Abstract
Introduction
Retinoic Acid Related Orphan Nuclear Receptor gamma T (RORγT) is a lineage specifying transcription factor for IL-17 expressing cells, which may contribute to the pathogenesis of Inflammatory Bowel Disease (IBD). VPR-254 is a selective in vitro inhibitor of RORγT.
Aims
The main goals of our study were twofold: (1) To determine if ex vivo treatment with VPR-254 reduced relevant cytokine (IL-17 and IL-21) secretion from colonic strips of mice with colitis; (2) To determine if treatment of mice with VPR-254 attenuated parameters of colitis, using three murine IBD models.
Methods
VPR-254 was evaluated ex vivo in a colonic strip assay, using tissue from mice with Dextran sulfate sodium (DSS)-induced colitis. In vivo, VPR-254 was evaluated for efficacy in DSS, Trintirobenzenesulfonic acid (TNBS) and Anti-CD40 antibody-induced murine models of colitis.
Results
VPR-254 reduced the production of key pro-inflammatory cytokines (e.g., IL-17) in ex vivo and in vivo models of colitis. This small molecule inhibitor of RORγT also improved various morphometric and histological parameters associated with three diverse murine models of IBD.
Conclusion
Our results support the concept that an inhibitor of ROR-gamma T may have potential utility for the treatment of IBD.
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Acknowledgements
This work was supported by: NIH SBIR Grant numbers 1R43 DK099896 & 2R44 DK098896. We would also like to acknowledge the research personnel at Antibodies Incorporated (Davis, CA) for their help with the animal husbandry associated with our ex vivo DSS colitis study. We also aknowledge the technical contributions of Laura Lingardo and Chris Buhr (Visionary Pharmaceuticals, San Diego, CA).
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10787_2019_643_MOESM1_ESM.tif
Supplemental Fig. 1A and 1B: Panel A shows the antagonist activity of VPR-254 against RORγt Ligand Bindind Domain (LBD) in a GAL4 luciferase reporter assay. VPR-254 inhibition of RORγt driven (open red circle, EC50 = 0.28 µM) and VP-16 driven (open blue diamond, EC50 ≥ 10 µM) luciferase activity are shown. Panel B shows that VPR-254 specifically inhibits the production of IL-17 and not that of other inflammatory cytokines, TNF-α and IFN-γ. Error bars represent the standard deviation seen between 3 replicate measurements of cytokine in cell supernatants. (TIF 127 kb)
10787_2019_643_MOESM2_ESM.tif
Supplemental Fig. 2: Panel A illustrates a negative colonic Immunohistochemistry (IHC) control from a histology slide an animal treated with VPR 254/CD40 antibody. The slide was not exposed to the primary antibody for IL-17. No brown immunostaining is evident in the figure. Similar negative control staining was obtained for GM-CSF (not shown). Panel B shows an example of the grid overlay method that was used to detect the area of immunostaining for IL-17. The picture was taken from a colonic histology slide of a Naïve (untreated) mouse. The area of mucosal GM-SCF staining (34.3%) was calculated from 12 boxes with detectable brown staining divided by 35 evaluable colonic mucosal boxes. The same type of method was used to detect the area of IL-17 immunostaining from colonic histology slides (image(s) not shown). (TIF 1191 kb)
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Fitzpatrick, L.R., Small, J., O’Connell, R. et al. VPR-254: an inhibitor of ROR-gamma T with potential utility for the treatment of inflammatory bowel disease. Inflammopharmacol 28, 499–511 (2020). https://doi.org/10.1007/s10787-019-00643-z
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DOI: https://doi.org/10.1007/s10787-019-00643-z