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E. Haring et al.
gen and activate T cells in the context of GvHD.42 Little is known about the connection of bile acids and their effect on antigen-presentation, especially in non-hematopoietic cells. In a model of Schistosoma mansoni infection, 24-nor- UDCA but not UDCA itself reduced surface MHC class II expression on macrophages and dendritic cells and the acti- vation as well as proliferation of T lymphocytes in vitro.43 Microbial diversity is a key factor in regulating the home- ostasis of the intestine. GvHD has been previously linked to alterations in the microbiome with loss of bacterial diversi- ty. In order to prove that TUDCA regulates aGvHD severi- ty by acting directly on the intestinal epithelium and not by changing the microbiome,44 we analyzed fecal samples of TUDCA-treated animals developing aGvHD. Diversity and abundancy analyses showed comparable results between vehicle- and TUDCA-treated mice underlining the hypoth- esis that TUDCA protects the intestinal epithelium in a cell- specific manner.
Current GvHD prophylaxis and treatment mostly rely on suppression of T-cell activation and cytokine release which on the downside increases the risk of malignancy relapse. While TUDCA treatment resulted in a decreased expres- sion of genes related to T-cell activation in the intestine, it did not impair systemic T-cell proliferation or the activity against tumor cells. This difference might be explained by the fact that bile acid levels are much more abundant in the intestine than in the systemic blood circulation (0.5 mg/mL in the serum vs. 1,000 mg/100 mg ileal content). Furthermore, the expression of antigen presentation-related proteins on leukemic cells was not reduced showing that the recognition of malignant cells by the immune system remains intact upon bile acid administration. In line with this concept, we showed preserved GvL activity in in vitro, ex vivo and in vivo models.
One potential explanation for the divergent effects of bile acids on intestinal and leukemic cells is the differen- tial expression of bile acid receptors and their affinities to different species of bile acids. Intestinal epithelial cells are equipped with various bile acid receptors, FXR and TGR5 amongst others.45 We show that mRNA expression of both receptors changes upon GvHD induction with an increase of FXR and decrease of TGR5. One could specu- late that these alterations in receptor expression make bile acid signaling a potential target for regulating intes- tinal homeostasis. Both bile acid receptors are able to bind bile acids with a high affinity and subsequently induce different signaling pathways including inflamma- tion and apoptosis signaling. Other receptors of bile acids are more likely to be acknowledged as bile acid “sensors” which rather chemically convert bile acids and can act on bile acid transporters.46 FXR activation in the intestine has several anti-inflammatory properties. OCA, as a strong semi-synthetic FXR agonist, was shown to ameliorate intestinal mucosal inflammation in several mouse colitis models via induction of antimicrobial peptide production and reinforcement of epithelial barrier function in intes- tinal epithelial cells.47 Since OCA and CDCA, the bile acids with the highest affinity to FXR, showed only minor protective effects compared to TUDCA on intes- tinal epithelium in our experiments, we hypothesize that there must be other underlying mechanisms than the engagement of FXR signaling. One other candidate is the high affinity receptor TGR5 which also induces anti- inflammatory responses similar to FXR48 but has the high- est affinity to secondary bile acids such as UDCA and its
taurine conjugate TUDCA and is expressed in the liver and intestine.49,50
Amongst our tested bile acids, TUDCA was the one with the strongest anti-apoptotic effects both in vitro as and in vivo. Active re-absorption of bile acids occurs in the terminal ileum and is dependent on their conjugation status. During this process, bile acids come in contact and modulate the mucosal immune system.19,20 Our results indicate that for GvHD prevention, bile acids have to be conjugated in order to fulfill their cytoprotective properties.
In summary, we show that allo-HCT induces a depletion of the bile acid pool. Exogenous application of bile acids and in particular TUDCA reduces aGvHD. We propose a model, in which this positive effect is achieved by two dis- tinct mechanisms: enhancing the viability during exposure to pro-inflammatory cytokines and reduction of antigen presentation in the intestine with a consequent decrease in apoptosis (Figure 8). A major advantage of TUDCA is that it is already approved by the Food and Drug Administration and that it possesses a good safety profile. These findings pave the way for a prospective clinical trial using TUDCA to improve the outcome of allo-HCT recipients by prevent- ing aGvHD and preserving the GvL effect.
Disclosures
ELP is the founder of Rheos Medicines and is a SAB member of Immunomet.
Contributions
EH and FMU helped to develop the concept, performed experiments, analyzed data and helped to write the manuscript; GA analyzed microarray data; MP and AB performed the microbiome experiments and analysis; BS performed experi- ments and analyzed data; LMB and EVG performed research; PRE and SFM helped to develop the overall concept and to write the manuscript; DP performed and analyzed microarray exper- iments; MF helped with microscopy experiments, imaging and data analysis; ASG performed histopathological analysis; JB and ELP performed metabolomics experiments and helped with data analysis; JD and BG helped to design the study and ana- lyze the data; MB analyzed microarray data and helped to write the manuscript; RZ and PA designed the study, planned the experiments and analyzed data; PA also performed experiments and wrote the manuscript.
Acknowledgments
The authors would like to acknowledge Dr. D. Kaiserlian (INSERM, France) who kindly provided the MODE-K cells. The authors would further like to acknowledge Michal Rössler for designing the graphical abstract.
Funding
This study was supported by the Else Kröner-Fresenius-Stiftung (EKFS 2015_A147 to PA), the INTERREG V European regional development fund (European Union) program (project 3.2 TRIDI- AG to RZ), Wilhelm Sander Stiftung (grant 2008.046.5 to RZ), Deutsche Forschungsgemeinschaft, Germany, SFB1160 (RZ) and SFB850 (MB), ERC Consolidator grant (681012 GvHDCure European Union, to RZ and by the Excellence Strategy of the German Federal and State Governments (CIBSS - EXC 2189). PA is supported by a scholarship from the Berta Ottenstein Program for Physician Scientists, Faculty of Medicine, Medical Center – University of Freiburg, Germany. MB is supported by the German Federal Ministry of Education and Research (BMBF) within the framework of the e:Med research and funding concept
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