On the potential role of intestinal microbial community in hepatocarcinogenesis in chronic hepatitis B

Abstract The chronic infection of hepatitis B virus (HBV) is the most potent risk factor for the development of cirrhosis and hepatocellular carcinoma (HCC). The association of intestinal microbiota alteration with progressive liver disease has been investigated in recent studies. Overgrowth of potentially pathogenic bacteria of gram‐negative species and, in particular, a significant increase in the fecal count of Escherichia coli (E. coli) are characterized in the presence of HCC. This study was conducted to describe the characteristics of the intestinal microbiota related to the presence of HCC in HBV‐carrier patients. The available literature indicates the colonization of E. coli as principal source of portal vein lipopolysaccharide (LPS), in the gut may contribute to the carcinogenesis process by inducing chronic inflammation. This understanding could help to predict the clinical outcomes in HBV‐carrier patients and innovative strategies to reduce the virulence of liver disease from intestinal dysbiosis.


| INTRODUCTION
The intestinal microbiota in healthy adults creates a complex ecological system that plays an important role in the digestion of a wide variety of foods, in the synthesis of vitamins and in the breakdown of toxins and drugs. 1 In addition, the microbial components of the intestine, which includes tryptophan catabolites, short-chain fatty acids and bile acids, act as functional signaling molecules for well-balanced microbial effects in the liver-intestine axis. The normal composition of the intestinal flora, the maturation of the immune system, and the intestinal mucosal barrier organize the healthy intestinal functional barrier which breaks down into microbial dysbiosis. 2 Chronic hepatitis with either hepatitis B or hepatitis C viruses (HBV or HCV) infections is associated with a greater translocation of the intestinal microbiota. 3,4 Intestinal microbiota alteration appears to have a significant role in the progression of hepatocarcinogenesis in HBVcarrier patients. 5,6 This review summarizes the composition of the intestinal microbiota in chronic hepatitis B (CHB). We have also referred to the mechanisms that Escherichia coli (E. coli) may imply in hepatocarcinogenesis.

| CURRENT KNOWLEDGE OF THE INTESTINAL MICROBIOTA
The intestinal microbiota is the most heterogeneous group of microorganisms that is rich in species, but unique as a fingerprint for each person. Half of the stools are virtually microbial biomass with about 10 13-14 microorganisms that are packed so densely with an extraordinary 10 11 cells/cm. 7 The intestinal microbiota includes about 100 times more genes than the number of human genes. This characterizes human as meta-organisms and the intestinal microbiota as our second genome. 8,9 Microbial diversity in the human gastrointestinal tract represents a challenge in characterizing microbial biomass as they are unidentified by current culture methods. 10 However, the use of modern culture-independent techniques based on variations in 16S rRNA genes revealed the presence of different species of more than 70 genera in the human gut. 11 The taxonomic classification of the intestinal microbiota according to the ordinary nomenclature (phylumclass-order-family-genus-species) has characterized the most common phyla Firmicutes, Bacteroidetes, Actinobacteria and Proteobacteria. Firmicutes, the main group, includes gram-positive anaerobic bacteria with a low G + C content in the gastrointestinal tract. The Bacteroidetes are rodshaped anaerobic bacteria characterized in gram-negative and nonspore. Actinobacteria such as gram-positive and Proteobacteria as gram-negative bacteria are also dominated in the gut. Despite the relatively stable intestinal microbiota, dysbiosis occurs in immune reactions, in inappropriate environmental and nutritional circumstances that are associated with a number of pathological situations. 9,12,13

HBV-CARRIER LIVER DISEASE
Recent study in the diversity and composition of the intestinal microbiota in HBV-induced chronic liver disease revealed alterations in the colonization of opportunistic intestinal bacteria. The intestinal microbiota of CHB patients has significant difference compared to healthy controls with increased bacterial charge in taxonomy: Actinomyces, Clostridium sensu stricto, unclassified Lachnospiraceae and Megamonas that influence on disease progression. 14 Complicated patients with liver cirrhosis and liver cancer demonstrate intestinal dysbiosis that is also approved in animal studies. 4,15 In general, the composition of intestinal microbiota in HBV-carrier patients shows an increase in the growth rate of E. coli. [15][16][17] The intestinal microbiota profile of end-stage liver disease in CHB patients showed an obvious increase in Enterobacteriaceae, Enterococcus faecalis and Faecalibacterium prausnitzii. 1,6 Intestinal bacteria with high frequency in CHB patients are illustrated in Table 1. Reciprocally, the lower number of intestinal lactic acid species such as Lactobacillus, Pediococcus, Weissella and Leuconostoc has been reported in CHB patients. 1,6 Furthermore, the amount of Clostridium spp cluster XI and XIVa as well as Bacteroides, Prevotella, and Lactobacillus spp. such as L. rhamnosus and L. fermentus was also adjusted with a concomitant decrease in fecal samples of HBV-related cirrhosis. 6,18 The Bifidobacteria/Enterobacteriaceae (B/E) ratio also decreased significantly. 6 Table 2 presented bacteria with lower frequency in HBV-carrier patients. Cirrhotic patients with abundant intestinal E. coli are more susceptible to the development of HCC. 17 Similarly, patients with liver cirrhosis and HCC showed higher levels of endotoxin The advantage of intestinal bacteria under normal physiological condition in the synthesis of essential products such as short-chain fatty acids (SCFA) and secondary bile acids in metabolic homeostasis is well known. The produced SCFAs by commensal and probiotic bacteria carry out the transcriptional pathways through epigenomic modifications. 14,22 The blood circulation of intestine with microbiota-derived nutrients and metabolites is directed to the liver via the hepatic portal vein and the liver, in turn, has a reactive impact on intestinal functions beyond the biliary secretion in the intestinal lumen. 22 Bacteria are mainly active in the proximal colon, where the availability of the substrate is in the maximum capacity. In the proximal colon, the nondigestible carbohydrates are hydrolyzed by the bacteria into oligosaccharides and subsequently into monosaccharides. The anaerobic environment of the distal colon, due to the removal of water and the abundance of substrate, is the appropriate situation for fermentation. Saccharolytic bacteria, such as Bacteroidetes, ferment the processed monosaccharides within the glycolysis and pentose-phosphate pathways, to produce SCFAs in addition to the gases CO 2 and H 2 . 23,24 From the main shortchain fatty acids, acetate, propionate, and butyrate are present in an estimated ratio of 60:20:20 in the large intestine. Butyrate is produced by beneficial colonic bacteria and acts as the main source of energy for the colonocytes. Butyrate is beneficial to its metabolic effects on thermogenesis and inducing fibroblast growth factor 21 transcription to increase the oxidation of fatty acid and production of ketone bodies. 25 Furthermore, butyrate has the capacity to improve the growth of lactobacilli and bifidobacteria in the colon. 26 Patients with chronic hepatitis have shown a decrease in polyunsaturated lipids by disrupting the integrity of the intestinal barrier in the alteration of the gut microbiota composition; however, the total lipid has been increased. 14 In addition, CHB patients have significantly lower levels of fecal bile acids with positive association with F. prausnitzii, bifidobacteria and lactic acid-producing bacteria. 27,28 It has also been reported that intestinal dysbiosis in CHB patients was closely associated with the accumulation of serum metabolites, including aromatic amino acids phenylalanine and tyrosine that play a pathogenic role in liver disease. 14

STIMULATION OF IMMUNE RESPONSES IN LIVER INJURY
Overproduction of lipopolysaccharide (LPS) as an endotoxin from the outer cell membrane of gram-negative bacteria has been reported in acute or chronic hepatitis B patients. [29][30][31] LPS stimulate inflammation in Toll-like receptor 4 (TLR4) signaling pathway by ligation to the LPSbinding protein (LBP), CD14, and the MD2. 32 TLRs are responsible for recognizing specific pathogen-associated molecular patterns (PAMP) to activate innate immune responses by the expression of proinflammatory cytokines. 33 Upon the TLR4 ligation, both MyD88-dependent transduction pathways and MyD88 induce an inflammatory response. 33 In HBV transgenic mice, TLR activation induces intrahepatic IFN-a/b production to suppress viral replication. 34 However, in the tolerant immune phase of chronic hepatitis B, HBeAg suppresses TLR signaling result in inhibition of tumor necrosis factor alpha (TNF alpha) expression. HBeAg through its precore-specific sequence binds to the Toll/IL-1 receptor (TIR) motif to stop TLR signaling that induced by TLR2 or TLR4 ligands. 34,35 It is known that HSC and Kupffer cells express TLR4 which makes them sensitive to LPS. 15 TLR4 signaling is a significant mediator of hepatic fibrosis in quiescent HSC by expression of proinflammatory factor-kappa B (NF-kB) factor in downstream of TLR4 activation. 36

MECHANISM FOR DEVELOPMENT OF HCC
Patients in the end stage of liver disease show higher growth of E. coli and increase level of LPS ligand in circulation. 6,15,37 The E. coli is a predominantly aerobic organism in the adult intestine, estimated at 10 7 -10 8 cfu (colony-forming unit) in the colon and a minor number within the terminal ileum. E. coli dominates during the first year of life and becomes the main facultative anaerobic bacterium within the mature intestinal flora. 38 This bacterium also contributes to the generation of an anaerobic environment that favors the further settlement of Bacteroides, Bifidobacterium, and Clostridium species. It also seems to be a harmless commensal that uses its host for a continuous source of nutritive, existing, and survivor. However, E. coli with a notable phenotypic variety of intestinal and extra intestinal pathogenic strains are associated with over 2 million human deaths per year. 39 This toxin is like DNase, in structure and activity, which breaks the double-strand DNA and induce cellular apoptosis. 41,45,46 Colonization by colibactin-producing B2 phylogroups at the early life of animal experiments associated with the reduction in regulatory T-cell populations which is a susceptibility factor for the development of immunemediated diseases and the predisposition of cancer. [47][48][49] The potential effect of intestinal E. coli on colorectal cancer development is extensively evaluated in animal and human models. 50 E. coli LPS in conjunction with TLR4 triggers a powerful cytokine cascade that results in septic shock and death. 51 Recent evidence has firmly confirmed the dynamic effect of intestinal dysbiosis on activation of TLR4 pathway in advanced liver disease. However, a number of tumor suppressors may elucidate the mechanism between the development of HCC and colonization of intestinal E. coli. As indicated above, colonic microbiota of patients with cirrhosis has revealed a growth of Enterobacteriaceae and Enterococcus but a decrease in Bifidobacterium species leading to endotoxemia with portal hypertension and damage to hepatocytes. 17,19 LPS derived from the microbiota binds to TLR4 and CD14, as a coreceptor, to activate the intracellular signaling pathways in upregulation of antiviral components. 30,31,33 Kupffer cells and sinusoidal endothelial cells (LSEC) of nonparenchymal hepatic cells of mice in TLR4 stimulation, inhibit replication of HBV independently of MyD88, so TLR agonists may be useful for the treatment of chronic HBV infection. 52