Gut Microbiota, Inflammation and Hepatic Encephalopathy: A Puzzle with a Solution in Sight

When things are investigated, then is true knowledge achieved.

—Confucius

Development of the gut microbiota

The human gastrointestinal tract is sterile at birth. However, several factors, such as mode of delivery (vaginal versus Caesarean delivery), gestational age (pre- versus full-term), type of feeding, hygiene, etc. play an important role in the colonization of the gut by a variety of microorganisms after birth. Contact with the mother's vaginal and intestinal flora is an important source of Lactobacillus, Prevotella and other Bifidobacterium that colonize the gut in a new born delivered vaginally while, in a Caesarean delivery, exogenous bacteria not derived from the mother are the main source of bacteria colonizing the gut. Gut flora in the latter is less diverse, is mainly derived from bacteria found on the skin surface and is dominated by Staphylococcus. Hence intestinal colonization by Lactobacillus, Bifidobacterium and Bacteroides is delayed. Feeding practices also modifies the gut microflora.

Based on the predominant dietary habit, Arumugam and colleagues have identified three distinct enterotypes, which are named after the genera – Bacteroides, Prevotella and Ruminococcus.¹ While presence of Bacteroides correlates with high-fat high-protein consumption, Prevotella is linked to intake of fibers, simple sugars and carbohydrates.

Gut flora, systemic inflammation and hepatic encephalopathy

Hepatic encephalopathy (HE) is characterized by cognitive impairment and is usually seen in patients with advanced liver disease or porto-systemic shunts. The onset of HE indicates a poor prognosis. Ammonia plays a key role in the pathogenesis of HE by producing astrocyte swelling and cerebral edema. However, recent evidences suggest that oxidative stress, inflammation and neurosteroids have synergistic role in pathogenesis of HE.² In the setting of intestinal barrier dysfunction and systemic inflammation, gut flora and their by-products such as ammonia, indoles, oxindoles and endotoxin play an important role in the pathogensis of HE (Figure 1). Bacterial overgrowth and impaired intestinal barrier integrity results in endotoxemia, i.e. release of bacterial endotoxins in circulation, mainly consisting of lipopolysaccharides (LPS), flagellin, peptidoglycan, and microbial nucleic acids.^3,4 They are also termed as ‘pathogen-associated molecular patterns (PAMPs)’. Endotoxemia initiates liver damage through its interaction with Toll-like receptors (TLRs), which are specific recognition receptors exhibiting a direct relationship between intestinal flora changes, endotoxemia and liver damage leading to the activation of immune response and regulation of inflammation.

Figure 1.

Gut microbiota, systemic inflammation and hepatic encephalopathy. PAMPs, pathogen-associated molecular patterns; TLRs, Toll-like receptors; NH₃, ammonia.

There is mounting evidence that systemic inflammation causes exacerbation of the symptoms of HE in patients with cirrhosis of liver across all grades of HE.^5–7 Proinflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF) and IL-6 modulate the cerebral effect of ammonia. Hence, proinflammatory mechanisms may act synergistically with ammonia in producing cognitive impairment of varying degree.⁸

In an elegant study, Shawcross et al⁵ demonstrated that the presence and severity of minimal HE in cirrhosis was not related to the severity of liver disease and plasma ammonia concentration. However markers of inflammation were significantly higher in those with minimal HE compared to those without. Recently the same group also demonstrated significant association between infection or systemic inflammatory response and grade 3/4 HE, while HE grade or coma score did not correlate with ammonia, biochemistry and model for end-stage liver disease (MELD) score.⁷

There is now a substantial body of evidence from studies in patients and animal models demonstrating that gut flora is altered in patients with liver cirrhosis and HE and may be responsible for formation or release of products such as ammonia, endotoxin, indoles, oxindoles, etc. that may lead to cognitive impairment.^9,10 Current treatments for HE, such as lactulose, antiobiotics and others also rely on manipulation of the gut flora.^11,12 Culture-based techniques were used for the characterization of gut flora in prior HE studies.^9,13

Recently, Bajaj and co-workers have published 3 studies related to gut microbiota, inflammation and cognition in hepatic encephalopathy. They used a systems biology approach, which could be useful to identify novel functional hypothesis and new therapeutic targets for HE. They also used a correlation-network analysis to link features within each treatment group to dissect out functionality in the system. The main findings of these 3 studies are summarized below.^14–16

Gut microbiota and its linkage with inflammation and cognition in hepatic encephalopathy

The authors hypothesized that the gut microbiome composition would be correlated with cognition and inflammation in cirrhotic patients with HE and that this association would be different from those who have never developed HE.¹⁴ Hence, Bajaj et al conducted this study to 1) link the gut microbiome with cognition and inflammation in cirrhotic patients with and without HE using a systems biology approach, 2) identify differences in the microbiome of healthy controls and cirrhotic patients, and 3) define the effect of lactulose withdrawal on microbiome of cirrhotic patients.¹⁴ They performed multitag pyrosequencing (MTPS) on stool of cirrhotic patients and age-matched controls. All cirrhotic patients also underwent cognitive testing, inflammatory cytokines, and endotoxin analysis.

Microbial analysis between controls and patients with cirrhosis

Fecal microbiome differed significantly between cirrhotic patients with and without HE and controls. There was a significantly greater abundance of Lachnospiraceae and Ruminococcaceae in the control group while Enterobacteriaceae, Fusobacteriaceae, Alcaligenaceae, Lactobacillaceae, and Leuconostocaceae were significantly lower in the controls compared with cirrhotic patients. Patients with cirrhosis and HE had higher concentration of Enterobacteriaceae, Alcaligenaceae, Lactobacillaceae, and Streptococcaceae. Further analysis demonstrated that there was no significant difference between the clinical, inflammatory, or cognitive profile between HE patients on lactulose alone compared with those on lactulose and rifaximin.

Alcaligenaceae abundance was significantly associated with poor cognitive performance. Enterobacteriaceae, responsible for most of the life-threatening infections associated with advanced cirrhosis, was found to be associated with worsening inflammation and increasing severity of liver disease in the cirrhosis group. Also, there was a negative correlation between Ruminococcaceae and endotoxemia and MELD score indicating a protective role on infections and liver disease severity. This study for the first time also demonstrated a direct correlation between Porphyromonadaceae and Alcaligeneceae and poor cognitive function. Alcaligeneceae are Proteobacteria that are associated with opportunistic infections and degrade urea to produce ammonia, which may explain the association of Alcaligeneceae with poor cognitive function. The results of this study indicate that future trials with targeted prebiotics and probiotics should aim at enhancing cognition through modulation of these microbiome components.^3,11

There were no significant differences in the fecal microbiome families between the HE and no HE groups except a significantly higher abundance of Veillonellaceae in the HE group. Also, lactulose withdrawal did not change the microbiome significantly except for reduction in Faecalibacterium sp.

Correlation-network analysis

Fusobacteriaceae, Veillonellaceae, and Enterobacteriaceae were positively- and Ruminococcaceae negatively-related to inflammation. Correlation-network analysis comparison showed robust correlations (all P < 10⁻⁵) only in the HE group between the microbiome, cognition, and IL-23, IL-1b, IL-2, IL-4 and IL-13, which indicate a synergy between inflammation, cognition and microbiome changes. IL-23/IL-17 cytokine pathway may be important for intestinal inflammation in HE and cirrhosis.

The influence of lactulose withdrawal on the gut microbiota

Lactulose, a prebiotic, has been used to treat HE. However its mechanism of action remains incompletely understood and is likely to be related, in part, to alterations in gut flora and their by-products.¹⁵ In the previous report, Bajaj et al have convincingly demonstrated that lactulose withdrawal does not exert a very significant effect on the composition of the fecal microbiome except for a reduction in Faecalibacterium sp.¹⁴ These observations suggest that gut bacterial function, rather than a change in the microbiome composition, may be responsible for the effects of lactulose.¹⁵ Authors hypothesized that cirrhotic patients with HE controlled on daily lactulose can be withdrawn from therapy without cognitive deterioration and those who did experience an episode of HE recurrence have different metabiome interactions than those who remained recurrence-free. They tested this hypothesis by taking a longitudinal metabiome approach to relate gut-derived metabolites to the activation of systemic inflammatory and metabolomic pathways and an assessment of cerebral metabolites by in-vivo brain MR spectroscopy (MRS).

Patients whose HE recurred post-withdrawal were compared to those without recurrence. Of 7 cirrhotic patients, HE recurred in 3 men ∼32 days after the withdrawal of lactulose. While HE recurrence after lactulose withdrawal can be predicted by baseline cognitive tests (inhibitory control test lures and block design test), there is relatively minor change in the content of the microbiome i.e. reduction in Faecalibacterium spp. (pre 6%–1% post-withdrawal, P = 0.026) and Veillonella decrease (2%–0%, P = 0.06). Faecalibacterium spp. are also associated with disrupted choline metabolism that was elegantly demonstrated in this study and can result in liver dysfunction. In this study, a shift in choline metabolism from gut bacterial metabolism to the formation of glycine, creatinine and ammonia was associated with the recurrence of HE.

The colonic mucosal microbiome versus stool microbiome: linkage to cognition and inflammation

Although HE is linked to substantial derangements in the gut microecology, with significant fecal overgrowth of potentially pathogenic Escherichia coli and Staphylococcal spp.,¹³ small intestinal bacterial overgrowth⁴ and a pro-inflammatory milieu,^5–7 Bajaj et al did not find differences in the fecal microbiome families between the HE and no-HE patients except a significantly higher abundance of Veillonellaceae in the HE patients.¹⁴ Therefore they hypothesized that the colonic mucosal microbiome rather than stool microbiome may be associated with these derangements in cirrhotic patients with HE.¹⁶

Changes in the intestinal microbiome in the gut mucosa compared with the stool have been demonstrated in non-cirrhotic populations but this has not been studied in cirrhotic patients to date.^17,18 In this study, Bajaj et al¹⁶ hypothesized that there would be significant differences in the colonic mucosa compared to the stool microbiome composition in cirrhotic patients with HE compared to those without HE and that these differences in the mucosal microbiome would be associated with changes in cognition and inflammation.

To prove their hypothesis, authors recruited 60 cirrhotic patients [36 HE and 24 no-HE] performed cognitive testing and analyzed stool sample, cytokine profile (Th1, Th2, Th17 and innate immunity) and endotoxin level in them. 36 patients (19 HE and 17 no-HE) and 17 age-matched controls underwent sigmoid biopsies. MTPS was performed on stool and mucosa. Correlation network analyses were performed to find differences in stool and mucosal microbiome within and between the groups.

Patients with HE had worse MELD score and cognition as well as higher IL-6 and endotoxin levels than those with no-HE. Controls had significantly higher autochthonous (i.e. Dorea, Subdoligranulum and Incertae sedis_other) and lower pathogenic genera (Enterococcus, Proteus, Clostridium and Burkholderia) compared to cirrhotic patients, especially HE patients. Principal Component Analysis (PCO) (Bray Curtis) confirmed a greater variance in the microbiome composition between controls and HE patients compared to controls and no HE patients.

Mucosal microbiota was different from stool within both HE and no-HE groups. While there was no difference in stool microbiota between HE/no-HE patients, the mucosal microbiome was different with lower Roseburia and higher Enterococcus, Veillonella, Megasphaera and Burkholderia abundance in HE. Correlation network analysis demonstrated that autochthonous genera (Blautia, Faecalibacterium, Roseburia and Dorea) were associated with good cognition and decreased inflammation in both HE/no-HE while genera over-represented in HE (Enterococcus, Megasphaera and Burkholderia) were linked to poor cognition and inflammation. Altered synthesis and secretion of bile acids and other biliary components may explain these differences; a decrease in bile acids, antibacterial peptides, and mucins in the colon may result in the selection of potentially pathogenic bacteria to adhere and grow in association with the colonic mucosa of patients with cirrhosis. This may allow for increased translocation of bacterial toxic products and may explain an increase in serum endotoxin and proinflammatory cytokines (especially IL-6) documented in cirrhotic patients with HE.

These 3 recently published studies are likely to be landmark studies and may prove to have given true knowledge about role of gut microbiome in the pathogenesis of HE. They lay the foundation for future studies in patients with liver cirrhosis for studying the effect of therapeutic interventions (probiotics, prokinetics, antibiotics such as rifaximin, etc) on both the colonic mucosal and stool microbiome and related changes in markers of inflammation, on translocation of bacterial toxic products, etc. Such studies are likely to provide newer and specific therapeutic targets – a major goal for patient healthcare – and thus pave the way for improved therapy of the cirrhotic patient with HE.