The Hypoxia Inducible Factor-2/Arginase-1 Axis Plays An Adaptive Role In Gvhd

The intestinal epithelium is a primary target of graft-versus-host disease (GVHD). The hypoxia-signaling pathway has been implicated as an adaptive response in the intestinal epithelium in numerous models of inflammatory bowel disease, such as colitis and T-cell induced diarrhea. The transcription factor family, hypoxia-inducible factor (HIF) has emerged as master regulators of the transcriptional response to hypoxic stress in normal and transformed cells. HIF heterodimers consist of an oxygen-labile α subunit (HIF-1α, HIF-2α) and a constitutively expressed HIF-1β subunit that mediate a wide spectrum of physiological and cellular adaptive responses, including angiogenesis, metabolic adaption, and erythropoiesis. HIF-1 has recently been implicated as a gut-protective factor in inflammatory bowel disease models by maintaining intestinal homeostasis. HIF-1 can also skew the differentiation of T cells to regulatory T cells (Treg) via the induction of FoxP3, thereby attenuating T-cell driven colitis. Although HIF-1 and HIF-2 share many overlapping functions, HIF-1 has been implicated in the inflammatory phenotype of M1 macrophages via inducible nitric oxide synthase (iNOS) whereas HIF-2 is involved in the anti-inflammatory phenotype of M2 macrophages via arginase-1 (Arg1). Based on these findings and that mucosal inflamed tissues are hypoxic, we hypothesized that the induction of the hypoxia-signaling pathway may limit GVHD-induced mucosal inflammation and injury.

To determine the adaptive roles of HIF-1 and HIF-2 in GVHD, we first tested the major histocompatibility complex (MHC)-haploidentical C567BL/6 (B6,H2b) -> B6xDBA/2 (B6D2)F1 (H2b/d) model in which donor spenocytes (2x10⁷) and anti-Thy1 + C’ treated (T- cell depleted) bone marrow (ATBM) cells (5x10⁶) are transplanted into B6D2F1 recipients after exposure to lethal irradiation (11Gy, split dose). B6 ATBM cells transplanted alone into B6D2F1 mice served as a negative control for all comparisons. Realtime PCR analysis demonstrated a modest increase in ileal mucosal HIF-2α expression 8 days (d) post-transplant (p<0.027). In contrast, HIF-1α mRNA levels were not induced. However, both HIF-1α and HIF-2α protein levels were upregulated 2-fold and 5-fold, respectively, in the ileal mucosa of B6D2F1 recipients receiving ATBM plus splenocytes, as determined by western blotting. Notably, Arg1 mRNA levels (HIF-2 target) were markedly upregulated during GVHD (p<0.018), whereas iNOS mRNA levels (HIF-1α target) were downregulated (p<0.01). Increased HIF-2α and Arg1 expression in the ileum as a consequence of GVHD was also observed in two MHC H2b-matched, minor histocompatability antigen (miHA)-mismatched models. BALB.B and CXB-2 mice were exposed to lethal irradiation (9Gy, split dose) and transplanted with B6 ATBM cells alone or along with host-presensitized B6 CD4⁺ T cells. After 8d, HIF-2α and HIF-1α mRNA ileal levels were increased and decreased, respectively, in both models undergoing GVHD. Similarly, Arg1 transcripts were increased by 12-fold (p<0.03) and 6.1 fold (p<0.007), respectively in B6->BALB.B and B6->CXB-2 models. However, after 20d post-transplant, a 4- and 3-fold decrease in Arg1 mRNA levels occurred in both models. Likewise, two anti-inflammatory, Treg-associated cytokines, interleukin-10 (IL-10) and transforming growth factor beta (TGFβ) mRNA expression were elevated by 2-and 7-fold, respectively, after d8 in B6->BALB.B mice. TGFβ levels returned to baseline (p<0.05 vs d8) after 20d and IL-10 mRNA levels were reduced by 2.5 fold (p<0.029 vs d8). Lastly, in an ELISpot assay, the addition of a prolyl hydroxylase inhibitor, dimethyloxaloylgylcine (DMOG), a HIF activator, reduced the alloreactive interferon-γ response to vehicle levels (p>0.001) in a B6 anti-B6D2F1 mixed lymphocyte reaction.

Taken together, our data suggest that HIF-2/Arg1 axis confers an anti-inflammatory response in the ileum after 8d of GVHD. However, after 20d, this response is inversely correlated with the lethality of the GVHD response. The amelioration of alloreactivity by DMOG suggests that the persistent activation of HIF may be necessary to dampen GVHD. Further studies will delineate the contribution of the HIF-2 response in the maintenance of intestinal homeostasis and limiting T cell alloreactivity.