Intestinal Epithelial HIF-1 Plays a Protective Role in Gut Graft Versus Host Disease

Acute graft-versus-host disease (GVHD) is a major cause of nonrelapse morbidity and mortality following allogeneic blood and marrow transplantation (BMT). The majority of studies delineating the mechanisms involved in GVHD have focused on the alloreactive T cell response and their downstream cellular and inflammatory effector phases. While these efforts have provided important mechanistic insights that have been translated into therapies that target the T cell compartment, they ignore the role that the target organ plays in both initiating and propagating this disease. Acute gut GVHD affects more than 60% of patients and is a leading cause of death. The mechanisms for the early, conditioning-related, intestinal injuries that are the precipitating event leading to GVHD or how subsequent gut-specific GVHD actually disrupts intestinal homeostasis have not been fully explored. The transcription factor, hypoxia inducible factor-1 (HIF-1) has emerged as a common denominator for hypoxia and inflammation. Given that inflammatory bowel disease (IBD) and GVHD share many pathogenic mechanisms and intestinal epithelial HIF-1α afforded protection in IBD models, we hypothesize that the persistent activation of HIF-1 will protect the intestinal epithelium from radiation/chemotherapy and alloreactive T cell-induced damage. Since crypt damage is a hallmark of gut GVHD, we first determined whether loss of intestinal epithelial HIF-1α impaired intestinal regeneration after GVHD damage using conditional HIF-1α (HIF-1α^IE) knockout mice that lack HIF-1α in the intestinal epithelium. In a major MHC mismatched B10.BR (H2^k) into B6 (H2^b) GVHD model, 8 days post-BMT, histopathologic assessment of H&E stained ileums of HIF-1α^IE mice showed more severe crypt damage, loss of Paneth cells and fewer regenerating crypts as compared to wild-type (WT) mice. A two-fold increase of aberrant mitoses (p<0.02 vs WT) was observed in crypts of HIF-1α^IE mice. Consistent with these findings, real-time PCR analysis demonstrated that loss of epithelial HIF-1α did not induce the expression of Lgr5 mRNA levels (p<0.05) and reduced Reg3γ mRNA levels in the ileum by 2.4-fold (p<0.05) relative to WT mice, post-BMT. Since intestinal crypts have been shown to undergo extensive proliferation to repair damage following injury, we examined the crypt proliferative response 21 days post-BMT using immunohistochemistry for the proliferative marker Ki67. Hyperplastic elongated crypts that are characteristic of regenerating crypts after radiation-induced damage were observed in Ki67-stained ileal sections of WT mice post-BMT whereas smaller "aberrant" appearing crypts with asymmetrically distributed Ki67-labeled cells and fewer regenerating crypts were evident in HIF-1α^IE mice post-BMT. In control WT and HIF-1α^IE (receiving T cell depleted BM), Ki67⁺-proliferating cells resided at the crypt base. The extent of irregular distribution of Ki67 within 50 crypts/ileum was graded independently with a grading score ranging 0 to 3 (0 = limited to the base of crypt, 1= symmetric distribution at crypt base and along each side of the elongated crypt, 2 = asymmetric distribution in bottom 2/3 of crypt and 3 = asymmetric distribution of Ki67-labeled cells in entire crypt without lumen). A modest but significant increase in the aberrant proliferative score was 1.72 in the HIF-1α^IE post-BMT group versus 1.46 in the WT post-BMT group (p<0.05). Taken together, our findings suggest that intestinal epithelial HIF-1 may protect the intestinal stem cell niche and preserve intestinal regeneration in response to gut GVHD.