Role of ferroptosis in aGVHD-induced liver tissue damage: Mediation by Acsl4-regulated PUFA-pls Free

Introduction: Acute graft-versus-host disease (aGVHD) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation affecting diverse organs, including the skin, liver, and gastrointestinal (GI) tract. Patients with acute liver GVHD usually response poorly to the first-line treatment with steroids. Moreover, the response rate for acute liver GVHD to ruxolitinib, an oral Janus kinase (JAK) 1/2 inhibitor and the optimal treatment option for steroid-refractory aGVHD (SR-aGVHD), is only 26.7%. Although aGVHD is triggered by donor T cells, the pathogenesis of the destruction of the recipient tissues and organs may provide valuable clues for developing novel approaches to protect and regenerate injured tissues without impeding the donor immune system. Ferroptosis, a form of regulated cell death characterized by iron-dependent lipid peroxidation, has emerged as a critical player in various pathological processes, including liver injury and inflammatory diseases. The role of ferroptosis in aGVHD-induced liver injury remains unexplored.

Methods: To investigate whether ferroptosis play roles in liver tissue damage during development of aGVHD, we established a murine model of aGVHD, via tail vein transplantation of 5 × 10⁶T cell-depleted bone marrow (TCD BM) alone (Control groups), or with 1× 10⁶ splenic T cells (aGVHD groups) from C57BL/6 (B6) mice to BALB/c mice after being lethally irradiated. Serum iron, tissue iron, Malondialdehyde (MDA), Thiol, Superoxide dismutase (SOD) activity and 4-Hydroxynonenal (4-HNE) in liver tissue were detected as well as RNA-seq sequencing analysis and lipid metabolomics detection. Moreover, feasibility of a dietary intervention with low-iron diet (being initated one month before transplantation) and therapeutic potential of rosiglitazone, which has the ability to inhibit lipid peroxidation and ferroptosis, for aGVHD as well as liver tissue damage were evaluated.

Results: Mice exhibited typical aGVHD symptoms on day 21 post-HSCT, including alopecia, hunched posture, and reduced activity. Serum iron levels were significant higher in aGVHD mice than controls from day 21 post-HSCT, with significant iron accumulation as well as a marked increase levels of ferrous iron in aGVHD liver. However, no significant differences in iron levels were observed in the small intestine or spleen bwtween aGVHD mice and control mice. Intriguing, the iron-restricted diet failed to ameliorate aGVHD symptoms. Then, we conducted RNA-sequencing analysis for liver tissues obtained from aGVHD and control groups to explore alternative molecular mechanisms. KEGG pathway enrichment analysis revealed significant activation of the ferroptosis pathway, and Gene Set Enrichment Analysis (GSEA) further demonstrated a trend of upregulation of the ferroptosis pathway in aGVHD liver. Liver affected by aGVHD is marked by ferroptosis with upregulated Ptgs2, downregulated GPX4, elevated MDA and 4-HNE, and reduced Thiol levels and SOD activity, indicating the involvement of ferroptosis in the pathogenesis of aGVHD-liver. Regarding the ferroptosis mechanism pathway, Acsl4, a key enzyme in generating polyunsaturated fatty acid-phospholipids (PUFA-PLs) whose accumulation triggers ferroptosis, was upregulated in aGVHD liver. And lipid metabolomics analysis revealed decreased fatty acids and increased PUFA-PLs, indicating dysregulated lipid metabolism. Additionally, rosiglitazone, an Acsl4 inhibitor, alleviated hepatic injury and restoring ferroptosis indicators. Finally, treatment with ruxolitinib combined with rosiglitazone contributed to reduced GvHD severity as well as superior survival, which was better than treatment with ruxolitinib or rosiglitazone alone.

Conclusions: Collectively, our findings highlight the importance of ferroptosis in the pathogenesis of aGVHD-induced liver tissue damage, which is regulated by the Acsl4 mediated PUFA-PLs accumulation. Modulation of ferroptosis during transplantation may be an effective approach for preventing or treating acute liver GVHD. More importantly, we provide a scientific rationale for ruxolitinib combined with rosiglitazone in patients with SR-aGVHD involved liver.