In vitro and in vivo studies in mouse models elucidate the protective role of bile acids during intestinal aGvHD

Graft-versus-host disease (GvHD) is a severe complication that can result from allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients are commonly given immunosuppressive agents as prophylaxis; however, this also decreases the ability of the immune system to target and kill tumor cells, the graft-versus-leukemia effect, and increases the likelihood of relapse. Intestinal acute GvHD (aGvHD) is one of the most severe manifestations and is responsible for a high mortality rate, especially if refractory to corticosteroid treatment. Ameliorating intestinal GvHD through novel therapies that regenerate the gut epithelium has the potential to allow for decreased use of immunosuppressive agents and, as a result, it may decrease the risk of relapse.

In a preclinical study published in Haematologica, Eileen Haring and colleagues working under the supervision of GvHD steering committee member Robert Zeiser,  investigated the effect of allo-HSCT on the composition of bile acid metabolites in GvHD murine models.¹ In addition, the researchers tested whether exogenous administration of bile acids can protect intestinal cells from aGvHD-induced damage and analyzed the molecular mechanisms regulating this protective function.

Results

Effect of allo-HSCT on bile acid pool and receptor expression

Bile metabolites extracted from the ileum, liver and serum of untransplanted mice and mice that had received allo-HSCT, were analyzed using liquid chromatography-mass spectrometry. In general, the bile acid pool was reduced in the mice examined following aGvHD induction. The level of most bile acids significantly deceased in the ileum and ileal contents (p = 0.007 and p = 0.05, respectively), however, ursodeoxycholic acid (UDCA) increased. Intestinal levels of Nr1h4 mRNA, which encodes for Farnesoid X receptor (FXR), initially increased at day 4, while the levels of Gpbar1, which encodes for transmembrane G protein-coupled bile acid receptor 5 (TGR5), decreased.

Bile acids and apoptosis in intestinal organoids and cell lines

In vitro intestinal organoids were used to test the effect of different bile acids on epithelial cell structure and viability. For the analysis, 4 bile acids belonging to different groups were selected: a primary conjugated bile acid, chenodeoxycholic acid; a secondary conjugated bile acid, tauroursodeoxycholic acid (TUDCA); a secondary unconjugated bile acid, UDCA; a synthetic compound known as the strongest FXR receptor agonist, obeticholic acid. The intestinal organoids and the murine small intestine cell line MODE-K were used to find non-toxic doses of bile acids for further studies. Then, the organoids were treated with tumor necrosis factor α (TNF-α) or interferon γ (IFN-γ) with or without bile acids. TNF-α treatment resulted in cell damage and reduced viability of the organoid system, which was significantly reversed by TUDCA (p = 0.04) and partially reversed by chenodeoxycholic acid. IFN-γ-induced damage was also reversed by TUDCA. Similar results were found in MODE-K cells.

Effect of bile acid administration on aGvHD severity and mode of action

TUDCA was selected for further testing in a murine model in vivo, as it showed the strongest effects in vitro. First it was confirmed that exogenous administration of TUDCA increased the concentration of this bile acid in the serum and ileal contents. Two murine models (BALB/c donor to C57BL/6 recipient and the converse) were treated with 200 mg/kg body weight TUDCA administered as an intraperitoneal injection once daily. Survival was significantly increased in the TUDCA group compared with control mice in both transplant models (BABLB/c donor, p = 0.0004 and C57BL/6 donor, p < 0.0001). This result was confirmed by histopathology showing significantly reduced aGvHD scores in the liver, small intestines and colon (p < 0.0001, p = 0.0004 and p < 0.0001, respectively). The levels of proinflammatory cytokines in the serum were measured and TNF-α was significantly decreased (p = 0.01), while IFN-γ decreased but the change was not significant (p = 0.09).

aGvHD and the microbiome

Alterations in the microbiome, such as reduced microbial diversity, have been noted in mice with aGvHD. Therefore, whether the protective effect of TUDCA occurred through modifying the microbiome or was due to direct action on the intestinal cells was investigated. Microbiome analysis showed the expected reduction of microbial diversity in transplanted mice. However, TUDCA treatment did not affect the microbial diversity, indicating that it works potentially by directly acting on intestinal cells. The beneficial effect of TUDCA was maintained even after antibiotic treatment, to further support this conclusion.

Gene analysis and antigen presentation

Gene expression analysis of small intestine samples from TUDCA-treated animals and controls was performed 14 days after allo-HSCT. Upon TUDCA treatment, the most significantly downregulated pathways were associated with immune activation and antigen processing and presentation. Transporter associated with antigen processing (Tap)1 and Tap2, which are involved in the transport of degraded cytosolic proteins across the endoplasmic reticulum membrane for antigen major histocompatibility complex (MHC) class I molecule assembly, were downregulated. Other downregulated genes included TAP binding protein (Tapbp) and Tapasin-related protein (Tapbpl), which aid the association of the newly formed MHC class I complexes and TAP proteins. Furthermore, transcription of class II MHC transactivator (Ciita) was decreased, implying that  class I and class II MHC antigen presentation were both reduced in the intestines. This was confirmed in animals treated with TUDCA, where downregulation of MHC class I and II expression was found on nonhematopoietic stem cells. The expression analysis data were also confirmed at the mRNA and protein level in a second independent cohort.

Bile acids and T-cell transcriptional profile

The effect of TUDCA application on T-cell activation was also investigated. While T-cell numbers in the lamina propria of the small intestine and IFN-γ, IL-6 and TNF-α levels were not affected by application of TUDCA, a transcriptional signature suggestive of downregulation of immune cell activation was found. Many genes associated with the gene ontology term ‘T-cell activation’ were significantly downregulated in the TUDCA-treated aGvHD mice.

The effect of TUDCA on general alloreactive T-cell expansion was examined using bioluminescence-based trafficking and flow cytometry, which showed that expansion was similar in the spleen of bile acid-treated and nontreated animals. The transcriptional data support the hypothesis that bile acid treatment is associated with reduced local T-cell activation but does not negatively impact on T-cell expansion.

TUDCA, apoptosis and the graft-versus-leukemia effect

Microarray analysis showed that there was significant downregulation of apoptosis-related genes in mice treated with TUDCA. aGvHD induction significantly upregulated intestinal apoptosis whereas TUDCA treatment reversed this in both small intestine and colon, which was confirmed by TUNEL staining.

Human and murine cell lines were treated with TUDCA and analysis showed that the expression of human leukocyte antigen (HLA)-A, -B, -C and MHC class I was not altered. This, along with the finding that the expression of Tap1 and Tap2 genes was unaltered, suggests that the graft-versus-leukemia effect is not impaired by reducing the presentation of MHC molecules on tumor cells.

The effect of TUDCA treatment on the ability of T cells to induce cell death in leukemic cells was examined in vivo. In vivo-primed T cells, re-isolated from the spleens of vehicle- and TUDCA-treated mice 14 days after bone marrow transplant, showed comparable killing capacity towards A20 lymphoma cells in vitro. Further in vivo experiments showed that alloreactive T cells reduced malignant cells expansion in the bone marrow and the spleen, demonstrating the persistence of the graft-versus-leukemia effect in TUDCA-treated animals.

Conclusion

The use of bile acids, particularly TUDCA, shows great promise for treating intestinal aGvHD. TUDCA protects intestinal epithelial cells and increases survival in a murine model of aGvHD without reducing the ability of T cells to target leukemic cells. TUDCA is currently under investigation in clinical trials as supplement for various diseases, such as ulcerative colitis, and has demonstrated a favorable safety profile which makes it an attractive candidate to take forward into clinical trials in patients with GvHD.