European Review for Medical and Pharmacological Sciences 2020; 24: 2750-2775 Biliary tract microbiota: a new kid on the block of liver diseases? A. NICOLETTI1, F.R. PONZIANI2, E. NARDELLA1, G. IANIRO2, A. GASBARRINI1, L. ZILERI DAL VERME2 1Internal Medicine, Gastroenterology and Hepatology, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Università Cattolica del Sacro Cuore, Rome, Italy 2Internal Medicine, Gastroenterology and Hepatology, Fondazione Policlinico Universitario Agostino Gemelli IRCCS, Rome, Italy Abstract. – The microbiome plays a crucial man body1,2. Indeed, a resident microbiota has recent- role in maintaining the homeostasis of the or- ly been described in several human environments ganism. Recent evidence has provided novel previously described as devoid of microorganisms, insights for understanding the interaction be- such as the urinary tract and the stomach3-9. Even tween the microbiota and the host. However, the 10 vast majority of such studies have analyzed the healthy placenta hosts microbial communities . interactions taking place in the intestinal tract. Bile has traditionally been considered sterile The biliary tree has traditionally been consid- under normal conditions11-14. ered sterile under normal conditions. However, The physical and chemical features of bile and the advent of metagenomic techniques has re- its antimicrobial activity were supposed to create vealed an unexpectedly rich bacterial communi- a hostile environment for bacteria. Moreover, the ty in the biliary tract. Associations between specific microbiolog- difficulty in collecting bile samples, coupled with ical patterns and inflammatory biliary diseases the lack of sensibility of culture techniques in and cancer have been recently described. Hence, detecting microbes in low-charge samples, sus- biliary dysbiosis may be a primary trigger in the tained this hypothesis for a long time. pathogenesis of biliary diseases. In particular, In 1967, while studying the microbial flora of recent studies have suggested that microorgan- patients undergoing percutaneous cholangiog- isms could play a significant role in the develop- raphy, Flemma et al15 observed that a consistent ment of gallstones, pathogenesis of autoimmune cholangiopathies and biliary carcinogenesis. number of patients had a positive bile culture with- Moreover, the intimate connection between out having had any signs, symptoms or history of the biliary tract, liver and pancreas, could reveal cholangitis. Ahead of their time, they hypothesized hidden influences on the development of diseas- that bacteria could exist in bile without causing es of these organs. any symptoms attributable to their presence. They Further studies are needed to deepen the named this condition “asymptomatic bactibilia”15. comprehension of the influence of the biliary microbiota in human pathology. This knowl- About 40 years later, the advent of 16S ribo- edge could lead to the formulation of strategies somal RNA sequencing confirmed the presence for modulating the biliary microbiota in order to of microbes in bile samples otherwise consid- treat and prevent these pathological conditions. ered sterile with culture-based techniques16. This knowledge has introduced the concept of “biliary Key Words: microbiota”. Biliary microbiota, Gallstones, Cholelithiasis, Primary At any level, the interplay between the micro- sclerosing cholangitis, Primary biliary cholangitis, Bili- ary tract cancer, Cholangiocarcinoma, Gallbladder car- biota and the host plays a pivotal role in the main- cinoma, Personalized medicine. tenance of homeostasis. However, quantitative or qualitative changes in the composition of the mi- crobial community can derange this equilibrium, 17 Introduction favoring the development of diseases . Recent evidence has revealed rich microbial An increasing number of studies about the human communities in the biliary tract of patients affect- microbiota have dismissed the classical postulate ed by biliary tract diseases. A remarkable associ- which states that there are sterile sites within the hu- ation has been observed between certain microbi- 2750 Corresponding Author: Alberto Nicoletti, MD; e-mail: [email protected] Biliary tract microbiota: a new kid on the block of liver diseases? al strains and each pathology. Thus, possible roles the bile-duct epithelium. Cholangiocytes, which for bacteria in such pathogenic processes have are highly heterogeneous in both structure and been hypothesized18-22. function 23,32,33, modify bile through a sequence The understanding of the interplay between of secretory and absorptive processes in order to the microbiota and the host at this level may fa- regulate its flow and alkalinity according to the cilitate the formulation of novel strategies for the physiological functions24. Along the biliary tree, prevention and treatment of such pathological glandular elements called peribiliary glands or conditions. accessory glands are also present34. Ductal se- cretion is regulated by a wide range of factors, Overview of the Biliary System: including gastrointestinal hormones and choliner- Anatomical and Cellular Determinants gic nerves35. The final secretory product is deliv- for the Production and Secretion of Bile ered to the gallbladder and then to the duodenum. The biliary system represents a complex net- Although the gallbladder is not essential for the work of ducts and organs that are involved in secretion of bile, it helps its storage to prepare for the production and transportation of bile23. Bile fat digestion30. During fasting, the gallbladder is production is a complex biological process that filled with bile31. Only about 50% of the hepatic begins in the bile canaliculi, which are formed by bile reaches the gallbladder for concentration and the apical membranes of two adjacent pericentral storage, while the remaining bile bypasses the hepatocytes linked by tight junctions24. The he- gallbladder to enter the duodenum and undergo patocyte apical membrane is provided with both continuous enterohepatic cycling36. During diges- bile salt-dependent and independent transport tion, cholecystokinin stimulates the contraction systems, which are series of adenosine triphos- of the gallbladder and the common bile duct and phate-binding cassette transport proteins that the relaxation of the SO, resulting in the discharge function as export pumps for bile salts and other of up to 80% of the gallbladder contents into the organic solutes25. These transport systems create duodenum37,38. osmotic gradients in the bile canaliculi, which give the driving force for the flow into the lumen The Mutual Interaction Between Bile and through aquaporins24. Tight junctions hold the the Microbiota hepatocytes together and form a physical barrier Bile is a vital aqueous solution composed of between the blood and canalicular lumen, facil- ∼95% water in which organic and inorganic sol- itating “paracellular permeability”24,26. Bile can- utes, including bile acids, cholesterol, phospho- aliculi conduct the flow of bile countercurrent to lipids, bilirubin and amino acids, are dissolved24. the direction of the portal blood and connect with Bile acids (BAs) are the most prevalent organic the initial branches of the biliary tree, i.e., the compounds in bile, constituting approximately canals of Hering27,28. These structures continue 50% of the organic components of bile. BAs are into ducts that progressively increase in diameter: 24-carbon water-soluble products of cholester- small bile ductules (diameter <15 μm), interlobu- ol metabolism24,39. There are two processes and lar ducts (15-100 μm), septal ducts (100-300 μm), anatomical sites for the biosynthesis of BAs: the area ducts (300-400 μm), segmental ducts (400- primary BAs are first synthesized de novo from 800 μm), and hepatic ducts (>800 μm) as original- cholesterol in the liver and then are modified ly defined by Ludwig23,29. The confluence of the by bacterial enzymes in the intestine38. The two right and left hepatic ducts at the hepatic hilum primary BAs synthesized in the liver are cholic forms the common hepatic duct that is joined by acid (CA), a trihydroxylated bile salt, and cheno- the cystic duct from the gallbladder to form the deoxycholic acid (CDCA), a dihydroxy bile salt39. common bile duct. The common bile duct runs These salts can be conjugated at the side chain through the head of the pancreas and ends in the with taurine or glycine, a process that metaboliz- sphincter of Oddi (SO), while penetrating the du- es BAs into stronger acids limiting their passive odenal wall to form the ampulla of Vater, which reabsorption at the biliary tree24. Intestinal bac- connects it to the pancreatic duct30. SO is a seg- teria, a consortium of a small number of species ment of circular and longitudinal smooth muscle belonging to the class Clostridia40, produce “sec- that incorporates the distal common bile duct and ondary BAs” by removal of the hydroxyl group at pancreatic duct, contained in the duodenal wall31. C7, transforming cholic acid to deoxycholic acid Once bile is secreted into the biliary tree, it is (DCA) and CDCA to lithocholic acid (LCA)38,39,41. exposed to cholangiocytes that form the lining of During transit through the caecum and colon, 2751 A. Nicoletti, F.R. Ponziani, E. Nardella, G. Ianiro, A. Gasbarrini, L. Zileri Dal Verme conjugated BAs can also be “deconjugated” from crease in the production of inflammatory cyto- the link with glycine or taurine by enzymes kines and in innate immune cells