SEARCH

SEARCH BY CITATION

Keywords:

  • Bacterial translocation;
  • cirrhosis;
  • endotoxins;
  • epithelial barrier;
  • intestine;
  • liver;
  • microbiota;
  • permeability;
  • tight junctions

Abstract

  1. Top of page
  2. Abstract
  3. Conclusion
  4. References

Recent evidence suggests that translocation of bacteria and bacterial products, such as endotoxin from the intestinal lumen into the systemic circulation is a contributing factor in the pathogenesis of chronic liver diseases and the development of complications in cirrhosis. In addition to alterations in the intestinal microbiota and immune system, dysfunction of the intestinal epithelial barrier may be an important factor facilitating bacterial translocation. This review aims to provide an overview of the current evidence of intestinal epithelial barrier dysfunction in human chronic liver diseases and cirrhosis, and to discuss possible contributing factors and mechanisms. Data suggest the presence of intestinal epithelial barrier dysfunction in patients with chronic liver diseases, but are more convincing in patients with cirrhosis, especially in those with complications. The barrier dysfunction can result from both direct and indirect effects of aetiological factors, such as alcohol and obesity, which can cause chronic liver diseases and ultimately cirrhosis. On the other hand characteristics of cirrhosis itself, including portal hypertension, alterations in the intestinal microbiota, inflammation and oxidative stress can affect barrier function of both small and large intestine and may contribute to the development of complications. In conclusion, there are indications for intestinal epithelial barrier dysfunction in patients with chronic liver diseases and especially in patients with cirrhosis, which can be caused by various factors affecting both the small and large intestine.

Cirrhosis is an advanced stage of liver fibrosis accompanied by vascular remodelling. It is the end result of any chronic liver disease for which liver transplantation is the only curative option. The natural history of cirrhosis is usually characterized by an asymptomatic stage of compensated cirrhosis followed by a symptomatic stage of decompensated cirrhosis. In clinical practice, decompensated cirrhosis is defined by complications owing to portal hypertension and impaired liver function, including ascites, variceal haemorrhage, hepatic encephalopathy and jaundice [1]. According to a report from the National Center for Health Statistics, cirrhosis and chronic liver diseases were the twelfth leading cause of death in the United States in 2011 [2].

The most common causes of cirrhosis are chronic liver diseases related to alcohol consumption [alcoholic liver disease (ALD)], hepatitis virus infection and obesity and/or other components of the metabolic syndrome [non-alcoholic fatty liver disease (NAFLD)]. Increasing evidence from animal studies indicates that the translocation of bacteria and bacterial products (e.g. endotoxin) from the intestinal lumen into the systemic circulation, resulting in endotoxemia, is a contributing factor in the pathogenesis of several chronic liver diseases by inducing inflammatory changes in the liver [3, 4]. In cirrhosis, bacterial translocation is considered to play an important role in the pathogenesis of its complications [5]. In addition to alterations in the intestinal microbiota and the immune system, dysfunction of the intestinal epithelial barrier is an important mechanism contributing to enhanced bacterial translocation [6]. Barrier dysfunction may be affected by putative aetiological factors, such as alcohol and obesity, as well as by characteristics of cirrhosis itself. Better understanding of the role of the intestinal epithelial barrier function in human chronic liver diseases and cirrhosis may provide further insight into the pathogenesis of cirrhosis and the development of its complications. The role of bacterial translocation, intestinal microbiota and the immune system [e.g. toll-like receptors (TLRs)] in chronic liver diseases and cirrhosis have recently been reviewed elsewhere [6-9].

In the current review, we aimed to summarize the available evidence of intestinal epithelial barrier dysfunction in patients with chronic liver diseases, and in patients with compensated and decompensated cirrhosis. In addition, possible contributing factors and mechanisms of intestinal epithelial barrier dysfunction in chronic liver diseases and cirrhosis will be discussed.

The intestinal epithelial barrier

The intestinal epithelial barrier consists of multiple defence mechanisms, and can mainly be subdivided into an epithelial (i.e. the mucus layer and epithelial cells) and an immunological barrier (i.e. the epithelial secretions and immune cells). In this review, we focus on the epithelial barrier, in particular on the single layer of epithelial cells, which are connected to each other by junctional complexes. The epithelium on the one hand facilitates absorption of luminal nutrients, water and electrolytes, whereas on the other hand serves as a barrier to prevent translocation of potential harmful substances via transcellular and paracellular transport [10]. Paracellular transport is regulated by the apical junctional complex, consisting of the tight junction (TJ) and the subjacent adherens junction (AJ) [11].

The TJs seal the paracellular space and form a selective barrier that allows transport via at least two pathways: a high capacity, charge selective pore pathway for small ions and uncharged molecules and a low-capacity leak pathway for larger molecules, regardless of charge [12]. TJs are considered to be highly dynamic and open and close continuously in response to a variety of stimuli [13]. They consist of several transmembrane proteins, such as occludin, members of the claudin family and junction adhesion molecules, as well as cytoplasmic plaque proteins, such as the zonula occludens proteins (i.e. ZO-1, ZO-2 and ZO-3), which connect the transmembrane proteins with the perijunctional actomyosin ring [11, 14]. Contraction of this ring is important in regulating paracellular permeability and is mainly mediated by activation of myosin light chain kinase (MLCK), which phosphorylates myosin II regulatory light chain (MLC) [11, 14].

The AJs primarily consist of the interaction between the transmembrane protein E-cadherin and the cytoplasmic protein β-catenin. Similar to the TJs, the AJs are connected with the perijunctional actomyosin ring and thereby may also play a role in regulating paracellular permeability [15].

Methods to assess the intestinal epithelial barrier

The intestinal epithelial barrier integrity can be assessed by evaluating the structure and function of the TJs. The morphological structure of the TJs can be studied by freeze fracture electron microscopy using intestinal biopsies. Hereby, the TJs are identified as a network of anastomosing linear fibrils or chains of protein particles, termed TJ strands, which seal adjacent cell membranes [16]. The number of TJ strands was found to correlate positively with the integrity of the epithelium [17]. The TJ structure can also be evaluated in thin sections of tissue or epithelial cells using transmission electron microscopy, where membrane fusions and increased electron density between adjacent cells can be observed [18]. Using the aforementioned techniques, the intercellular space was found to be occluded and therefore the epithelial TJs were considered to be static. However, more advanced techniques using fluorescently labelled TJ proteins, such as fluorescence recovery after photobleaching (FRAP), have demonstrated that, for example, occludin and ZO-1 are highly mobile at the TJ [19, 20].

Furthermore, the expression of TJs and its associated proteins in tissue samples and cell cultures can be assessed on the protein and gene level by means of immunohistochemistry, Western blotting and quantitative polymerase chain reaction (qPCR) [21, 22].

In addition to structural data, functional analyses are also often used to evaluate intestinal epithelial barrier integrity. In vivo culture of intestinal epithelial cell monolayers or ex vivo mounting of live excised intestinal mucosa tissue in Ussing chambers with subsequent measurement of transepithelial resistance (TEER) and/or permeation of specific markers (e.g. FITC-dextran or sucralose) across the epithelium are widely accepted [23, 24]. TEER measures the overall epithelial ion fluxes, whereas permeation of most markers points to the paracellular leak pathway [12, 25]. Recently, three-dimensional culture of Caco-2 and T84 cells in synthetic extracellular matrix proteins have also shown to be suitable for the assessment of intestinal epithelial barrier function [26-28].

In vivo, the most widely accepted method to evaluate TJ function is analysing the urinary recovery of orally administered inert test markers. Combining two test markers that differ in size and transport (i.e. paracellular or transcellular) enables correction for differences in, for example, gastric emptying, intestinal transit, dilution by secretions, tissue distribution and renal function [29]. Examples of these test markers include sugars (monosaccharides and disaccharides, i.e. rhamnose and lactulose respectively), polyethylene glycols (PEGs: 400, 1000, 4000) and radiolabeled chelates (51Cr-ethylenediaminetetraacetic acid (51Cr-EDTA), 99mTc-diethylenetriaminepentaacetic acid (99mTc-DTPA)). Because of their size, most of these markers reflect the leak pathway [12]. TJ function can also be evaluated by measuring the presence of intraluminal substances, such as endotoxin and D-lactate, in the systemic circulation [30].

In addition to loss of TJ integrity, intestinal epithelial barrier dysfunction can result from damage to intestinal epithelial cells. Intestinal cell damage can be assessed non-invasively by measuring endogenous cytosolic enterocyte proteins in plasma or urine, such as intestinal fatty acid binding protein (I-FABP) [30]. Intestinal biopsies provide the possibility for histological evaluation of intestinal epithelial cell damage and apoptosis.

Intestinal epithelial barrier dysfunction in chronic liver diseases and cirrhosis

Several studies have been published assessing intestinal permeability in patients with chronic liver diseases that may eventually progress towards cirrhosis as well as in patients with cirrhosis in comparison with healthy subjects by measuring the urinary excretion of orally administered test markers.

In Table 1, studies in patients with chronic liver diseases are presented, grouped by aetiology. Studies performed in chronic alcoholics, of which at least a subgroup had evidence of liver disease, showed a significant increased gastroduodenal and/or whole intestinal permeability compared with healthy subjects [31-33](Table 1).

Table 1. Studies comparing intestinal permeability in patients with chronic liver diseases vs. healthy controls, either as a primary or secondary outcome
ReferenceN of pt with CLD analysed Aetiology /degree of CLDN of HC analysedIP results (P-value)Duration of urine collection
  1. CLD, chronic liver diseases; h, hours; HC, healthy controls; IP, intestinal permeability; LD, liver disease; L/R ratio, lactulose/rhamnose ratio; L/M ratio, lactulose/mannitol ratio; N, number; NASH, non-alcoholic steatohepatitis; np, not provided; ns, not significant; pt, patients.

  2. a

    27 with cirrhosis.

  3. b

    7 with cirrhosis.

  4. c

    16 with cirrhosis.

  5. d

    16 with cirrhosis.

  6. e

    Patients abstaining from alcohol ≤ 4 days (n = 26) or 5–14 days (n = 20) or > 14 days (n = 9).

  7. f

    In NAFLD, sig. difference between mild vs. moderate/severe steatosis.

Alcoholic liver disease
Bjarnason et al. [31] 1984

26e

20

9

Alcoholics without cirrhosis (degree np.)34

[UPWARDS ARROW]51Cr-EDTA (P < 0.001)

[UPWARDS ARROW]51Cr-EDTA (P < 0.02)

51Cr-EDTA ns. (P > 0.1)

24 h
Parlesak et al. [32] 2000

17

18

Alcoholics with mild LD

Alcoholics with moderate LD

30

PEG 400 ns. (np), [UPWARDS ARROW] PEG 1500 + 4000 (P < 0.01)

PEG 400 ns. (np), [UPWARDS ARROW] PEG 1500 + 4000 (P < 0.01)

24 h
Farhadi et al. [33]201069Alcoholics with/without LD49[UPWARDS ARROW] sucrose (P = 0.004)5–12 h
Non-alcoholic fatty liver disease
Wigg et al. [36] 200118NASH16L/R ratio ns. (P = 0.37)5 h
Farhadi et al. [37] 2008

10

6

NASH

Steatosis

12

L/M ratio ns. (np), sucralose ns. (np)

([UPWARDS ARROW] sucralose after aspirin in NASH (P = 0.002)

L/M ratio ns. (np), sucralose ns. (np)

5 h/24 h

5 h/24 h

Miele et al. [35] 200935NAFLDf24[UPWARDS ARROW]51Cr-EDTA (P < 0.001)24 h
Volynets et al. [34] 201220NAFLD10 (NAFLD-free)[UPWARDS ARROW] L/M ratio (P < 0.05)6 h
Other chronic liver diseases
Resnick et al. [39] 199033Hepatobiliary disordersa1199mTc-DTPA ns. (P > 0.05)24 h
Keshavarzian et al. [40] 199910HCV + PBCb20sucrose ns. (np), L/M ratio ns. (np)5 h
Feld et al. [38] 2006

86

69

PBCc

HCVd

101-155 15 new

[UPWARDS ARROW] sucrose (P = 0.0001), [UPWARDS ARROW] L/M ratio (P < 0.0001)

Sucrose ns. (np), [UPWARDS ARROW] L/M ratio (P < 0.0001)

8 h
Cariello et al. [41] 201043Chronic hepatitis (HCV/ALD/NASH)134[UPWARDS ARROW] L/M ratio (P < 0.01)5 h

In patients with NAFLD, results are conflicting (Table 1). Two studies showed a significant increased small and whole intestinal permeability comparing 20 and 35 NAFLD patients, respectively, with healthy subjects [34, 35], whereas two smaller studies could not find significant differences [36, 37]. However, the latter study by Farhadi et al. [37] did observe a significantly increased 0–24 h and 6–24 h urinary excretion of sucralose (indicating whole intestinal and large intestinal permeability respectively) in non-alcoholic steatohepatitis (NASH) patients after an aspirin challenge.

In addition, several studies have assessed intestinal permeability in patients with chronic liver diseases as a result of hepatitis C virus infection (HCV) [38], primary biliary cirrhosis (PBC) [38] or mixed aetiologies [39-41]. Results were not always consistent and no definite conclusion can be drawn from these single studies. Furthermore, in most of them also patients with cirrhosis were included [38-40] (Table 1).

Studies assessing intestinal permeability in patients with cirrhosis are given in Table 2. Most studies found a significantly increased small intestinal [40-54], gastroduodenal [40, 54] and whole intestinal [32, 55-58] permeability in patients with cirrhosis when compared with healthy subjects, despite different methods used (e.g. test markers) and patients included (e.g. aetiology and severity) (Table 2). Studies investigating specifically the large intestine in cirrhosis are scarce; one recent study reported a significantly increased 5–26 h urinary excretion of sucralose in cirrhotic patients vs. healthy subjects [54].

Table 2. Studies comparing intestinal permeability in cirrhotic patients vs. healthy controls, either as a primary or secondary outcome
ReferenceN of CP analysed (Child–Pugh: A/B/C) AetiologyN of HC analysedIP results (P-value)Duration of urine collection
  1. a

    One P-value for multiple group comparisons.

  2. A, alcohol; AV, alcohol and viral; CP, cirrhotic patients; h, hours; HC, healthy controls; IP, intestinal permeability; L/M ratio, lactulose/mannitol ratio; L/R ratio, lactulose/rhamnose ratio; N, number; np, not provided; ns, not significant; O, other; sig, significant; V, viral.

Budillon et al. [155] 198511 (np)A1/V4/O68[DOWNWARDS ARROW] L/R ratio (P < 0.05)5 h
Romiti et al. [42] 199024 (12/12/1)A8/O1720[UPWARDS ARROW] L/R ratio (P < 0.05)5 h
Campillo et al. [43] 199980 (13/26/41)A77/V2/O128[UPWARDS ARROW] L/M ratio (P < 0.01)6 h
Ersoz et al. [55] 199944 (17/10/17)A6/V30/O810[UPWARDS ARROW]99mTc-DTPA (P < 0.0001)24 h
Keshavarzian et al. [40] 199910 (10/0/0)A1020

[UPWARDS ARROW] sucrose sig. (np)

[UPWARDS ARROW] L/M ratio sig. (np)

5 h
Parlesak et al. [32] 200019 (np)A1930

PEG 400 ns. (np)

[UPWARDS ARROW] PEG 1500 + 4000 (P < 0.05)

24 h
Fujii et al. [44] 200135 (23/8/4)A1/V30/O46[UPWARDS ARROW] L/R ratio (P < 0.05)8 h
Huglo et al. [59] 200132 (1/19/15)A321451Cr-EDTA ns. (np)24 h
Xu et al. [45] 200220 (5/11/4)A/V/O20[UPWARDS ARROW] L/M ratio (P < 0.01)6 h
Liu et al. [46] 200443 (np)np11[UPWARDS ARROW] L/M ratio (P < 0.001)a6 h
Zuckerman et al. [47] 200466 (8/32/26)

A40/V8/

AV12/O6

74[UPWARDS ARROW] L/R ratio (P < 0.001)5 h
Kalaitzakis et al. [56] 200620 (8/2/10)A9/V3/O820[UPWARDS ARROW]51Cr-EDTA (P < 0.05)24 h
Spahr et al. [48] 200719 (6/7/6)A12/O512[UPWARDS ARROW] L/M ratio (P < 0.05)5 h
Dastych et al. [49] 200826 (0/26)A2620[UPWARDS ARROW] L/M ratio (P < 0.0001)5 h
Lee et al. [50] 200853 (23/21/9)A31/V21/AV126

PEG 3350/400 ratio:

[UPWARDS ARROW] in CP with ascites (P < 0.05)

ns. in CP without ascites (np)

8 h
Scarpellini et al. [57] 200952 (18/19/15)A14/V36/O248[UPWARDS ARROW]51Cr-EDTA (P < 0.0001)24 h
Cariello et al. [41] 201040 (30/10)A11/V29134[UPWARDS ARROW] L/M ratio (P < 0.01)5 h
Thalheimer et al. [51] 20107 (4/3/0)A3/V3/AV114[UPWARDS ARROW] L/M ratio (np)5 h
Choi et al. [52] 201127 (12/8/7)V2745[UPWARDS ARROW] PEG 3350/400 ratio (P = 0.019)8 h
Van Vlerken et al. [58] 201212 (9/3/0)A4/V5/O39

PEG 400 ns. (P = 0.16)

[UPWARDS ARROW] PEG 1500 + 3350 (P = 0.02)

24 h
Benjamin et al. [53] 201380 (0/41/39)

A46/V10/

AV3/O21

50[UPWARDS ARROW] L/M ratio (P = 0.007)5 h
Norman et al. [54] 201264 (0/15/49)A4863

[UPWARDS ARROW] sucrose (P = 0.002)

[UPWARDS ARROW] L/M ratio (P < 0.0001)

[UPWARDS ARROW] sucralose (P < 0.0001)

5 h/26 h

As mentioned above, in most studies with cirrhotic patients, mixed groups of patients were included with regard to aetiology, severity of cirrhosis and the presence of complications. Most of the studies assessing intestinal permeability in relation to the severity of cirrhosis could not find significant differences among patients with Child A, B and C cirrhosis [41-44, 47, 53-56, 59, 60]. However, several of them observed a non-significant increase in permeability as the severity changed from Child A to B and C cirrhosis [41, 43, 44, 47, 56] and three others did find a significantly higher permeability in patients with Child C vs. those with Child A and B cirrhosis [50, 57, 61]. Overall, these observations indicate that an increased intestinal permeability may be more pronounced in the most severe degree of cirrhosis.

Studies have also compared intestinal permeability in cirrhotic patients with vs. those without complications, either as primary or secondary outcome (Table 3). Most studies did not observe a significant difference in cirrhotic patients with ascites vs. those without ascites [32, 41, 43, 54, 59, 60], but several of them performed these analyses as secondary outcomes only. Four well-designed studies (based on primary outcome and/or large numbers) did find a significantly higher intestinal permeability [47, 50, 57, 61]. This is further supported by studies showing a significantly increased intestinal permeability in patients with ascites, but not in patients without ascites when compared with healthy controls [47, 50, 56]. These findings therefore suggest that especially the presence of ascites is associated with changes in intestinal permeability in cirrhosis. Results on the association between intestinal permeability and spontaneous bacterial peritonitis (SBP) are difficult to interpret because of methodological differences, such as the time of assessing intestinal permeability, and the small number of patients with SBP [43, 46-48, 53, 55, 57, 60-62]. Contrasting results have been reported on the association between intestinal permeability and hepatic encephalopathy (HE) [50, 53, 59, 61].

Table 3. Studies comparing intestinal permeability in cirrhotic patients with vs. those without complications, either as a primary or secondary outcome
ReferenceN of CP analysedN of CP with complicationsN of CP without complicationsIP results (P-value)Duration of urine collection
  1. a

    One P-value for multiple group comparisons.

  2. CP, cirrhotic patients; h, hours; HE, hepatic encephalopathy; IP, intestinal permeability; L/M ratio, lactulose/mannitol ratio; L/R ratio, lactulose/rhamnose ratio; N, number; np, not provided; ns, not significant; sig, significant.

Ascites
Campillo et al. [43] 1999804931L/M ratio ns. (P = 0.07)6 h
Parlesak et al. [32] 200019811PEG 1500 + 4000 ns. (np)24 h
Huglo et al. [59] 20013227851Cr-EDTA ns. (np)24 h
Di Leo et al. [60] 2003392514

Sucrose ns. (np)

L/M ratio ns. (np)

6 h
Pascual et al. [61] 2003795524[UPWARDS ARROW] L/M ratio (P = 0.03)5 h
Zuckerman et al. [47] 2004664818[UPWARDS ARROW] L/R ratio (P < 0.001)a5 h
Kalaitzakis et al. [56] 2006201010

51Cr-EDTA:

[UPWARDS ARROW] in CP with ascites vs. HC (P < 0.05)

ns. in CP without ascites vs. HC (P > 0.1)

24 h
Lee et al. [50] 2008532627[UPWARDS ARROW] PEG 3350/400 ratio (P < 0.05)8 h
Scarpellini et al. [57] 2009522230[UPWARDS ARROW]51Cr-EDTA (P < 0.001)24 h
Cariello et al. [41] 2010403010Correlation between L/M ratio and degree of ascites ns. (np)5 h
Norman et al. [54] 2012645014L/M ratio ns. (np)5 h/26 h
Spontaneous bacterial peritonitis
Campillo et al. [43] 19998016 (8 SBP)64[UPWARDS ARROW] L/M ratio (P < 0.01)6 h
Ersoz et al. [55] 199944133199mTc-DTPA ns. (np)24 h
Di Leo et al. [60] 200339npnp

Sucrose ns. (np)

L/M ratio ns. (np)

6 h
Pascual et al. [61] 200379574[UPWARDS ARROW] L/M ratio (P = 0.03)5 h
Liu et al. [46] 2004433211[UPWARDS ARROW] L/M ratio (P < 0.001)a6 h
Zuckerman et al. [47] 200448 ascites741L/R ratio ns. (np)5 h
Spahr et al. [48] 200719613L/M ratio ns. (P = 0.17)5 h
Scarpellini et al. [57] 200922 ascites1012Impaired 51 Cr-EDTA (P < 0.01)24 h
Kim et al. [62] 201111326 (2 SBP)87[UPWARDS ARROW] PEG 3350/400 is sig. predictor for development of bacterial infections (P < 0.01)24 h
Benjamin et al. [53] 2013711259No sig. difference in incidence of SBP between pt with abnormal and normal L/M ratio (P = 0.173)5 h
Hepatic encephalopathy
Huglo et al. [59] 20013243151Cr-EDTA ns. (np)24 h
Pascual et al. [61] 2003791564[UPWARDS ARROW] L/M ratio (P = 0.005)5 h
Lee et al. [50] 2008531142[UPWARDS ARROW] PEG 3350/400 ratio (P < 0.05)8 h
Benjamin et al. [53] 2013712348No sig. difference in incidence of HE between pt with abnormal and normal L/M ratio (P = 0.464)5 h

Overall, there are indications for an increased intestinal permeability in patients with chronic liver diseases, especially in patients with ALD and possibly also in patients with NAFLD, but the number of studies is very limited, a variety of test markers is used and in some of them also patients with cirrhosis are included. The majority of studies in cirrhotic patients point to the presence of an increased intestinal permeability. Changes seem to be most pronounced in patients with advanced cirrhosis and/or complications, especially in those with ascites.

It is important to bear in mind that the evaluation of the intestinal epithelial barrier function by the urinary excretion of orally administered test markers has several pitfalls. The possibility of a delayed gastrointestinal transit, differences in volume distribution (for example as a result of the presence of ascites) and renal dysfunction in cirrhotic patients, for example, can affect intestinal permeability results when using a single test marker. The urinary excretion ratio of two test markers that differ in size and transport pathway will therefore more accurately reflect intestinal permeability in these patients. Furthermore, medication often used by cirrhotic patients such as NSAIDs or lactulose may influence test results.

Findings of increased endotoxin levels in patients with chronic liver diseases [32, 34, 37] and cirrhosis [52, 63, 64], and also the presence of bacterial DNA in blood and ascites in 30–40% of cirrhotics [65-67] support the relevance of the increased intestinal permeability observed in these patients. Direct measures of bacterial translocation in humans are too invasive and therefore scarce. One study has been published reporting a significantly increased prevalence of bacterial translocation, defined as the isolation of enteric organisms from mesenteric lymph nodes (MLNs) in patients with Child C cirrhosis compared with Child A and B cirrhosis [68].

The above-mentioned findings of an increased intestinal permeability suggest an impairment of TJs. However, studies assessing TJ structure in patients with chronic liver diseases and cirrhosis are limited. A small study by Tang et al. [69] showed that patients with ALD had a significantly reduced protein expression of ZO-1 in sigmoid biopsies compared with healthy subjects, which was accompanied by a significantly higher miR-212 expression in ALD patients. The mRNA expression of ZO-1, however, was not significantly different between both groups. Protein expression of ZO-1 was also found to be significantly lower in duodenal biopsies of NAFLD patients than in healthy subjects [35]. In addition, in this latter study intestinal permeability was increased.

Cirrhotic patients with and without ascites were studied by Assimakopoulos et al. [63]. They showed a significantly reduced expression of the TJ proteins occludin and claudin-1 in duodenal biopsies of the total patient group compared with healthy controls and this correlated inversely with the increased plasma endotoxin levels. In addition, the cirrhotic patients with ascites showed a significantly reduced expression of occludin and claudin-1 compared with those without ascites. Recently, increased duodenal protein levels of claudin-2, and a decreased TEER and increased permeability was detected in duodenal biopsies mounted in Ussing chambers from patients with decompensated cirrhosis [70]. However, no differences in mRNA and proteins levels of occludin, claudin-1, ZO-1 and connexion-43 were found between the cirrhotic patients and controls. Application of electron microscopy in this study, further showed an intact epithelial barrier in patients with decompensated cirrhosis, suggesting that the epithelial barrier in cirrhosis is functionally altered, but structurally normal [70]. In contrast with the latter finding, Such et al. [71] found distended intercellular spaces with morphologically intact TJs in duodenal biopsies of six cirrhotic patients with complications.

An increased intestinal permeability may also be related to epithelial cell damage. Histological changes in the intestinal mucosa have been reported in ALD patients, such as villous atrophy, increase in lamina propria infiltrate and intraepithelial lymphocytes, as well as changes in the cellular functions (i.e. brush border, membrane and cellular enzymes) [72]. Changes were also found in the intestinal mucosa of cirrhotic patients with portal hypertension and included shortened and wider microvilli, a decreased villus/crypt ratio, oedema of lamina propria, fibromuscular hyperplasia, thickened muscularis mucosae, increased apoptosis and capillary abnormalities [71, 73-75].

Although differences in TJ structure and expression supporting functional permeability analyses have been demonstrated in cirrhotic patients, overall conclusions on specific TJ proteins or subgroups of patients cannot be drawn as a result of methodological differences and the relative small number of studies.

Possible contributing factors and mechanisms of intestinal epithelial barrier dysfunction in chronic liver diseases and cirrhosis

Non-alcoholic fatty liver disease; obesity

Non-alcoholic fatty liver disease is a common cause of chronic liver disease in developed countries that can progress to cirrhosis. An increase in intestinal permeability has been observed in some studies with NAFLD patients, but not in all (Table 1). However, an increased intestinal permeability in animal models and elevated endotoxin levels in both animals and NAFLD patients [34, 37, 76-79], further support the role of a decreased barrier function.

The majority of patients with NAFLD are obese [80], and though two small studies could not find a significant difference in intestinal permeability by the urinary excretion of sugars between obese subjects and lean controls [81, 82], Moreno-Navarrete et al. [83] recently showed that circulating zonulin levels were significantly increased in obese subjects and considered this to be a marker of intestinal permeability. Furthermore, evidence from animal models of obesity clearly show an increased intestinal permeability, changes in the expression of TJ proteins/genes and elevated endotoxin levels [3, 84-86].

Several possible factors may contribute to the barrier dysfunction in obesity. Results from both animal and human studies have suggested that especially the diet (e.g. high-fat intake) itself played a role [87-92], for example, by altering intestinal microbiota composition [92, 93].

In addition, obesity is considered a state of chronic, low-grade inflammation, characterized by increased production of pro-inflammatory cytokines and adipokines. The systemic inflammation may lead to inflammation and epithelial barrier dysfunction of the small and/or large intestine, as TNF-α and IFN-γ, for example, have been shown to increase permeability by disrupting TJ integrity in vitro [94-96].

Finally, both quantitative and qualitative alterations of the intestinal microbiota have been reported in obesity [97]. For example, small intestinal bacterial overgrowth (SIBO) has been shown in some obese subjects [98, 99] and although not confirmed in all studies [100, 101], a shift in the faecal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been found in obese subjects [102] and in animal models of obesity [103, 104]. Changes in intestinal microbiota (mainly SIBO) have also been reported in NAFLD patients [105].

The microbial alterations can affect the intestinal epithelial barrier function indirectly, for example, via increased endotoxin levels [106] or directly by altering TJ expression and integrity [107, 108]. The association between intestinal microbiota, intestinal epithelial barrier function and obesity is further supported by intervention studies in mice, showing that modulation of the intestinal microbiota composition with prebiotic and/or antibiotic treatment improved intestinal permeability, changed expression of TJ proteins/genes and reduced endotoxin levels [84, 109]. However, Fouts et al. [110] showed that increased intestinal permeability and bacterial translocation can also be independent of changes in the intestinal microbiota.

Alcoholic liver disease; alcohol

Alcohol abuse is one of the major causes of chronic liver disease. In the United States, almost 50% of all deaths from cirrhosis and chronic liver diseases were found to be attributed to ALD in 2011 [2].

An increase in intestinal permeability has been found in chronic alcoholics with and without liver disease [31-33, 111], as well as in non-alcoholics after administration of a single dose of alcohol [112, 113]. Furthermore, endotoxin levels were found to be higher in patients with different stages of ALD than in healthy subjects [32, 64, 114]. Several animal studies further support that alcohol can increase intestinal permeability and induce endotoxemia, subsequently leading to liver injury [4, 115, 116]. Not only alcohol itself but also its oxidative and non-oxidative metabolites, such as acetaldehyde and fatty acid ethyl esters, respectively, may reach the small as well as the large intestine and can contribute to intestinal epithelial barrier dysfunction by disrupting TJ and AJ integrity through several mechanisms involving, for example, MLCK activation and increased generation of reactive oxygen species (ROS) [117].

They may also impair the intestinal barrier function by inducing direct cell damage [118, 119]. Finally, recent evidence found alcohol-induced changes in the intestinal microbiota [120, 121], which were associated with intestinal epithelial barrier dysfunction [122-124].

The increased intestinal permeability observed in patients with cirrhosis can be the result of the (on-going) effects of obesity and alcohol. On the other hand, cirrhosis itself can also lead to barrier dysfunction via the effects of portal hypertension, the intestinal microbiota, inflammation and oxidative stress, each of which will be discussed below.

Portal hypertension

Portal hypertension is a severe consequence of cirrhosis and may lead to ascites, variceal haemorrhage and hepatic encephalopathy. It results from an increased intrahepatic vascular resistance, mainly caused by architectural distortion of the liver secondary to fibrous tissue and regenerative nodules, and an increased portal blood inflow secondary to splanchnic vasodilatation. Splanchnic vasodilatation and the consequent decrease in effective arterial blood volume lead to activation of neurohumoral systems (i.e. renin–angiotensin aldosterone, sympathic nervous system and antidiuretic hormone), which are responsible for the elevated plasma volume [125]. The latter has been suggested to cause dilatation of epithelial intercellular spaces, probably through the formation of intestinal wall oedema, which thereby may alter TJ integrity and increase intestinal permeability [45, 71, 126]. Histological studies did show dilatation of the intercellular spaces [71, 126] and oedema of the lamina propria in cirrhotic patients with portal hypertension [73].

Several studies have shown a significantly higher gastroduodenal and/or intestinal permeability in cirrhotic patients with endoscopic signs of portal hypertension than in those without [41, 60, 127, 128]. The portal pressure was found to correlate significantly with intestinal permeability, and placement of a transjugular intrahepatic portosystemic shunt resulted in a significant decrease in both portal pressure and intestinal permeability in cirrhotic patients [45]. Consistent with these findings, Reiberger et al. [129] recently reported a significant correlation between portal pressure and gastroduodenal and intestinal permeability, plasma levels of LPS-binding protein and IL-6 in cirrhotic patients with portal hypertension. The significant improvement of intestinal permeability and bacterial translocation after treatment with non-selective beta-blockers in these patients further supports a role for portal hypertension in affecting intestinal barrier function [129].

A disturbed microcirculation of the intestinal mucosa could lead to an increase in intestinal permeability [45, 129]. Another possible mechanism is nitric oxide (NO), which has been proposed to play a key role in the haemodynamic changes associated with cirrhosis and portal hypertension [130], but can also increase intestinal permeability [131].

The intestinal microbiota

As stated before, alterations in the intestinal microbiota are an important factor, which can affect TJ integrity and facilitate bacterial translocation. SIBO is a frequent finding in cirrhotic patients when using the glucose-hydrogen breath test and/or culture of jejunal aspirates and has partly been attributed to an impaired intestinal motility [125]. However, Steed et al. [132] reported no evidence of bacterial overgrowth by real-time qPCR, except for enterococci, in the duodenal mucosa of cirrhotic patients, and suggested to shift focus to the large intestine. In faecal samples of cirrhotic patients, significantly increased numbers of Escherichia coli and Staphylococcus spp. were found when compared with healthy controls [133]. Studies using 16S rRNA-based molecular methods, also showed differences in faecal microbiota composition between cirrhotic patients and healthy controls, including, for example, a significant increase in the numbers of Enterobacteriaceae [134-137]. This is in line with E. coli being a major cause of SBP and urinary tract infections in these patients [138]. Recently, the mucosal microbiota composition of the sigmoid of cirrhotic patients was also found to be significantly different from healthy controls (with a lower abundance of potentially beneficial and higher abundance of potentially pathogenic genera) [139]. The difference was most prominent in patients with HE.

Inflammation and oxidative stress

Cirrhosis is associated with systemic inflammation as shown by elevated levels of inflammatory cytokines, NO and its metabolites in blood of these patients [41, 140-144]. Consequently, this may lead to inflammation and epithelial barrier dysfunction in the small and/or large intestine. Evidence of intestinal inflammation in cirrhotic patients is still limited, but increased faecal concentrations of polymorphonuclear elastase [145] and calprotectin [146] have been reported.

Oxidative stress plays also a role in the systemic inflammation and is increased in cirrhosis. It has been hypothesized that inducers of oxidative stress produced in the liver, may cause intestinal oxidative stress upon transferal to the intestine via bile excretion and blood circulation [147, 148]. The decreased antioxidant status observed in cirrhotic patients [149], may further facilitate intestinal oxidative stress. Studies in cirrhotic rats have indeed shown evidence of oxidative stress in the small and large intestine [150-153], but data in cirrhotic patients are still lacking. Intestinal oxidative stress can impair epithelial barrier function by inducing direct cell damage and/or disrupting TJ and/or AJ integrity [148, 154].

Conclusion

  1. Top of page
  2. Abstract
  3. Conclusion
  4. References

Dysfunction of intestinal epithelial barrier may enhance the risk of translocation of bacteria and bacterial products into the systemic circulation, and thereby contribute to the pathogenesis of chronic liver diseases, cirrhosis and the development of complications.

Current evidence indicates the presence of intestinal epithelial barrier dysfunction in patients with chronic liver diseases, but is more convincing in patients with cirrhosis, especially in those with complications. Most evidence is derived from studies using the urinary excretion of orally administered test markers (mainly sugars), focusing on small and whole intestinal permeability. However, it should be noted that the increased permeability in these studies does not necessarily prove the occurrence of bacterial translocation, which most likely also involves other mechanisms. Furthermore, reliable analyses of bacterial translocation in humans are difficult and often based on indirect measurements. Additional studies investigating both TJ structure and function of the small and large intestine in patients with cirrhosis are necessary to clarify whether the impaired barrier function is supported by structural TJ alterations, epithelial cell damage and/or bacterial translocation.

Dysfunction of the intestinal epithelial barrier in cirrhosis could be because of the on-going effects of aetiological factors that can cause chronic liver diseases and ultimately cirrhosis, such as obesity and alcohol (Fig. 1). Clinical and experimental evidence suggests that both can affect barrier function directly and indirectly through mechanisms involving alterations of the intestinal microbiota, inflammation and oxidative stress. Cirrhosis itself is associated with portal hypertension as well as alterations in the intestinal microbiota, chronic on-going inflammation and oxidative stress, each of which can affect barrier function in both small and large intestine and may contribute to the development of complications (Fig. 1).

image

Figure 1. Possible contributing factors and mechanisms of intestinal epithelial barrier dysfunction in chronic liver diseases and cirrhosis.

Download figure to PowerPoint

In conclusion, there are indications for intestinal epithelial barrier dysfunction in patients with chronic liver diseases and especially in patients with cirrhosis, which can be caused by various factors affecting both the small and large intestine and may contribute to the development and progression of these liver diseases.

References

  1. Top of page
  2. Abstract
  3. Conclusion
  4. References