Metabolic reprogramming by itaconate: Haescs-derived therapy targets fibroblast GLUD1 to alleviate ocular graft versus host disease Free

Backgroud：Ocular chronic graft-versus-host disease (ocGVHD) is a significant complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT), with an incidence rate of 40-60%. Its primary manifestations include dry eye syndrome and visual impairment, with severe cases potentially progressing to blindness. Human amniotic epithelial stem cells (hAESCs) are characterized by low immunogenicity, robust immunosuppressive properties, and a strong safety profile, making them a promising candidate for ocGVHD treatment. In this study, we evaluated the safety and efficacy of hAESC-based eye drops for treating ocGVHD and explored the underlying mechanisms of their therapeutic effects.

Method: For ocGVHD mdoel, the BALB/C recipients were conditioned with a total body irradiation of 7.5Gy and transplanted with bone marrow cells and splenocytes from B10.D2 donors.The treatment groups received hAESCs eye drop with the concentration 2×10⁶four times a day from day7 to day 21 post-HSCT. Corneal fluorescein staining and laser confocal microscopy were utilized to evaluate corneal epithelial, endothelial, and nerve damage, as well as inflammatory cell infiltration, in ocGVHD patients and mice. Single cell RNA sequencing was employed to analyze the changes in cell types and numbers, as well as transcriptional variations, during the pathogenesis of cGVHD in ocGVHD.

Result: Here hAESCs eye drop significantly alleviated damage to the corneal epithelium, endothelium, and nerves, while also reducing inflammatory cell infiltration. To investigate the pathogenesis and pathological of ocular cGVHD, as well as the therapeutic mechanisms of hAESCs, we identified eight main cell clusters through single-cell sequencing. These clusters include corneal cells, limbal stem cells(LSC), corneal basal transient amplifying cells(TAC) ,iris cells, retinal cells, fibroblasts, and immune cells. LSCs are essential for maintaining corneal homeostasis and facilitating corneal repair. We performed further subclustering analyses of LSCs and identified two clusters. Compared with bone marrow (BM) and treatment groups, the LSC2 cluster was significantly elevated in the GVHD group and exhibited features of senescence and cell cycle arrest. Meanwhile, KEGG analysis demonstrated significant enrichment of GVHD and antigen presentation pathways in LSC2 cluster. To investigate the underlying reasons for the dysfunction of LSCs, we conducted a cell communication analysis and spatial transcription, which revealed a close interaction between fibroblasts and LSCs. Subclustering analysis of fibroblasts indicated significant activation of inflammation-associated fibroblasts in the cGVHD group, characterized by features of secreting inflammatory factors, including PTGS2 and MMP2. Spatial transcriptomic analysis revealed that PTGS2+ fibroblasts induces corneal stromal remodeling as well as facilitating inflammatory cell infiltration. Further analysis revealed significant enrichment of the IL-17 response pathway in PTGS2+ fibroblasts. Immunofluorescence and flow cytometry confirmed markedly elevated IL-17 levels in cGVHD corneas, primarily secreted by γδT cells. RNA sequencing and C₁₃ tracing metabolic flux analysis demonstrated that IL-17 induces metabolic reprogramming in fibroblasts by suppressing the tricarboxylic acid (TCA) cycle, mainly conversion of isocitrate to a-ketoglutarate(a-KG) . Consequently, fibroblasts exhibit reduced demethylation capacity and increased histone methylation. To investigate the therapeutic mechanism of hAESCs in ocGVHD, we observed that the hAESC supernatant retained therapeutic efficacy. The mass spectrometry analysis of the supernatant revealed a significant increase of itaconic acid. Using an itaconate-specific probe, we identified glutamate dehydrogenase 1 (GLUD1) as a potential target of itaconation. We further demonstrated that itaconate enhanced GLUD1's catalytic activity in generating a-KG from glutamate, reversing the IL-17-induced metabolic reprogramming in fibroblasts.Notably, treatment with HAESCs significantly inhibited the inflammatory activation of fibroblasts.

Conclusion: HAESCs secrete itaconic acid and inhibit the activation of fibroblasts into inflammation-associated fibroblasts in ocGVHD, thereby reducing the secretion of PGE2 from PTGS2+ fibroblasts. This modulation supports the restoration of LSC functionality, alleviates corneal inflammation, facilitates corneal repair, thus reducing ocGVHD.