Recent advances in basic science Leaky gut: mechanisms, measurement and clinical

implications in humans Gut: first published as 10.1136/gutjnl-2019-318427 on 10 May 2019. Downloaded from Michael Camilleri‍ ‍

Division of Gastroenterology Abstract There is much folklore about the leaky gut and and Hepatology, Mayo Clinic, The objectives of this review on ’leaky gut’ for clinicians its relationship to microbial balance within the gut. Rochester, Minnesota, USA are to discuss the components of the intestinal barrier, One of the first ‘hits’ in searching information on leaky gut on the internet provides comprehensive Correspondence to the diverse measurements of intestinal permeability, their Professor Michael Camilleri, perturbation in non-inflammatory ’stressed states’ and advice, contrasting what happens when the balance Mayo Clinic, Rochester MN the impact of treatment with dietary factors. Information is ‘right’ and when ‘out of whack’, and advice on 55905, USA; on ’healthy’ or ’leaky’ gut in the public domain requires how to get the gut microbes back into balance camilleri.​ ​michael@mayo.​ ​edu confirmation before endorsing dietary exclusions, (box 1). Received 1 February 2019 replacement with non-irritating foods (such as fermented There is no controversy regarding barrier dysfunc- Revised 22 April 2019 foods) or use of supplements to repair the damage. The tion in diseases resulting in intestinal inflammation Accepted 23 April 2019 intestinal barrier includes surface mucus, epithelial layer and damage such as coeliac or Crohn’s disease, or Published Online First and immune defences. Epithelial permeability results ulceration from non-steroidal anti-inflammatory 10 May 2019 from increased paracellular transport, apoptosis or drugs (NSAIDs) resulting in structural abnormali- transcellular permeability. Barrier function can be tested ties of the epithelium. The ‘leaky gut’ is a simplistic in vivo using orally administered probe molecules or in term reflecting intestinal permeability, a function vitro using mucosal biopsies from humans, exposing that was extensively studied in these diseases and the colonic mucosa from rats or mice or cell layers to reported in the scientific literature from 1970 to 1–3 extracts of colonic mucosa or stool from human patients. 1990. Assessment of intestinal barrier requires measurements There may be several reasons for this resurgence beyond the epithelial layer. ’Stress’ disorders such as of interest in the ‘leaky gut’. First, there is frus- endurance exercise, non-steroidal anti-inflammatory tration about the lack of perceived advances in drugs administration, pregnancy and surfactants (such the management of common GI symptoms such as bile acids and dietary factors such as emulsifiers) as pain, diarrhoea and bloating; thus, a cause, increase permeability. Dietary factors can reverse such as leaky gut, is sought. Second, the scientific intestinal leakiness and mucosal damage in the ’stress’ literature has promulgated ‘dysbiosis’ in diverse disorders. Whereas inflammatory or ulcerating intestinal states, from obesity to autism, despite evidence http://gut.bmj.com/ that this documented parabiosis4 diseases result in leaky gut, no such disease can be cured does not neces- sarily result in any metabolic or other changes in by simply normalising intestinal barrier function. It is still mucosal functions, including barrier function, or unproven that restoring barrier function can ameliorate the role in pathogenesis of these diseases.5 Third, clinical manifestations in GI or systemic diseases. there is scientific research using diverse methods Clinicians should be aware of the potential of barrier that documents alterations in human intestinal

dysfunction in GI diseases and of the barrier as a target on October 2, 2021 by guest. Protected copyright. barrier function in disorders such as IBS or food for future therapy. allergy. However, there is no current gold standard with clear performance characteristics of the tests for barrier function, the diverse methods available actually measure very different endpoints and the Introduction clinical significance and relevance are unclear. The objective of this review is to address three ques- Fourth, as explained by Quigley,6 there are popular tions based on data almost exclusively acquired in perceptions of the barrier as a single cell thick, the humans: what does the ‘leaky gut’ mean? Should epithelial layer having disruptions of intercellular clinicians diagnose leaky gut and, if so, how can it connections leading to increased permeability and be diagnosed? Is the leaky gut treatable? There are consequent access to the blood stream for various vast numbers of papers that link ‘leaky gut’ with noxious chemicals, intact bacteria and a host of altered microbiota in disease models in exper- dietary and microbial components and designation imental animals, from allergy to non-alcoholic as the primary abnormality in diverse diseases such steatohepatitis to depression and amyotrophic as food intolerance, fibromyalgia, chronic fatigue lateral sclerosis. This review focuses almost exclu- syndrome and autism (all unsupported by any © Author(s) (or their sively on the evidence from human studies, since data). employer(s)) 2019. No the clinicians need to address the three questions in Despite the recommendation to not self-treat, commercial re-use. See rights the context of their patients. In addition, it focuses there are many resources, books and articles with and permissions. Published on the evidence of leaky gut in non-GI diseases, recommendations on restoring the ‘healthy balance’ by BMJ. which are the focus of many diseases or disorders in including eating dirt, curing with candida and To cite: Camilleri M. Gut which leaky gut and microbiota are considered to education about the microbiome, the ‘human super 2019;68:1516–1526. be aetiopathologically important mechanisms. organism’ and ‘the good gut’.

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A (IgA) secretion and by the physical barrier provided by the Box 1 Leaky gut glycocalyx and mucus. Epithelial cells, connected by apical junc- tional complexes, have the ability to transport luminal content, Gut: first published as 10.1136/gutjnl-2019-318427 on 10 May 2019. Downloaded from https://www.healthyway.com/content/the-truth-about-leaky-gut- but they also react to noxious stimuli by secretion of chloride syndrome-what-it-is-and-why-you-want-to-avoid-it and antimicrobial peptides. The Paneth cells in the epithe- Balance: when the microbial balance in your gut is right, lial layer, where they are most numerous in the crypts, also your whole body functions the way it is supposed to. But produce high quantities of defensins and several other antibiotic when that balance gets out of whack—say because of chronic peptides and proteins when exposed to Gram positive and nega- stress, chronic constipation, exposure to environmental toxins tive bacteria or bacterial products such as lipopolysaccharide. like pesticides, eating a poor diet or taking an antibiotic that Beyond the epithelium, the lamina propria provides defence wipes out a lot those microbes—the ‘bad’ bacteria cut holes based on innate and acquired immunity cells secreting IgA, cyto- in the fence and some of them, along with food particles and kines, chemokines and mast cell proteases, as well as endocrine toxins, leak into the bloodstream. When your immune system and secretomotor mechanisms mediated by the enteric nervous sees organisms where they do not belong, it attacks, causing system, which result in intestinal propulsive motility.7 Some of irritation and inflammation. the important transmitters are serotonin (5-HT), histamine and Causes: leaky gut has so many possible causes, so many cannabinoids. possibly symptoms The mucus layer consists of two components: an inner firmly Consequences: ‘The leakage in leaky gut may be responsible adherent layer where bacteria are sparse and secreted peptides for a huge variety of health issues, ranging from minor are protective with antibacterial functions (eg, defensins and (bloating, cramps, fatigue, food allergies and sensitivities, gas, lysozyme) and a thicker and loosely adherent outer layer where and headaches) to ‘bigger things’: autoimmune conditions, bacteria and bacterial products are abundant. The mucus layer depression and other mood disorders, diabetes, inflammatory is thicker in the colon than in the small bowel and may reach bowel disease, and multiple sclerosis’. a depth of over 800 microns, which is not much less than the Treatment: ‘Functional medicine’: get gut microbes back into height of an entire villus (range 500–1600 microns). There balance: multistep programme is regional variation in the barrier along the gut; in the small ►► Remove foods that create problems for example, gluten, intestine, pore size increases from 4 Å to 5 Å at the villus tip sugar and dairy. to over 20 Å at the base of the crypt. In addition, the micro- ►► Replace with foods less likely to irritate gut: fermented foods, biota influences the barrier and elements of the barrier impact for example, sauerkraut, kimchi, yoghourt, kefir, and pickles, the microbiota.6 There are several examples in the literature are healing foods. demonstrating diverse effects of bacteria and their products ►► Repair the damage with supplements: L-glutamine (heal the on intestinal barrier structure or function. Thus, Bifidobacteria intestinal lining), vitamin D, zinc and omega-3 fatty acids enhance barrier function in experimental necrotising enteroco- (such as fish oil). litis in mice,8 the yeast Saccharomyces boulardii has beneficial ►► Repopulate your good gut bacteria: probiotics or get a effects on altered intestinal microbiota and epithelial barrier

transplant from another person. defects in different pathologies,9 and different strains of Esch- http://gut.bmj.com/ ►► One of the biggest leaky gut red flags is having issues with a erichia coli have opposite effects on the barrier: E. coli Nissle variety of foods. 1917 stimulates the tight junction protein ZO-2,10 11 whereas a ►► Talk with your healthcare provider: you might have leaky gut prototypic translocating bacterium, E. coli strain C25, increases syndrome. permeability.12 Products of the bacteria such as bacterial toxins, ►► Do not, however, try to treat it yourself. or bacterial dehydroxylation producing secondary bile acids and short-chain fatty acids produced by bacterial fermentation protect against bacteria or enhance the barrier function.13–15 on October 2, 2021 by guest. Protected copyright. Given the current perceptions on ‘leaky gut’ that appear At the level of the epithelial cells, from the apical to the basal informed mostly by folklore or overreaching conclusions based domains of enterocytes, there are three sets of intercellular junc- on limited data, it is important to provide a balanced view of the tions: tight junction (zonula occludens (ZO)), adherens junction scientific data to facilitate the role of clinicians in addressing the (zonula adherens) and desmosome. Together they comprise the nature, diagnosis and treatment of abnormal intestinal barrier apical junctional complex, which supports the dense micro- function in humans. To address this, it is important to charac- villus brush border and regulates epithelial barrier function terise the intestinal barrier, the pathways between and through and intercellular transport.16 The anatomy and composition of epithelial cells and measurement of intestinal permeability in the mucosal barrier and its intercellular junctions are shown in humans. This provides the basis for examination of non-in- figure 1.17 testinal diseases characterised as ‘stress’ can really break the In general, it is recognised that there are three distinct para- barrier and to review possible treatments including diet, natural cellular epithelial permeability pathways: ‘leak’ and ‘pore’ path- substances and medications. ways, which are regulated by tight junctions and define intestinal permeability; and an ‘unrestricted’ pathway, which is associated The intestinal barrier with apoptotic leaks in pathological states, is independent of The intestinal barrier is a dynamic entity interacting with and tight junctions and provides access of luminal antigens to the responding to various stimuli. It consists of multiple elements. In lamina propria. In the presence of erosions or ulcers, bacteria the lumen, there is degradation of bacteria and antigens by bile, gain access to the mucosa (figure 2).16 18 gastric acid and pancreatic juice, and commensal bacteria that inhibit the colonisation of pathogens by production of antimi- crobial substances. The next element of the barrier is the micro- The importance of the mucus component of the barrier climate consisting of the unstirred water layer, glycocalyx and Mucus is secreted by the goblet cells and serves as the first physical mucus layer that prevent bacterial adhesion by immunoglobulin defense in the barrier, preventing antigens, toxins and bacteria

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Figure 1 Anatomy of the mucosal barrier. Left panel: in the human intestinal mucosa, composed of columnar epithelial cells, lamina propria (with its immune cells) and muscular mucosa, the goblet cells synthesise and release mucin, and the unstirred layer is immediately above the epithelial cells. The tight junction (TJ) is a component of the apical junctional complex, and it seals paracellular spaces between epithelial cells. Middle and right panels show an electron micrograph and the corresponding line drawing of the junctional complex of an intestinal epithelial cell. The key elements of the TJ are the zona occludens and zona adherens, each of which is made up of different components. Just below the base of the microvilli (Mv), the plasma membranes of adjacent cells seem to fuse at the TJ, where claudins, zonula occludens 1 (ZO1), occludin and F–actin interact. E-cadherin, α-catenin 1, β-catenin, catenin δ1 (also known as p120 catenin; not shown) and Factin interact to form the adherens junction (AJ). Myosin light chain kinase (MLCK) is associated with the perijunctional actomyosin ring. Desmosomes, located beneath the apical junctional complex, are formed by interactions between desmoglein, desmocollin, desmoplakin and keratin filaments. In general, diffusion through claudins and occludin within the membrane is energy independent, whereas ZO-1 facilitates exchange between TJ and cytosolic pools through energy-dependent mechanisms. Reproduced from ref. 17.

23 from directly contacting the epithelial cells. The elements of mucus. In fact, bile salts impact the ability of mucus to serve http://gut.bmj.com/ the mucus layer are highly glycosylated mucin proteins with a as a barrier to hydrophilic and lipophilic compounds.24 Secre- central protein core (abundant in serine, threonine and proline tion of mucus is a prelude to the epithelial damage that leads (Pro) amino acid residues) and O-glycosylation with hexoses and to colonic secretion by the secretagogue bile acids, chenodeoxy- hexosamines oriented almost perpendicular to the protein core cholic acid25 26 and deoxycholic acid,27 28 and these effects can be like a bottle brush, forming a gel-like sieve overlying the intes- partly inhibited by prostaglandins. Intra-arterial prostaglandin tinal epithelium.19 E2 (PGE2) evokes the secretion of intestinal mucus,29 and PGE2 In the small and large intestine, mucin 2 (MUC2) is the reverses NSAID enteropathy, in part, by inducing mucus secre- on October 2, 2021 by guest. Protected copyright. most abundant mucus protein secreted by goblet cells. Intes- tion.30 Other goblet cell secretagogues are cholinergic agonists, tinal epithelial cells (IECs) also express transmembrane mucins histamine, peptide tyrosine tyrosine (peptide YY) and serotonin. (MUC1, MUC3, MUC4, MUC12, MUC13 and MUC17) that Overall, there is a role for the enteric nervous system, the entero- remain attached to the apical surface and form the glycocalyx endocrine cells and resident immune cells in mediation of colonic together with glycolipids. Other major mucus proteins secreted mucus release.31 Cholinergic inhibition of mucin secretion with by goblet cells are chloride channel regulator, calcium-acti- intra-arterial atropine reduced the epithelial damage and fluid vated-1, Fc globulin-binding protein, which covalently binds and secretion secondary to 5 mM sodium chenodeoxycholate in the cross-links mucus proteins, zymogen granule protein 16 (ZG16, rabbit colon in vivo.32 a small lectin-like protein that binds to Gram+organisms), and Dietary emulsifiers, like bile acids, are amphipathic, that is, antibodies, especially IgA. Secreted mucus mixes with Paneth cell they are molecules with hydrophilic and lipophilic sections that secretions containing antibacterial peptides, lysozyme, deleted in maintain fat molecules in liquid suspension or water-soluble malignant brain tumours 1, and also MUC2.19 components in a hydrophobic environment. Dietary emulsifiers Immune regulators, such as antimicrobial proteins and IgA interact with the multilayered endogenous mucus secretions that molecules, are released in the mucus gel in a gradient from the coat the luminal surfaces of the intestinal tract and may compro- epithelium to the lumen, thereby reinforcing the defence against mise the ability of human mucus to prevent contact between the luminal microbes.20 The composition of the mucus layer can micro-organisms and IECs.33 Numerous synthetic surfactant affect the microbiota in the gut, while the microbiota also deter- food additives (anionic, cationic or non-ionic) are used in the mines the properties of the mucus gel.21 Muc2 knockout mice food industry (such as mono- and diglycerides or esters of fatty spontaneously develop colitis.22 acids (E471, E473 and E475)), as reviewed elsewhere.34 35 Surfactants, including bile salts (chenodeoxycholate (10 mM) Some of these have been shown to increase intestinal permea- and hyodeoxycholate (10 mM), but not cholate (10 mM), bility through paracellular and/or transcellular mechanisms, and ursodeoxycholate (10 mM) or Tween-20] induce secretion of some of them were also shown to inhibit P-glycoprotein or have

1518 Camilleri M. Gut 2019;68:1516–1526. doi:10.1136/gutjnl-2019-318427 Recent advances in basic science Gut: first published as 10.1136/gutjnl-2019-318427 on 10 May 2019. Downloaded from

Figure 2 Intestinal barrier and its dysfunctions. The intestinal barrier includes the mucus layer preventing bacterial adhesion by secretion of chemicals such as α-defensins and IgA secretion, epithelial cells, connected at the tight junctions (TJ) by junctional complexes, having the ability to transport luminal content and react to noxious stimuli by secretion of chloride and antimicrobial peptides, and the lamina propria innate and acquired immunity cells secreting Ig and cytokines. Intestinal permeability measurements are determined by the marker molecules used for measurement, since http://gut.bmj.com/ the type of molecules that pass the intestinal barrier depends on the type of lesion. Reproduced from ref. 18. mucolytic activity, such as the two emulsifiers, carboxymethyl- intestinal permeability, as well as an increased short-circuit cellulose and polysorbate 80 (Tween).36 current measured across the epithelial layer in Ussing cham- Additionally, based on the general characteristics of surfac- bers. There were also increased intestinal bacteria at systemic tants, it can be predicted that they decrease the hydrophobicity sites. Control experiments showed that these effects were due on October 2, 2021 by guest. Protected copyright. of the mucus layer, which has also been shown to be associated to hyperglycaemia rather than obesity or alterations in leptin with increased intestinal permeability.34 Dietary emulsifiers signalling. may interact with gut microbiota and altered mucus thick- ness to promote colitis in Il10 and Tlr5 knockout mice, which 37 Intestinal permeability: pathophysiological mechanisms and are predisposed to development of spontaneous colitis and increase translocation of E. coli across IECs.38 methods of analysis In this section, diverse methods of measurement and analysis are discussed, and the strengths and weaknesses are addressed. It is Example of increased transcellular permeability also important to note that intestinal permeability is influenced The examples provided above focused predominantly on by several factors, which are not always considered in the publi- the intercellular barrier and mucus barrier. However, there is cations, including the circadian cycle40 and stress.41 evidence that there may be altered expression of cellular trans- port mechanisms that may ultimately lead to intercellular barrier dysfunction and systemic inflammation. An excellent example Orally administered probe molecules of the potential impact of increased transcellular permeability Intestinal permeability is most commonly measured indirectly without apoptosis or intestinal ulceration is provided by the in humans by the fractional urinary excretion of orally ingested demonstration, in mouse models of obesity and diabetes, that probes that cross the intestinal epithelium by the paracellular hyperglycaemia drives intestinal barrier permeability through pathway, enter the bloodstream, are filtered by the glomerulus glucose transporter 2-dependent transcriptional reprogram- and excreted in the urine without active reabsorption in the ming of IECs and alteration of tight and adherence junction kidney.42 Fractional urinary excretion can, therefore, be used as integrity.39 These findings were demonstrated by reduced ZO-1 an indirect measure of intestinal permeability. expression and increased fluorescein isothiocyanate (FITC)-dex- The most commonly used probe molecules are saccharides. tran in serum after oral administration, indicating increased Although regional differences for preferred absorption sites

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the monosaccharide, mannitol, compared with the disaccha- Table 1 Molecular sizes of the sugar and other probe molecules ride, lactulose. In practice, modern methods of assay based on Mol. weight, Mol. diameter, Å, Mol. diameter, liquid chromatography-mass spectrometry accurately measure Gut: first published as 10.1136/gutjnl-2019-318427 on 10 May 2019. Downloaded from Probe molecule Da reported Å, estimated* the saccharides, and the most commonly used combinations are Rhamnose 164 8.2 6.9 therefore lactulose or sucralose (as disaccharides) with mannitol Mannitol 182 6.7 7.2 or rhamnose (as monosaccharides). Because of potential ‘contam- Lactulose 342 9.5 9.7 ination’ by environmental exposures to mannitol and sucralose, 13 Cellobiose 342 10.5 9.7 the lactulose and rhamnose or C-mannitol saccharides are Sucralose 398 NA 10.4 increasingly used for in vivo permeability measurements. In Cr-EDTA 340 10.5 9.6 addition, as indicated above, 0–2 hour urine collection reflects Dextran 4 kDa 4000 NA 30.0 predominantly small intestinal permeability and 8–24 hour collection almost exclusively colonic permeability. *Calculated based on the formula: radius=0.33*(MM^0.46). Å, ångström; Cr-EDTA, chromium complexed with ethylene diamine tetracetic acid, Overall, these tests still have limited validity based on uncer- Da, dalton; MM, molecular mass. tainty of the normal values, lack of standardisation of test procedure and lack of validation including responsiveness of a standardised test to treatment. have been suggested,43 it is important to note that sucrose is only useful in the first hour postingestion, as it is rapidly metabo- In vitro or tissue measurements of intestinal barrier lised (to glucose and fructose) and, therefore, at best, provides Several methods are used to assess intestinal permeability on information about gastric and duodenal permeability. Moreover, biopsies taken from the human gut and measured by the transfer other monosaccharides, such as mannitol and rhamnose, and of probe molecules across mucosal biopsies in Ussing chambers disaccharides, such as lactulose and sucralose, are all absorbed (in association with measurements of transepithelial resistance in the small bowel and colon, and the timing of urinary excre- and short circuit current measurements). Other approaches tion provides the best way to differentiate regional analyses: quantitate the tight junction proteins in the mucosal biopsies, or 0–2 hours reflects predominantly small intestinal permeability they assess the faecal supernatant in cellular monolayers or rat or and 8–24 hours reflects almost exclusively colonic permea- 44 mouse colonic mucosa in vitro. bility. Also, among these saccharides, sucralose is the one There are differences in in vivo compared with in vitro not metabolised by colonic bacteria; similarly, polyethylene measurements of barrier functions. The molecular size of probe glycol 400 and radioactive chromium complexed with ethylene 51 molecules that can cross the epithelial barrier in humans in vivo is diamine tetracetic acid ( Cr-EDTA) are not degraded by colonic at least 10-fold smaller than in vitro, which shows that molecules bacteria. However, the utility of these probe molecules is some- of approximately 4–40 kDa (eg, dextran 4 or 40) easily traverse what compromised by the ‘background’ ingestion of some of the the intestinal mucosa in a Ussing chamber in vitro. There are sugars, particularly mannitol and sucralose. also differences that may reflect additional functional barriers Until relatively recently, interpretation of permeability tests in vivo that are excluded in the in vitro studies, including the was based on the following assumptions. Lactulose is a relatively lamina propria, innervation by submucosal neurons and perme- http://gut.bmj.com/ large molecule, can only cross via the leak pathway or at sites of ability of end capillaries that constitute other potential barriers epithelial damage and is considered a marker of barrier integ- impeding passage of the probe molecules into the circulation in rity. Mannitol, which is reputedly one-third as large as lactulose, vivo. For example, intercellular complexes are under neurohu- is assumed to cross the pore pathway, which allows passage of moural control, such as from vasoactive intestinal peptide (VIP) sodium ions, water and small solutes; therefore, mannitol and and cholinergic neurons, from the submucosal plexus,47 48 and other monosaccharides such as rhamnose used to be regarded these are lost in biopsied mucosa. as measures of surface area.42 The inference was that the lact- on October 2, 2021 by guest. Protected copyright. ulose:mannitol ratio might measure the sum of leak pathway permeability and epithelial damage normalised to surface area. Endoscopic measurements of intestinal barriers in humans However, a review of the molecular sizes of the sugar probe Two techniques are available: first, confocal endomicroscopy, which shows leaks of intravenously administered fluorescein molecules (table 1) suggests that there is no relevant difference 49 in the reported or estimated molecular diameters and, therefore, into the gut lumen during endoscopy (eg, in response to it appears unlikely that they traverse the epithelium through food-associated changes in the intestinal mucosa of patients with different pathways. diarrhoea-predominant IBS); and second, endoscopic mucosal In fact, the mass of saccharides that is absorbed during 1 hour impedance, in which a 2 mm diameter catheter is passed through in the healthy gut following ingestion orally is typically up to 2% an endoscope and placed in contact with the duodenal mucosa of rhamnose and 0.07% of lactulose administered in children in under direct visualisation, and two circumferential sensors, USA.45 In healthy adults, the fractional excretion over 24 hours placed 2 mm apart on the mucosa for 0.10 s, in the four quad- 13 rants of the duodenum with a decompressed lumen, and all fluid is 31.2%±3.4% (SEM) for C-mannitol and 0.32%±0.03% for 50 lactulose.46 Therefore, despite the similarity in molecular diam- aspirated. The studies of food-associated changes during these eters, there is a 100-fold difference in the per cent of recovery challenge tests provide some evidence that there can be barrier of the monosaccharides and disaccharides; however, if the larger changes that may indeed support the concept of transient ‘leak- molecule traverses the ‘leak’ pathway, the latter might also iness’ of the gut. allow passage of the small molecule, and there is no compelling evidence that the different sugars actually traverse the intestinal Abnormal barrier function in intestinal disease states barrier via different pathways. It is clear that inflammatory or ulcerating diseases result in Other factors, such as tertiary molecular structure, are likely abnormal intestinal barrier function. However, this is not the relevant to explain the marked difference in absorption ratios category of disease that is being associated with leaky gut, as of monosaccharides and disaccharides which is 30–100-fold for discussed in the next section. The abnormal barrier function

1520 Camilleri M. Gut 2019;68:1516–1526. doi:10.1136/gutjnl-2019-318427 Recent advances in basic science is well described for conditions such as IBD, as well as in first though the mechanism whereby this results in the eosinophilic degree relatives of patients with IBD,3 51 coeliac disease and infiltration of the oesophagus is unclear57; the second group gluten sensitivity without overt coeliac disease in patients with did not document increased small bowel permeability, but they Gut: first published as 10.1136/gutjnl-2019-318427 on 10 May 2019. Downloaded from HLA-DQ2/8 (genotype associated with coeliac disease,52 53 reported improvement in the eosinophilic oesophagitis with an intestinal graft versus host disease, enteric infections and infes- elemental diet.58 All of these diseases and disorders associated tations and HIV infection and AIDS).42 There is also extensive with possibly altered intestinal barrier function are pathological literature54 documenting abnormal intestinal permeability in IBS diseases, not what is usually associated with non-specific ‘leaky and the association of abnormal permeability with pain in IBS, gut’. although the degree of altered barrier function is clearly lower than in inflammatory bowel or coeliac diseases. Longitudinal Leaky gut: the pro arguments studies in patients with IBD suggest that increased intestinal The concept of a leaky gut in non-GI diseases is supported by permeability preceded relapsed of Crohn’s disease,55 suggesting evidence of dysfunctional gut mucosal barrier in stress-associated a pathogenetic role of the epithelial barrier in the pathogen- conditions and the response of the altered barrier to non-drug esis of gut inflammation; in addition, IBS is highly prevalent in interventions; associations of disease states with altered intes- first degree relatives of IBD patients,56 suggesting that intestinal tinal permeability and microbiome; and alterations in intestinal permeability could be a relevant factor in the determination of permeability as a result of gut-directed therapy in diverse condi- symptoms. tions including healthy people or diverse groups of children in Several other non-GI diseases have been associated with central African countries. leaky gut, based on limited or no supporting data,6 42 including asthma, autism, Parkinson’s disease, multiple sclerosis, eczema, psoriasis, eosinophilic oesophagitis, environmental enterop- Dysfunctional gut mucosal barrier due to endurance exercise and athy, kwashiorkor, fibromyalgia, depression, chronic fatigue effects of non-drug intervention syndrome, multiorgan failure syndrome (shock, burns and Table 2 summarises the literature on two types of conditions that trauma), non-alcoholic fatty liver disease (NAFLD), alcoholic result in stress to the intestinal barrier, with documented effects cirrhosis, obesity, metabolic syndrome, pancreatitis and rheuma- on intestinal permeability or biochemical evidence of mucosal toid arthritis. Two separate groups independently studied small damage, and restoration with a dietary, non-pharmacological inter- bowel permeability in patients with eosinophilic oesophagitis; vention. These studies suggest that there are ‘stress’ states in which one group documented increased small bowel permeability, there is documentation of altered barrier function and examples of

Table 2 Summary of the literature on conditions that result in stress to the intestinal barrier, with documented effects on intestinal permeability or biochemical evidence of mucosal damage, and restoration with a dietary, non-pharmacological intervention Effects on barrier function Barrier stressor; clinical scenario Specific study Intestinal permeability Mucosal damage Dietary intervention and its effects Reference # http://gut.bmj.com/ Endurance exercise Biking challenge Urine iohexol (MW 821 Da) ↑ Serum I-FABP ↑, 70 Marathon runners with zonulin ↓ faecal occult blood or bloody Running challenge LRR ↑ and correlated with core ND 71 diarrhoea temperature (eg, >39°C) Biking challenge LRR ↑ Serum I-FABP ↑ Citrulline (vs alanine) reversed ↑serum 72 I-FABP and gastric hypoperfusion without effect on LRR. on October 2, 2021 by guest. Protected copyright. Biking challenge ND Serum I-FABP ↑ Sucrose (vs nitrate) reversed ↑ serum 73 I-FABP no Δ gastric hypoperfusion. Seven runners, two boxers, LRR ↑ ND Nurticeutical colostrum versus placebo 74 three rugby reduced LRR ↑ and apoptosis of cell lines in vitro. NSAID enteropathy: NSAIDs Diverse NSAIDs, for example, 51CrEDTA and saccharides ND 75–77 cause small bowel ulcers indomethacin and inflammation Indomethacin LRR ↑ ND Zinc carnosine (vs placebo) reduced LRR 78 and increased HT29 cell proliferation (vs ZnSO4). Aspirin increased colon permeability: ND Bifidobacterium BB-12 and adolescentis 79 urine SLR and sucralose (IVS-1) and galacto-oligosaccharide prebiotic reduce colon permeability. Pregnancy with or without Increased serum zonulin and ND 80 obesity increased serum LPS, associated with metabolic risk markers. High serum zonulin. ND n-3 PUFAs, fibre and a range of vitamins 81 and minerals: reduced high serum zonulin; greater richness of gut microbiota. No effects on serum zonulin ND Probiotics and/or LC-PUFA. 82 or LPS. Da, dalton; I-FABP, intestinal fatty-acid binding protein; IVS-1, first intervening sequence; LC-PUFA, long chain polyunsaturated fatty acids; LPS, lipopolysaccharide; LRR, lactulose rhamnose ratio; MW, molecular weight; ND, not determined; NSAIDs, non-steroidal anti-inflammatory drugs; PUFA, polyunsaturated fatty acids; SLR, sucralose-to- lactulose ratio.

Camilleri M. Gut 2019;68:1516–1526. doi:10.1136/gutjnl-2019-318427 1521 Recent advances in basic science ). 119 LPS, LPS, 104 Gut: first published as 10.1136/gutjnl-2019-318427 on 10 May 2019. Downloaded from ; 58 106 mannitol excretion ratio;

87 64 91 LMR, lactulose LMR, Effects of treatment no effect of exclusion diets on oesophageal microbiome. Increased LMR in children with food allergy despite dietary exclusion. No effect of elemental diet on duodenal mucosa or TJ protein expression LMR or Reduced cirrhosis severity with Lactobacillus and VSL#3 probiotics. Successful use of faecal microbial transplant for the treatment of recurrent D-lactic acidosis. showed any benefit Only 2/6 trials of omega-3 FA typically in those with mild on cognitive decline, cognitive impairment. Probiotics reduce depression scores in six randomised, Probiotics reduce depression scores in six randomised, placebo-controlled trials (reviewed in ref. ; permeability;

107 ; 113 ; 103 and Lachnospira 98 92 88 IgA, immunoglobulin A; IP, intestinal IP, A; immunoglobulin IgA, 102 61 110 ; psychological stress increases pro- ; 117 ). 118 112 ther effects O Lactobiota fermentation leads to increased risk of d-encephalopathy. 3/5 patients had elevated inflammatory markers in inflammatory markers 3/5 patients had elevated stool. relative abundance of Enterobacteriaceae positively associated with severity of postural instability and gait difficulty. Postulated mast cell and IgE-mediated increase in Postulated inflammatory cytokines. Bacterial TLR4 load TLR2, and TLR9 and TLR1, were overexpressed and mucin genes underexpressed on biopsies with active EoO. Increased endogenous ethanol production by gut bacteria in NAFLD. independent predictor IgA antibodies against F-actin, of poor disease outcome. microbes or LPS reaching liver TPN liver disease, In cells Kupffer and activating Correlation of increased intestinal permeability in disease with intestinal alpha-synuclein Parkinson’s Decreased levels of butyrate-producing bacteria; decreased levels of micro-organisms the genera Oscillibacter , Anaerostipes serum IgM and IgA against LPS in Elevated depression inflammatory cytokines (extensive literature reviewed in ref. fatty acid binding protein;

116 I-FABP, intestinal I-FABP, ZO, zonula occludens. ZO, 112 85 or Neisseria and 105 . 90 97 96 . isothiocyanate;

102 parenteral nutrition;

109 91 ; ; 92 ; 94 115 108 (F/B) ratio at phylum level

95 ; TLR, Toll-like receptor; TPN, total TPN, receptor; Toll-like TLR, 101 junction;

http://gut.bmj.com/ reduced butyrate-producing bacteria from the genera Blautia , Coprococcus and Roseburia ; putative ‘proinflammatory’ Proteobacteria of the genus Ralstonia significantly more abundant in mucosa disease. patients with Parkinson’s Microbiota changes Microbiota , Anaerostipes Differences in Bacteroides , Faecalibacterium Prevotella , Escherichia and Lachnospira at genus level decreased Firmicutes/Bacteroidetes Significant microbiota change in liver cirrhosis enhanced mucosal immune response to various microbial antigens enhanced mucosal immune response to various associated with IgA antibodies against F-actin. review documents show diverse microbiota changes (variable in review documents show diverse microbiota changes (variable different studies). Corynebacterium in active EoO. Firmicutes, Bifidobacteria, Faecalibacterium prausnitzii . Bifidobacteria, ↓ Firmicutes, Clostridia and facultative anerobes . ↑ Bacteroidetes, increased haemophilus Oesophageal microbiome: especially Gram negative ↑ SIBO (37.5%) in patients with NAFLD, bacteria and Escherichia coli 1/22 had SIBO ( Enterobacter ) in microbial diversity patients with small bowel Wide variability resections coli in epithelium and lamina Significantly more intense staining of E. propria of sigmoid mucosa low Low diversity of the microbiome compared with healthy cohorts; in 3/5 patients with low F/B ratio. Ruminococcus spp. A review documents extensive literature on cross-sectional and longitudinal studies documenting association between stool microbiota and anxiety depression. A review documents studies of the microbiome and microbial translocation in patients with schizophrenia and bipolar disorder. patients with short bowel on TPN have ‘lactobiota’ enriched in the TPN have patients with short bowel on depleted in anaerobic micro-organisms Lactobacillus/Leuconostoc group, (especially Clostridium and Bacteroides ). FABP2, fatty acid-binding protein 2; FITC, fluorescein FITC, fatty acid-binding protein 2; FABP2, TJ, tight TJ, 108 94 acid binding protein;

ability and altered microbiota intestinal bacterial overgrowth;

; on October 2, 2021 by guest. Protected copyright. 93 98 FA, fatty acids; FABP, fatty FABP, fatty acids; FA, 84 oesophagitis;

erum biomarkers S ↑ LPS in NAFLD associated with SIBO. ↑ Zonulin ↑ LPS in 42% of NASH with IgA antibodies Higher serum I-FABP associated against F-actin. an Lower plasma levels of LPS binding protein, indirect measure of systemic endotoxin exposure. er 114 111 83 97 87 or in 57 58 100 108 86 62 ; hours urinary sucralose (mark

99 58 , zonulin and FABP2 were each zonulin and FABP2 , ; but not LMR in adults 107 Cr-EDTA in 39% of 139 patients with Cr-EDTA 57 51 ; ex vivo assessment of duodenal mucosal ; however, flux of sulfonic acid and however, 89 NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; SIBO, small SIBO, non-alcoholic steatohepatitis; NASH, non-alcoholic fatty liver disease; NAFLD, mall intestinal or colonic barrier function LPS in most severe amyotrophic lateral sclerosis. No difference in LMR or most TJ protein expression, TJ protein expression, No difference in LMR or most but increased claudin 2 expression and decreased transepithelial resistance in ileal biopsies ex vivo. IP probe molecules or epithelial damage IP probe NAFLD (SRMA five studies). integrity was normal. integrity was children ↓ ZO-1 and villus height in mice. ↑ LMR threefold versus health. absorption horseradish peroxidase not abnormal with or without Lewy bodies in depression/anxiety patients significantly elevated compared with non-depressed or anxious controls. S ↑ LMR or ex vivo; ↑ FITC-Dextran I.P. E-cadherin and claudin-4 in unfed segments ↓ ZO-1, in paediatric patients ↑ LRR normal (83% (19/22) with quiescent IBD). Downregulation of occludin not ZO-1 in colonic mucosa; Plasma levels of LPS ↑ LMR 38% in children with food allergy. of total intestinal permeability). LMR normal, but ↑ 24 LMR normal, rhamnose ratio;

ation Summary of diseases or disorders with increased intestinal perme

 NAFLD/NASH Cirrhosis Sclerosing cholangitis Parkinson Psychiatric diseases TPN or enteral depriv  Alzheimer ALS Ageing lipopolysaccharide; LRR, lactulose LRR, lipopolysaccharide; Condition allergy Food Eosinophilic oesophagitis Increased small bowel IP based on lactulose Liver diseases Neurological diseases it is infrequent for the altered barrier dysfunction and microbiota to be documented in same human study. In each category, eosinophilic EoO, amyotrophic lateral sclerosis; ALS, ethylenediamine tetraacetic acid; , chromium-51 51Cr-EDTA Table 3 Table

1522 Camilleri M. Gut 2019;68:1516–1526. doi:10.1136/gutjnl-2019-318427 Recent advances in basic science

Table 4 Examples from the literature of in vivo human studies showing alterations in intestinal permeability as a result of gut-directed therapy with nutrient, supplement or fibre

Therapy Patients with Comments Reference # Gut: first published as 10.1136/gutjnl-2019-318427 on 10 May 2019. Downloaded from Glutamine intravenous Nutritional depletion presurgery. Patients with increased intestinal permeability did not improve after glutamine- 120 enriched total parenteral nutrition. Enteral glutamine 50%–80% burns. Urinary LMR in enteral glutamine group lower than standard enteral formula. 121 Enteral glutamine 30%–75% burns. Plasma DAO activity and urinary LMR in enteral glutamine group were lower than 122 in untreated burn group. Enteral glutamine Crohn’s disease patients. Glutamine and active control (whey) both reduced LMR. 123 Oral glutamine Postinfectious IBS. Elevated urinary LMR was normalised in the glutamine but not the control group. 65 Galactooligosaccharides Obesity. Postaspirin sucralose:lactulose ratios and sucralose excretion reduced indicating 79 improvements in colonic permeability. Inulin-enriched pasta Healthy young volunteers. Reduced serum zonulin and urine LMR but not mannitol excretion. 124 Psyllium Children with IBS. No effect on sucrose and sucralose recovery and LMR. 125 Fibre NAFLD patients undergoing diet Rx for Increasing nutritional fibre from 19 to 29 g/day reduced serum ZO levels and 126 obesity. improved hepatic steatosis. Rhubarb Day 3 postburns. Plasma DAO activity in rhubarb-treated group were lower than in controls. 127 Gelling complex of tara Healthy participants. Reduced LMR and reduuced sucralose concentration suggesting improvement in 128 gum+exopoly saccharides of Strep. Thermos- both small intestinal and colonic permeability. philus ST10 Ascorbic acid Healthy female participants. Excretion of lactulose over 6 hours augmented after consumption of either aspirin 129 or ascorbic acid compared with that after consumption of placebo. Fermented and amylase-digested weaning Tanzanian children aged 6–25 months Reduction in L/M ratio between admission and day 3 of hospitalisation was 130 foods with acute diarrhoea. significantly greater in the fermented and amylase-digested weaning group (89%) than the conventional or high-energy density amylase digested porridge groups (44% and 75%, respectively). Common beans Rural children in second year of life Additional beans in diet reduced lactulose excretion without effect on LMR 131 (Malawi) Micronutrient-fortified complementary/ Zambian infants from 6 to 18 months LMR (adjusted for baseline urinary L:M ratio, socioeconomic status, mother’s 132 replacement food old. education, season of birth and baseline stunting and stratified by maternal antenatal HIV status, child’s sex, concurrent breastfeeding status and anaemia at baseline) worse in group with fortified diet especially among boys and among the infants of HIV-negative mothers. DAO, diamine oxidase; L/M, lactulose to mannitol ratio; LMR, lactulose mannitol excretion ratio; NAFLD, non-alcoholic fatty liver disease; Rx, treatment; ZO, zonula occludens.

61 normalisation that would support the concept of a transient leak- bacteria or their products. An alternative hypothesis is that the http://gut.bmj.com/ iness of the gut barrier. There is also recent evidence suggesting liver disease causes a systemic inflammatory response that leads to that changes in intestinal microbiota composition (characterised increased intestinal permeability, with bacterial translocation and by increased α-diversity and changes in the relative abundance further hepatic damage.62 63 of >50% of identified genera, including increased abundance of In many studies in the literature, particularly in human studies, less dominant taxa at the expense of more dominant taxa such the three focal points of the relationship are not all examined, and as Bacteroides) and metabolism (reduced serum interleukin-6 and ‘triangulation’ is therefore based on hypothesis or inference based reduced stool cysteine) coincide with increased intestinal permea- on the association between two of these three factors. Table 3 on October 2, 2021 by guest. Protected copyright. bility (documented by increased sucralose excretion in urine after summarises information garnered from studies in ageing, food oral load) in young adults under prolonged physiological stress in allergy, liver disease, parenteral nutrition (or enteral exclusion) and the form of a 4-day cross-country ski march.59 Discussion of the neuropsychiatric diseases in humans or in animal models where role of the microbiota in intestinal barrier function is beyond the there are no human data available. In general, the data should be scope of the current article. Further studies are required to explore regarded as hypothesis generating, that is, the leaky gut may be the hypothesis that epithelial barrier dysfunction associated with a cause or an effect of the disease (as in the case of liver disease), mucosal enrichment of specific bacterial strains may predispose and there may be either normal or dysbiotic microbiota that lead to a shift to disease-associated microbiota that eventually leads to to inflammatory or other consequences that have impact on the pathological consequences such as IBD in individuals with genetic disease. In some situations, alteration of the microbiota may result predisposition.60 in reduced severity of the disease in humans, as demonstrated with hepatic cirrhosis in response to treatment with Lactobacillus casei Associations of disease states with altered intestinal permeability strain Shirota or VSL#3, which contains eight lyophilised bacterial 64 and microbiome strains. From a detailed review of the literature, it is clear that animal models of disease have documented a three-point relationship: Alterations in intestinal permeability as a result of gut-directed disease phenotype, barrier change and altered microbiota. As exem- therapy plified best with chronic liver disease, the directionality of the rela- A third proargument is provided by examples from the litera- tionship is controversial, even from animal studies. For example, ture of in vivo human studies showing alterations in intestinal one hypothesis argues that increased endogenous production of permeability as a result of gut-directed therapy, as summarised in ethanol by gut bacteria (eg, E. coli) caused by small intestinal bacte- table 4. One study worthy of specific comment is the randomised, rial overgrowth results in increased intestinal permeability, bacte- placebo-controlled trial of oral glutamine in about 100 patients rial translocation and hepatic inflammation due to the translocated showing normalisation of LMR in association with improvement

Camilleri M. Gut 2019;68:1516–1526. doi:10.1136/gutjnl-2019-318427 1523 Recent advances in basic science in the IBS-symptom severity score, stool frequency and consis- manifestations in local GI disease or systemic diseases is as tency.65 However, most of the other studies are small, used yet unproven. Clinicians should be aware of the potential different nutrients and diverse methods and require replication. of barrier dysfunction in GI diseases and the potential as a Gut: first published as 10.1136/gutjnl-2019-318427 on 10 May 2019. Downloaded from There is also hope that probiotics or commensal organisms target for future therapy. improve intestinal barrier function66; however, the evidence to date is sparse, often based on animal models rather than Acknowledgements The author would like to thank Mrs Cindy Stanislav for excellent secretarial assistance. human studies, the beneficial effect may be through the effects of butyrate and the documented effects have been reported for Contributors MC: authorship of manuscript. the organism Akkermansia municiphila.67 There is a microin- Funding MC is supported by grants R01-DK67071 and R01-DK115950 from tegral membrane protein (MIMP) that is the smallest domain National Institutes of Health. of surface layer protein from Lactobacillus plantarum. MIMP Competing interests None declared. had a significant anti-inflammatory effect in an experimental Patient consent for publication Not required. 68 model of dextran sodium sulfate (DSS)-induced colitis. 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