methods to determine intestinal permeability and bacterial translocation (1)

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Review

Methods to determine intestinal permeability and bacterialtranslocation during liver disease

Lirui Wang a,b, Cristina Llorente a,b, Phillipp Hartmann a, An-Ming Yang a,Peng Chen a, Bernd Schnabla,b,a Department of Medicine, University of California San Diego, La Jolla, CA, United Statesb Department of Medicine, VA San Diego Healthcare System, San Diego, CA, United States

a r t i c l e i n f o a b s t r a c t

Article history:

Received 28 November 2014Accepted 4 December 2014Available online xxxx

Liver disease is often times associated with increased intestinal permeability. A disruption of thegut barrier allows microbial products and viable bacteria to translocate from the intestinal lumento extraintestinalorgans. The majority of the venous blood from the intestinal tract is drained intotheportal circulation, which is part of the dual hepatic blood supply. The liver is therefore the firstorgan in the body to encounter not only absorbed nutrients, but also gut-derived bacteria andpathogen associated molecular patterns (PAMPs). Chronic exposure to increased levels of PAMPshas been linked to disease progression during early stages and to infectious complications duringlate stages of l iver disease (cirrhosis). It is therefore important to assess and monitor gut barrierdysfunction during hepatic disease.We review methods to assess intestinal barrier disruption anddiscuss advantages and disadvantages. We will in particular focus on methods that we have usedto measure increased intestinal permeability and bacterial translocation in experimental liver

disease models. 2015 Elsevier B.V. All rights reserved.

Keywords:

EndotoxinIntestinal injuryMicrobiomeMicrobiotaGutliver axisIntestinal leakiness

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Evaluation of intestinal integrity and mucosal tight junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Functional methods to assess intestinal permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3.1. Methods assessing theow from the intestinal lumen to the blood . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Methods assessing theow from the blood to the intestinal lumen . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Measurement of translocated microbial PAMPs and bacteria in extraintestinal space . . . . . . . . . . . . . . . . . . . . . . . 05. Biomarkers mainly used in humans to assess intestinal inammation and permeability . . . . . . . . . . . . . . . . . . . . . . 06. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

Journal of Immunological Methods xxx (2015) xxxxxx

Abbreviations:LPS, lipopolysaccharide; H&E, Hematoxylin & Eosin; PAMPs, pathogen associated molecular patterns; TLR, Toll-like receptor; FITC, fluoresceinisothiocyanate-conjugated; PEG, polyethylene glycols; GFP, green fluorescent protein; MLN, mesenteric lymph nodes; BDL, bile duct ligation; BSA, bovine serumalbumin; A1AT, Alpha-1-Antitrypsin; FABPs, fatty acid binding proteins; DAO, diamine oxidase. None of the authors has anancial, personal or professional conict of interest to disclose. Corresponding author at: Department of Medicine,University of California San Diego, MC0063, 9500Gilman Drive, La Jolla, CA 92093, United States. Tel.:+ 1 858

822 5311; Fax: +1 858 822 5370.E-mail address: [email protected](B. Schnabl).

JIM-11958; No of Pages 10

http://dx.doi.org/10.1016/j.jim.2014.12.0150022-1759/ 2015 Elsevier B.V. All rights reserved.

Contents lists available atScienceDirect

Journal of Immunological Methods

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / j i m

Please cite this article as: Wang, L., et al., Methods to determine intestinal permeability and bacterial translocation during liverdisease, J. Immunol. Methods (2015),http://dx.doi.org/10.1016/j.jim.2014.12.015
http://dx.doi.org/10.1016/j.jim.2014.12.015http://dx.doi.org/10.1016/j.jim.2014.12.015http://dx.doi.org/10.1016/j.jim.2014.12.015mailto:[email protected]://dx.doi.org/10.1016/j.jim.2014.12.015http://www.sciencedirect.com/science/journal/00221759http://dx.doi.org/10.1016/j.jim.2014.12.015http://dx.doi.org/10.1016/j.jim.2014.12.015http://www.sciencedirect.com/science/journal/00221759http://dx.doi.org/10.1016/j.jim.2014.12.015mailto:[email protected]://dx.doi.org/10.1016/j.jim.2014.12.015


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1. Introduction

The luminal side of the intestine is lined by epithelial cells,which promote water and nutrient absorption; the epitheliumalso provides a dynamic and semi-permeable barrier betweenthe luminal microbiota and the host. The barrier is formedby individual epithelial cell membranes and tight junctionproteins that seal the paracellular space between adjacent cells.Thus, the permeability of this barrier is regulated by theintegrity of cellular plasma membranes and tight junctions,as well as by epithelial cell processes mediating secretionand absorption. Small molecules (b300 Da) and electrolytespassively cross the tight junction barrier (Sun et al., 1998). Bothphysiological and pathological stimuli change the barrierpermeability. During homeostasis the intestinal epitheliumabsorbs nutrients while effectively preventing translocationof intraluminal bacteria. However, pathological conditions(e.g. toxins or intestinal inflammation) can increase theparacellular pathway and adversely affect barrier permeability,which poses the risk of an ineffective nutrient absorption and afailure to prevent thetranslocation of luminal bacteria andtheirproducts (also called pathogen associated molecular patternsor PAMPs). This can result in chronic intestinal diseases, but itmight also affect other distant organs that drain and filtertranslocated bacteria and associated PAMPs (Sun et al., 1998;Turner, 2006; Marchiando et al., 2010a; Fouts et al., 2012).

The majority of the intestinal venous blood reaches the livervia the portal vein. Due to this unique blood supply system,the liver is vulnerable to exposure of bacterial productstranslocated from the gut lumen when intestinal epithelialbarrier functions are disrupted (Seki and Schnabl, 2012). Theliver represents therefore the first organ in the body thatencounters not only nutrients from the diet, but also othermolecules thatare able to translocate from the intestinal lumento the blood stream. The amount of translocated PAMPs isusually low during health (Bode et al., 1987; Fukui et al., 1991;Lin et al., 1995). However, liver diseases are associated withincreasedintestinal barrier permeability in humans(Bode et al.,1987; Fukui et al., 1991; Lin et al., 1995) and animal models(Yan et al., 2011; Hartmann et al., 2012; Hartmann et al., 2013).Increased levels of lipopolysaccharide (LPS or endotoxin) andbacterial DNA resulting from increased intestinal barrierpermeability are elevated in the serum of patients with liverdiseases (Fukui et al., 1991). Translocated bacterial productscontribute to liver disease progression by binding to specificpathogen recognition receptors. In particular, Toll-like receptor

4 (TLR4), the major receptor for LPS has been implicated inthe progression of many liver diseases and induces hepaticinflammation (Schnabl and Brenner, 2014). Increased translo-cation of microbial products due to a disrupted intestinalbarrier will also lead to an activation of the mucosal immunesystem and secretion of inflammatory mediators, which in turnmight increase barrier dysfunction. Such an inflammatoryprocess might eventually also affect the quantity (overgrowth)and composition of the luminal microbiota (Marchiando et al.,2010a). Although intestinal permeability can increase witha rise in transcellular transport processes, the relevance andimportance of transcytosis for liver disease have not beendetermined.

In addition, enhanced translocation of viable bacteria tomesenteric lymph nodes and extra-intestinal sites is commonly

seen in patients with end-stage liver disease (cirrhosis) (Schnabl,2013). The hepatic immune system might also be compromisedin liver cirrhosis, so that translocated viable bacteria cannotbe effectively cleared (Balmer et al., 2014). However, whetherand how viable bacteria affect liver disease progression requirefurther investigation.

Given the significance of monitoringintestinal permeabilityin thesettingof acute and chronicliver diseases, we will reviewmethods to assess gut permeability in mostly animal models.

2. Evaluation of intestinal integrity and mucosal

tight junctions

Histology is important to initially evaluate the integrity ofthe intestinal barrier. Standard light microscopy of Hematox-ylin & Eosin (H&E) stained intestinal sections is able to detectintestinal pathology including ulcerations of the mucosa andsevereintestinal inflammation that will cause andcontribute toincreased intestinal permeability.

As mentionedabove, tight junctionsplayan essential role inmaintaining the integrity of the membrane barrier in theintestine. Tight junctions locate in the apical end of the lateralmembrane and are composed of couples of transmembraneproteins such as occludins and claudins interacting withintracellular anchor proteins such as zonula occludens proteinswhich in turn are connected to the actin cytoskeleton. Tightjunctions are rate-limitingfor the paracellularleakage pathway(Menard et al., 2010).

Electron microscopy led to the discovery of tight junctionsin epithelial barriers. The zonula occludens (tight junction) ischaracterized by fusion of the adjacent cell membranes with adense outer leaflet of the adjoining cell membranes, whichconverge to form a single intermediate line. A diffuse bandof dense cytoplasmic material is often associated withthis junction (Farquhar and Palade, 1963). In addition, manyreports have employed mostly immunofluorescent stainingmethods for visualization using specific antibodies directedagainst various tight junction proteins (Hartmann et al., 2012;Chen et al., 2014; Chen et al., 2015). Protein and mRNAtranscript levels of tight junction molecules can be assessedwith western blotting and quantitative PCR, respectively (Chenet al., 2015). Tight junction complexes are composed ofmultiple proteins. Because the importance of single moleculesfortheintegrity andfunction of tight junctionsis ratherobscureat this moment, functional assays might be necessary toelucidate their role.

An example for the importance of evaluating gut barrierintegrity in culture and in vivo is alcoholic disease. A directcytotoxic effect of high concentrations of ethanol (N40%)increases intestinal permeability by causing vascular andmucosal damage, which can be best seen on H&E stained slides(Szabo et al., 1985). However, subsequent Caco-2 monolayercell based studies showed that even lower, non-cytotoxicdoses of ethanol may alter the structure and function of tightjunctions through activating myosin light chain kinase (MLCK)(Ma et al., 1999). Differentiated intestinal epithelial cells suchas Caco-2 cells are commonly used to functionally analyze tightjunction dynamics. Although ethanol has been reported todisrupt tight junctions in Caco-2 cells, acetaldehyde, a product

of ethanol metabolism, is a much stronger inducer of tightjunction dysfunction. Detailed protocols using acetaldehyde

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have been published (Rao, 2008; Chen et al., 2015). Ethanoland acetaldehyde have also been used in three dimensionalCaco-2 cell culture systems to disrupt tight junction integrity(Elamin et al., 2012). Other polarized cell culture systemsare currently being developed to overcome the use of coloncancer cell lines. Isolated primary intestinal stem cells can bedifferentiated into crypt forming enterocytes that are alsocalled enteroids (Foulke-Abel et al., 2014).

Ethanol is oxidized to acetaldehyde in the intestine andaccumulation of acetaldehyde has been reported to be associ-ated with alcohol related tissue injury (Salaspuro, 1996).Bacterial overgrowth may increase the oxidation of ethanol toacetaldehyde and result in accumulation of acetaldehyde inintestine. Because the colonic mucosa and microbiome have alow capacity in oxidizing acetaldehyde, acetaldehyde accumu-lates in the colon (Salaspuro, 1996) and acetaldehyde mayredistribute tight junction proteins mediated by a tyrosinekinase-dependent mechanism with a subsequent increase inintestinal permeability.

Patients with cirrhosis show a decreased expression in tightjunction proteins in duodenal biopsy. Patients with decom-pensated cirrhosis had less tight junction protein expressionthan patients with compensated cirrhosis (Assimakopouloset al., 2012). However, another study showed that patientswith compensated liver cirrhosis showed no alteration in tightjunction protein expression in gastroduodenal and smallintestinal mucosa but downregulation of these proteins in thecolon (Pijls et al., 2014). This again suggests that functionalassays are required to determine intestinal permeability.

3. Functional methods to assess intestinal permeability

3.1. Methods assessing the ow from the intestinal lumen to the

blood

Intestinal permeability can be assessed through enteraladministration of non-digestible markers, which ideally shouldcross the mucosal barrier by non-mediateddiffusion(Sun et al.,1998). The principle of this method is based on assessing theflow from the intestinal lumen to extraintestinal space such asblood, specific organs or urine. There are several types ofmarkers including sugars, radioisotopes (e.g. 51Cr-EDTA) andpolyethylene glycols (PEG).

The obvious advantage is that intestinal permeability canbe tested under in vivo conditions. However, the locationof gut barrier dysfunction cannot always be accurately

assessed. In addition, there are factors that might affect theabsorption, the metabolism and the excretion of the sugars,e.g. gastrointestinal motility including intestinal transit timeand surface area, mucosal blood flow, the distribution of themarkers in the body, use of interfering drugs and kidneyfunction (Bjarnason et al., 1984a,b; Peeters et al., 1994).Other factors might affect the urinary excretion of ingestedmolecules, such as the urine volume and/or the duration ofthe collection. A careful monitoring of the test and precisemeasurement of the parameters is therefore warranted(Mattioli et al., 2011).

We have used fluorescent-labeled dextrans for assessmentof intestinal permeability during liver disease. Dextrans are

polysaccharides and are available in different molecular sizes(3 kD to 2000 kD) and conjugated to various fluorophores. Using

a larger size will mimic bigger endogenous macromolecules,although dextran is still an inert test probe. It is important thattested tissue or blood does not have an autofluorescence thatinterferes with the emission of the fluorescent labeled probe.For example, during cholestatic liver disease, increasedbilirubinin the plasma has a similar emission wavelength as fluoresceinisothiocyanate-conjugated (FITC)-dextran. Choosing differentfluorophores might overcome this problem. We are typicallyadministering 200 l of FITC-dextran 4 kD (600 mg/kg bodyweight) to mice by gavage, and the blood is collected 4 h later.Varying the time after harvesting will depend on which part ofthe intestinal tract is to be investigated. The serum concentra-tion of the FITC-dextran is then determined using a fluorimeterwith an excitation wavelength of 490 nm and an emissionwavelength of 530 nm. Serially diluted FITC-dextran is used toestablish a standardcurve, andtheconcentration of serum FITC-dextrancan thenbe calculated. Ideally, dilutionsof FITC-dextranshould be performed with non-hemolytic serum from healthy,non-gavaged mice. Intestinal permeability will be presentedas the concentration of serum FITC-dextran (Napolitano et al.,1996; Furuta et al., 2001; An et al., 2007; Fouts et al., 2012;Hartmann et al., 2013).

The role of other sugars was first investigated in 1899 byHober who found that dogs absorbed galactose fasterthan glucose (Hober, 1899). Since then many changes haveoccurred. In the 1970s, non-metabolizable oligosaccharideswere introduced to develop reliable methods to assess the gutpermeability (Menzies, 1974). The combined administration ofa larger and a smaller molecule yields a specific large/smallmolecule ratio in the urine which is a reflection of the intestinalpermeability and has greater clinical value than the adminis-tration of one marker alone (Menzies, 1974; Menzies, 1984).The most common dual-sugar test in clinical practice is thelactulosemannitol test (van Elburg et al., 1995; Dastych et al.,2008), however L-rhamnose is sometimes used instead ofmannitol (van Nieuwenhoven et al., 1999; van Wijck et al.,2013). Common characteristics of these sugars are that they arepassively absorbed from the gut without considerable metab-olism and that they are excreted in an unaltered form into theurine in a direct correlation to their absorbed amount from theintestine (Sequeira et al., 2014). Mannitol is a monosaccharidewith a molecular weight (MW) of 182 Da and demonstrates atranscellular permeation with high appearance in urine afteroral application, and a decrease in the presence of villousatrophy as it occurs in celiac disease (Cobden et al., 1978; Jubyet al., 1989). Similarly, L-rhamnose another monosaccharide of

164 Da

shows a reduced absorption in celiac disease, whereaslactulose is absorbed at an increased rate hence leading to aheightenedlactulose/L-rhamnose excretion ratio(Menzieset al.,1979). This is believed to occur since the larger disaccharidelactulose (MW 342 Da) is transported via a paracellularroute through the gut wall versus the transcellular route ofthe aforementioned monosaccharides (Bjarnason et al., 1986;Maxton et al., 1986). The routinely employed method of thelactulosemannitol test consists of the simultaneous ingestionof the sugars in water, and after fasting for 2 h the collectionof the urine over a 24-hour period. The lactulose/mannitol ratiofrom the urine collection of the first 6 h is used to measure thesmall intestinal permeability (Spiller et al., 2000). More rarely,

the urine collections at 0

3, 3

5, and 5

24 h are carried out toassess the permeability of the proximal small intestine, distal

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small intestine, and colon, respectively (Bjarnason et al., 1983a;Bjarnason et al., 1983b; Maxton et al., 1986).

Sugar absorption tests are employed in the diagnosticworkup of several diseases, such as irritable bowel syndrome(IBS) (Rao et al., 2011), inflammatory bowel disease (IBD)(Munkholm et al., 1994; Halme et al., 2000), and autoim-mune diseases (van Elburg et al., 1993; Vogelsang et al.,1995). However, studies using small inert markers to assessintestinal permeability in vivo do not necessarily correlatewith the uptake of larger macromolecules (Menard et al.,2010). And indeed an intestine that is permeable to smallsugar molecules can be impermeable to larger molecules(Vojdani, 2013).

Creating isolated intestinal loops is a powerful model toassess intestinal permeability in individual intestinal segments.The concept again relies on the flow of markers from theintestinal lumen of the loop to extraintestinal space. For thisreason, non-digestible markers as described above, labeledbacterial products or even live bacteria are injected into anisolated gut segment that has blind ends on each side.Translocation of these markers is then measured in plasma orother tissues that the reagent/bacteria might penetrate into.Several studies used this method to investigate intestinalpermeability.

Example A

We have assessed intestinal permeability in various intes-tinal segments of alcohol- or isocaloric diet-fed mice (Chenet al., 2014). Mice were fully anesthetized, and a midlinelaparotomy incision was made. Gentle manipulation of theintestine using sterile cotton swabs helped with the identifica-tion of each intestinal segment (e.g. jejunum, ileum, cecum,and colon). An approximately 4 cm long segment of the

intestinal tract wasisolated withtwo sterile vascular hemoclips(Jorgensen Laboratories) without disrupting the blood supply.Care must be taken that themesenteric vascular arcades arenotinjured (Fig. 1). FITC-dextran 4 kD (50 l, 100 mg/ml) is thencarefully injected intothe isolated loop of the intestine using aninsulin syringe, and the abdomen is closed with sutures. Onehour later, mice are sacrificed and fluorescence in theplasma ismeasured as described above. By using different fluorescent-labeled dextrans, this method can assess intestinal permeabil-ity simultaneously in multiple intestinal segments of the samemouse.

Example B

Besides fluorescent-labeled markers, live bacteria can beinjected into the loop to quantify bacterial translocation. Forexample, we used green fluorescent protein (GFP)-plasmidtransfected Escherichia coli (E. coli) serotype O74:K:H39(carrying a chloramphenicol resistance) to show that disrup-tion of the colon barrier caused labeled bacteria to translocateto mesenteric lymph nodes following bile duct ligation (BDL) ascompared with sham treatment. Loop surgery was performedas described above. The bacteria suspension (approximately106 CFUs in a total volume of 50l) is injected into theloopandmice were sacrificed 4 h later. Mesenteric lymph nodes (MLN)are then harvested in a sterile fashion, homogenized andcultured on nutrient-broth agar plates (containing chloram-phenicol) for 4872 h. GFP expression is confirmed in coloniesusing fluorescence microscopy (Hartmann et al., 2012).

Example C

We also use this model to assess survival rate of biolumines-centE. coliin vivo. Non-pathogenicE. coliwere transfected withthe pXen13 plasmid (Caliper), which is a vector carrying theoriginal Photorhabdus luminescens luxCDABE operon for engineer-ing bioluminescent bacteria. The bacteria suspension (approxi-mately106 CFUsina total volumeof50l) is then injected into theisolated intestinal loop. Bioluminescence imaging was performedusing IVIS Spectrum (Caliper). Mice were kept anesthetized, theabdominal cavity was left open and bioluminescence wasrecorded serially over time (Fig. 2) (Hartmann et al., 2013). Sincethe abdominalcavityis left open in theheated (37 C)IVIS imager,intestinal surfaces exposed to the air require intermittent mildmoisturization. Survival in percentage and effective killing ofinjected bacteria can be calculated. Although the purpose of our

experiment was to investigate survival of bacteria in the isolatedintestinal loop, this model could possibly be altered to imagetranslocation of bioluminescent bacteria into the adjacent tissuelike mesenteric lymph nodes. And indeed, after injecting labeledLPS into the loop we were able to assess translocation andphagocytosis by macrophages(Grivennikov et al.,2012). Similarly,intravital microscopy was able to visualize the transit of GFP-tagged E. coli from the lumen into the mucosal stroma andmuscularis of the terminal ileum in rats with developing cirrhosis(Palma et al., 2007).

Taken together,thisintestinal loop model is a very powerfulmethod to assess intestinal permeability and it can serve withmodifications a wide variety of purposes. It is also independent

from intestinal motility as compared with gavage of macromo-lecular tracers.

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Loop in the

distal small intestine

Fig. 1.Intestinal loop model in the distal small intestine. Shown is a typicalphotograph of the in vivo loop model surgery.




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3.2. Methods assessing theow from the blood to the intestinal

lumen

The intestinal permeability is influenced not only bythe epithelial but also by the endothelial barrier. Capillarypermeability is restricted to large solutes, such as red bloodcells (40,000 radius). The capillary endothelium partlyrestricts molecules such as albumin, which has a molecularradius of 36 (Granger and Taylor, 1980).

The use of models that assess the flow from the blood to theintestinal lumen offers a variety of advantages. Ideally, onewould like to useendogenous markers thatare restrictedto theblood compartment during healthy conditions and are notpresent in theintestinal lumen. Following the onset of a barrierdysfunction this endogenous marker moves from blood vesselsacross the mucosal barrier into the intestinal lumen by non-mediated diffusion. Using such a method is non-invasive, doesnot require any manipulation of the animal and could beassessed serially over timeprovided that concentrationscan bemeasured in fecal pellets.

Measurement of albumin in fecal pellets is an example forsuch a flow directed towards the intestinal lumen. Albuminrepresents approximately 50% of the total protein content inhuman blood. Albumin is a small globular protein (molecularweight: 66.5 kDa) consisting of a single chain of 585 aminoacids, produced by the hepatocytes with none or very lowintracellular storage (Nicholson et al., 2000; Evans, 2002).3040% of the albumin is maintained in theblood stream, whilethe remainder is distributed in the interstitial space, where itsconcentration is low (1.4 g/dl). The protein leaves the circula-tion, returning to it via the lymphatic system. Albumin can becatabolized in many tissues, but mainly in the muscles, liverand kidneys (Nicholson et al., 2000; Evans, 2002; Fanali et al.,2012; Garcia-Martinez et al., 2013). Enhanced capillary per-meability increases the release of albumin into the interstitialspace (Peters, 1984). However, for this test to be accurate,serum albumin levels need to be normal. Conditions with lowserum levels of albumin due to decreased synthesis (e.g. end-

stage liver disease) or due to albumin loosing diseases(e.g. kidney disease) might result in false negative results.

Usually an impermeable macromolecule in the healthybeing, isotope-labeled albumin has been used to measureintestinal permeability in disease states. A healthy intestinalepithelial and endothelial barrier prevents the spilling over ofalbumin into the interstitial space. However, in conditionswhere the intestinal barrier integrity may be injured, endothe-lial and epithelial permeability is increased (Iqbal et al., 1996).As mentioned above, a prominent feature of alcohol abuse isdisruption of the intestinal barrier. Animal models of alcoholicliver disease lead to leaky gut (Hartmann et al., 2013) andpatients with alcohol abuse display an impaired intestinalbarrier (Bodeetal.,1987). Thereby, measurement of albumin infecal samples is a good indicator of a disrupted intestinalbarrier. We have measured albumin concentrations in freshlycollected fecal pellets from mice by a standard ELISA test(Bethyl Lab). Mice are single housed in cages without beddingfor several hours to collect fecal pellets from individual mice(Hartmann et al., 2013). We compared intestinal permeabilityresults using a fecal albumin ELISA to the plasmatic fluores-cence of orally administered FITC-Dextran 4 kD (as describedabove) in mice fed an alcohol or isocaloric control diet. Theresults indicative of a gut barrier dysfunction in ethanol fedmice were comparable, and correlated well between the twomethods (Hartmann et al., 2013).

Intestinal permeability can also be measured by an in vivoperfusion system assessing the flow from the blood to theintestinal lumen. Mice are subjected to the model or treatmentthat is relevant to assess intestinal permeability. Then the miceare anesthetized and injected intravenously with Alexa 488-conjugated bovine serum albumin (BSA). The abdomen isopened by a midline incision, and a 5-cm loop of jejunum iscannulated at the proximal and distal ends with 0.76-mminternal diameter polyethylene tubing. Then the flushingsolution (NaCl 140 mM, HEPES 10 mM, pH 7.4) and testsolution (NaCl 50 mM, HEPES 5 mM, sodium ferrocyanide2 mM, KCl 2.5 mM, glucose 20 mM, pH 7.4) will be perfused

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Fig. 2. Bioluminescent imaging after injection of luciferase expressing Escherichia coli into a jejunal loop. A representative photograph of the jejunal loop and a

corresponding bioluminescent image following injection of luciferase expressingE. coliusing the IVIS imager is shown.




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through the jejunal loop using a peristaltic pump. Aliquots ofthe test solution are collected at the beginning and end of theperfusion. Theintestin,al permeability, which is reflectedby theBSA flux from the blood into the lumen of a perfused segmentof small intestine will be measured by the concentration offluorescent-tagged BSAin the perfusate. The BSA concentrationin the perfusate is detected using a fluorescent microplatereader (Clayburgh et al., 2005; Clayburgh et al., 2006;Marchiando et al., 2010b). This in vivo perfusion system couldalso be used to test potential effects of drugs on intestinalpermeability.

4. Measurement of translocated microbial PAMPs and

bacteria in extraintestinal space

Liver diseases are commonly associated with increasedintestinal permeability. Translocation of gut-derived PAMPshas been implicated into the pathogenesis of several chronicliver diseases including alcoholic liver disease and NASH(Schnabl and Brenner, 2014). It is therefore important todirectly assess levels of gut-derived and translocated PAMPs inextraintestinal space such as the portal and systemic bloodcirculation, mesenteric lymph nodes, liver and spleen. It isimportant to harvest animals in a sterile fashion. In addition, allinstruments used for harvesting need to be sterile, DNA- andRNA-free. Various ELISA assays and other kits are available todetermine serum or tissue levels of specific PAMPs. Forexample, LPS can be measured by the traditional Limulusassay, but a competitive ELISA with increasedsensitivity is nowalso available (Hartmann et al., 2012; Chen et al., 2014).Microbial proteins can be detected with western blotting fromsterile tissue extracts. We have used anE. coliantibody (Dako)to determine direct hepatic translocation of PAMPs (Chen et al.,2015). Similarly, bacterial DNA can be amplified from tissueusing common 16S ribosomal primers (unpublished results) ordeep 16 rRNA sequencing (Cuenca et al., 2014).

Although the importance of translocation of viable bacteriafrom the gut lumen to extraintestinal space for the progressionof liver disease is not completely understood, translocatedliving bacteria play an important role in advanced stages ofliver disease and cirrhosis. A significant percentage of patientswith alcoholic hepatitis succumbs to bacterial infections withinfection-attributed mortality of 12% to 54% (Coffin and Sharpe,2007) underscoring the importance of a leaky gut withsubsequent translocation of bacteria to extraintestinal sites.To detect viable bacteria in blood and tissues standard aerobic

and anaerobic culture techniques are used (Fouts et al., 2012).We typically use beads (zirconia/silica, 1.0 mm; SpectrumLaboratory Products) and a mechanical beads beater to releasebacteria from tissue specimens (MagNA Lyser (Roche); speedsetting: 6000; time setting: 30 s to 60 s). Given that theminority of intestinal bacteria can be cultured by traditionalculture techniques, this method will obviously not be able todetect non-culturable translocated bacteria.

Liver disease, in particular advanced stages of liver disease,results in an impairment of the immune system. The diseasedliver is not able to clear bacteria and bacterial PAMPs aseffectively as a healthy organ does (Balmer et al., 2014).Increased levels of translocated bacteria and bacterial products

might therefore not necessarily be the result of increasedintestinal permeability alone, but rather a combination

between a dysfunction of the gut barrier and the immunesystem. Methods to detect bacteria andPAMPs in the blood andtissues are therefore considered more indirect evidence for gutleakiness. Nevertheless, they are important to estimate the riskof liver disease progression and bacterial infections.

The above mentioned methods with their advantages anddisadvantages are summarized inTable 1.

5. Biomarkers mainly used in humans to assess intestinal

inammation and permeability

Zonulin is a 47 kDa protein which modulates intestinalpermeability by disassembling intercellular tight junctionsbetween epithelial cells in the digestive tract (Wang et al.,2000; Fasano, 2001; Vanuytsel et al., 2013). The effect ofzonulin on increased intestinal permeability is mediatedthrough activation of EGF receptor (EGFR) via proteinase-activated receptor 2 (PAR2) activation (Tripathi et al., 2009).Patients with type 1 diabetes, an autoimmune disease in whichthe finely tuned regulation of intestinal tight junctions is lost,have increased serum zonulin levels. Serum zonulin correlateswith increased intestinal permeability. Moreover, this studyalso revealed that zonulin upregulation seems to precede theonset of the disease, suggesting the important role of increasedintestinal permeability in the pathogenesis of this disease(Sapone et al., 2006). Other diseases with increased levels ofzonulin are celiac disease and obesity (Fasano, 2012). Althoughfurther studies are needed for the application of serum zonulinin the human diseases, an important role for zonulin inregulation of intestinal permeability has been established andwill hold much promise for future applications.

Calprotectin is a 36-kDa calcium- and zinc-binding proteincomplex which consists of onelight and twoheavy polypeptidechains (Dale et al., 1983). It constitutes up to 60% of thecytosolic proteins in human neutrophil granulocytes (Johneet al., 1997) but it is also expressed in activated macrophagesand monocytes (Dale et al., 1985; Johne et al., 1997). In thecondition of intestinal diseases, activated granulocytes migrat-ing into the intestinal wall will overexpress and releasecalprotectin into feces (Bjerke et al., 1993; Costa et al., 2005).There is substantial evidence to prove that thefecal calprotectinis a sensitive marker of intestinal inflammation (Konikoffand Denson, 2006; Langhorst et al., 2008; Xiang et al., 2008;Schoepfer et al., 2009). Fecal calprotectin has shown excellentdiagnostic accuracy in distinguishing inflammatory boweldisease (IBD) from irritable bowel syndrome (IBS), and has

been applied to monitor therapy andassess treatmentresponse(Sipponen, 2013; Burri and Beglinger, 2014).Alpha-1-Antitrypsin (A1AT) is a protease inhibitor, which

protects tissues from enzymes of inflammatory cells, especiallyneutrophil elastase. A1AT is oneof the principal serum proteinsand has a reference range in serum of 1.53.5 g/l, but theconcentration can increase to a very high level during acuteinflammation. A1AT is highly resistant to proteolysis in theintestine and can be excreted intact in the feces (Sharp, 1976).A1AT can extravasate from serum into the gut in the conditionof increased intestinal permeability, and finally be detected inthe feces. This supports fecal A1AT as a biomarker of intestinalpermeability (Crossley and Elliott, 1977; Laine et al., 1993;

Alam et al., 1994). There is a commercial kit to measure thefecal A1AT and researchers are exploring the application of this




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assay especially in infants with intestinal disorders (Kelleret al., 1997; Kosek et al., 2013).

Fatty acid binding proteins (FABPs) are small (1415 kDa)cytosolic proteins which can bind and transport fatty acids.There are several immunologically distinct types of FABPdepending on the different tissues, e.g. heart, intestine, liver,muscle, and adipocyte (Niewold et al., 2004). Intestinal fattyacid binding protein (IFABP) is a 15-kDa protein uniquelylocated in mature small-intestinal enterocytes. The location ofIFABP in the matureepithelium of villi facilitates its leakage intothe circulation from enterocytes when intestinal mucosaldamage occurs (Kanda et al., 1992). Therefore, it is easilydetected in plasma or urine in thesetting of intestinal ischemia(Gollin et al., 1993). Measurement of plasma I-FABP concen-trations was a highly specific and sensitive method for

assessing the severity of mucosal injury in rats (Kanda et al.,1992). Recent studies also suggested that IFABP in serum orurinemight be a useful biochemical marker for the diagnosis ofintestinal ischemic injury in humans(Kanda et al.,1996; Thuijlset al., 2011). Compared with uninfected individuals, individualswith chronic hepatitis B and C infection had higher plasmalevels of IFABP indicative of enterocyte death. IFABP levelsbecame undeletable in patients with successful treatment ofthe hepatitis infection (Sandler et al., 2011). It is expected thatthe availability of commercialkits detecting the IFABP in serumor urine might lead to a wider application of IFABP measure-ment in the human intestinal diseases.

Diamine oxidase (DAO) activity in serum correlates

inversely with intestinal permeability of the small intestine(Luk et al., 1980; Honzawa et al., 2011). DAO is the main

enzyme to catalyze the oxidation of diamines such ashistamine, putrescine, and cadaverine (Shakir et al., 1977).The expression of DAO occurs predominantly in humanintestinal mucosa as well as the placenta, kidney and thymus(Rangachari, 1992). However, serum DAO appears to comeprimarily from the small intestine (Buffoni, 1966). In theintestine, DAO is specifically located in tips of enterocyte villi,and its activity reflects the integrity and maturity of the smallintestinal mucosa (Luk et al., 1980). Serum DAO activity issignificantly decreased in patients with Crohn's disease andulcerative colitis regardless of the level of disease activity(Honzawa et al., 2011). Considering that serum DAO activitycould be easily measured with Enzyme Immunoassay using acommercial kit, it might thus become a convenient methodfor evaluating small intestinal permeability in patients with

intestinal diseases in the future.

6. Conclusion

Maintenance of the physical barrier in the intestine isdependent on the physical integrity of barrier components.Increased paracellular and (possibly) transcellular permeabil-ity, and epithelial cell damage will result in a gut barrierdysfunction. Subsequent translocation of PAMPs and viablebacteria is key event during liver disease. Dysfunction of theimmune system might contribute to persistently elevatedsystemic levels of PAMPs and chronic liver disease. It istherefore important to measure and monitor intestinal perme-

ability during liver disease. Most of the methods assessing theflow from theintestinal lumen to the blood use inert markers of

Table 1

Methods to assess intestinal permeability.

Methods Assay Advantage and disadvantage References

Morphology analysis H&E staining of intestinal sections Histology is easy to perform and to interpretNo functional analysis

Szabo et al. (1985)

Assessing intestinal tight junctions(electron microscopy, gene and proteinexpression)

No functional analysis Hartmann et al. (2012);Chenet al. (2014)

Functional analysisMethods to assess ow

from lumen to bloodEnteral administration of non-digestiblemarkers such as sugars, radioisotopes(e.g. 51Cr-EDTA) and polyethyleneglycols (PEG)

Performed under in vivo conditions, butaffected by factors such as gastrointestinalmotility, mucosal blood ow and thedistribution of the markers in the bodyInert markers are being used

Sun et al. (1998);Bjarnason et al.(1984a);Bjarnason et al. (1984b);Peeters et al. (1994)

Creating isolated intestinal loops andinjection of labeled bacterial products,markers or live bacteria

Serves with modications a wide variety ofpurposes and are independent from intestinalmotilityAble to determine the site of increased leakinessSurgery required

Chen et al. (2014);Hartmannet al. (2012);Hartmann et al.(2013)

Methods to assess owfrom blood to lumen

Fecal albumin measurement Non-invasive, does not require anymanipulation of the animal.Requires normal blood albumin levels

Hartmann et al. (2013)

In vivo perfusion system Can be used to test the effects of drugs onintestinal permeabilitySurgery required

Clayburgh et al. (2006);Marchiando et al. (2010b)

Microbiology tests Measurementof translocated microbialPAMPs

Direct assessment of gut-derived andtranslocated PAMPs in extraintestinal spaceLevels are dependent on the immune system

Hartmann et al. (2012);Chenet al. (2014);Chen et al. (2015);Cuenca et al. (2014)

Culturing translocated live bacteria Direct assessment of gut-derived andtranslocated bacteriaNumbers are dependent on the immune systemNot able to detect non-culturable translocatedbacteria

Fouts et al. (2012)




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different sizes that do not necessarily correlate with the uptakeof larger macromolecules. And even measurement of electricresistance, which has been considered as gold standard toassess permeability for a long period of time (Li et al., 2003;Klingberg et al., 2005; Ulluwishewa et al., 2011), is not linearlycorrelated with permeation of small inert sugars or othermolecules (Menard et al., 2010). This suggests that an idealpermeability assay will use labeled probes (such as bacteria,proteins or macromolecules). However, no universal markerprovides a definitive answer on the leakiness of the intestine,and a combination of methods assessing the flow from theblood to the intestinal lumen might be useful.

Acknowledgments

Studies described were supported in part by NIH grantsK08 DK081830, R01 AA020703, and U01 AA021856 (to BS).The project described was also supported by Award Number1I01BX002213 from the Biomedical Laboratory Research &Development Service of the VA Office of Research and

Development (to BS).

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