Page 104 - 2021_06-Haematologica-web

E. Haring et al.
Introduction
Allogeneic hematopoietic cell transplantation (allo-HCT) is a potentially curative treatment for leukemia and lym- phoma. However, approximately 50% of acute myeloid leukemia patients develop a malignancy relapse after allo- HCT.1 One contributing factor is the administration of immunosuppression as a prophylaxis or treatment for acute graft-versus-host disease (aGvHD). aGvHD is a severe trans- plant-associated complication which affects half of all allo- HCT recipients.2 Compared to other clinical manifestations of aGvHD, intestinal GvHD causes the highest mortality, especially if it is refractory to corticosteroid treatment.3 The pathogenesis of aGvHD is marked by activation of donor T cells and their expansion and migration to the target organs liver, skin and gastrointestinal tract where they cause tissue damage either by direct cytotoxicity or via cytokine secre- tion. A hallmark of intestinal aGvHD is the damage of intestinal stem cells (ISC) and Paneth cells.4,5 Recent work has shown that interferon γ (IFNγ), a cytokine commonly increased in aGvHD, is responsible for intestinal tissue damage.6,7 Current approved GvHD prophylaxis strategies are based on immunosuppressive drugs that deplete T cells or modulate their activity and cytokine signaling. Novel developments aiming at regenerating the intestinal epitheli- um would allow a reduction of broad-spectrum immuno- suppression and potentially decrease the relapse rates after allo-HCT.
There are two main classes of bile acids. Primary bile acids are generated in the liver via cholesterol catabolism. In the final step of their synthesis, they are conjugated to the amino acids glycine and taurine. This step stabilizes their amphipathic structure that is necessary to keep their full emulsifying activity in the intestine. After fulfilling their digestive role, most bile acids (appoximately 90-95%) are actively taken up by the intestinal epithelial cells and trans- ported back to the liver.8 Primary bile acids, which are not re-absorbed, can be converted into secondary bile acids via modification and metabolization by microbial enzymatic activity thereby influencing the microbiome composition.9 Bile acids have cytoprotective and anti-apoptotic properties for hepatocytes10,11 via the preservation of mitochondrial glutathione.12 Moreover, they modulate the translocation of apoptosis-regulating proteins BCL-2-associated X protein (BAX) and BCL-2-antagonist-of-cell-death (BAD) from the cytosol to the mitochondria and vice versa.13,14 Also immunomodulatory effects of bile acids and their receptors have been described. The two most prominent bile acid receptors are the Farnesoid X receptor (FXR) and the trans- membrane G protein-coupled bile acid receptor 5 (TGR5). FXR is expressed in both liver and intestine and was shown that mice lacking FXR were more prone to development of acute and chronic colitis.15 Intestinal damage during GvHD has been previously linked with bile acid malabsorption.16 In a recent study, metabolomic analysis of serum samples from allo-HCT recipients showed alterations in bile acid levels among other metabolites.17 So far, no data on bile acid levels in liver, intestines or in intestinal content in the con- text of GvHD have been available.
The aim of this study was to investigate whether allo- HCT causes alterations in the bile acid composition and whether exogenous bile acid application can positively influence aGvHD development. We show that the bile acid pool was depleted after allo-HCT. Administration of tau- roursodeoxycholic acid (TUDCA) improved aGvHD out-
come by direct protection of the intestinal epithelium from cytokine damage and by lowering antigen presentation by non-hematopoietic cells. This makes it an attractive com- pound for combination therapy with already clinically available immunosuppressive strategies.
Methods
Mice
BALB/c (H-2Kd) and C57BL/6 (H-2Kb) mice were purchased from the animal facility at Freiburg University Medical Center or from Janvier Labs (Le Genest-Saint-Isle, France). Luciferase-trans- genic C57BL/6 mice (H-2Kb) were bred in the animal facility of the Center for Clinical Research at Freiburg University Medical Center (Freiburg i.Br., Germany). B6.129P2-Lgr5tm1(cre/ERT2)Cle/J mice on the C57BL/6 background (H-2Kb) were a kind gift from Prof. Roland Schüle (Center for Clinical Research, Freiburg i.Br.). All animals were housed under specific pathogen-free conditions at the ani- mal facility of the Center for Clinical Research (ZKF, Freiburg i.Br., Germany). All animal protocols (G-13/045, G-16/018, G-17/063; X-13/07J; X-15/10A) were approved by the Federal Ministry for Nature, Environment and Consumer Protection of the state of Baden-Württemberg, Germany.
All other materials and methods are provided in the Online Supplementary Appendix.
Results
Allogeneic hematopoietic cell transplantation alters the levels of bile acids and the expression of bile acid receptors
We first investigated how allo-HCT influences the enterohepatic circulation (Figure 1A) and the bile acid pool. We extracted metabolites from liver, ileum, ileal contents and serum of untreated mice and mice developing aGvHD (Figure 1B). We measured the levels of eight major bile acid metabolites by liquid chromatography - mass spectrometry (LC-MS) (Figure 1C) and observed that taurocholic acid (TCA) was the predominant bile acid in all four compart- ments. Induction of aGvHD resulted in reduced bile acid pools. While the levels of most bile acids significantly decreased, the absolute amount of ursodeoxycholic acid (UDCA) increased (Figure 1D; Online Supplementary Figure S1A to C).
Another important determinant of bile acid signaling is the expression of bile acid receptors. We found that intes- tinal Nr1h4 mRNA levels (encoding for FXR) increased and Gpbar1 levels (encoding for TGR5) decreased with a peak on day 4 after bone marrow transplantation (BMT) (Figure 1E; Online Supplementary Figure S1D). Collectively, our results show that allo-HCT causes a reduction of the bile acid pool in liver, intestine and intestinal content as well as a deviation in bile acid receptor expression.
Treatment with bile acids prevents cell death of intestinal organoids and cell lines
Small intestinal organoids mimic the structure of the intestine.18 We used this in vitro system to test the impact of different bile acids on the viability and morphology of the intestinal epithelium. We selected bile acids belonging to different groups for our analysis: chenodeoxycholic acid (CDCA) as a primary bile acid, UDCA as a secondary unconjugated bile acid, TUDCA as a secondary conjugated
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haematologica | 2021; 106(8)