Abstract

Graft-versus-host disease (GVHD) remains the major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Impaired mucosal epithelial barrier contributes significantly to amplifying systemic inflammatory responses characteristic of GVHD. Emerging evidence demonstrates that protecting or restoring intestinal barrier function represents a feasible approach to mitigate GVHD.

There is growing interest in how vitamin D and the vitamin D receptor (VDR) signaling regulate immune responses in intestinal inflammatory diseases. The role of VDR signaling in regulating alloimmunity is unknown. We therefore tested the novel hypothesis that VDR signaling plays an important role in maintaining intestinal epithelial barrier function during GVHD. In initial studies, we examined whether intestinal VDR signaling is altered in recipient mice undergoing either syngeneic or allogeneic bone marrow transplantation (BMT). We found a progressive decline in VDR gene expression over time in colon tissues of allogeneic, but not syngeneic, BMT recipients compared with that of naïve animals. Notably, the expression of CYP27B1, the key enzyme participating in the biosynthesis of the active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25VitD3), was also significantly decreased in the intestines of allogeneic BMT recipients compared to that of naïve animals. Caco-2 is an epithelial cell line that is widely used as a model for the intestinal barrier. We showed that proinflammatory cytokines TNF-α and IL-6 reduced Caco-2 VDR protein levels in a dose and time dependent manner by western blot analysis. To assess monolayer barrier function, an in vitro permeability assay was performed using FITC-dextran. We found 1,25VitD3 significantly attenuated the increase in epithelial monolayer permeability induced by TNF-α and IL-6. Tight junctions are important components of the intestinal barrier. We also found proinflammatory cytokines reduced gene expression of tight junctions such as occludin in Caco-2 cells. Remarkably, 1,25VitD3 treatment significantly restored occludin expression.

We then examined how vitamin D/VDR pathway regulates the development of GVHD. 1,25VitD3 supplementation after BMT significantly increased the survival of recipient mice in a well-established C57Bl/6→Balb/c GVHD model (p=0.0045). 1,25VitD3 treatment did not appear to modify donor T cell alloreactivity. Specifically, donor T cell activation, proliferation, and polarization were not affected by 1,25VitD3 treatment. Notably, infusion of donor T cells from VDR-WT (VDR+/+) or VDR-deficient (VDR-/-) mice caused similar GVHD-associated mortality in Balb/c recipient mice (p=0.98), further suggesting that the beneficial effects of 1,25VitD3 treatment is not due to its effects on donor T cells. 1,25VitD3 also did not appear to affect host and donor antigen presenting cells to modulate GVHD severity. Specifically, dendritic cells from VDR+/+ or VDR-/- mice showed equal ability to stimulate the proliferation of alloreactive T cells in mixed lymphocyte reactions. There was also no difference in post-transplant survival when recipient mice were reconstituted with donor T-cell-depleted bone marrow cells from either VDR+/+ or VDR-/- mice (recipients were transplanted with the same donor T cells from VDR-WT animals; p=0.58). Instead, 1,25VitD3 treatment significantly increased tight junction protein expression in the gut, suggesting its effect on intestinal epithelial cells. Finally, transgenic mice selectively overexpress VDR in the intestinal epithelium (villin promoter-driven FLAG-tagged human VDR gene expression) were used as BMT recipients in gain-of-function studies. We found a significantly decreased GVHD-associated mortality in these mice compared to that of control littermates (p=0.0002), providing direct evidence that intestinal epithelial VDR signaling protects against GVHD. Our results provide new evidence supporting the notion that impaired mucosal barrier function is a major pathogenic factor in the development of GVHD. Intestinal VDR signaling plays an important role in maintaining intestinal barrier integrity and enhancing this pathway protects against GVHD. Thus, our study provides pre-clinical data supporting the use of vitamin D or its analogs as a simple and inexpensive therapeutic modality with minimal side effects for mitigating GVHD.

Disclosures

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.