Abstract

Background: The liver is a tolerising organ, to prevent continuous immune activation following the portal influx of antigens from the gut. Regulatory T cells (Treg) dampen inflammation and are important to prevent liver injury. We discovered that hepatocytes engulf and delete live Treg cells by enclysis, which was distinct from known cell-in-cell structure processes and thus can be targeted specifically (PMID: 31693899). Understanding how enclysis is modulated will enable us to toggle Treg cell frequencies in the liver and dampen inflammation or boost immunity as required. We investigated the role of Peroxisome proliferator-activated receptor alpha (PPARα) in the modulation of enclysis.

Methods: FoxP3, Tbet and PPARα were assessed in non-cirrhotic livers, AIH and MASH (n=30) by immunohistochemistry (IHC). To test pharmacological inhibitors of enclysis in vitro, we set up a fluorescence-based high content assay using Huh-7 cells and Jurkat cells as models for hepatic epithelia and CD4+ T cells respectively, and tested titrations of Clofibric Acid, Fenofibrate, and GW6471. Cell-in-cell structures were quantified using custom rule-based algorithms in collaboration with Celentyx Ltd. Hits were validated in concentration-response assays using confocal microscopy.

Results: Enclysis for Treg cells, but not Th1 cells, was reduced in AIH compared to MASH livers (p=0.0019). PPARα agonists Clofibric Acid (p=0.0156) and Fenofibrate (p=0.0156) reduced enclysis, and PPARα antagonist GW6471 increased enclysis in vitro (p=0.02). In non-cirrhotic livers, PPARα was expressed in periportal hepatocytes and less in mid-lobular hepatocytes. MASH was similar, with high PPARα in peri-fibrous regions and low in mid-lobular hepatocytes. Conversely, AIH liver explants showed reduced PPARα in all hepatocytes, consistent with increased enclysis (Fig.1).

Conclusion: The natural deletion of Treg cells by hepatocytes was increased in AIH compared to MASH, revealing enclysis as a new target for therapeutic intervention to dampen inflammation. The robust modulation of enclysis by PPARα agonist and antagonist compounds suggests that enclysis can be toggled pharmacologically to control Treg cell frequencies specifically in the liver. The reduction of hepatocyte PPARα expression in AIH compared to healthy livers and to MASH, proposes a role for aberrant PPARα expression in the pathogenesis of AIH, which needs further investigation.

Keywords: Enclysis, inflammation, regulatory T cells, fibrates, PPARalpha, fenofibrate, clofibrate, AIH, MASH