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

Abstract

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-1αIE) knockout mice that lack HIF-1α in the intestinal epithelium. In a major MHC mismatched B10.BR (H2k) into B6 (H2b) GVHD model, 8 days post-BMT, histopathologic assessment of H&E stained ileums of HIF-1αIE 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-1αIE 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-1αIE mice post-BMT. In control WT and HIF-1αIE (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-1αIE 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. Disclosures No relevant conflicts of interest to declare.

A Metabolic Roadmap for Somatic Stem Cell Fate

“…reduced Paneth cell numbers (Feinman et al, 2014). These results support the hypothesis that oxygen availability may regulate stem cell function in the gut, but given that HIF-1a is common for both an inflammatory response and hypoxia, the precise role of oxygen availability in CBC stem cell function remains to be determined.…”