The gut microbiome and intestinal failure-associated liver disease

Lu Jing ,Jun Xu ,Si-Yng Cheng ,Ying Wng ,c,Wei Ci ,,∗

a Division of Pediatric Gastroenterology and Nutrition, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 20 0 092, China

b Shanghai Institute for Pediatric Research, Shanghai 20 0 092, China

c Shanghai Key Laboratory of Pediatric Gastroenterology and Nutrition, Shanghai 20 0 092, China

d Department of Pediatric Surgery, Xinhua Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 20 0 092, China

Keywords: Parenteral nutrition Microbiota Bile acid Short-chain fatty acid Prebiotics Probiotics

ABSTRACT Intestinal failure-associated liver disease (IFALD) is a common hepatobiliary complication resulting from long-term parenteral nutrition (PN) in patients with intestinal failure. The spectrum of IFALD ranges from cholestasis,steatosis,portal fibrosis,to cirrhosis. Development of IFALD is a multifactorial process,in which gut dysbiosis plays a critical role in its initiation and progression in conjunction with increased intestinal permeability,activation of hepatic immune responses,and administration of lipid emulsion.Gut microbiota manipulation including pre/probiotics,fecal microbiota transplantation,and antibiotics has been studied in IFALD with varying success. In this review,we summarize current knowledge on the taxonomic and functional changes of gut microbiota in preclinical and clinical studies of IFALD. We also review the function of microbial metabolites and associated signalings in the context of IFALD. By providing microbiota-targeted interventions aiming to optimize PN-induced liver injury,our review provides perspectives for future basic and translational investigations in the field.

Introduction

Parenteral nutrition (PN) was introduced since the 1960s as a way to maintain growth and development in infants and adults,and it has been used as a life-saving therapy for patients with intestinal failure [1] . However,long-term PN is often accompanied by complications such as central venous thrombosis,catheter-related bloodstream infections,and intestinal failure-associated liver disease (IFALD) [ 2,3 ]. Typical clinical signs of IFALD include cholestasis,steatosis,and gallbladder stones that eventually may lead to liver cirrhosis [4] . Overall,IFALD is characterised by cholestasis in infants and children and steatohepatitis in adults [5] . Unfortunately,there is no pharmacological treatment for this disease,and when it progresses to end-stage,combined liver and intestinal transplantation is the only option.

In the last few decades,the role of gut-liver axis has been addressed in various liver diseases,such as alcoholic and nonalcoholic fatty liver disease [6],liver fibrosis [7],cirrhosis [8],and hepatocellular carcinoma [9] . The pathogenesis of IFALD is a multifactorial process that is mainly attributed to intestinal failure and PN administration. Changes in gut microbiota composition,microbial metabolism,and gut barrier function are additional co-factors that can contribute to progression of IFALD. Although preclinical studies have reported an important role of the gut microbiota in modulating IFALD,studying the microbiome in the controlled setting of preclinical models still has several limitations and the causal relationships are elusive in the very complex human ecological situation. In this review,we summarize current studies elucidating the role of gut microbiota and microbial metabolites in IFALD. We focus on the pathogenic pathways in humans,and the potential to target gut microbiome as for the treatment of IFALD.

Lipids and IFALD

Lipids in PN solutions are essential to maintain proper growth and health. However,preclinical and clinical studies provide evidence that intravenous lipid emulsions (ILEs) are the key contributors to IFALD pathogenesis compared with nonlipid components [10] . In general,the major source of lipids is soybean-based-ILEs (SO-ILEs),which are featured by the proinflammatoryω-6 polyunsaturated fatty acids (PUFAs) and phytosterols [ 11,12 ]. In comparison,fish oil (FO)-based ILEs are enriched inω-3 PUFAs,which has been shown to reverse PN-associated cholestasis (PNAC)in children on long-term PN [13–15] .

In addition toω-6 PUFAs,SO-ILEs are highly abundant in phytosterols,which are plant-derived sterol compounds. In enteral feeding,only a small proportion of phytosterols can be absorbed.However,it can be fully delivered to the liver by PN administration [16] . A previous study suggested that high phytosterol level in PN-dependent children is associated with more severe cholestasis [16] . It has been shown that phytosterols disrupt bile acid homeostasis that leads to cholestasis by regulating FXR signaling [16] . Addition of stigmasterol to the FO-ILEs leads to marked increase in markers of liver injury,bile acids,hepatic macrophage activation,and downregulation of bile acid transporters to a similar level to SO-ILEs [17] .Invitro,stigmasterol is shown to activate macrophages that may contribute to the hepatic innate immune activation. In mice pretreated with dextran sodium sulfate(DSS) before PN administration,expression of ABCG5/G8,which is responsible for hepatocyte secretion of phytosterols,is downregulated by IL-1β,leading to accumulation of phytosterols and interruption of FXR signaling [18] . Taken together,these data suggest that lipid emulsions in combination with impaired intestine synergize to result in IFALD.

Gut barrier dysfunction in IFALD

The gut barrier is the most important line of preventing pathogens from attaching to and invading the host tissue [19],and it comprises three major lines of defense: a physical barrier,a biochemical barrier,and an immune system barrier [ 20,21 ]. There is a bidirectional communication between gut and liver through the portal vein,biliary tract and systemic circulation. Blood flows from the intestine through the portal vein to the liver before returning to the heart and lungs. The liver also communicates with the gut via hepatic bile flow and can release mediators into the circulation [22] . Therefore,the gut barrier protects liver from being attacked by toxins,microorganisms,and microbial products when translocating from intestine.

PN-induced gut barrier dysfunction leads to increased intestinal permeability and absorption of bacterial products and other microbial-associated molecular patterns (MAMPs) from injured intestine that induces innate immune responses in the liver [23] . Pediatric patients with intestinal resection showed marked decline in tight junction proteins [zonula occludens-1 (ZO-1),occludin,Ecadherin,and claudin-4] as well as significant increases in tumor necrosis factor-α(TNF-α) and Toll-like receptor 4 (TLR4) levels in unfed segments of bowel compared to fed segments from the same individual [24] . Mice receiving total parenteral nutrition (TPN) for 7 days showed that the complex interplay between TNF-αand its two receptors (TNFR1 and TNFR2) was responsible for the TPNassociated gut barrier dysfunction [25] . Further,Lin 28A expression was aberrantly increased in the intestinal epithelial cells of TPN rats. It disrupted intestinal barrier function through a sustained translational repression of occludin [26] .

Researchers revealed immune system reactions to TPN since 1988 when Alverdy and co-workers demonstrated increased bacterial counts in mesenteric lymph nodes in rats [27] . PN impairs innate mucosal immunity by suppressing mucin2 (MUC2) produced by goblet cells and secretory phospholipase A2 (sPLA2) secreted by Paneth cells that can be reversed by exogenous interleukin-25 (IL-25),suggesting that IL-25-induced effects augment the barrier defense mechanisms [28] . Previous studies suggest that PN reduces polymeric immunoglobulin receptor (pIgR) expression and luminal IgA production by impairing mucosal immunity. The mechanisms involve suppression of IL-4 and Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling that can be reversed by IL-25 administration [29–32] . In addition,Takeda G protein receptor 5 (TGR5) signaling is also associated with IFALD progression,and loss of TGR5 exacerbates cholestasis by increasing levels of IL-6 and TNF-αin TPN-fed mice [33] . Last,PN leads to an expansion of Gram-negative Proteobacteria and increases release of lipopolysaccharide (LPS),which activates NF-κB and decreases intraepithelial lymphocyte (IEL)-derived IL-10 and epidermal growth factor (EGF) levels. Eventually,these changes lead to breakdown of tight junctions,loss of epithelial barrier function,increase of bacterial translocation,and sepsis [34] .

Gut microbiota in IFALD

Gut microbial diversity develops as feeding and dietary patterns mature until the age of 3-5 years old [35] . The gut microbiota plays an important role in maintaining homeostasis and health by interacting with host and contributing to different processes,such as bile acid metabolism [36],carbohydrate digestion [37],and vitamin synthesis [38] . The gut microbiome comprises trillions of bacteria,fungi,and viruses that encode more genes than the human genome. Although the composition of gut microbiota is similar in healthy individuals,there are distinct features in each person’s microbial profile. The methodologies to characterize the taxonomic composition of gut microbiota have rapidly evolved since the application of 16S rDNA and metagenomic shotgun sequencing. Under normal conditions,the gut microbiota is balanced in a state that promotes human physiological resilience [39],while perturbation of gut homeostasis resulting from challenges can cause many chronic diseases [40] .

PN is associated with an overall decrease in diversity and a general shift from Firmicutes to LPS-producing Proteobacteria and Bacteroidetes [41] . Interestingly,PN induces different alterations of gut microbial profile in adults,infants,and animals (mouse,rat,piglet) [42] . In PN-fed piglets,the bacterial diversity and concentrations were reduced compared with the enteral nutrition (EN)group. Translocation of bacteria from the intestinal tract to tissues or blood showed similar levels in PN-fed piglets compared with the EN group,while the PN group was at higher risk of colonization byClostridiumdifficile[43] . A study compared the effects of different lipid emulsions,ω-3 PUFAs (SMOFlipid) versusω-6 PUFAs (Intralipid),on gut microbiota composition and mucosal surfaces in TPN-fed neonatal piglets. They showed that piglets in the SMOFlipid group had a similar microbial profile as the sow-fed group (control),while the Intralipid group showed a specific and dramatic increase inParabacteroides. Genes associated with intestinal tight junction (claudin 1) and inflammation (IL-8) were increased in the TPN groups,especially in the SO group [44] . In addition,Call et al. [45] compared the bile acid and gut microbiota composition of different body compartments in TPN-fed piglets. Interestingly,theClostridiumcluster XIVa was significantly increased in the TPN group compared with controls [45] .

Compared with adults,infants are more susceptible to various challenges since their gut microbiota is more dynamic and can change greatly after weaning [ 38,46 ]. In a longitudinal analysis,a group of infants received TPN for 4 weeks and fecal samples were analyzed for 16S rDNA sequencing. Overall,bacterial diversity was significantly lower in the TPN group at week 4. At phylum level,the TPN group had significantly lower Bacteroidetes and higher Verrucomicrobia abundance compared to controls,and these differences became more pronounced over time. At genus level,abundance ofBacteroidesandBifidobacteriumwas lower in the TPN group at all time points. Further analysis showed that infants who developed cholestasis are predominated by Proteobacteria,suggesting a possible link between Proteobacteria expansion and liver injury [47] . As PN is associated with increased risk of bloodstream infections,antibiotics are often used for bacterial infections. Antibiotics treatment with metronidazole and gentamicin in a rabbit model of TPN reduced hepatic apoptosis and TPN-induced liver injury [48] . Jia et al. [49] found that prolonged use of antibiotics and PN causes a significant decrease inLactobacillusandBifidobac-terium. Moreover,gestational age,sex,and birth weight are factors impacting specific genera in preterm infants.

Patients with short bowel syndrome (SBS) are at increased risk of developing hepatobiliary complications when receiving PN.Based on the anatomy of remaining intestine,SBS can be divided into three categories,type I (end-jejunostomy),type II (jejunocolic anastomosis),and type III (jejunoileal anastomosis) [50] . As type II SBS patients do not have ileocecal valve and intact colon,their gut microbiota,intestinal adaption,and clinical symptoms are affected [ 51,52 ]. Several studies have shown that the disruption of gut microbiota leads to prolonged PN,poor growth,and predisposes to liver diseases in SBS [53–55] . Huang et al. [56] compared the gut microbial signatures among three different types of SBS in a small cohort. They showed that a decreased bacterial diversity was positively correlated with remaining small intestine length.Overall,commensal bacteria from Lachnospiraceae,Ruminococcaceae and Bacteroidaceae families were declined in SBS patients.SBS II patients were enriched in Proteobacteria but deficient in Firmicutes and Bacteroidetes. In comparison,LactobacillusandPrevotellaare dominated in SBS II. The PN duration of SBS patients was positively correlated with the proportion of Enterobacteriaceae and negatively correlated withLactobacillus.Functional analysis of citrate cycle and branched-chain found that the biosynthesis of aromatic amino acids was abundant in SBS II patients,while functional profiles of pyrimidine and purine metabolism were dominant in SBS III patients [56] . Another study from Budinska et al. [57] characterized gut microbiota and metabolites in fecal samples from different types of SBS patients. They showed that SBS I patients were characterized by the abundance of oxygen-tolerant microorganisms and depletion of strict anaerobes. Non-PN SBS patients showed markers of partial fecal microbiota normalization.

Microbial products in IFALD

Bile acids

Bile acids are signaling molecules that are synthesized in the liver via cytochrome P450-mediated oxidation of cholesterol. Bile acid synthesis can occur through two biosynthetic pathways: the“classical pathway” and the “alternative pathway”. Primary bile acids are conjugated and secreted to the bile via bile salt export pump (BSEP) [58] or multidrug resistance-associated protein 2(MRP2) [ 59,60 ]. During cholestatic conditions,excessive bile acids and bilirubin can be excreted into the systemic circulation through MRP3 and MRP4,as well as organic anion transporting polypeptide 2 (OATP2) and organic solute transporter-α/β(OST-α/β) [ 59,60 ].Following lipid absorption in the small intestine,both conjugated and unconjugated bile acids are reabsorbed in the distal ileum by the apical sodium dependent bile acid transporter (ASBT) [ 61,62 ].Hepatic FXR regulates bile acid synthesis and enterohepatic circulation,while limiting bile acid accumulation in the liver. Intestinal FXR activation upregulates fibroblast growth factor 15 (FGF15)in mice and FGF19 in humans to inhibit bile acid synthesis in the liver by activating FGFR4.

Gut microbiota,bile acids,and the host interact closely to modulate host immune and metabolic homeostasis. Gut microbiota modulates bile acid metabolism in the gut [63],and bile acids shape the gut microbial community [64] . A previous study showed that total bile acid pool shrinks due to impairment of bile acid absorption in pediatric SBS patients [65] . Occurrence of bile acid malabsorption is the result of decreased anatomic and functional intestinal surface area,especially the terminal ileum where bile acids are reabsorbed. In patients with intestinal failure,hepatic bile acid synthesis is increased to compensate for the bile acid loss,while bile acid transport is disrupted. In SBS patients with PN support,the serum level of C4 was elevated,suggesting an increased bile acid synthesis [66] . In pediatric intestinal failure patients with liver injuries,FGF19 levels were decreased in patients without remaining ileum and were correlated with remaining ileum length and cholesterol synthesis markers. Further,FGF19 levels were negatively correlated with liver inflammation,fibrosis,and injury [67] . TGR5 is widely expressed in the liver and gut. A previous study suggested that TGR5 was associated with liver steatosis and inflammation [68] . In human newborns with PN support,TGR5-specific bile acids were negatively correlated with worsening clinical diseases markers. In TGR-/- mice,PN significantly increased liver weight,cholestasis,and serum hepatic stress enzymes. Mechanistically,PN increased unconjugated primary bile acids and secondary bile acids in TGR-/- mice,while increasing conjugated primary bile acid levels in wildtype mice. The gut microbiota of TGR-/- mice displayed highly elevated levels ofBac-teroidesandParabacteroides,possibly responsible for the elevated levels of secondary bile [33] .

Short-chain fatty acids

Short-chain fatty acids (SCFAs) are end products of intestinal microbial fermentation of indigestible foods,such as resistant starch or dietary fiber [69] . Acetate,propionate,and butyrate are the main SCFAs that constitute>95% SCFAs. Most SCFAs are produced in the cecum and proximal colon [70] . SCFA production can be affected by microbiota diversity and food consumed by the host. SCFAs function by binding to G-protein coupled cell surface receptors 41 (GPR41) and GPR43 to stimulate epithelial cell proliferation and differentiation in rats [71] . Using radiolabeled13C-SCFAs infused into the cecum of mice,a study showed that acetate and butyrate were involved in liver cholesterol synthesis. In comparison,propionate infusion was used fordenovogluconeogenesis,but not lipogenesis [72] . Budinska et al. [57] analyzed fecal metabolomic profile in PN-dependent SBS patients and found that they were characterized by high saturated aldehydes and medium-chain fatty acids with reduced SCFAs (butanoic and pentanoic acids) compared with non-PN-dependent SBS patients and healthy controls. In children with intestinal failure,the relative abundance of butyrate-producing Clostridia was reduced as well as intestinal butyrate level,emphasizing the therapeutic role of butyrate in intestinal failure [73] . Considering the beneficial roles of SCFAs,especially butyrate,in improving liver function and intestinal barrier,several preclinical studies have been performed to investigate the role of butyrate in parenteral nutrition-associated liver disease (PNALD). In PN-fed rats,butyrate supplementation in PN solution partially alleviated PN-induced intestinal barrier impairment and normalized IL-4,IL-10,and IgA mRNA expression. In addition,PN was associated with an increase in Tregs in mesenteric lymph nodes,which was normalized by butyrate [74] . In neonatal piglets that underwent 80% jejunoileal resection,supplementation of butyrate in TPN augmented intestinal adaptation by increasing proliferation and decreasing apoptosis of intestinal epithelial cells. Plasma glucagon-like peptide 2 (GLP-2) concentration was also increased by butyrate [75],suggesting an increase of intestinal epithelial cell proliferation and improved gut barrier function. Collectively,these data suggest that butyrate administration may promote enteral feeding transition in PN-dependent SBS patients.Invitro,sodium butyrate treatment attenuated SO-based lipid emulsion-induced increase in intestinal permeability of LPS by modulation of P-glycoprotein,which restricted the absorption of harmful substances including LPS from the gut [76] .

Modulation of the gut microbiota in IFALD

To prevent or reverse the development of IFALD,the most recommended approach is to discontinue PN and initiate enteral feeding either by oral or tube feeding. High concentrations of proinflammatory fatty acids and phytosterols are present in traditional SO-ILE,modification of which has been tested over the past decades in IFALD. In surgical infants who required long-term PN,lipid restriction to 1 g/kg/day significantly reduced the incidence of PNALD/IFALD compared with infants who received standard lipid doses [77] . A multicenter study compared the effects of low- and traditional-dose SO-ILE on the development of liver injury. They found that the low-dose SO-ILE group had a slower increase in direct bilirubin levels compared with the traditional-dose group [78] . However,caution should be taken when reducing lipids since neonates are at high risk of developing essential fatty acid deficiency.

As many patients with IFALD are not responsive to changes of lipid emulsions or cannot initiate enteral feeding,other options to improve their prognosis are needed. On the basis of growing evidence of an important role in IFALD,modulating gut microbiota by targeted or untargeted approaches could be therapeutically beneficial. Targeted approaches include using bacteria and host metabolites as targets,while untargeted approaches include modulation by diet,antibiotics,prebiotics,probiotics,and fecal microbiota transplantation (FMT) [79] . Probiotics such as members fromBifidobacteriumandLactobacillusare widely used and they exhibit beneficial effects by producing lactic and acetic acids to inhibit growth of pathogens. In addition,they can cross-feed other commensal microbiota to increase SCFAs [80] . Nevertheless,the effects of prebiotics and probiotics remain controversial in different studies. In children with SBS,Lactobacillusrhamnosustreatment had no effect on intestinal permeability compared with the placebo group [81] . Uchida et al. [82] performed a case-control study to evaluate the effects of synbiotics (Bifidobacteriumbreve,Lactobacil-luscasei,and galacto-oligosaccharides) on SCFA levels in SBS patients. They showed that symbiotic treatment improved SCFA levels in 3 out of 4 patients. Since SBS patients are deficient in butyrateproducers,supplementing anerobic butyrate producers could be potentially effective. Small intestinal bacterial overgrowth (SIBO)is a common complication of intestinal failure that is defined as the overgrowth (>105CFU/mL) of bacteria in a small intestinal aspirate [83] . For patients who develop SIBO,oral antibiotic regimens are commonly used for treatment. Previous studies showed that antibiotics such as gentamicin and metronidazole may reduce cholestasis occurrence in PN-dependent neonates [ 84,85 ]. As antibiotics are nonspecific,they may cause the development of drug resistance in bacteria. Therefore,management of IFALD with antibiotics requires further validation.

Future perspectives and conclusions

Relevant animal models are needed to recapitulate human IFALD to investigate the mechanisms of disease progression. Compared with rodent models,piglets are more comparable to humans in the aspect of physiological characteristics of IFALD. In addition,intestine resection or drug-induced intestinal injury is needed before administration of PN to better recapitulate IFALD in clinic. Last,modulation of gut microbial composition and function using precise medicine techniques is needed to avoid adverse effects. This can tailor specific therapies based on the stage of liver diseases and gut microbial profile of patients with IFALD.

In summary,although PN provides life-saving therapies for patients with intestinal failure,the side effects are not negligible. The gut microbiota is intricately linked with the initiation and propagation of liver injuries in patients who receive PN ( Fig. 1 ). Although an increasing number of studies have revealed the associations between gut microbiota and IFALD,future preclinical and large clinical studies are needed to adequately translate these microbial findings into practice.

Fig. 1. Schematic overview of the role of gut microbiota in the pathogenesis of intestinal failure-associated liver disease (IFALD). Parenteral nutrition (PN) and intestinal failure lead to alterations of gut microbiota and microbial products that translocate from gut to the liver via portal vein,resulting in liver injuries including cholestasis,steatosis,inflammation,fibrosis,cirrhosis,and eventually liver failure. SCFAs: short-chain fatty acids; PAMPS: pathogen-associated molecular patterns.

Acknowledgments

None.

CRediT authorship contribution statement

LuJiang:Conceptualization,Funding acquisition,Writing - original draft.JuanXu:Writing - review & editing.Si-YangCheng:Writing - review & editing.YingWang:Writing - review & editing.WeiCai:Supervision,Writing - review & editing.

Funding

This study was supported by grants from the National Natural Science Foundation of China (82100950) and Shanghai Natural Science Foundation (23ZR1452600).

Ethical approval

Not needed.

Competing interest

No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.