Abstract
Abstract 341
Damage to the gastrointestinal tract during graft-versus-host disease (GVHD) is one of the major causes of morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. However, the molecular mechanism(s) responsible for colonic GVHD induction remain obscure. All-trans retinoic acid (RA) is an active metabolite of vitamin A and has recently emerged as a critical regulator of gut immunity. We hypothesized that administration of RA might be a strategy to reduce inflammatory responses during GVHD, particularly within the colon microenvironment. In a B6→Balb/c MHC-mismatched murine allogeneic bone marrow transplantation (BMT) model, we found unexpectedly that exposure of recipient mice to exogenous RA resulted in significantly accelerated GVHD-associated mortality compared with DMSO-treated animals (p<0.0001). Histological examination of GVHD target organs 5 weeks after transplantation revealed a significant increase in pathologic damage in the colons of RA-treated mice compared with DMSO-treated animals (9.7 ± 0.8 versus 3.3 ± 0.9, p<0.001), whereas there was no significant difference in pathology scores in either the lung or liver. RA treatment resulted in significantly increased expression of the gut-homing molecules, CCR9 and α4β7, on donor T cells in mesenteric lymph nodes early after BMT. Notably, expression of other chemokine receptors that have been implicated in GVHD pathogenesis, such as CCR5, CXCR3, and CCR10, was not affected by RA treatment, demonstrating a selective role of RA in imprinting gut-homing donor T cells. Examination of GVHD target organs three weeks after transplantation revealed a significant increase (eight-fold) in the absolute numbers of both CD4+IFN-γ+ (TH1) and CD4+IL-17+ (TH17) cells in the colon of RA-treated mice versus DMSO-treated animals, whereas there were no statistical differences in TH1 or TH17 cells in the spleen, liver and lung. There was also a significant increase in the absolute number of regulatory T cells (Tregs) in the colon of RA-treated mice. However, the increase in Tregs was noted to be more modest (three-fold) than was observed for TH1 and TH17 cells, suggesting a pro-inflammatory role of RA. To further confirm a role for RA in imprinting gut-homing donor T cells during GVHD, we generated vitamin A-deficient mice and employed them as recipients. Depletion of RA in recipient mice by vitamin A deprivation resulted in a dramatic reduction of gut homing molecule expression on donor T cells after allogeneic BMT. Retinoic acid receptor-α (RAR-α) signaling appears to be critical in mediating the effects of RA on alloreactive T cells. In mixed lymphocyte culture, when B6 T cells were stimulated with Balb/c dendritic cells, the presence of an RAR-α agonist was able to significantly enhance the expression of gut-homing molecules on T cells. In contrast, the addition of a selective RAR-α antagonist abolished the effects of RA on T cells. We then measured endogenous RA levels in the colons of mice undergoing either syngeneic or allogeneic BMT by mass spectrometry. These studies revealed that RA was significantly increased in the colon tissue of allogeneic transplant recipients compared with syngeneic transplant recipients early after BMT (day 7). In addition, Real-Time q-PCR analysis revealed a mean 3.5-fold increase in RAR-α expression in purified donor splenic CD4+ T cells 4 days post transplantation when compared to purified naïve CD4+ T cells obtained from original donor animals. Finally, we examined the ability of RAR-α-deficient T cells to cause GVHD. We found that recipients of RAR-α-deficient marrow grafts had significantly decreased GVHD-associated mortality when compared with the recipients of wild type T cells (93% versus 36% at day 90, p=0.002). Histological examination of GVHD target organs on days 10–12 after transplantation revealed a significant reduction in pathologic damage in the colon of mice that received RAR-α-deficient T cells compared with control mice (7.3 ± 2.5 versus 0.8 ± 1.0, p<0.01). In summary, we have identified RA as a novel and critical molecule in the induction of gastrointestinal GVHD. These findings provide evidence that blockade of the RA signaling pathway may represent a novel strategy to mitigate the severity of colonic GVHD.
Disclosures:
No relevant conflicts of interest to declare.
The role of retinoic acid in embryonic and post-embryonic development