliferation of MOG-specific splenocytes. Splenocytes from groups (n = 5) of MOG-immunized mice were harvested at day 28 and analyzed ex vivo for proliferative responses to MOG at the concentrations indicated. Cells were cultured in triplicate in 96-well plates for 60 hours, and proliferation was measured by 3H-thymidine incorporation during the final 8 hours of culture. Values represent the mean ± SE of triplicate for each peptide concentration. Ovalbumin (OVA) peptide was used as specificity control since the mice were immunized with MOG peptide. Vehicle group consisted of MOG immunized mice treated with vehicle (sesame seed oil). CFA group was mock immunized with CFA only (without MOG peptide) and was not treated with any ligands. (b) Effect of VDR ligands on cytokine elaboration in MOG-immunized animals. For dendritic cell IL-10 production, CD11c+ cells were purified on day 28 from splenocytes obtained from MOG-immunized animals. The effect of VDR ligands on IL-10 levels from splenic CD11c+ cultures stimulated for 24 hours with 100 ng/mL LPS is shown. VDR ligands decreased IFN-γ elaboration from splenocyte cultures stimulated for 48 hours with 80 μg/mL MOG peptide. IL-10 and IFN-γ protein levels were measured by ELISA.

Mentions: To determine if compound A can modulate antigen T cell function in EAE, total splenocytes from diseased mice were stimulated ex vivo with either MOG peptide or ovalbumin peptide at the indicated concentrations, and T cell proliferation was measured by 3H-thymidine incorporation. Compared with vehicle-treated mice, in vivo treatment with compound A suppressed the specific recall response to the encephalitogenic MOG peptide used in the EAE model (Figure 9(a)). The recall response of MOG-immunized animals was specific for the MOG peptide and was not observed for ovalbumin (OVA) peptide (Figure 9(a)). Furthermore, both 1,25-(OH)2D3 and compound A decreased Th1 cytokine IFN-γ production in MOG-stimulated splenocytes (Figure 9(b)). Interestingly, both 1,25-(OH)2D3 and compound A also induced IL-10 cytokine production in isolated CD11c+ dendritic cells stimulated with 100 ng/mL LPS (Figure 9(b)), indicating that compound A modulated immune response in vivo in autoimmune pathogenic conditions. Th17 cells have recently been demonstrated to be the essential pathogenic cells involved in EAE model. In order to test whether 1,25-(OH)2D3 and compound A had the direct effect on Th17 differentiation or Th17 secretion, we performed Th17 differentiation assay in vitro in the presence of these compounds. As shown in Figure 10, both 1,25-(OH)2D3 and compound A significantly inhibited both IL-17 and IL-22 expression, indicating that Th17 differentiation was efficiently inhibited by these compounds. Interestingly, once Th17 cells were fully differentiated, the restimulation of these differentiated Th17 cells by anti-CD3 mAb to produce IL-17 and IL-22 was only slighly affected by these compounds (data not shown), indicating that VDRMs were mainly involved in Th17 differentiation stage.

A Nonsecosteroidal Vitamin D Receptor Modulator Ameliorates Experimental Autoimmune Encephalomyelitis without Causing Hypercalcemia

Na S, Ma Y, Zhao J, Schmidt C, Zeng QQ, Chandrasekhar S, Chin WW, Nagpal S - Autoimmune Dis (2011)

Bottom Line: However, the side effect of 1,25-(OH)(2)D(3) and its synthetic secosteroidal analogs is hypercalcemia, which is a major impediment in their clinical development for autoimmune diseases.Hypercalcemia develops as a result of the action of VDR agonists on the intestine.Here, we describe the identification of a VDR modulator (VDRM) compound A that was transcriptionally less active in intestinal cells and as a result exhibited less calcemic activity in vivo than 1,25-(OH)(2)D(3).

Affiliation: Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA.

ABSTRACT
Vitamin D receptor (VDR) agonists are currently the agents of choice for the treatment of psoriasis, a skin inflammatory indication that is believed to involve an autoimmune component. 1,25-dihydroxyvitamin D3 [1,25-(OH)(2)D(3)], the biologically active metabolite of vitamin D, has shown efficacy in animal autoimmune disease models of multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and type I diabetes. However, the side effect of 1,25-(OH)(2)D(3) and its synthetic secosteroidal analogs is hypercalcemia, which is a major impediment in their clinical development for autoimmune diseases. Hypercalcemia develops as a result of the action of VDR agonists on the intestine. Here, we describe the identification of a VDR modulator (VDRM) compound A that was transcriptionally less active in intestinal cells and as a result exhibited less calcemic activity in vivo than 1,25-(OH)(2)D(3). Cytokine analysis indicated that the VDRM not only modulated the T-helper cell balance from Th1 to Th2 effector function but also inhibited Th17 differentiation. Finally, we demonstrate that the oral administration of compound A inhibited the induction and progress of experimental autoimmune encephalomyelitis in mice without causing hypercalcemia.

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