Rapid detection and online analysis of microbial changes through flow cytometry.

Short-read 16 S rRNA gene sequencing is the dominating technology to profile microbial communities in different habitats. Its uncontested taxonomic resolution paved the way for major contributions to the field. Sample measurement and analysis, that is, sequencing, is rather slow-in order of days. Alternatively, flow cytometry can be used to profile the microbiota of various sources within a few minutes per sample. To keep up with high measurement speed, we developed the open source-analyzing tool FlowSoFine. To validate the ability to distinguish microbial profiles, we examined human skin samples of three body sites (N = 3 × 54) with flow cytometry and 16 S rRNA gene amplicon sequencing. Confirmed by sequencing of the very same samples, body site was found to be significantly different by flow cytometry. For a proof-of-principle multidimensional approach, using stool samples of patients (N = 40) with/without inflammatory bowel diseases, we could discriminate the health status by their bacterial patterns. In conclusion, FlowSoFine enables the generation and comparison of cytometric fingerprints of microbial communities from different sources. The implemented interface supports the user through all analytical steps to work out the biological relevant signals from raw measurements to publication ready figures. Furthermore, we present flow cytometry as a valid method for skin microbiota analysis.

The small chain fatty acid butyrate antagonizes the TCR-stimulation-induced metabolic shift in murine epidermal gamma delta T cells.

The metabolic requirements change during cell proliferation and differentiation. Upon antigen-stimulation, effector T cells switch from adenosine-triphospate (ATP)-production by oxidative phosphorylation in the mitochondria to glycolysis. In the gut it was shown that short chain fatty acids (SCFA), fermentation products of the microbiota in colon, ameliorate inflammatory reactions by supporting the differentiation of regulatory T cells. SCFA are a major energy source, but they are also anabolic metabolites, histone-deacetylase-inhibitors and activators of G protein receptors. Recently, it was reported that a topical application of the SCFA butyrate promotes regulatory T cells in the skin. Here we ask if the SCFA butyrate, propionate and acetate affect the energy metabolism and inflammatory potential of dendritic epidermal T cells (DETC), the innate resident skin γδ T cell population. Using the Seahorse™ technology, we measured glycolysis and oxidative phosphorylation (OXPHOS) in a murine DETC cell line, 7-17, upon TCR-stimulation by CD3/CD28 crosslinking, with or without SCFA addition. TCR engagement resulted in a change of the ratio glycolysis/OXPHOS. A similar metabolic shift has been described for activated CD4 T cells. Addition of 5 mM SCFA, in particular butyrate, antagonized the effect. Stimulated DETC secrete cytokines, e.g. the pro-inflammatory cytokine interferon-gamma (IFNγ), and thereby regulate skin homeostasis. Addition of butyrate and propionate to the cultures at non-toxic concentrations decreased secretion of IFNγ by DETC and increased the expression of the immunoregulatory surface receptor CD69. We hypothesize that SCFA can dampen the inflammatory activity of DETC.

AHR Signaling Dampens Inflammatory Signature in Neonatal Skin γδ T Cells.

Background Aryl hydrocarbon receptor (AHR)-deficient mice do not support the expansion of dendritic epidermal T cells (DETC), a resident immune cell population in the murine epidermis, which immigrates from the fetal thymus to the skin around birth. Material and Methods In order to identify the gene expression changes underlying the DETC disappearance in AHR-deficient mice, we analyzed microarray RNA-profiles of DETC, sorted from the skin of two-week-old AHR-deficient mice and their heterozygous littermates. In vitro studies were done for verification, and IL-10, AHR repressor (AHRR), and c-Kit deficient mice analyzed for DETC frequency. Results We identified 434 annotated differentially expressed genes. Gene set enrichment analysis demonstrated that the expression of genes related to proliferation, ion homeostasis and morphology differed between the two mouse genotypes. Importantly, with 1767 pathways the cluster-group "inflammation" contained the majority of AHR-dependently regulated pathways. The most abundant cluster of differentially expressed genes was "inflammation." DETC of AHR-deficient mice were inflammatory active and had altered calcium and F-actin levels. Extending the study to the AHRR, an enigmatic modulator of AHR-activity, we found approximately 50% less DETC in AHRR-deficient mice than in wild-type-littermates. Conclusion AHR-signaling in DETC dampens their inflammatory default potential and supports their homeostasis in the skin.

Modulation of the intestinal bile acid/farnesoid X receptor/fibroblast growth factor 15 axis improves alcoholic liver disease in mice.

Alcoholic liver disease (ALD) is associated with changes in the intestinal microbiota. Functional consequences of alcohol-associated dysbiosis are largely unknown. The aim of this study was to identify a mechanism of how changes in the intestinal microbiota contribute to ALD. Metagenomic sequencing of intestinal contents demonstrated that chronic ethanol feeding in mice is associated with an over-representation of bacterial genomic DNA encoding choloylglycine hydrolase, which deconjugates bile acids in the intestine. Bile acid analysis confirmed an increased amount of unconjugated bile acids in the small intestine after ethanol administration. Mediated by a lower farnesoid X receptor (FXR) activity in enterocytes, lower fibroblast growth factor (FGF)-15 protein secretion was associated with increased hepatic cytochrome P450 enzyme (Cyp)-7a1 protein expression and circulating bile acid levels. Depletion of the commensal microbiota with nonabsorbable antibiotics attenuated hepatic Cyp7a1 expression and reduced ALD in mice, suggesting that increased bile acid synthesis is dependent on gut bacteria. To restore intestinal FXR activity, we used a pharmacological intervention with the intestine-restricted FXR agonist fexaramine, which protected mice from ethanol-induced liver injury. Whereas bile acid metabolism was only minimally altered, fexaramine treatment stabilized the gut barrier and significantly modulated hepatic genes involved in lipid metabolism. To link the beneficial metabolic effect to FGF15, a nontumorigenic FGF19 variant-a human FGF15 ortholog-was overexpressed in mice using adeno-associated viruses. FGF19 treatment showed similarly beneficial metabolic effects and ameliorated alcoholic steatohepatitis. CONCLUSION: Taken together, alcohol-associated metagenomic changes result in alterations of bile acid profiles. Targeted interventions improve bile acid-FXR-FGF15 signaling by modulation of hepatic Cyp7a1 and lipid metabolism, and reduce ethanol-induced liver disease in mice. (Hepatology 2018;67:2150-2166).

Intestinal fungi contribute to development of alcoholic liver disease.

Chronic liver disease with cirrhosis is the 12th leading cause of death in the United States, and alcoholic liver disease accounts for approximately half of all cirrhosis deaths. Chronic alcohol consumption is associated with intestinal bacterial dysbiosis, yet we understand little about the contribution of intestinal fungi, or mycobiota, to alcoholic liver disease. Here we have demonstrated that chronic alcohol administration increases mycobiota populations and translocation of fungal ß-glucan into systemic circulation in mice. Treating mice with antifungal agents reduced intestinal fungal overgrowth, decreased ß-glucan translocation, and ameliorated ethanol-induced liver disease. Using bone marrow chimeric mice, we found that ß-glucan induces liver inflammation via the C-type lectin-like receptor CLEC7A on Kupffer cells and possibly other bone marrow-derived cells. Subsequent increases in IL-1ß expression and secretion contributed to hepatocyte damage and promoted development of ethanol-induced liver disease. We observed that alcohol-dependent patients displayed reduced intestinal fungal diversity and Candida overgrowth. Compared with healthy individuals and patients with non-alcohol-related cirrhosis, alcoholic cirrhosis patients had increased systemic exposure and immune response to mycobiota. Moreover, the levels of extraintestinal exposure and immune response correlated with mortality. Thus, chronic alcohol consumption is associated with an altered mycobiota and translocation of fungal products. Manipulating the intestinal mycobiome might be an effective strategy for attenuating alcohol-related liver disease.

Effects of Liver Fibrosis Progression on Tissue Relaxation Times in Different Mouse Models Assessed by Ultrahigh Field Magnetic Resonance Imaging.

Recently, clinical studies demonstrated that magnetic resonance relaxometry with determination of relaxation times T1 and T2⁎ may aid in staging and management of liver fibrosis in patients suffering from viral hepatitis and steatohepatitis. In the present study we investigated T1 and T2⁎ in different models of liver fibrosis to compare alternate pathophysiologies in their effects on relaxation times and to further develop noninvasive quantification methods of liver fibrosis. MRI was performed with a fast spin echo sequence for measurement of T1 and a multigradient echo sequence for determination of T2⁎. Toxic liver fibrosis was induced by injections of carbon tetrachloride (1.4 mL CCl4 per kg bodyweight and week, for 3 or 6 weeks) in BALB/cJ mice. Chronic sclerosing cholangitis was mimicked using the ATP-binding cassette transporter B4 knockout (Abcb4 -/-) mouse model. Untreated BALB/cJ mice served as controls. To assess hepatic fibrosis, we ascertained collagen contents and fibrosis scores after Sirius red staining. T1 and T2⁎ correlate differently to disease severity and etiology of liver fibrosis. T2⁎ shows significant decrease correlating with fibrosis in CCl4 treated animals, while demonstrating significant increase with disease severity in Abcb4 -/- mice. Measurements of T1 and T2⁎ may therefore facilitate discrimination between different stages and causes of liver fibrosis.

Vitamin D modulates biliary fibrosis in ABCB4-deficient mice.

PURPOSE: Impaired vitamin D receptor signaling represents an aggravating factor during liver injury, and recent studies suggest that vitamin D might exert a protective role in chronic hepatobiliary diseases. We hypothesized that vitamin D supplementation would ameliorate liver fibrosis in ATP-binding cassette transporter B4 knockout (Abcb4 (-/-)) mice as a preclinical model of sclerosing cholangitis. METHODS: Abcb4 (-/-) and wild-type mice were fed a regular chow diet (600 IU vitamin D/kg food) or diets with lower (100 IU/kg) and higher (2,400 IU/kg) vitamin D concentrations for 12 weeks. Serum 25-hydroxyvitamin D concentrations were measured by chemiluminescence immunoassays. Liver injury and biliary fibrosis were assessed by liver enzyme activities, histopathology and hepatic collagen contents. Hepatic mRNA expression of markers for fibrosis, vitamin D and bile acid metabolism were analyzed by quantitative PCR. RESULTS: Different vitamin D concentrations were observed depending on genotype and diet group, with Abcb4 (-/-) mice on the control diet showing lower vitamin D concentrations compared to wild-type mice. Abcb4 (-/-) animals on the low vitamin D diet demonstrated the most advanced liver fibrosis and highest hepatic collagen contents. Feeding Abcb4 (-/-) mice a high vitamin D diet enriched serum vitamin D levels, lowered liver enzyme activities, altered expression levels of profibrogenic genes and ameliorated, in part, liver injury. CONCLUSIONS: This is the first report to demonstrate that fibrogenesis in the established Abcb4 (-/-) model is influenced by vitamin D supplementation. Since vitamin D modulates sclerosing cholangitis in vivo, we speculate that sufficient vitamin D intake might improve liver damage and induce antifibrotic effects in chronic cholestasis in humans.

Systems genetics of liver fibrosis: identification of fibrogenic and expression quantitative trait loci in the BXD murine reference population.

The progression of liver fibrosis in response to chronic injury varies considerably among individual patients. The underlying genetics is highly complex due to large numbers of potential genes, environmental factors and cell types involved. Here, we provide the first toxicogenomic analysis of liver fibrosis induced by carbon tetrachloride in the murine 'genetic reference panel' of recombinant inbred BXD lines. Our aim was to define the core of risk genes and gene interaction networks that control fibrosis progression. Liver fibrosis phenotypes and gene expression profiles were determined in 35 BXD lines. Quantitative trait locus (QTL) analysis identified seven genomic loci influencing fibrosis phenotypes (pQTLs) with genome-wide significance on chromosomes 4, 5, 7, 12, and 17. Stepwise refinement was based on expression QTL mapping with stringent selection criteria, reducing the number of 1,351 candidate genes located in the pQTLs to a final list of 11 cis-regulated genes. Our findings demonstrate that the BXD reference population represents a powerful experimental resource for shortlisting the genes within a regulatory network that determine the liver's vulnerability to chronic injury.

Modeling hepatic osteodystrophy in Abcb4 deficient mice.

Hepatic osteodystrophy (HOD) denotes the alterations in bone morphology and metabolism frequently observed in patients with chronic liver diseases, in particular in case of cholestatic conditions. The molecular mechanisms underlying HOD are only partially understood. In the present study, we characterized the bone phenotypes of the ATP-binding cassette transporter B4 knockout mouse (Abcb4(-/-)), a well-established mouse model of chronic cholestatic liver disease, with the aim of identifying and characterizing a mouse model for HOD. Furthermore, we investigated the influence of vitamin D on bone quality in this model. The bone morphology analyses revealed reduced bone mineral contents as well as changes in trabecular bone architecture and decreased cortical bone densities in Abcb4(-/-) mice with severe liver fibrosis. We observed dysregulation of genes involved in bone remodeling (osteoprotegerin, osteocalcin, osteopontin) and vitamin D metabolism (7-dehydrocholesterol reductase, Gc-globulin, Cyp2r1, Cyp27a1) as well as alterations in calcium and vitamin D homeostasis. In addition, serum RANKL and TGF-ß levels were increased in Abcb4(-/-) mice. Vitamin D dietary intervention did not restore the bone phenotypes of Abcb4(-/-) animals. We conclude that the Abcb4(-/-) mouse provides an experimental framework and a preclinical model to gain further insights into the molecular pathobiology of HOD and to study the systemic effects of therapeutic interventions.

The hepatic phosphatidylcholine transporter ABCB4 as modulator of glucose homeostasis.

The hepatic phosphatidylcholine (PC) transporter ATP-binding cassette (ABC) B4 flops PC from hepatocytes into bile, and its dysfunction causes chronic cholestasis and fibrosis. Because a nuclear receptor-dependent PC pathway has been determined to exert antidiabetic effects, we now analyzed the role of ABCB4 in glucose metabolism. We bred congenic Abcb4-knockout (Abcb4(-/-)) mice on the fibrosis-susceptible BALB/cJ background. Knockout mice and wild-type controls were phenotyped by measuring plasma glucose concentrations, intraperitoneal glucose tolerance, hepatic RNA expression profiles, and liver histology. In addition, 4 procholestatic ABCB4 gene variants were correlated with blood glucose levels in 682 individuals from 2 independent European cohorts. Systemic glucose levels differ significantly between Abcb4(-/-) mice and wild-type controls, and knockout mice display improved glucose tolerance with significantly lower area under the curve values on intraperitoneal glucose challenge. Of note, hepatic expression of the antidiabetic nuclear receptor 5A2 (LRH-1) is induced consistently in Abcb4(-/-) mice, and its specific rare PC ligands are detected in liver by mass spectrometry imaging. In humans, serum glucose levels are associated significantly with the common ABCB4 variant c.711A>T. In summary, ABCB4 might play a critical role in glucose homeostasis in mice and humans. We speculate that the effects could be mediated via LRH-1-dependent PC pathways.

Identification of RARRES1 as a core regulator in liver fibrosis.

Genetic factors contribute to progression and modulation of hepatic fibrosis. High throughput genomics/transcriptomics approaches aiming at identifying key regulators of fibrosis development are tainted with the difficulty of separating essential biological "driver" from modifier genes. We applied a comparative transcriptomics approach and investigated fibrosis development in different organs to identify overlapping expression changes, since these genes may be part of core pathways in fibrosis development. Gene expression was analysed on publicly available microarray data from liver, lung and kidney fibrosis. RARRES1, AGER and S100A2 were differentially regulated in all fibrosis experiments. RARRES1 was extensively analysed by means of advanced bioinformatics analyses and functional studies. Microarray and Western Blot analysis of a standard liver fibrosis model (CCl(4)) demonstrated an early induction of RARRES1 mRNA and protein expression. In addition, quantitative RT-PCR in tissue samples from patients with advanced liver fibrosis showed higher expression as compared to non-fibrotic biopsies. Microarray analysis of RARRES1 overexpressing cells identified an enrichment of a major signature associated with fibrosis. Furthermore, RARRES1 expression increased during in vitro activation of hepatic stellate cells. To further verify the pro-fibrogenic role across organs, we demonstrated an increase in RARRES1 expression in a rat lung fibrosis model induced by adenoviral TGF-ß1 induction. We have performed a comparative transcriptomics analysis in order to identify core pathways of liver fibrogenesis, confirmed a candidate gene and enlightened the up- and downstream mechanisms of its action leading to fibrosis across organs and species.

Common genetic variation in vitamin D metabolism is associated with liver stiffness.

UNLABELLED: Recently, genome-wide studies identified genetic variants that affect serum 25-hydroxyvitamin D levels in healthy populations (rs12785878, near dehydrocholesterol reductase, DHCR7; rs10741657, at CYP2R1; and rs7041, at vitamin D binding protein, GC). Because vitamin D deficiency is associated with advanced liver disease, we hypothesized that these variants are associated with 25(OH)-vitamin D levels and liver fibrosis. Overall, 712 Caucasian patients with chronic liver diseases were included. Liver fibrosis was assessed by transient elastography (TE) and/or histology. Serum levels of 25(OH)-vitamin D were correlated with TE and fibrosis stages. Genotypes were determined using TaqMan assays and tested for association with vitamin D and liver stiffness. Serum 25(OH)-vitamin D levels were inversely correlated with liver stiffness and histology (P < 0.001). Homozygous carriers of the rare DHCR7 allele or the common CYP2R1 allele presented with reduced 25(OH)-vitamin D levels (P < 0.05). The variant rs12785878 in the DHCR7 locus was associated with liver stiffness in both patients with TE <7.0 kPa and TE between 7.0 and 9.5 kPa. 25(OH)-vitamin D levels correlated with sunshine hours at the time of inclusion (P < 0.001). CONCLUSION: Common variation in 25(OH)-vitamin D metabolism is associated with liver stiffness in patients presenting with low to moderately increased elasticity. Although the susceptible DHCR7 genotype confers small risk, we speculate that the observed stiffness differences indicate a stronger influence of 25(OH)-vitamin D on initiation rather than progression of hepatic fibrosis.

Expression of the megalin C-terminal fragment by macrophages during liver fibrogenesis in mice.

The low-density-lipoprotein receptor megalin (LRP2, gp330) is strongly expressed in the kidney, where it is responsible for the resorption of metabolites from primary urine. One of the main ligands is the complex of retinol and retinol binding protein. Megalin has been hypothesized to be part of the retinol storage system in liver. Considering the role of hepatic stellate cells in retinol storage and fibrogenesis we investigated mouse strains that developed different degrees of fibrosis after challenge with CCl(4). Immunoblotting revealed the invariable expression of the megalin C-terminal fragment independent of liver damage in all strains. However, only a specific cell population in centrilobular areas of fibrotic livers from DBA/2J mice, which were most susceptible for CCl(4)-induced fibrogenesis in our study, was stained using megalin-specific antibodies. Double immunostaining indicated that a subset of hepatic macrophages might represent the megalin-expressing cells in fibrotic liver. Fluorescence activated cell sorting based isolation of hepatic macrophages and megalin specific expression analysis demonstrated the transcription of the whole megalin gene in liver macrophages. We argue that megalin might exhibit a proinflammatory effect by the uptake of retinoids in recruited monocytes, which thereby differentiate to liver macrophages and potentiate fibrogenesis by the release of proinflammatory mediators. Otherwise, megalin might be activated in macrophages during advanced fibrogenesis and act as a negative regulator of proinflammatory genes.