Evaluating the immunomodulatory role of celastrol in murine acute graft versus host disease after allogeneic hematopoietic stem cell transplantation

Introduction: Allogenic hematopoietic stem cell transplantation (HSCT) is used to treat various hematological malignancies. Graft versus host disease (GVHD) is a serious complication arising after HSCT and acts as a major limiting factor of bone-marrow transplantation. Acute GVHD (aGVHD) primarily targets the gastrointestinal tract, skin, and liver, driven by alloreactive T-cells. We hypothesize that Celastrol, a bioactive triperpenoid possessing anti-inflammatory and anticancer properties, can mitigate aGVHD symptoms. Methods: A well-defined murine aGVHD MHC-mismatched model was used, recipient (C57BL/6) mice received total body irradiation of 850 cGy and received bone marrow and splenocytes from either C57BL/6 (syngeneic) or BALB/c (allogeneic). They were then treated with Celastrol (2mg/kg) or vehicle control from day 1. On day 7, the animals were scored for clinical GVHD, euthanized, and their organs were collected for further analysis. The gut and colon pathology score was evaluated to assess inflammation, apoptosis, and disease severity. Results: The clinical GVHD score showed a significant reduction in disease burden (p < 0.0001) in celastrol-treated mice compared to the allogenic control. The colon pathology scores also showed a significant decrease in gut injury in celastrol-treated (p < 0.01) mice compared to allogenic control. To assess the anti-inflammatory effect of Celastrol, we performed CD3+ staining of colon tissue, which showed significantly reduced (p < 0.05) T-cell infiltration in Celastrol-treated mice compared to allogenic control. A significant reduction in TUNEL-positive apoptotic cells (p < 0.01) was observed in the Celastrol-treated mice compared to allogenic recipients. To further validate and identify the pathways involved in pathophysiology, we performed RNASeq analysis. RNA-seq analysis of the colon identified differentially expressed genes (972 upregulated; 536 downregulated) between the Celastrol-treated and allogenic control group. Furthermore, gene set enrichment analysis revealed significant downregulation of genes Mpeg1, Lyz2, CX3CR1, Laptm5, and Pld4, which are relevant to the involvement of GM-CSF in the Celastrol-treated group. This suggests that GM-CSF-driven alloantigen presentation in the gut is reduced, thereby alleviating GVHD symptoms. Conclusion: Celastrol acts as an anti-inflammatory immunomodulator, attenuating alloreactive immune cell-mediated injuries and protecting gut barrier integrity in aGVHD after hematopoietic stem cell transplantation.