Su1387 F1F0-ATPase Modulation Is Efficacious in Models in IBD and Induces Apoptosis of Human CCR9+ Gut-Tropic T-cells

Abstract

Background: Targeting immune metabolism may represent a novel approach for therapeutic intervention in autoimmunity. The mitochondrial F1F0-ATPase is the final complex in the electron transport chain. Small molecule, allosteric modulators of this complex cause increase in superoxide formation and apoptosis of susceptible cells. Because of their distinctive metabolic phenotype, chronically activated lymphocytes are preferentially sensitive to ATPase Modulator-induced apoptosis vs resting or acutely activated lymphocytes. In inflammatory bowel disease (IBD), mucosal lymphocytes are chronically activated, overexpress anti-apoptotic proteins and show resistance to activation-induced cell death (AICD). Thus, restoring lymphocyte AICD may be a potential therapeutic strategy. Here a set of F1F0-ATPase Modulators was evalulated in murine IBD models and in gut-tropic T cells from IBD patients. Methods: Acute IBD was induced by sensitizing mice with topical TNBS followed 7 days later by intracolonic TNBS challenge. ATPase Modulators were dosed orally on Day1 and Day2 after TNBS challenge. On Day4 mice were sacrificed and the colon was collected for histological analysis or isolation of lamina propria mononuclear cells which were analyzed for viability and inflammatory molecules by flow cytometry or RT-PCR. Peripheral blood mononuclear cells (PBMCs) from healthy donors or IBD patients were treated with ATPase Modulators for 20 hours. Cell death of activated, gut-tropic T cells (CD3+CCR9+) vs other T cells (CD3+ CCR9-) was detected by 7ADD uptake and Annexin V staining. Statistical analyses used unpaired Student's T test. Results: In acute murine TNBS-induced colitis, oral dosing at of ATPase Modulators attenuated weight loss, improved histologic assessments, decreased IFNγ and TNF production and significantly increased LP lymphocyte cell death (%viability: vehicle 15.8±3.3 vs LYC-51194 27.3±6.6, p<0.05). In PBMC from healthy subjects (n=24), ATPase Modulators were able to induce cell death but only in CCR9+ gut-tropic T cells (%cell death: CD3+CCR9+ vehicle 22.9±11.7 vs LYC-53661 56.6±11.7, p<0.0001; CD3+CCR9vehicle 0.47±0.51 vs LYC-53661 3.2±2.97). The induction of cell death in gut-tropic T cells was preceded by an increase in reactive oxygen species and was prevented by the ROS scavenger vitamin E. Similar experiments were performed with PBMC from IBD patients (CD n=15, UC n=15). LYC-51661, and similarly LYC-51194, induced cell death in CCR9+ T cells (%cell death: CD vehicle 17.9±13 vs LYC-51661 61.3±16.6 p< 0.0001; UC vehicle 18.8±9.5 vs LYC-51661 55.2±16.1 p< 0.0001) while sparing CD3+CCR9negative cells. Conclusions: The apparently selective effect on chronically activated lymphocytes may provide an advantage over current immunosuppressive treatments and suggest that ATPase Modulators may be a promising approach for treating IBD.