Dae Joong Kang

Bile salt biotransformations by human intestinal bacteria

Secondary bile acids, produced solely by intestinal bacteria, can accumulate to high levels in the enterohepatic circulation of some individuals and may contribute to the pathogenesis of colon cancer, gallstones, and other gastrointestinal (GI) diseases. Bile salt hydrolysis and hydroxy group dehydrogenation reactions are carried out by a broad spectrum of intestinal anaerobic bacteria, whereas bile acid 7-dehydroxylation appears restricted to a limited number of intestinal anaerobes representing a small fraction of the total colonic flora. Microbial enzymes modifying bile salts differ between species with respect to pH optima, enzyme kinetics, substrate specificity, cellular location, and possibly physiological function. Crystallization, site-directed mutagenesis, and comparisons of protein secondary structure have provided insight into the mechanisms of several bile acid-biotransforming enzymatic reactions. Molecular cloning of genes encoding bile salt-modifying enzymes has facilitated the understanding of the genetic organization of these pathways and is a means of developing probes for the detection of bile salt-modifying bacteria. The potential exists for altering the bile acid pool by targeting key enzymes in the 7α/β-dehydroxylation pathway through the development of pharmaceuticals or sequestering bile acids biologically in probiotic bacteria, which may result in their effective removal from the host after excretion.

Bile Acids and the Gut Microbiome

Purpose of review We examine the latest research on the emerging bile acid-gut microbiome axis and its role in health and disease. Our focus revolves around two key microbial pathways for degrading bile salts, and the impact of bile acid composition in the gut on the gut microbiome and host physiology. Recent findings Bile acid pool size has recently been shown to be a function of microbial metabolism of bile acids in the intestines. Recent studies have shown potential mechanisms explaining how perturbations in the microbiome affect bile acid pool size and composition. Bile acids are emerging as regulators of the gut microbiome at the highest taxonomic levels. The role of bile acids as hormones and potentiators of liver cancer is also emerging. Summary The host and microbiome appear to regulate bile acid pool size. The host produces a large, conjugated hydrophilic bile acid pool, maintained through positive-feedback antagonism of farnesoid X receptor (FXR) in intestine and liver. Members of the microbiome utilize bile acids and their conjugates resulting in agonism of FXR in intestine and liver resulting in a smaller, unconjugated hydrophobic bile acid pool. Hydrophilicity of the bile acid pool is associated with disease states. Reduced bile acid levels in the gut are associated with bacterial overgrowth and inflammation. Diet, antibiotic therapy, and disease states affect the balance of the microbiome-bile acid pool.

Consequences of bile salt biotransformations by intestinal bacteria

ABSTRACT Emerging evidence strongly suggest that the human “microbiome” plays an important role in both health and disease. Bile acids function both as detergents molecules promoting nutrient absorption in the intestines and as hormones regulating nutrient metabolism. Bile acids regulate metabolism via activation of specific nuclear receptors (NR) and G-protein coupled receptors (GPCRs). The circulating bile acid pool composition consists of primary bile acids produced from cholesterol in the liver, and secondary bile acids formed by specific gut bacteria. The various biotransformation of bile acids carried out by gut bacteria appear to regulate the structure of the gut microbiome and host physiology. Increased levels of secondary bile acids are associated with specific diseases of the GI system. Elucidating methods to control the gut microbiome and bile acid pool composition in humans may lead to a reduction in some of the major diseases of the liver, gall bladder and colon.

Salivary microbiota reflects changes in gut microbiota in cirrhosis with hepatic encephalopathy

Altered gut microbiome is associated with systemic inflammation and cirrhosis decompensation. However, the correlation of the oral microbiome with inflammation in cirrhosis is unclear. Our aim was to evaluate the oral microbiome in cirrhosis and compare with stool microbiome. Outpatients with cirrhosis (with/without hepatic encephalopathy [HE]) and controls underwent stool/saliva microbiome analysis (for composition and function) and also systemic inflammatory evaluation. Ninety‐day liver‐related hospitalizations were recorded. Salivary inflammation was studied using T helper 1 cytokines/secretory immunoglobulin A (IgA), histatins and lysozyme in a subsequent group. A total of 102 patients with cirrhosis (43 previous HE) and 32 age‐matched controls were included. On principal component analysis (PCA), stool and saliva microbiome clustered far apart, showing differences between sites as a whole. In salivary microbiome, with previous HE, relative abundance of autochthonous families decreased whereas potentially pathogenic ones (Enterobacteriaceae, Enterococcaceae) increased in saliva. Endotoxin‐related predicted functions were significantly higher in cirrhotic saliva. In stool microbiome, relative autochthonous taxa abundance reduced in previous HE, along with increased Enterobacteriaceae and Enterococcaceae. Cirrhotic stool microbiota demonstrated a significantly higher correlation with systemic inflammation, compared to saliva microbiota, on correlation networks. Thirty‐eight patients were hospitalized within 90 days. Their salivary dysbiosis was significantly worse and predicted this outcome independent of cirrhosis severity. Salivary inflammation was studied in an additional 86 age‐matched subjects (43 controls/43 patients with cirrhosis); significantly higher interleukin (IL)−6/IL‐1β, secretory IgA, and lower lysozyme, and histatins 1 and 5 were found in patients with cirrhosis, compared to controls. Conclusions: Dysbiosis, represented by reduction in autochthonous bacteria, is present in both saliva and stool in patients with cirrhosis, compared to controls. Patients with cirrhosis have impaired salivary defenses and worse inflammation. Salivary dysbiosis was greater in patients with cirrhosis who developed 90‐day hospitalizations. These findings could represent a global mucosal‐immune interface change in cirrhosis. (Hepatology 2015;62:1260‐1271)

Colonic inflammation and secondary bile acids in alcoholic cirrhosis

Alcohol abuse with/without cirrhosis is associated with an impaired gut barrier and inflammation. Gut microbiota can transform primary bile acids (BA) to secondary BAs, which can adversely impact the gut barrier. The purpose of this study was to define the effect of active alcohol intake on fecal BA levels and ileal and colonic inflammation in cirrhosis. Five age-matched groups {two noncirrhotic (control and drinkers) and three cirrhotic [nondrinkers/nonalcoholics (NAlc), abstinent alcoholic for >3 mo (AbsAlc), currently drinking (CurrAlc)]} were included. Fecal and serum BA analysis, serum endotoxin, and stool microbiota using pyrosequencing were performed. A subgroup of controls, NAlc, and CurrAlc underwent ileal and sigmoid colonic biopsies on which mRNA expression of TNF-α, IL-1β, IL-6, and cyclooxygenase-2 (Cox-2) were performed. One hundred three patients (19 healthy, 6 noncirrhotic drinkers, 10 CurrAlc, 38 AbsAlc, and 30 NAlc, age 56 yr, median MELD: 10.5) were included. Five each of healthy, CurrAlc, and NAlc underwent ileal/colonic biopsies. Endotoxin, serum-conjugated DCA and stool total BAs, and secondary-to-primary BA ratios were highest in current drinkers. On biopsies, a significantly higher mRNA expression of TNF-α, IL-1β, IL-6, and Cox-2 in colon but not ileum was seen in CurrAlc compared with NAlc and controls. Active alcohol use in cirrhosis is associated with a significant increase in the secondary BA formation compared with abstinent alcoholic cirrhotics and nonalcoholic cirrhotics. This increase in secondary BAs is associated with a significant increase in expression of inflammatory cytokines in colonic mucosa but not ileal mucosa, which may contribute to alcohol-induced gut barrier injury.

Gut microbiota, cirrhosis, and alcohol regulate bile acid metabolism in the gut.

The understanding of the complex role of the bile acid-gut microbiome axis in health and disease processes is evolving rapidly. Our focus revolves around the interaction of the gut microbiota with liver diseases, especially cirrhosis. The bile acid pool size has recently been shown to be a function of microbial metabolism of bile acid, and regulation of the microbiota by bile acids is important in the development and progression of several liver diseases. Humans produce a large, conjugated hydrophilic bile acid pool, maintained through positive-feedback antagonism of farnesoid X receptor (FXR) in the intestine and liver. Microbes use bile acids, and via FXR signaling this results in a smaller, unconjugated hydrophobic bile acid pool. This equilibrium is critical to maintain health. The challenge is to examine the manifold functions of gut bile acids as modulators of antibiotic, probiotic, and disease progression in cirrhosis, metabolic syndrome, and alcohol use. Recent studies have shown potential mechanisms explaining how perturbations in the microbiome affect bile acid pool size and composition. With advancing liver disease and cirrhosis, there is dysbiosis in the fecal, ileal, and colonic mucosa, in addition to a decrease in bile acid concentration in the intestine due to the liver problems. This results in a dramatic shift toward the Firmicutes, particularly Clostridium cluster XIVa, and increasing production of deoxycholic acid. Alcohol intake speeds up these processes in the subjects with and without cirrhosis without significant FXR feedback. Taken together, these pathways can impact intestinal and systemic inflammation while worsening dysbiosis. The interaction between bile acids, alcohol, cirrhosis, and dysbiosis is an important relationship that influences intestinal and systemic inflammation, which in turn determines progression of the overall disease process. These interactions and the impact of commonly used therapies for liver disease can provide insight into the pathogenesis of inflammation in humans.

Gut Microbiota Alterations can predict Hospitalizations in Cirrhosis Independent of Diabetes Mellitus

Diabetes (DM) is prevalent in cirrhosis and may modulate the risk of hospitalization through gut dysbiosis. We aimed to define the role of gut microbiota on 90-day hospitalizations and of concomitant DM on microbiota. Cirrhotic outpatients with/without DM underwent stool and sigmoid mucosal microbial analysis and were followed for 90 days. Microbial composition was compared between those with/without DM, and those who were hospitalized/not. Regression/ROC analyses for hospitalizations were performed using clinical and microbial features. 278 cirrhotics [39% hepatic encephalopathy (HE), 31%DM] underwent stool while 72 underwent mucosal analyses. Ultimately, 94 were hospitalized and they had higher MELD, proton pump inhibitor (PPI) use and HE without difference in DM. Stool/mucosal microbiota were significantly altered in those who were hospitalized (UNIFRAC p< = 1.0e-02). Specifically, lower stool Bacteroidaceae, Clostridiales XIV, Lachnospiraceae, Ruminococcacae and higher Enterococcaceae and Enterobacteriaceae were seen in hospitalized patients. Concomitant DM impacted microbiota UNIFRAC (stool, p = 0.003, mucosa,p = 0.04) with higher stool Bacteroidaceae and lower Ruminococcaeae. Stool Bacteroidaceaeae and Clostridiales XIV predicted 90-day hospitalizations independent of clinical predictors (MELD, HE, PPI). Stool and colonic mucosal microbiome are altered in cirrhotics who get hospitalized with independent prediction using stool Bacteroidaceae and Clostridiales XIV. Concomitant DM distinctly impacts gut microbiota without affecting hospitalizations.

Gut microbiota drive the development of neuroinflammatory response in cirrhosis in mice

The mechanisms behind the development of hepatic encephalopathy (HE) are unclear, although hyperammonemia and systemic inflammation through gut dysbiosis have been proposed. The aim of this work was to define the individual contribution of hyperammonemia and systemic inflammation on neuroinflammation in cirrhosis using germ‐free (GF) and conventional mice. GF and conventional C57BL/6 mice were made cirrhotic using CCl4 gavage. These were compared to their noncirrhotic counterparts. Intestinal microbiota, systemic and neuroinflammation (including microglial and glial activation), serum ammonia, intestinal glutaminase activity, and cecal glutamine content were compared between groups. GF cirrhotic mice developed similar cirrhotic changes to conventional mice after 4 extra weeks (16 vs. 12 weeks) of CCl4 gavage. GF cirrhotic mice exhibited higher ammonia, compared to GF controls, but this was not associated with systemic or neuroinflammation. Ammonia was generated through increased small intestinal glutaminase activity with concomitantly reduced intestinal glutamine levels. However, conventional cirrhotic mice had intestinal dysbiosis as well as systemic inflammation, associated with increased serum ammonia, compared to conventional controls. This was associated with neuroinflammation and glial/microglial activation. Correlation network analysis in conventional mice showed significant linkages between systemic/neuroinflammation, intestinal microbiota, and ammonia. Specifically beneficial, autochthonous taxa were negatively linked with brain and systemic inflammation, ammonia, and with Staphylococcaceae, Lactobacillaceae, and Streptococcaceae. Enterobacteriaceae were positively linked with serum inflammatory cytokines. Conclusion: Gut microbiota changes drive development of neuroinflammatory and systemic inflammatory responses in cirrhotic animals. (Hepatology 2016;64:1232‐1248)

Continued Alcohol Misuse in Human Cirrhosis is Associated with an Impaired Gut-Liver Axis

BACKGROUND Cirrhosis and alcohol can independently affect the gut-liver axis with systemic inflammation. However, their concurrent impact in humans is unclear. METHODS Our aim was to determine the effect of continued alcohol misuse on the gut-liver axis in cirrhotic patients. Age- and MELD-balanced cirrhotic patients who were currently drinking (Alc) or abstinent (NAlc) and healthy controls underwent serum and stool collection. A subset underwent upper endoscopy and colonoscopy for biopsies and duodenal fluid collection. The groups were compared regarding (i) inflammation/intestinal barrier: systemic tumor necrosis factor levels, intestinal inflammatory cytokine (duodenum, ileum, sigmoid), and ileal antimicrobial peptide expression; (ii) microbiota composition: 16SrRNA sequencing of duodenal, ileal, and colonic mucosal and fecal microbiota; and (iii) microbial functionality: duodenal fluid and fecal bile acid (BA) profile (conjugation and dehydroxylation status), intestinal BA transporter (ASBT, FXR, FGF-19, SHP) expression, and stool metabolomics using gas chromatography/mass spectrometry. RESULTS Alc patients demonstrated a significant duodenal, ileal, and colonic mucosal and fecal dysbiosis, compared to NAlc and controls with lower autochthonous bacterial taxa. BA profile skewed toward a potentially toxic profile (higher secondary and glycine-conjugated BAs) in duodenal fluid and stool in Alc patients. Duodenal fluid demonstrated conjugated secondary BAs only in the Alc group. There was a greater expression of all ileal BA transporters in Alc patients. This group also showed higher endotoxemia, systemic and ileal inflammatory expression, and lower amino acid and bioenergetic-associated metabolites, without change in antimicrobial peptide expression. CONCLUSIONS Despite cirrhosis, continued alcohol misuse predisposes patients to widespread dysbiosis with alterations in microbial functionality such as a toxic BA profile, which can lead to intestinal and systemic inflammation.

Gut microbial composition can differentially regulate bile acid synthesis in humanized mice

We previously reported that alcohol drinkers with and without cirrhosis showed a significant increase in fecal bile acid secretion compared to nondrinkers. We hypothesized this may be due to activation by alcohol of hepatic cyclic adenosine monophosphate responsive element‐binding protein 3‐like protein 3 (CREBH), which induces cholesterol 7α‐hydroxylase (Cyp7a1). Alternatively, the gut microbiota composition in the absence of alcohol might increase bile acid synthesis by up‐regulating Cyp7a1. To test this hypothesis, we humanized germ‐free (GF) mice with stool from healthy human subjects (Ctrl‐Hum), human subjects with cirrhosis (Cirr‐Hum), and human subjects with cirrhosis and active alcoholism (Alc‐Hum). All animals were fed a normal chow diet, and none demonstrated cirrhosis. Both hepatic Cyp7a1 and sterol 12α‐hydroxylase (Cyp8b1) messenger RNA (mRNA) levels were significantly induced in the Alc‐Hum and Ctrl‐Hum mice but not in the Cirr‐Hum mice or GF mice. Liver bile acid concentration was correspondingly increased in the Alc‐Hum mice despite fibroblast growth factor 15, fibroblast growth receptor 4, and small heterodimer partner mRNA levels being significantly induced in the large bowel and liver of the Ctrl‐Hum mice and Alc‐Hum mice but not in the Cirr‐Hum mice or GF mice. This suggests that the normal pathways of Cyp7a1 repression were activated in the Alc‐Hum mice and Ctrl‐Hum mice. CREBH mRNA was significantly induced only in the Ctrl‐Hum mice and Alc‐Hum mice, possibly indicating that the gut microbiota up‐regulate CREBH and induce bile acid synthesis genes. Analysis of stool bile acids showed that the microbiota of the Cirr‐Hum and Alc‐Hum mice had a greater ability to deconjugate and 7α‐dehydroxylate primary bile acids compared to the microbiota of the Cirr‐Hum mice. 16S ribosomal RNA gene sequencing of the gut microbiota showed that the relative abundance of taxa that 7‐α dehydroxylate primary bile acids was higher in the Ctrl‐Hum and Alc‐Hum groups. Conclusion: The composition of gut microbiota influences the regulation of the rate‐limiting enzymes in bile acid synthesis in the liver. (Hepatology Communications 2017;1:61–70)