Page 107 - 2021_06-Haematologica-web

Bile acids reduce GvHD and preserve the GvL effect
bile acid and 6-ethylchenodeoxycholic acid (obeticholic acid, OCA) as a synthetic compound and the strongest ago- nist of the FXR receptor. We evaluated the toxicity of these bile acids on organoids and the intestinal cell line MODE-K to select non-toxic doses for further experiments (Online Supplementary Figure S2A and B). Organoids were treated with tumor necrosis factor (TNF) or IFNγ in combination with or without bile acids (Figure 2A). We observed that TNF treatment, performed to induce cellular damage, reduced organoid viability as shown by changes in cell mor- phology and organoid structure. This effect was partially reversed by addition of CDCA and more significantly of TUDCA (Figure 2B to E). We confirmed the beneficial effect of TUDCA on organoid viability using IFNγ to induce organoid damage (Figure 2F and G). As TUDCA was the most potent bile acid to protect intestinal organoids from cytokine-induced cell death, we performed quantitative polymerase chain reaction (qPCR) analysis from organoids treated with TNF ± TUDCA in order to evaluate which cell types were particularly preserved. We observed a strong upregulation of the anti-microbial peptides defensin α1 and 4 (Defa1 and Defa4) which are produced by Paneth cells (Online Supplementary Figure S2C). Additionally, we identi- fied an elevated expression of the intestinal stem cell (ISC) marker leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5). Although there was a trend towards an increase in the goblet cell marker mucin 2 (Muc2) and the enteroendocrine cell marker chromogranine A (Chga), these changes were not significant (Online Supplementary Figure S2C). We confirmed the hypothesis that bile acids protect intestinal cells from cell death via performing additional experiments using the murine small intestine cell line MODE-K. TNF treatment reduced the viability of MODE- K cells. This was reversed partly by CDCA and even more by TUDCA (Figure 2H and I). Together, these data indicate that exposure to bile acids reduces intestinal cell damage induced by pro-inflammatory cytokines.
Prophylactic administration of bile acids reduces acute graft-versus-host disease severity in mice through a cell-intrinsic effect independent of the microbiome
Our in vitro studies provided evidence that bile acids pro- tect the intestinal epithelium. Since TUDCA showed the strongest effects, we used this compound for further studies in a murine in vivo BMT model (Figure 3A and C). We deter- mined bile acid concentrations and confirmed an increase of TUDCA levels after exogenous administration. Levels increased from 0.5% to 35% of the measured bile acids in the serum and from 3.2% to 57% in the ileal content reach- ing a concentration of almost 1 mg per 100 mg (Online Supplementary Figure S3A and B). Survival was significantly improved in animals treated with TUDCA in two different transplantation models using total body irradiation (TBI) as the conditioning treatment (Figure 3B and D). As a confir- mation, histopathological analysis of the liver, small intes- tine and colon of recipient mice demonstrated significantly reduced aGvHD scores in the TUDCA-treated group (Figure 3E). This was further validated in an irradiation-free model with chemotherapy conditioning (Online Supplementary Figure S3C and D). Pro-inflammatory cytokines contribute to tissue damage and T-cell activation during aGvHD. We observed reduced serum cytokine lev- els in the treatment group with significant differences for TNF and a trend towards lower levels of IFNγ (Figure 3F).
These data demonstrate a protective effect of TUDCA in the prophylactic setting with improved survival rates, reduced histological aGvHD scores and lower concentra- tions of pro-inflammatory cytokines.
Since we found in our in vitro system an effect on intestin- al cell viability by other bile acids as well, we assessed the impact of CDCA and UDCA as a prophylaxis for aGvHD (Online Supplementary Figure S3E). Histopathological analy- sis revealed a slight improvement upon CDCA administra- tion (Online Supplementary Figure S3F) and a more pro- nounced effect after treatment with UDCA (Online Supplementary Figure S3G).
Intestinal bacteria play an essential role in bile acid metabolism. They generate secondary bile acids by remov- ing glycine or taurine residues from primary bile acids, which were not reabsorbed from intestinal epithelial cells and recycled in the enterohepatic circle.19,20 The microbio- me composition and the bile acid pool influence each other.21 Recent data show that aGvHD is associated with changes in the microbiome including a diminished micro- bial diversity.22-24 We therefore investigated whether TUDCA potentially mediates its protective effects by act- ing directly on the intestinal cells or by modifying the intes- tinal microbiome composition. Microbiome analysis was conducted in mice developing aGvHD. We could detect the expected reduction in microbial diversity in both groups compared to untreated mice. This reduction was independ- ent of TUDCA treatment as depicted by similar Shannon indices and inversed Simpson indices (Figure 3G and H). Detailed analysis of the relative abundance of different species confirmed that the microbiome is altered during aGvHD development. In line with previous reports, Lactobacillus numbers increased and certain anaerobic species (in our case Muribaculum and Sporobacter) decreased. TUDCA- and vehicle-treated mice exhibited similar relative abundance profiles suggesting that TUDCA protects the intestinal epithelium by directly affecting the intestinal cells and not by changing the microbial composition (Figure 3I). Supporting this concept, TUDCA preserved its beneficial effects on the survival of mice developing aGvHD even after decontaminating antibiotic treatment that was admin- istered prior to BMT (Figure 3J and K). Of note, antibiotic treatment did not lead to the general reduction in the bile acid pool of the ileal content seen as a result from GvHD induction (Online Supplementary Figure SH3). Together, these data suggest that although bile acids and the microbiome are closely connected in the intestinal tract, the beneficial effect of TUDCA on GvHD outcome is not mainly mediat- ed by regulation of the intestinal bacteria.
Bile acid treatment decreases intestinal antigen presentation
In order to better understand the mechanism by which bile acid administration protects the intestine from aGvHD- induced damage, we performed gene expression analysis of small intestinal samples on day 14 after BMT. Using sam- ples from TUDCA-treated animals and controls, we per- formed a hypergeometric test of significant regulated genes (q-value <0.05) to find differentially regulated terms from the Gene Ontology database. The most significantly down- regulated pathways upon TUDCA treatment included pathways related to immune activation, among others the gene set ‘antigen processing and presentation’ (Figure 4A). Expression analysis in a second independent cohort con- firmed the data on the mRNA and protein level (Figure 4B
haematologica | 2021; 106(8)
2135