Introduction: Allogeneic hematopoietic cell transplantation (HCT) is increasingly utilized in the treatment of hematologic malignant and non-malignant diseases. Therapy-related toxicity and graft versus host disease (GVHD) remain major challenges as they significantly contribute to treatment related morbidity and mortality. Prolyl hydroxylase inhibitors (PHI) interfere with signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection resulted in the hypothesis, that use of the PHI dimethyl oxalyl glycine (DMOG) reduces GVHD after murine allogeneic HCT. We already showed that DMOG treatment of allogeneic recipients resulted in significantly improved survival by day +50 compared to allogeneic controls, and was associated with significant decrease in organ pathology and proinflammatory cytokines (data not shown). We now expanded our study to better understand the underlying mechanisms for this observation.
Methods: Acute GVHD was induced in lethally irradiated BALB/c mice followed by the infusion of 5 million bone marrow cells mixed with 4 million splenocytes from allogeneic C57BL/6 donors. DMOG dissolved in PBS was given at 8mg per mice intraperitoneally while controls received PBS only. Animals were monitored for clinical GVHD and survival and analyzed at day +7 and at day +50 post HCT. Formalin-fixed paraffin sections were processed for immunohistochemistry with CD3, Hif-1 alpha, or FADD pSer191 antibodies. Western blot analysis was performed after CD3/CD28 antibody activation of T cells to see the effect of DMOG treatment on Hif-1 α expression. Effect of DMOG on apoptosis and CD137 expression was analyzed by flow cytometry.
Results: Our results demonstrate that the protective effect of DMOG is mainly caused by two independent pathways of apoptosis. DMOG treatment of allogeneic recipients resulted in decreased T cells infiltration (p=0.0534) and (Fas associated death domain) FADDpSer191 level (p<0.05) in small intestine sections at day +50. Decreased FADD pSer191 indicates decreased TNF-a induced epithelial cell apoptosis. This was associated with increased Hif1- α expression at day+7 (p<0.05) as well day+50 (p<0.05) as shown by histochemical analysis of small intestine sections. DMOG treatment of activated T cells showed increased expression of CD137 (p<0.005) and Annexin V FITC (p<0.005) staining suggesting that DMOG subjects T cells to activation induced cell death (AICD) via induced CD137 expression. Microscopic scanning of DMOG treated T cells showed condensed chromatin along with numerous fragmented nuclei, indicative of apoptotic cell. In vitro proliferative T cell responses were suppressed (p<0.05) in the presence of DMOG in a dose-dependent manner along with increase in Hif1- α level (p<0.05) as analyzed by western blot.
Conclusion: Our data demonstrates protective effects of DMOG on the GI tract early after allogeneic HCT which is associated with improved survival and reduced lung injury at later time point. DMOG treatment results in the induction of CD137 on T cells subjecting them to AICD and represses FADD level in epithelial cells regulating their apoptosis. Taken together, DMOG treatment reduces GVHD via a two pronged mechanism, possibly allowing for novel strategies to prevent and treat this deleterious disease.
Palaniyandi:Pharmacyclics: Research Funding; Takeda Pharmaceutical International, Inc.: Research Funding; JAZZ: Research Funding.
Asterisk with author names denotes non-ASH members.