Abstract 255: Peroxidasin deficiency recruits pro-healing macrophages into the liver and inhibits NAFLD progression to HCC
Authors: Sojoodi Mozhdeh , Barrett Stephen C. , Erstad Derek J. , Salloum Shadi , Zhu Shijia , Qian Tongqi , Colon Selene , Gale Eric , Jordan Veronica Clavijo , Wang Yongtao , Li Shen , Lanuti Michael , Zukerberg Lawrence , Caravan Peter , Hoshida Yujin , Chung Raymond T. , Bhave Gautam , Lauer Georg M. , Fuchs Bryan C. , Tanabe Kenneth K. 
Published: June 2022

Introduction: During liver fibrosis, tissue repair mechanisms replace necrotic tissue with highly stabilized extracellular matrix (ECM) proteins. ECM stabilization influences the speed of tissue recovery. Here, we used a mouse model of nonalcoholic fatty liver disease (NAFLD) to study the function of peroxidasin (PXDN), a peroxidase that uses H2O2 to cross-link collagen IV, during liver fibrosis progression to hepatocellular carcinoma (HCC).

Method: Pxdn-/- and Pxdn+/+ mice were fed with a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) for 16 weeks to create a NAFLD-HCC preclinical model. Liver histology, collagen content, flow cytometry, immunostaining of immune cells, RNA-seq, and liver function tests were analyzed. In vivo imaging of liver reactive oxygen species (ROS) was performed using a redox-active iron complex, Fe-PyC3A.

Results: Using Fe-PyC3A as an MRI contrast agent, we detected a higher content of ROS in Pxdn-/- livers (healthy) that was not necessarily directly toxic but could activate hypoxia-related molecular pathways. Genome-wide expression analysis of liver tissue and differential gene expression (DGE) combined with Gene Ontology (GO) analysis identified significant upregulation of genes associated with hypoxia and TNFα signaling pathways already in Pxdn-/- sham livers (without injury). In addition, we observed an upregulation of genes involved in the innate immune response, leukocyte activation, and chemotaxis. After 16 weeks of CDAHFD, gross analysis of collected liver showed no HCC nodule formation in Pxdn-/- mice while 60% of the WT mice had HCC tumors. Collagen deposition showed less collagen accumulation in Pxdn-/- mice. Flow cytometry of macrophages showed Pxdn-/- mice had increased pro-healing M2 macrophages recruitment in early- and mid-stage NAFLD (4 weeks and 8 weeks on CDAHFD) compared to WT controls. In addition, we observed a significant decrease in the number of CD3+ T cells and CD8+ cytotoxic T cells in the late-stage of NAFLD in Pxdn KO mice. DGE analysis revealed that IL-12 is highly expressed in Pxdn-/- injured livers. Additionally, multiple other T cell-related molecules such as IL-10, IL-6, CCL2, IL-7, and CD4 were elevated in Pxdn-/- injured liver. Elevation of these cytokines is an indicator for higher recruitment of pro-healing and anti-HCC macrophage to the site of injury.

Conclusion: Our findings demonstrate that PXDN deficiency is associated with the induction of the hypoxia and TNFα signaling pathways and the recruitment of pro-healing and anti-HCC macrophages to the liver. This results in significantly decreased collagen stabilization during liver fibrosis and accelerates fibrosis reversal. In addition, recruited macrophages-controlled T cell response and inhibited HCC formation in Pxdn-/- mice.

Citation Format: Mozhdeh Sojoodi, Stephen C. Barrett, Derek J. Erstad, Shadi Salloum, Shijia Zhu, Tongqi Qian, Selene Colon, Eric Gale, Veronica Clavijo Jordan, Yongtao Wang, Shen Li, Michael Lanuti, Lawrence Zukerberg, Peter Caravan, Yujin Hoshida, Raymond T. Chung, Gautam Bhave, Georg M. Lauer, Bryan C. Fuchs, Kenneth K. Tanabe. Peroxidasin deficiency recruits pro-healing macrophages into the liver and inhibits NAFLD progression to HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 255.