Review
`Molecular Pathophysiology of Epithelial Barrier
`Dysfunction in Inflammatory Bowel Diseases
`
`3
`
`2
`
`Jessica Y. Lee 1, Valerie C. Wasinger 1,2,*, Yunki Y. Yau 3,4 ID , Emil Chuang 5, Vijay Yajnik 5,6 and
`Rupert WL. Leong 3,4
`1
`School of Medical Sciences, Faculty of Medicine, The University of New South Wales, Sydney 2052, NSW,
`Australia; jessyouminlee@gmail.com
`Bioanalytical Mass Spectrometry Facility, Mark Wainwright Analytical Centre,
`The University of New South Wales, Sydney 2052, NSW, Australia
`South West Sydney Clinical School, Faculty of Medicine, The University of New South Wales, Sydney 2052,
`NSW, Australia; yunki.yau@health.nsw.gov.au(Y.Y.Y.); rupert.leong@health.nsw.gov.au (R.W.L.)
`4 Department of Gastroenterology, Concord Repatriation General Hospital, Sydney 2139, NSW, Australia
`5
`Translational Medicine and Early Clinical, Takeda Pharmaceuticals, Cambridge, MA 02139, USA;
`Emil.Chuang@takeda.com (E.C.); Vijay.Yajnik@takeda.com (V.Y.)
`6 Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Boston,
`MA 02114, USA
`* Correspondence: v.wasinger@unsw.edu.au; Tel.: +61-2-9385-7739; Fax: +61-2-9385-5473
`
`Received: 11 January 2018; Accepted: 26 March 2018; Published: 31 March 2018
`
`Abstract: Over the years, the scientific community has explored myriads of theories in search of the
`etiology and a cure for inflammatory bowel disease (IBD). The cumulative evidence has pointed to
`the key role of the intestinal barrier and the breakdown of these mechanisms in IBD. More and more
`scientists and clinicians are embracing the concept of the impaired intestinal epithelial barrier and its
`role in the pathogenesis and natural history of IBD. However, we are missing a key tool that bridges
`these scientific insights to clinical practice. Our goal is to overcome the limitations in understanding the
`molecular physiology of intestinal barrier function and develop a clinical tool to assess and quantify it.
`This review article explores the proteins in the intestinal tissue that are pivotal in regulating intestinal
`permeability. Understanding the molecular pathophysiology of impaired intestinal barrier function in
`IBD may lead to the development of a biochemical method of assessing intestinal tissue integrity which
`will have a significant impact on the development of novel therapies targeting the intestinal mucosa.
`
`Keywords: intestinal barrier function; inflammatory bowel disease
`
`1. Introduction
`
`The Inflammatory Bowel Diseases (IBD) including ulcerative colitis (UC) and Crohn’s disease (CD)
`are chronic relapsing disorders of the gastrointestinal tract [1]. The intestinal epithelium is a dynamic
`ecosystem that maintains a perpetual cycle of death and renewal of the epithelial lining while preserving
`an elegant balance of immune education, immune response, and immune tolerance to the microorganisms
`in the intestinal lumen [2]. Evaluation of barrier function in IBD has been shown to reflect disease activity
`and may have the potential to predict disease course [2–13]. However without a standard validated
`method of intestinal barrier function assessment, it is difficult to compare and compile findings in this
`important field. Additionally, the exact mechanisms associated with defective barrier functions and
`IBD remains largely unknown. Better understanding of such phenomenon may unravel an important
`pathophysiological process of IBD as well as setting a foundation for the development of a biochemical
`method of assessing and measuring intestinal tissue integrity. This will have significant implications for
`directing and evaluating future research for novel therapies targeting the intestinal mucosa. This review
`
`Proteomes 2018, 6, 17; doi:10.3390/proteomes6020017
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`will examine the components of the epithelial barrier, the pathological changes that result in impaired
`intestinal permeability and its significance in IBD, and current methods of assessing barrier function.
`Barrier function is one side of the dichotomy of the host-environment interface, with the balance of these
`two elements being intrinsic to IBD pathogeneses [14]. The magnitude of the intestinal flora, together
`with its even more numerous and intricate pathogenic pathways, is a subject matter in itself that requires
`focused scrutiny. We have thus focused this review on the intestinal epithelium with limited illustrations
`of significant immunological and microbial interactions when required for context. Recent reviews on the
`microbial side of the intestinal barrier-environmental interface can be found elsewhere [14–16].
`
`2. The Intestinal Epithelial Barrier
`
`The intestinal mucosal barrier is a dynamic structure that separates the intestinal lumen and the
`sterile extracellular internal milieu of the body. Controlled communication between the intestinal
`lumen and the body is essential for the absorption of nutrients, electrolytes, and water, as well as for the
`immune system to greet the microbiota and defend against toxins and pathogens [17]. The intestinal
`barrier consists of the intestinal epithelium, the overlaying mucus layer containing mucin, and various
`antimicrobial peptides [2].
`The paracellular pathways are stringently regulated only to permit the passage of certain solutes
`and fluids, creating a selectively permeable barrier [18]. The junctional complexes, tight junctions
`(TJ), adherens junctions, and desmosomes with connections to the intracellular cytoskeleton, seal the
`paracellular space and provide structural support [18] (Figure 1).
`Claudins are a family of proteins that regulate paracellular pathways across the intestinal
`epithelial barrier [19]. They form charge- and size-specific channels that allow permeation of solutes,
`water, and macromolecules through the TJs [19]. Occludin is a part of the tight junction associated
`marvel protein (TAMP) group along with tricellulin and marvelD3, with a role in cell polarity and
`TJ maintenance by interacting with other TJ proteins and intracellular actin and kinases [19,20].
`Zonulaoccludens (ZO) are scaffolding membrane proteins that connect transcellular proteins to the
`intracellular cytoskeleton, therefore, have a role in assembly and maintenance of junctional proteins
`and paracellular permeability [21].
`Adherens junctions (AJs) are basolateral to the TJs, and have important roles in cell-cell adhesion
`and signalling [9]. Various cadherin proteins interact to regulate the intracellular actin cytoskeleton
`and contribute to the formation of the perijunctionalactomyosin ring [18,22].
`In conjunction with AJs, desmosomes provide the mechanical cohesion of intestinal epithelium,
`providing structural stability [17]. It is composed of various protein subunits including desmoglein,
`desmocolin, plakoglobin, plakophilin and desmoplakin. Its role in maintaining barrier function is
`largely unknown [23].
`Many of these proteins can be aberrant in their abundances and thereby contribute to a
`weaker intestinal barrier [18]. Structural analysis of junction proteins revealed that there are fewer
`horizontal TJ strands and frequent strand discontinuities in IBD tissues, creating a paracellular
`route for macromolecule uptake [24]. The overall pattern of TJ protein abundances show consistent
`upregulation of pore-forming claudin-2 [10,24,25] (up to 10-fold increase in UC [26]) and down
`regulation of several barrier-enhancing claudins (claudin-3 [10,27], -4 [10,25,28], -5 [24], -8 [24]) as
`well as occluding [9,24,29–31] and ZO-1 [9,10,28,30] in IBD [32]. E-cadherin is the main component
`of the adherens junction and genetic polymorphism of this protein was identified to be associated
`with CD [32]. This highlights the role of junctional proteins and barrier function in the pathogenesis
`of IBD. Although decreased expression of colonic E-cadherin was associated with active disease [30],
`E-cadherin expression of the TI had no bearing on disease severity or intestinal barrier function [33].
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`Figure 1. The junctional complexes of the intestinal barrier. Tight junctions are made up of the claudin
`and occludinmembrane proteins which bind together to seal the paracellular gap between epithelial cells.
`Zonulin-1 (ZO-1) binds the tight junction complex to the actin cytoskeleton and also regulates the selective
`passage of macromolecules through the tight junction. The lower junctional complex is the adherens
`junction which is made up of E-cadherin proteins that attach adjacent epithelial cells. These proteins are
`anchored by beta-catenin and alpha catenin to the actin cytoskeleton. The deepest junctional complexes
`at the baselateral end of the epithelial cells are the desmosomes and hemidesmosomes which attach
`epithelial cells to each other and also to the basement membrane, respectively. Desmosomes are made up
`of desmoglein and desmocollin partner proteins which are anchored to the filament lattice structure by
`plakogobin and plakophilin proteins, which in turn attach to desmoplakin. Whilst tight junctions have
`a primary role in regulating selective ion absorbance from the lumen to the extracellular internal milieu
`of the body, adherens junctions, desmosomes, and hemidesmosomesare principally responsible for the
`mechanical and tensile strength of the barrier. Please note: This figure is not to scale.
`
`3. Intracellular Regulators of Paracellular Permeability
`
`Selective paracellular permeability is a critical component of a functional gastrointestinal (GI) tract,
`and is distinctive from other modalities of absorption in that no molecular transporters are involved
`and thus the rate and concentration of absorption is largely determined by transmural potential
`differences and concentration gradients [34]. In the healthy GI tract, passive paracellular chloride
`absorption facilitates a normal stool concentration in the range of 10–15 mmol/L. A number of critical
`nutrients and minerals are also passively absorbed through the paracellular pathway, and principally
`regulated by epithelial junctions to maintain homeostasis [34]. Some of these include oxalate, calcium,
`phosphate, and magnesium. Many of these molecules are also transcellularly absorbed, with the
`balance in transport modalities summarily dependent on the amount of the solute in the lumen
`(dietary intake) and physiological demand [34]. The paracellular luminal-tissue transport channel
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`is largely regulated by the TJ and AJ complexes, and there is significant evidence of decreased
`expression and irregular distribution of TJ and AJ components including occludins, claudins and
`junctional adhesion molecules in IBD [35]. At the clinical level, IBD patients also appear to have
`higher rates of unregulated intestinal permeability via confocal endoscopic imaging [5]. TJ and AJ
`proteins are directly or indirectly (through protein-protein interaction) connected to the intracellular
`perijunctionalactomyosin ring [36]. Myosin Light Chain Kinase (MLCK) induces phosphorylation of
`myosin II regulatory light chain to cause contraction of the perijunctionalactomyosin ring, thereby,
`influencing the structure and function of the junctional proteins [37–39]. MLCK causes re-organisation
`of the perijunctional actin, occludin and ZO-1 [38], leading to the paracellular flux of uncharged
`macromolecules that is reversible with MLCK inhibition in experimental models [40]. There is an
`upregulation of MLCK in ileal biopsies of IBD patients, which correlates with disease activity [39].
`However, MLCK appears to be an effector of inflammatory cytokines. Its expression is induced by
`Tumor Necrosis Factor (TNF) [41], and inhibiting MLCK can reverse barrier loss in the presence
`of TNFα [42]. This prevents TNF-α-induced caveolin-1-dependent occluding endocytosis [43,44],
`which is one of the predominant ways TNF-α causes barrier loss.
`Furthermore, an experimental model showed that increase in permeability of macromolecules
`from MLCK activation leads to an increase in IL-13 and subsequent claudin-2 expression; therefore,
`an increase cation permeability [40]. Hence, MLCK interacts with various inflammatory cytokines
`to modulate paracellular permeability. However, constitutively-active-MLCK mice showed mucosal
`immune activation (increased TNF-α, IFN-Υ, IL-10, IL-13, and lamina propria T cells) but not
`spontaneous disease [45], suggesting that overactivation of MLCK alone is insufficient to cause IBD.
`The intracellular actin cytoskeleton itself can modify TJ function under the influence of various stimuli
`such as inflammatory cytokines, growth factors and microorganisms [22]. For example, Rho family of
`Guanosine Triphosphate hydrolase enzymes (GTPases) (a key molecule in intracellular actin signalling)
`can be inactivated by bacterial products (e.g., from Clostridium difficile and Clostridium botulinum),
`which results in re-organization of the F-actin in the perijunctionalactomyosin ring and alteration of
`TJ protein structures [44]. Other bacteria (e.g., E. coli) activate Rho GTPase to cause barrier loss via a
`different mechanism, sparing the perijunctionalactomyosin [22]. Also, depolymerisation of actin has
`been found to cause occludin re-distribution and internalisation via caveolae-mediated endocytosis
`which results in disruptions to the mucosal barrier [46]. In support of this finding, many studies have
`found TJ proteins in cytoplasmic vesicles following exposure to chemical and pathophysiological
`stimuli (e.g., calcium chelation, pathogenic E. coli infection, TNF) [22]. The Rho GTPasesignalling
`pathways have a complex interrelationship with MLCK that remains to be fully elucidated. Whilst both
`MLCK and Rho GTPase pathways phosphorylate MLC and appear to have complimentary roles in
`cell contractility and paracellular permeability, they seem to act at different sites of the cell—MLCK
`acts on the periphery of the cell to assemble microfilaments while Rho GTPases assemble stress fibres
`at the centre. MLCK is critical for maintaining basal stress fibres but does not affect late stress fibre
`reorganization [47]. Rho GTPases on the other hand, are critical in late stress fibre organization,
`and have been found to do so under TNF-α stress [47]. Rho GTPase subtype Cdc42 acts on PAK,
`a serine/threonine p21 activating kinase that phosphorylates MLCK and inactivates it, leading to
`tight junction disruption and intestinal barrier leak [43,48]. Whether MLCK and Rho GTPase MLC
`phosphorylation and Cdc42 induced phosphorylation of MLCK forms a part of a larger barrier function
`regulation loop remains an evolving subject.
`
`4. Epithelial Restitution and Healing
`
`The intestinal epithelial lining is continuously shed and replaced, maintaining the homeostasis
`between cell shedding and renewal [49]. Stem cells from the crypt differentiate and migrate to the villus
`tip in small bowel or colonic surface where they are shed. The dying cell signals to the surrounding
`cells to contract the actomyosin structure, which will extrude the dying cell out [50]. This is detected
`by a stretch-sensitive channel, which causes re-distribution of TJ proteins to transiently seal the gap
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`left by the dying cell to maintain an intact barrier [50]. Similarly, the intestinal epithelium restores
`tissue integrity following any injury or damage in two steps: epithelial restitution (re-organisation of
`adjacent cells and TJs) and wound-healing (maturation and differentiation of stem cells and cell
`migration) [51]. These processes are critical in IBD as recurrent and extensive mucosal damage
`occur with disease activation. The mucosal biopsy of IBD expresses activated caspase-1 and -3
`which is associated with intestinal barrier defects from a higher rate of epithelial cell extrusion [51].
`Various cytokines and growth factors affect epithelial restitution and wound healing (Table 1).
`Other elements involved are goblet cells, immune cells (e.g., macrophages and T cells produce IL-6
`and TNF, fibroblastsproduce hepatocyte growth factor to regulate epithelial cell regulation [52]),
`molecular pathways (e.g., canonical Wnt/β-catenin pathway in epithelial proliferation [2]), and the
`actin cytoskeleton and its regulators (e.g., Rho GTPase in epithelial restitution and toll-like-receptor
`function (TLR2 in synthesis of TTF3) [49,52].
`
`Table 1. Regulatory factors of epithelial restitution and wound healing [49,52].
`
`Action
`
`Inhibit cell proliferation
`
`Promote epithelial restitution via TGF-β dependent pathway
`
`Promote epithelial restitution via TGF-β independent pathway
`
`Decrease epithelial restitution velocity
`
`Promote epithelial proliferation
`
`Induce cell apoptosis
`Prevent cell apoptosis
`
`Regulatory Factors
`TGF-β
`Activin A
`Epidermal growth factor (EGF)
`Glucagon-like-peptide-2 (GLP-2)
`IL-1
`IFN-Υ
`IL-2
`HGF
`VEGF
`FGF
`Trefoil peptides
`Galectin-2
`Galectin-4
`Keratinocyte growth factor (KGF)
`IL-13 [32]
`Epidermal growth factor (EGF)
`TGF-α
`IL-6IL-22
`TNF-α
`Prostaglandin E2
`
`Increased rates of cell shedding and subpar epithelial restitution and healing observed in patients
`with IBD may explain the higher number ofepithelial gaps and microerosions, potentially creating a
`route for the uptake of luminal content [53]. However, the role of microerosions or epithelial gaps on
`the barrier function is uncertain [53].
`
`5. Clinical Implications of Impaired Intestinal Permeability in IBD
`
`Barrier dysfunction is defined as loss of the continuous layer of the intestinal epithelium with
`interruptions in the interepithelial junctions and epithelial gaps [54]. This allows the permeation of
`microorganisms, dietary antigens and other noxious particles into the laminalpropria, resulting in the
`activation of the mucosal immune system and the inflammatory sequela of IBD [54]. The importance
`of intestinal barrier function in IBD has been recognised for decades [2,8,55]. However, it is still
`under debate whether the mechanisms that result in barrier loss are the primary cause of IBD or the
`consequence of a separate underlying pathology.
`Increased permeability appears to correlate better with symptoms than endoscopic activity [56]
`and predicts relapse better than other clinical and blood markers [6]. The emergence of a novel
`imaging technique, confocal laser endomicroscopy (CLE), sparked the comprehensive exploration
`of the functional and structural features of the intestinal barrier [57] (Table 2). These studies have
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`consistently highlighted the pervasiveness of intestinal barrier dysfunction in the pathogenesis of
`IBD. Firstly, the features of impaired barrier function distinguish patients with IBD from healthy
`controls and these abnormalities persist in the absence of active clinical disease and affect the entire
`gastrointestinal tract [58–60]. The important features of impaired barrier function include fluorescein
`leakage, which is shown by an efflux of intravenous fluorescein contrast into the intestinal lumen and
`a loss of continuous epithelium (e.g., cell drop-out, epithelial gaps, or microerosions) [61]. Secondly,
`barrier loss has been found to be a reliable predictor of relapse and serious complications in IBD
`patients [13,60,62–65]. Within one year, an abnormal epithelial barrier is associated with an 80% risk
`of relapse and 45% risk of major events (such as hospitalisation or surgery), compared to 20% and
`5% for those with normal epithelial barrier function [62]. Structural changes and increased intestinal
`permeability of the colon also accurately predicted relapse over a 12-month follow-up period of UC
`patients [63,64]. Similarly, a defective mucosal barrier of TI is associated with a significant risk of
`relapse in both UC and CD [13,60,65]. Lastly, some but not all features of barrier dysfunction may
`be reversed with treatment [33]. Karstensen et al. followed up CLE features of patients with UC in
`response to medical therapy and found that structural features such as crypt changes improved in
`response to medical therapy but not fluorescein leakage, a functional parameter delineating intestinal
`permeability [33]. This finding furthersupports the idea that intestinal barrier dysfunction may be a
`primary pathologic feature of IBD, and current immunosuppressive and anti-inflammatory therapies
`maynot restore complete tissue integrity.
`On the other hand, leaky gut has been observed in healthy relatives [12] and spouses of patients
`with CD [66,67] and some experimental and animal models with defects in various barrier components
`do not result in spontaneous inflammation [45]. Therefore, the causes of barrier dysfunction may be
`multifactorial and may not be the predominant pathogenic process in IBD. Some of the molecular
`changes in TJs and increased paracellular permeability observed in IBD patients are limited to patients
`with active disease and absent in remission. Such findings suggest that the increased permeability may
`be secondary to another inflammatory cause [24]. As yet, we can only comprehend with certainty that
`IBD is a complex disease with multiple contributing factors that interact with one another to result
`in the disease phenotype. The exact place for the loss of mucosal barrier function in the puzzle of
`IBD pathogenesis is obscure; however, the evidence indicates that barrier dysfunction predisposes or
`enhances disease progression in IBD.
`
`6. Assessing Barrier Function in Clinical Practice Today
`
`Although the exact molecular pathogenesis behind barrier loss in IBD is uncertain, the intestinal
`epithelial barrier is an unequivocal source of important clinical information. However, the key obstacle
`in the assessment of intestinal barrier function in IBD is the lack of cost-effective and acceptable tools.
`The current methods for assessing intestinal barrier function and epithelial integrity have pronounced
`limitations, as outlined in Table 2.
`In the past, sugar tests and Ussing chambers have been commonly used in research studies to
`study intestinal permeability. The gold standard has been lactulose/mannitol testing, in which the
`urinary excretion of a large sugar (lactulose), which generally does not cross the intestinal barrier, and a
`small sugar (mannitol), which freely crosses the intestinal barrier, are measured. Sugar tests require
`strict dietary restriction of sugars for 5–6 h, which is inconvenient for patients, and where test accuracy
`is heavily reliant on their compliance. Small sugars and other molecular probes such as polyethylene
`glycols (PEG 4000, 1500, 400) and radioactively labelled Cr-EDTA have numerous confounding factors
`such as intestinal motility, transit time, renal excretion, and bacterial degradation. These tools are
`unable to discern intestinal permeability at distinct sites (e.g., inflamed versus non-inflamed tissue).
`CLE with intravenous fluorescein contrast is a functional endoscopic imaging technique that
`allows 1000× magnification of the intestinal wall to visualise the epithelial lining and vasculature. It is
`recognised to be an excellent tool for the assessment of the barrier function as it can show structural
`and functional features of the intestinal epithelium [61]. In recent years, CLE has been used to study
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`the intestinal barrier function of IBD patients [61]. The findings have accentuated the role of intestinal
`barrier function in the pathogenesis and natural history of IBD. Restoration of intestinal barrier function
`as assessed by CLE demonstrates cellular level evidence of remission and has been suggested as the
`new gold standard of mucosal healing [68].
`One of the biggest limitations of CLE is the lack of a single standardised and validated
`system for interpreting the measurable parameters. With growing research using CLE, there is
`increasing heterogeneity in study protocols and interpretation of images reference required. Numerous
`researchers have devised scoring systems to evaluate intestinal barrier function using CLE. Watson’s
`grade is a categorical grading classification based on functional and structural abnormalities of the
`TI [13]. On CLE, fluorescein leakage (FL) and microerosions (defined as an epithelial gap with a
`diameter greater than one cell) are graded as Watson’s grade 2 and 3, respectively, and grade 1 defines
`normal barrier function. Watson’s grade has been validated against clinical outcomes and histology and
`replicated by several studies and applied to other areas of the GI tract (GIT) [13,58,59]. The sensitivity,
`specificity and accuracy of Watson’s score to predict for relapse in IBD is 62.5%, 91.2% and 79%,
`respectively [60].
`The Confocal Leak Score (CLS) is a continuous scoring system [69]. It is calculated by the number
`of images showing three key features of leak proportional to the total number of images reviewed [69].
`These features are fluorescein leakage, cell junction enhancement (the accumulation of fluorescein
`between epithelial cells, representing TJ abnormalities) and cell dropout or an epithelial gap [69].
`CLS has a significant correlation to clinical symptoms in patients with mucosal healing (i.e., endoscopic
`remission). A CLS greater than 13.1 correlated to ongoing bowel symptoms, with every increase of
`CLS 1.9 associated with an extra diarrheal motion a day [5].
`The Chang-Qing scale classifies colonic features of UC into four types based on the regularity
`of crypt arrangement, crypt density, dilation of crypt openings and crypt destruction and FL [63].
`This has been validated against endoscopic and histologic assessment and clinical outcomes, with a
`sensitivity of 64%, specificity of 88.9% and accuracy of 74.4% at predicting relapse in UC [63].
`The significance of some of these CLE features has been questioned by some studies. For instance,
`epithelial cell extrusion and gap have been found to be higher in patients with IBD in several studies,
`and this has been validated against histology and clinical outcomes [51,62]. By contrast, a study
`found that although there were more epithelial gaps in IBD patients compared to controls, this did
`not correlate with disease activity nor correlate to risk of hospitalisation or surgery [53]. Similarly,
`Kiesslich et al. found that only microerosions and not cell shedding have a prognostic significance in
`IBD [13]. Other controversial CLE features include vascular changes and the presence of inflammatory
`infiltrates [57].
`The technical limitations of CLE have been listed in Table 2. Areas in need of further study include
`randomised control studies with standardized definitions of “barrier loss” and measures of disease
`activity [57], and direct comparison of CLE with conventional measures of intestinal permeability such
`as sugar tests and Ussing chambers.
`A few biomarkers of intestinal epithelial cell damage and inflammation have been discussed
`in the literature including plasma citrulline, fatty acid binding protein and faecal calprotectin [17].
`However, these are markers of epithelial damage or inflammation rather than measures of intestinal
`permeability [17]. It is questionable whether subtle changes in barrier function that may precede active
`disease would be accurately reflected by these biomarkers.
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`Technique
`
`General Principle
`
`Test Site
`Molecular Probes
`
`Test Method
`
`Limitations
`
`Table 2. Assessment of intestinal permeability [17,54].
`
`Lactulose/mannitol
`
`Oligosaccharides of different sizes
`
`Small intestine
`
`Sucralose
`
`Sucralose
`
`Colon
`
`Multi-sugar test
`
`Sucrose, lactulose, sucralose, erythritol, rhamnase
`
`Whole intestine
`
`51Cr-EDTA
`
`51Cr-EDTA crosses the intestinal barrier via the
`paracellular route and has similar physiological
`properties to oliogosaccharides.
`
`Whole intestine
`
`Urine
`
`Urine
`
`Urine
`
`Urine
`
`PEG4000/400
`
`Polyethylene glycol, an inert molecule of
`different sizes.
`
`Whole intestine
`
`Urine
`
`Gadolinium-based MRI
`contrast agent [71]
`
`Gadolinium (500–1000 Da)
`
`Whole intestine
`
`24-h urine collection
`
`Ussing chambers
`
`Ion transport across the intestinal epithelium
`tissue sample is measured using a short
`circuit current.
`
`Site-specific
`
`Biopsy
`
`Imaging
`
`Confocal laser
`endomicroscopy
`
`Intravenously-administered fluorescent contrast is
`seen to leak through the small intestinal mucosa
`under real time endoscopy.
`
`Terminal ileum, colon, duodenum
`
`Endoscopy
`
`Time-consuming.
`Metabolised in the colon so limited application in assessing the
`large intestine (e.g., ulcerative colitis (UC)).
`Does not show permeation of bacterial components.
`Mannitol is contraindicated with blood transfusions.
`Time-consuming.
`Does not show permeation of bacterial components.
`Time-consuming.
`Does not show permeation of bacterial components.
`Invasive and complex detection method.
`Not readily available.
`Radioactivity.
`Impractical in clinical setting.
`Does not show permeation of bacterial components.
`Time-consuming.
`The exact route of PEG is not well defined [70], thus implications
`in interpreting results.
`Does not show permeation of bacterial components.
`Lack of evidence in human studies.
`More expensive and may have higher toxicity than
`conventional sugars.
`Partial hepatobiliary elimination.
`Contraindicated in renal impairment.
`Invasive and complex detection method.
`Ex-vivo.
`Lack of correlation between Ussing chamber and other
`permeability assays.
`
`Invasive.
`Time-consuming (average of 46.5 minutes [60]).
`Validated measurement scores include the Watson grade
`(semi-quantitative [60]) and confocal leak score (quantitative) [5].
`Requires special training of the endoscopist.
`Does not show permeation of bacterial components.
`
`Claudin-3 [27]
`
`Epithelial tight junction protein
`
`Biomarkers of Intestinal Permeability
`NA
`
`Urine
`
`Limited data and lack of randomised trials.
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`Technique
`
`General Principle
`
`Table 2. Cont.
`
`Test Site
`Bacteria-Related Markers
`
`Test Method
`
`Lipopolysaccharide
`(LPS) assay
`
`Show endotoxemia from bacterial translocation
`due to barrier function failure.
`
`Colon
`
`Blood (portal venous)
`
`Circulating endotoxin
`core antibodies
`
`Plasma D-lactate
`
`Faecal butyrate
`concentrations
`
`Bacteria-derived
`haemolysin
`
`Assessment of fatty
`liver disease
`
`An indirect measure of translocation of bacterial
`products by quantifying immunoglobulins (IgG,
`IgM and IgA) against the inner core of endotoxin
`for acute phase of intestinal barrier damage and
`function [72].
`D-lactate is produced by the gut bacteria and
`translocated across the intestinal mucosa with
`barrier dysfunction.
`Butyrate is a barrier enhancing substance,
`modifying claudin-1 and -2 to preserve intestinal
`barrier function and preventing
`bacterial translocation.
`
`Toxin that impair the intestinal barrier.
`
`Inflammation and fatty liver disease result from
`translocation of bacteria and its products into the
`portal system.
`
`Limitations
`
`Technical limitation in detecting low levels of LPS in the
`peripheral blood.
`Requires careful standardization of the measurement.
`Evidence of use in Inflammatory Bowel Disease (IBD).
`
`Only study done on post-operative patients, not patients with
`chronic gastrointestinal disease.
`Evidence for use in IBD.
`
`False positive test with bacterial over growth.
`Limited use in critically ill patients (e.g., ischemic colonic injury,
`acute necrotizing pancreatitis).
`Poorly established.
`The test relies on the principle that butyrate as a single major
`component of the barrier function rather than a complex and
`interactive entity.
`Poorly established.
`Results are attributed to only haemolysin-producing bacteria.
`
`Colon
`
`Colon
`
`Colon
`
`Colon
`
`Blood
`
`Blood
`
`Faeces
`
`Whole intestine
`
`Imaging
`
`Poor specificity.
`
`

`

`Proteomes 2018, 6, 17
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`10 of 17
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`7. Biomarkers of Intestinal Barrier Function
`
`Barrier function is affected by various factors and some of these have been shown to be defective
`or altered in IBD, prompting the invasion of pathogenic organisms. The differential expression of
`proteins that are directly involved in or regulate the interactions between the intestinal epithelium,
`the immune system and the intestinal microbiota have potential as biomarkers of intestinal barrier
`function in IBD.
`A handful of studies have investigated the role of specific proteins in the regulation of intestinal
`permeability from human tissue samples (Table 3).Some affect the paracellular pathway by acting
`on the junctional proteins (e.g., Protein C pathway [73], prion protein (PrPc) [23], PECAM1) and the
`actin cytoskeleton (RTN-4B [74]), while others target epithelial homeostasis e.g., epithelial apoptosis
`(JAM-A). The studies included in this review focus on specific proteins previously known to contribute
`to the intestinal mucosal barrier, i.e., whole proteins have been quantified using immunohistochemistry
`rather than