REVIEW published: 28 October 2021 doi: 10.3389/fnut.2021.718093 Present and Future Therapeutic Approaches to Barrier Dysfunction Marina Fortea 1†‡ , Mercé Albert-Bayo 2†‡ , Mar Abril-Gil 2‡ , John-Peter Ganda Mall 2,3‡ , Xavier Serra-Ruiz 4‡ , Alejandro Henao-Paez 4‡ , Elba Expósito 2‡ , Ana María González-Castro 2‡ , Danila Guagnozzi 2,4,5,6‡ , Beatriz Lobo 2,4,5‡ , Edited by: Carmen Alonso-Cotoner 2,4,5,6*‡ and Javier Santos 2,4,5,6‡ Giovanni Barbara, 1 University of Bologna, Italy Laboratory for Enteric NeuroScience, Translational Research Center for GastroIntestinal Disorders, University of Leuven, Leuven, Belgium, 2 Laboratory of Neuro-Immuno-Gastroenterology, Digestive System Research Unit, Vall d’Hebron Institut de Reviewed by: Recerca (VHIR), Vall d’Hebron Hospital Universitari, Barcelona, Spain, 3 Department of Biomedical and Clinical Sciences, Laura Baldomà, Linköping University, Linköping, Sweden, 4 Department of Gastroenterology, Vall d’Hebron Hospital Universitari, Barcelona, University of Barcelona, Spain Spain, 5 Facultad de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain, 6 Centro de Investigación Biomédica en Linette Willemsen, Red de Enfermedades Hepáticas y Digestivas (CIBERHED), Instituto de Salud Carlos III, Madrid, Spain Utrecht University, Netherlands *Correspondence: Carmen Alonso-Cotoner There is converging and increasing evidence, but also uncertainty, for the role of abnormal carmen.alonso@vhir.org intestinal epithelial barrier function in the origin and development of a growing number † These authors have contributed of human gastrointestinal and extraintestinal inflammatory disorders, and their related equally to this work and share first complaints. Despite a vast literature addressing factors and mechanisms underlying authorship ‡ ORCID: changes in intestinal permeability in humans, and its connection to the appearance Marina Fortea and severity of clinical symptoms, the ultimate link remains to be established in many orcid.org/0000-0001-8885-7495 cases. Accordingly, there are no directives or clinical guidelines related to the therapeutic Mercé Albert-Bayo orcid.org/0000-0002-9726-8083 management of intestinal permeability disorders that allow health professionals involved Mar Abril-Gil in the management of these patients to carry out a consensus treatment based orcid.org/0000-0003-0318-6755 on clinical evidence. Instead, there are multiple pseudoscientific approaches and John-Peter Ganda Mall orcid.org/0000-0002-2120-7743 commercial propaganda scattered on the internet that confuse those affected and health Xavier Serra-Ruiz professionals and that often lack scientific rigor. Therefore, in this review we aim to orcid.org/0000-0003-2478-8923 Alejandro Henao-Paez shed light on the different therapeutic options, which include, among others, dietary orcid.org/0000-0001-6062-4753 management, nutraceuticals and medical devices, microbiota and drugs, and epigenetic Elba Expósito and exosomes-manipulation, through an objective evaluation of the scientific publications orcid.org/0000-0001-6088-3149 Ana María González-Castro in this field. Advances in the knowledge and management of intestinal permeability will orcid.org/0000-0003-3955-3318 sure enable better options of dealing with this group of common disorders to enhance Danila Guagnozzi orcid.org/0000-0002-6171-1901 quality of life of those affected. Beatriz Lobo Keywords: epithelial barrier function, intestinal permeability, nutrients, short chain fatty acids, prebiotics, orcid.org/0000-0003-3391-7125 probiotics, mast cell stabilizers, mucoprotectants Carmen Alonso-Cotoner orcid.org/0000-0002-3483-2919 Javier Santos orcid.org/0000-0002-4798-5033 INTRODUCTION Specialty section: This manuscript belongs to a series of articles dealing with the role of intestinal barrier dysfunction This article was submitted to in the origin of chronic inflammatory disorders. Previous papers in this monography review Nutritional Immunology, a section of the journal the anatomical, molecular, microbiological, immunological, and pathophysiological bases that Frontiers in Nutrition link intestinal permeability to the development of chronic conditions within the gastrointestinal tract. Some studies also point to a prominent role of abnormal responses to food and microbial Received: 31 May 2021 Accepted: 29 September 2021 antigens, and toxins, resulting from the alteration of the intestinal epithelial permeability, in the Published: 28 October 2021 generation of symptoms and signs common to functional diseases of the digestive tract. Although Frontiers in Nutrition | www.frontiersin.org 1 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction the theoretical basis for this hypothesis is apparently solid, route as the main target of the epithelial barrier breakdown it is nonetheless true that translation from pathophysiological and as an early event whose loss of functional integrity likely alterations to clinical manifestations relies mostly on in vitro facilitates transepithelial antigen penetration, and the stimulation and ex vivo studies and preclinical models. Therefore, more of immunological responses, further increasing paracellular evidence from clinical trials is needed to determine their role in epithelial permeability and promoting the development of low- the management of these diseases. grade mucosal inflammation (Figure 1) (1). Despite outstanding advances on the pathophysiology The potential market for intestinal permeability regulatory and molecular mechanisms underlying barrier abnormalities, products is unknown but intuitively ample. However, it remains currently we have no universal standards to accurately determine to be established the mechanistic link between alterations in the magnitude of the problem (see other papers in this intestinal permeability and specific diseases to estimate how monography). In this sense, functional barrier parameters such many patients could benefit from better therapies for intestinal as lactulose/mannitol ratio and maybe certain blood markers permeability and the direct and indirect cost derived from may be more indicative for intestinal barrier function than attending these people. secondary parameters such as levels of tight junction protein Finally, this manuscript is not intended as a systematic expression. This lack of standardization generates confusion review of the literature concerning intestinal permeability and its impeding further actions of regulatory agencies and many management. We just want to raise awareness on the potential health-care professionals doubt the validity, usefulness and of targeting intestinal permeability to improve gut mucosal clinical applicability of the different techniques used for the inflammation and related clinical manifestations. However, determination of intestinal permeability. we also want to make clear that improving barrier integrity Closely related to the scant clinical evidence linking does not mean that inflammation and immune activation are permeability alterations with inflammatory disorders of the interrupted because this deserves further evidence and possibly digestive system and other body systems is the insufficient complementary approaches to manage microbiome and immune development of molecules or drugs aimed at controlling this system defects. function. This is despite the large number of potential therapeutic targets in which a regulatory role has been evidenced in both the pore pathway and the leak pathway. In addition, the modulation APPROACH TO MANAGEMENT of the microbiota and its metabolites, through nutrition, can also play an important role in the therapeutic armamentarium Nutrients of altered intestinal permeability. Nutrition has a key role in shaping gut microbiota (2) whereas There are hundreds of publications that have investigated processing of food by gut microbiota releases byproducts and a huge number of molecules involved in intestinal barrier metabolites that influence the functioning of the intestinal barrier homeostasis, though many data are derived from in vitro or and mucus layer integrity (2, 3) in health and disease (4, 5) animal studies what may not well-represent the physiologic (Figure 2). situation in the human organism and may not correctly mimic human pathology. In this article, we will review the evidence Dietary Fibers, Prebiotics, and Short-Chain Fatty related to the use of those molecules or products that offer greater Acids potential for the clinical management of diseases that have been The international CODEX Alimentarius Commission defined more consistently associated with intestinal epithelial barrier in 2009 dietary fibers (DF) as “carbohydrate polymers with 10 (IEB) dysfunction. We will focus our review on the paracellular or more monomeric units which are not hydrolyzed by the endogenous enzymes in the small intestine of humans” (6). Abbreviations: AMPK, AMP-activated protein kinase; Arg, Arginine; CD, In terms of solubility, DF differ in their chemical properties Celiac Disease; ClC-2, Chloride channel type 2; CLDN, Claudin; CRF, (7, 8). Insoluble fibers mainly contribute to stool bulk whereas Corticotropin-releasing factor; CRF-R, CRF Receptor; DF, Dietary Fibers; soluble fibers are metabolized by the host microbiota, also DMM, Dexmedetomidine; DSCG, Disodium cromoglycate; DSS, Dextran contributing to maintain eubiosis (9). In fact, many of these sodium sulfate; EcN, Escherichia coli Nissle 1917; EV, extracellular vesicles; fibers can be considered as prebiotics because they are resistant FODMAP, Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols; GC, Glucocorticoids; GI, Gastrointestinal; Gln, Glutamine; GLP-2, to the acidic pH of the stomach, not hydrolyzed by mammalian Glucagon-like peptide 2; GLP-2R, Glucagon-like peptide 2 receptor; GOS, enzymes, not absorbed in the GI tract, but fermented by Galactooligosaccharide; GT, Gelatine tannate; IBD, Inflammatory Bowel Disease; intestinal microbiota, and selectively stimulate the growth and/or IBS, Irritable Bowel Syndrome; IBS-D, Irritable Bowel Syndrome with Diarrea; IE- activity of the intestinal microbiota, particularly Bifidobacteria IFG-1R, Epithelial insulin-like growth factor-1 receptor; IEB, Intestinal epithelial barrier; IFN-γ, Interferon gamma; Ig, Immunoglobulin; IL, Interleukin; LBP, and Lactobacilli (10, 11), to improve host’s health (12). This Lubiprostone; LPS, Lipopolysaccharide; LRH-1, Liver receptor homolog-1; MC, may be relevant as bacterial dysbiosis is highly associated Mast cell; MiRNA, Micro-RNA; MLC, Myosin light chain; MLCK, Myosin with intestinal barrier dysfunction and related pathologies such light chain kinase; NF-KB, Nuclear factor kappa B; NLRP3, NLR family as Inflammatory Bowel Disease (IBD) (13) (see Table 1). In pyrin domain containing 3; OCLN, Occludin; SCFA, Short-chain fatty acids; addition, the outer mucus layer is degraded to glycans by the TEER, Transepithelial electrical resistance; TJ, Tight Junction; TLR, Toll-Like Receptor; TNF-α, Tumor necrosis factor; UC, Ulcerative colitis; VDR, Vitamin glycan-consuming microbiota and glycans reused by bacteria in D receptor; Vit, Vitamin; XenomiRs, xeno-miRNAs; XG, Xyloglucan; Zn, Zinc; the absence of sufficient DF, as shown in a gnotobiotic mouse ZO, Zonula Occludens. model, what may lead to erosion of the colonic mucus barrier, Frontiers in Nutrition | www.frontiersin.org 2 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction FIGURE 1 | Intestinal barrier anatomy and its components in normal and impaired conditions. The intestinal mucosa comprises a layer of polarized, columnar epithelial cells next to a subepithelial region that contains the lamina propria, the enteric nervous system, connective tissue, and muscular layers. On top of columnar cells there is the mucus layer. Normal mucus is 98% water, being the rest glycosilated proteins (mucins) and glycolipids. In the colon, mucus has an outer layer, densely colonized by bacteria, fungus, virus, and able to retain toxins and allergens, and a mostly sterile inner layer where immunoglobulins (mostly secretory-IgA), and defensins such as lysozyme are present. The inner mucus layer is dense and attached an ∼50 µm thick and the outer layer is loose and unattached and about 100 µm thick. The small intestine has only one single mucus layer, which is much thinner than the mucus layer in the large intestine. The lamina propria includes a diffuse lymphoid tissue constituted by macrophages, dendritic cells, plasma cells, lamina propria lymphocytes, MCs, eosinophils and occasionally, neutrophils. (A) The intestinal barrier in homeostasis, where cells are closely attached by intercellular junctions (TJs, adherens junctions, desmosomes and GAP junctions) represented in detail in (A1). (B) Impaired intestinal barrier, with increased trans and paracellular passage of lumen contents. This increased transport activates the immune system and cell recruitment and degranulation in the lamina propria. MC and PC are able to modulate the ENS interacting with SMP/MP neurons and with EGCs. (A1) Representation of intercellular junctions. Intercellular junctions are primary responsible for nutrient absorption and water and chloride secretion. Intercellular junctional complexes, including TJs, adherens junctions, gap junctions, and desmosomes, are dynamic structures that restrict the passage of molecules: 4–5 Å at the villus tip to over 20 Å at the base of the crypt in the small bowel. The integrity and structure of epithelial cells are mostly modulated by the cytoskeleton, mainly by actin, myosin, and intermediate filaments. Cells adhere to the basement membrane through hemidesmosomes. TJs are primarily made up of CLDNs, OCLNs, and JAM proteins, which are connected through zonula occludens and cingulin to the cytoskeleton. Adherens junctions include cadherins such as E-cadherin, which binds catenins (α and β) connected to the cytoskeleton. Desmosomes are mainly comprised of desmocollin and desmoglein, which interact with desmoplakin, in turn connected to the intermediate filaments. AJ, Adherens junctions; BC, B Cell; CD4, Lymphocyte T helper CD4+ ; CLDN, Claudin; CM, Circular muscle; D, Desmosomes; DC, Dendritic cell; EGC, Enteric glial cell; ENS, Enteric nervous system; IL-13, Interleukin 13; JAM, Junctional adhesion molecule; LM, Longitudinal muscle; M0, Macrophages type 0; M1, Macrophages type 1; MC, Mast cell; MM, Muscularis mucosae; MP, Myenteric plexus; NK, Natural killer; NT, Neutrophil; OCLN, occludin; PC, Plasma cell; SMP, Submucous plexus; TJ, Tight junctions; TNF-α, Tumor necrosis factor alpha; Treg, T regulatory lymphocyte. promoting greater epithelial access, and lethal colitis by mucosal 1–6 carbons, being the most abundant acetate, propionate, and pathogens (38). butyrate (39). SCFAs show a wide range of biological functions These dietary compounds mainly include inulin-type fructans including anti-inflammatory responses, modulation of colonic (inulin, oligofructose, and fructooligosaccharides), galactans, contractility and maintenance of both mucosal immune cell galactooligosaccharides (GOS), and other heteropolysaccharides activity and integrity of the IEB, among others (see Table 2). such as chitosan, starch, alginate, pectin, or dextran, among SCFAs, specially at low concentrations, increased transepithelial others. These products have been shown to positively impact electrical resistance (TEER) in T84 and Caco-2 cells, what intestinal barrier function through different mechanisms immediately enhanced barrier function of the colonic epithelium after their fermentation by non-pathogenic colonic bacteria. through cholesterol-rich microdomain in the plasma membrane Fermentation of DF by gut microbiota releases short-chain fatty and decreased inulin permeability (42, 54–56). These effects acids (SCFAs). SCFAs are carboxylic acids with aliphatic tails of seem to be mediated by AMP-activated protein kinase (AMPK) Frontiers in Nutrition | www.frontiersin.org 3 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction FIGURE 2 | Dietary components involved in the regulation of intestinal permeability. Effects of minerals, vit, nutraceuticals, amino acids, soluble DFs (prebiotics), and SCFAs on the intestinal barrier. These dietary components are capable of affecting the microbiota by increasing α-diversity and the number of beneficial bacteria. DFs also enhance mucus secretion from the IEB and increase the expression of TJs proteins. In the lamina propria, dietary compounds increase T reg population, but also the production of anti-inflammatory cytokines restoring the Th1/Th2 balance. Fermentation of insoluble DF to SCFA (butyrate, acetate, and propionate) also increases the expression of SCFA receptors. DC, Dendritic cell; DF, Dietary fiber; IEB, Intestinal epithelial barrier; M0, Macrophage type 0; MC, Mast cell; NK, Natural Killer; Th1, T helper lymphocyte type 1; Th2, T helper lymphocyte type 2; Treg, T regulatory lymphocyte; SCFA, Short chain fatty acid; Vit, Vitamins. activity and the accelerated assembly of tight junction (TJ) barrier dysfunction (57). In this regard, a study performed proteins (43, 44). in intestinal epithelium cells IEC-6 showed that propionic In vitro studies have shown the ability of DF to attenuate acid inhibited NLRP3 inflammasome activation and preserved epithelial barrier dysfunction caused by bacterial infection (14, intestinal barrier function (48). 15) (Table 1). Similar in vitro studies with SCFAs (Table 2) The role of DF and SCFAs in modulating intestinal barrier indicate that main SCFAs, butyrate, propionate, and acetate, function and GI inflammation has been also tested in vivo in modulate contractile activity (40), to maintain the circadian several preclinical models and in multiple species (Tables 1, rhythm (47). SCFAs are also able to inhibit cytokine production 2). In this sense, sodium butyrate has been shown to revert (16, 49), activate Tregs (45), enhance IEB, by facilitating TJ colonic permeability in a rat model of Irritable Bowel Syndrome assembly via AMPK activation in Caco-2 cell monolayers (IBS) (50). In C57/BL6 mice submitted to chemotherapy- and through selective upregulation of claudin (CLDN) 3 induced mucositis, high fiber diet (pectin-based) decreased the and 4, and the activation of Akt/mTOR mediated protein influx of immune cells, improved histopathological parameters synthesis in IPEC-J2 cells (43), increasing TEER (41–44). and decreased intestinal permeability, compared to those that Interestingly, the activation of NLR family pyrin domain received the normal diet (58). In obese mice, prebiotics containing (NLRP) 3 inflammasome induces the secretion of exhibited lower plasma lipopolysaccharide (LPS) and cytokines, proinflammatory cytokines (46), which is linked to intestinal and lower intestinal permeability and improved TJ integrity Frontiers in Nutrition | www.frontiersin.org 4 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction TABLE 1 | Dietary fiber role in the recovery of impaired barrier function. Dietary fiber Sample Effect/Implicated mechanisms References In vitro β-galactomannan Salmonella enterica-infected • Reduction secretion of pro-inflammatory cytokine (14) Mannanoligosaccharide intestinal porcine cells IPI-2I Salmonella-induced Monosaccharide D-Mannose (ECACC 93100622) β-galactomannan S. enterica-infected Caco-2 cells • Prevention epithelial barrier function induced disruption induced by (15) the pathogen In vivo 5% Plantago ovata seeds HLA-B27 transgenic rats • Ameliorate development of colonic inflammation and (16) pro-inflammatory mediators • Improve the intestinal cytoarchitecture • Increase butyrate/propionate in intestinal content High-amylose corn starch TNBS-induced colitis male • Protection against colonic injuries. (17) Wistar rats • Improvement in the SCFA production. • Reduced colonic permeability. Dietary pectin IBD IL10−/+ mice • Reduced inflammatory response in colon (18) • Modulation of pro-inflammatory cytokines and Ig. Soluble fiber and corn starch IBD IL10−/+ mice • Amelioration of clinical disease and inflammatory ileal and colonic (19) lesions development. • Suppression of gut inflammation by Treg cells, IFN-γ, and colonic PPARγ expression Dietary cellulose DSS-induced colitis neonatal • Induction of colonic microbiome shifts. (20) C57BL/6J male mice • Improvement of intestinal inflammation. Psyllium fiber DSS-induced colitis ICR and • Amelioration or resolution of the colonic damage and inflammation. (21) BALB/c mice GG and PHGG Chronic kidney disease male • Restoration of colonic barrier. (22) induced ICR mice • Up-regulation of TJ. • Increase beneficial microflora composition. Plantago ovata Broiler chickens • Increase body weight and small intestine length. (23) • Reduction amount of E. coli. Grape peel powder Acute TNBS-induced colitis adult • Reduction of colitis. (24) male Wistar • Reestablishment intestinal barrier function. Lemon peel powder DSS-induced colitis male • Reduction of the intestinal damage. (25) BALB/c mice • Protection of TJ barriers. • Suppression inflammatory reaction. GOS Barrier damage LPS-induced • Attenuation of the intestinal barrier damage. (26) mice • Reduction of inflammatory responses in the jejunum and ileum. • Up-regulation of intestinal TJ. • Down-regulation of pro-inflammatory cytokines. Psyllium husk Chronic large-bowel diarrhea • Decrease defecation frequency. (27) induced dogs • Improvement stool consistency. • Weight gain promotion. AOS Weaned pigs • Increase TJ expression, cecal and colonic p-AMPKα, bacteria from (28) Bacteroidetes and Firmicutes phylum and fecal SCFA. • Decrease pro-inflammatory cytokines and NF-κB Sulfated polysaccharide BALB/c mice • Inhibit colon shortening and oedema forming (29) DSS-induced colitis • Down-regulation of TNF-α, IL-6, IL-1β • Up-regulation of CLDN-1, ZO-1, MUC-2 and SCFA receptors Clinical trials Plantago ovata seeds IBD patients (12 m) • Safe and effective. (30) • Maintenance UC remission. FOS Active CD patients (3w) • Increase bifidobacteria. (31) • Enhancement of DC IL-10 production. • Increase TLR expression in lamina propria. FOS Active CD patients (4w) • Not show clinical benefit. (32) • No significant differences in fecal concentration of bifidobacteria and F. Prausnitzii. Wheat bran CD patients (4w) • Consumption was feasible. (33) (Continued) Frontiers in Nutrition | www.frontiersin.org 5 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction TABLE 1 | Continued Dietary fiber Sample Effect/Implicated mechanisms References • No adverse effects. • Improvement health-related QoL and GI function. Low-FODMAP IBS-patients (3w) • Reduction GI symptoms. (34) Non-digestible polysaccharides Elderly population with GI • Reduction colonic hyperpermeability. (35) symptoms (biopsies, ex vivo) Controlled-fiber diet NAFLD patients (6 m) • Reduction serum biomarker of permeability. (36) • Positive influence in NAFLD-associated parameters. Low-FODMAP diet IBS-D patients (4w) • Low-FODMAP improved symptoms and QoL. (37) Traditional dietary advice AOS, Alginate oligosaccharide; CD, Celiac disease; DC, Dendritic cell; DSS, Dextran Sulfate Sodium; FODMAP, Fermentable oligosaccharides, disaccharides, monosaccharides, and polyols; FOS, Fructooligosaccharides; GG, Guar gum; GI, Gastrointestinal; GOS, Galactooligosaccharides; IBS, Irritable Bowel Syndrome; IBS-D, Diarrhea predominant Irritable Bowel Syndrome; IBD, Inflammatory Bowel Disease; IFN-γ, Interferon gamma; Ig, Immunoglobulin; IL, interleukin; m, Months; NAFLD, Non-alcoholic fatty liver disease; PHGG, Partially hydrolyzed GG; PPARγ, Peroxisome proliferator-activated receptor gamma; QoL, Quality of life; SCFA, Short-chain fatty acids; TEER, Transepithelial electrical resistance; TJ, Tight junction; TLR, Toll-like receptor; TNBF, Trinitrobenzene sulfonic acid; Treg, Regulatory T lymphocyte; UC, Ulcerative Colitis; w, Weeks. compared to controls (59). Dietary enrichment with psyllium reducing intestinal E. Coli in broiler chickens (23). Recent studies fiber (21), dietary cellulose (20), or lemon peel powder (25) also have shown that alginate oligosaccharide is able to increase TJ ameliorated colonic damage and inflammation and decreased expression, Bacteroidetes, and Firmicutes phylum bacteria and to TJ protein expression in the dextran sodium sulfate (DSS)- decrease pro-inflammatory cytokines in weaned pigs (28). induced colitis model in mice, particularly during the infancy. Regarding SCFA, in a rat model of irritable bowel syndrome Sulfated polysachharide not only reduced colonic inflammation, (IBS) (50), sodium butyrate has been shown to revert colonic but also but inhibited colon shortening and oedema in mice permeability. Also in this model, mixed or alone SCFA have been model (29). In this model, DF also ameliorated intestinal barrier reported improving IBS symptomatology (52). In neonatal IBS- dysfunction and inflammation (51). In specific pathogen-free and mice model, different concentrations of SCFA were able to reduce germ-free mice given DSS, psyllium, pectin, and cellulose fiber the colonic transit alteration in a dose-dependent manner (53). reduced the severity of colitis through microbiota-dependent and In humans, conclusions derived from dietary interventions microbiota-independent mechanisms, including restoration of with supplemental fiber have been often inconclusive and intestinal permeability (60). Similar studies in rats have disclosed weighted down by differences in the design and performance the ability of high-amylose cornstarch diet to protect against 2,4,6 of studies, as highlighted in a recent meta-analysis in IBD trinitrobenzene sulfonic acid (TNBS)-induced colonic injury, population (64). Thus, pectin supplementation (15/day) or and improve colonic permeability (17). In the same model, daily supplementation (12 g/day) with the DF β-glucan high rich DF containing grape peel powder also reduced colitis and wheat arabinoxylan did not affect baseline intestinal and reestablished intestinal barrier function in Wistar rats (24). barrier function in young and elderly healthy individuals Moreover, apple-derived pectin has been shown to modulate gut (65) or indomethacin-induced intestinal hyperpermeability in microbiota and CLDN-1 expression in obese rats submitted to vivo or gut microbiota composition in elderly, respectively high-fat diet, to attenuate metabolic endotoxemia, inflammation, (66). Furthermore, oligofructose-enriched inulin (8 g/day) did and weight gain (61). In other rat models, the addition of not improve intestinal permeability in children with diabetes cellulose fiber to elemental diet could ameliorate barrier failure mellitus (67) as did not either oligofructose (6 g/day) in in the ileum compared to total parenteral nutrition (62) and patients with burn injury (68). Plantago ovata seeds have been pectin supplementation significantly reversed the methotrexate- shown to maintain remission in UC (30), and FOS increased induced increase in permeability in the distal small bowel and bifidobacteria and IL-10 in CD patients (31), although it has colon (63). not been supported by further research (32). However, CD DF was able to restore colonic barrier integrity in a mice patients were shown to achieve an improvement in their model of chronic kidney disease (22) and GOS administration quality of life and GI function after wheat bran intake (33). attenuated intestinal barrier damage and inflammatory responses In other study, healthy male volunteers who ingested inulin induced by LPS in the jejunum and ileum of mice (26). In for 8 weeks (69), had significantly lower lactulose/mannitol other models, particularly in interleukin (IL)-10 knockout mice (L/M) ratio and serum zonulin and higher levels of mucosal with IBD, dietary pectin and cornstarch diets downregulated GLP-2. However, it is important to note that the methods the inflammatory response in colon, but its relation with the used were suboptimal. On the contrary, a non-digestible regulation of intestinal permeability was not established (18, 19). polysaccharide-enriched diet reduced colonic hyperpermeability DF, such as Psyllium husk, was able to decrease bowel induced by mast cell (MC) activation, as determined in movements, and improve stool consistency and weight gain in Ussing chambers, in elderly suffering constipation or diarrhea dogs (27). Moreover, Plantago ovata showed effectiveness in and elevated baseline colonic permeability (35). Similarly, in increasing body weight and small intestine length as well as in patients with non-alcoholic fatty liver disease, 6-months of Frontiers in Nutrition | www.frontiersin.org 6 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction TABLE 2 | Short-chain fatty acids role in the recovery of impaired barrier function. SCFAs Sample Effect/Implicated mechanisms References In vitro 10 or 100 mM mixed SCFA Ex vivo male Wistar rats • Modulation contractile activity. (40) 10 or 100 mM acetic, propionic or Cecum and colon butyric acids Butyrate and propionate THP-1 cells • Inhibition of TNF-α (16) Butyrate 2 mM [low] Caco-2 cells • [Low] promotion of intestinal barrier function, increase TEER and (41) Butyrate 8 mM [high] decrease inulin permeability. • [High] induce apoptosis, decrease cell viability. Mixed SCFA: acetate –propionate ex vivo colon Sprague-Dawley rats • TEER: increase by physiological SCFA mixture and individual SCFA (42) –butyrate (dose-dependent). 80:40:20, 40:20:10, and 20:10:5 (mmol/l) • Paracellular transport: dose-dependent reduced by mixed SCFA, acetate and propionate. 80 acetate, 40 propionate, and 20 Caco-2 cells • TEER increase with acetate (40 and 80 mmol/l) and propionate (20 butyrate (mmol/l) and 40 mmol/l). 80 acetate, 40 propionate, and 20 T84 cells • Acetate + propionate: increase TEER dose/time-dependent manner. butyrate (mmol/l) • Butyrate and formate do not change TEER. Formate, lactate and succinate (50 mmol/l) • Propionate, acetate and butyrate and lactate: TEER higher 30 min after (50 mmol/l). • Succinate reduces TEER. Butyrate 2 mM Caco-2 cells • Increase AMPK activity. (43) • Accelerated TJ assembly. • Increase TEER. Butyrate at 2, 5, or 8 mM Caco-2 cells • 2 mM: does not modify intestinal permeability. (44) SB203580: p38 MAPK inhibitor • 5- and 8-mM increase permeability. • 5 mM + SB203580 restore the permeability. Butyrate, propionate, and acetate YAMC and Caco-2 cells • SCFA triggers Aryl hydrocarbon receptor-responsive genes. (45) • AhR plays an important role in GI health and in the gut inflammation by the induction of Tregs. Acetate, propionate or butyrate with or • Caco-2 cells • Reduction NLRP3 inflammasome and autophagy. (46) without LPS • Decrease intestinal barrier disruption. Main fecal SCFA (acetate, propionate ex vivo C57BL/6J mice • Microbial SCFA are modulated by the circadian rhythm. (47) and butyrate) SCFA affects colon contractility. Propionic acid (PA) Intestinal epithelium cells (IEC-6) • Promotion of cell migration. (48) • Inhibition of NLRP3 inflammasome. • Activation and improvement of intestinal barrier function. • Suppression of TLR4/NF-κB pathway. SCFA produced by E. coli Cancer cell lines: colon (HT-29), • Lower cytotoxicity activity. (49) breast (MCF-7) and leukemia (THP-1) • Decrease production of inflammatory cytokines. In vivo Sodium butyrate Wistar rat • Dose-dependent inhibition of allodynia and (50) IBS model (WAS) colonic hyperpermeability. GG and PHGG DSS-induced colitis BALB/c mice • Improved clinical score. (51) • Up-regulation of colonic TJ. • High fecal SCFA Mixed SCFA: Sodium acetate (3 mM), Sprague-Dawley rat • SCFA alleviated colonic spontaneous motility (52) Sodium propionate (1 mM), Sodium IBS model (WAS) • Fecal SCFA reduction in WAS butyrate (1 mM) • Up-regulate SCFA colonic receptors in WAS. Acetate, propionate, and butyrate at 0.5, Neonatal BALB/c mice-IBS model • Dose-dependent reduction colonic transit rate. (53) 1, 5, 10, 30 mM rectal 1% acetic ac. AhR, Aryl hydrocarbon receptor; AOS, Alginate oligosaccharide; CLDN, Claudin; DSS, Dextran Sulfate Sodium; GG, Guar gum; IBS, Irritable Bowel Syndrome; IL, Interleukin; LPS, Lipopolysaccharide; MUC, Mucin; NF-κB, Nuclear factor – kappa beta; NLRP3, NLR family pyrin domain containing 3; p-AMPKα, Phosphorylated AMP-activated protein kinase alpha; PHGG, Partially hydrolyzed GG; SCFA, short-chain fatty acid; TEER, Transepithelial electrical resistance; TJ, Tight junction; TLR, Toll-like receptor; TNF-α, Tumor necrosis factor alpha; Treg, Regulatory T lymphocyte; WAS, Water avoidance stress; YAMC, Young adult mouse colonic cells; ZO, Zonula occludens. fiber intervention demonstrated a reduction in zonulin levels, in obese patients independently of its prebiotic effect (70). a purported serum biomarker of permeability (36), and GOS The combination of green banana and pectin showed good supplementation reduced aspirin-enhanced colonic permeability antidiarrheal properties in children with persistent diarrhea, Frontiers in Nutrition | www.frontiersin.org 7 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction activity that was linked to the reduction in small intestinal 1,25-Dihydroxyvitamin D3 -active form of vit D- restored the permeability (71). expression and localization of TJ proteins and reverted LPS- SCFAs are also able to modulate intestinal permeability induced down-regulated VDR expression (86). Likely, intestinal in humans. Indeed, decrease in gut-derived plasma SCFAs damage caused by LPS in IEC-18 line cells and organoids was correlated with increased colonic permeability in shift workers improved after vit D treatment, restoring permeability and TJ (72) and organoid studies based on human colonic mucosal (87). Similar findings have been reported in a model of alcoholic biopsies showed that fermentation of 2′ Ofucosyllactose which liver disease in Caco-2 challenged with ethanol (88). Moreover, led to an increase of Bifidobacteria and an increase of vit D deficiency may compromise mucosal barrier integrity, SCFAs, in particular butyrate, resulted in CLDN-5 significant raising susceptibility to develop IBD, as also shown in Caco-2 upregulation (73). cells (89). A diet low in fermentable oligosaccharides, disaccharides, In specific-pathogen-free rats, the deficiency of vit A monosaccharides, and polyols (FODMAP) is commonly used aggravates the severity of diarrhea and intestinal mucosal in the management of patients with IBS and overall, 52–86% damage. On the contrary, during the clinical course of diarrhea, of patients report significant improvement of their symptoms supplementation with vit A relieves diarrhea and improves (34, 37, 74). Moreover, this diet was more effective than intestinal damage, increasing the expression of TJ proteins (90). others (traditional dietary advice, modified National Institute When intestinal epithelial cells from VDR-deficient mice are for Health and Care Excellence guidelines, gluten-free diet complemented with a human VDR-encoding trans-gene, the and Mediterranean diet and a sham diet) and also non- integrity of the mucosal barrier prevents the hyperinflammatory dietary interventions (gut directed hypnotherapy or yoga) response that is otherwise seen in the lamina propria immune (75). Interestingly, this diet improved intestinal permeability cells of VDR-deficient mice (91). in patients with diarrhea-predominant IBS in relation with In humans, several reports indicate insufficient levels of vit increased circulating vitamin (vit) D levels (76). However, to date, D in many inflammatory conditions, including IBD (92, 93) there is no further demonstration of how low-FODMAP diet may and IBS (94), with more than 50% of patients affected by interfere with intestinal permeability. hypovitaminosis (95, 96). Vit D deficit has been also related Recently, a combination of herbs and nutrients including with clinical symptoms and quality of life, but the correlation curcumin, aloe vera, slippery elm, guar gum, pectin, peppermint between the intestinal expression of VDR and CLDN2 remains oil, and glutamine (Gln) significantly improved the frequency controversial (97–100). Patients with celiac disease (CD) in and severity of upper and lower GI symptoms by 60–80% in a remission received a supplementation of vit D -or placebo- small sample sized study. This improvement was accompanied during 3 months. The supplemented patients showed higher by reduction of intestinal permeability, as measured by lactulose- plasma levels of vit D, improved the results of quality-of-life mannitol ratios, and by beneficial changes in microbiota and kept intestinal permeability as it was at baseline, whereas composition (77). permeability increased in the placebo group (101). As previously Other dietary factors such as the non-sugar prebiotics mentioned, a recent study assessed the relation between vit D soy protein hydrolysates have shown promising effects to levels, intestinal permeability, and a 12-week intervention with strengthen the epithelial barrier in response to several barrier low-FODMAP diet in IBS with diarrhea (IBS-D). Those patients disruptors (78). with low vit D levels before the intervention, reduced small bowel permeability, increased vit D levels and also improved clinical Vitamins symptoms (76). Vit A, and vit D are micronutrients involved in the regulation of TJ molecule expression in the intestinal barrier (79) and mucosal immune system, shaping the microbial populations in the gut Amino Acids (80, 81). Both epithelial and immune cells in the GI tract, but not The pore pathway regulates, through IL-13-mediated expression the microbiota, express receptors for vit A (retinoic acid receptor) of CLDN-2, the selective paracellular transport of smalI-sized and vit D (vit D receptor, VDR) (4). VDR protects against (5–10 Å) ions (K+, Na+) and molecules (water) (102, 103). mucosal inflammation in experimental colitis and contributes In vitro studies in Caco-2 cells with deprived from Gln or after to systemic bile acid homeostasis by regulating expression of Gln synthetase inhibition, reported reduced TEER, increased fibroblast growth factor (82). Retinoic acid receptor enhances permeability and lower TJ protein expression (104–106) which Zonula Occludens (ZO)-2 expression by regulating Toll-Like could be reestablished after Gln addition. Gln, and to a lesser Receptor (TLR)-4 to improve IEB function in Caco-2 cells, as well extent arginine (Arg), also prevented methotrexate-induced as in rat and mouse models, but not in humans (83). barrier disruption in Caco-2 cells (107). Gln improved intestinal The presence of vit D increased TEER and preserved the barrier function in a rat model of biliary obstruction (108), structural integrity of the TJ in Caco-2 cells treated with and Gln and Arg prevented the mucosal injury in a model of DSS (84). In a model of intestinal barrier permeability using ischemia-reperfusion in rats (109, 110). Gln regulated TJ integrity IPEC-J2 cells, vit A reverted LPS-induced barrier dysfunction and distribution through calcium/calmodulin-dependent kinase through the enhancement of TEER and TJ protein expression 2 (CaMKK2)-AMP-activated protein kinase signaling in porcine (85). In Caco-2 cells treated with LPS, emulating the barrier epithelial cells (111). Recently, Gln alleviated IL-13-induced damage of necrotizing enterocolitis (NEC), the presence of barrier dysfunction by increasing CLDN-1 expression, via Frontiers in Nutrition | www.frontiersin.org 8 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction disruption of the phosphatidylinositol-3-kinase/Akt signaling ZO-1 proteins in Caco-2 cells (125). Depletion of Zn induced pathway (112). OCLN-3 proteolysis and decreased CLDN-3 transcription In humans, microRNA (miR)-29 has been shown to (126) while Zn supplementation increased TEER and ZO-1 regulate Gln synthetase, CLDN-1 expression, nuclear factor expression and decreased CLDN-2 and CLDN-7 expression kappa-light-chain enhancer of activated B cells (NF-κB) and (127, 128), facilitating OCLN and ZO-1 expression in Caco2 ultimately tumor necrosis factor (TNF-α), to regulate the and HT29 cells (90). In mice with bacterial infections, Zn leak paracellular pathway in a series of elegant experiments supplementation enhanced protection against toxin-induced performed in colonic tissues of IBS-D patients (113). Moreover, intestinal dysfunction and leakage (129). The ZRT/IRT-like in a subsequent randomized, placebo-controlled trial, the same protein 14, Zn transporter is expressed on plasma membranes authors showed that supplemental Gln (10 g/day) improved and mediates Zn influx into the cytosol. Mice lacking intestinal permeability and major symptoms in post-infectious ZRT/IRT-like protein 14 display increased intestinal permeability IBS-D patients (114). In addition, although disputed, enteral associated with altered expression of CLDN-1 and CLDN-2 Gln supplementation has been shown to improve intestinal (130). Other studies have shown that Cu and Zn supplementation permeability in severely thermally injured patients (115). A improved intestinal integrity during the Eimeria spp. Infection small randomized trial also showed that Gln and whey protein in broilers (131). Selenium has also been proposed as a good improved small intestinal permeability and morphology in candidate to prevent changes in intestinal permeability and patients with Crohn’s disease (116). mitochondrial damage in several species (132, 133). In humans, Arg is a semi-essential amino acid that can be metabolized by Zn supplementation is effective in the prevention of diarrhea host arginases and nitric oxide synthases or be consumed by gut (134), and has been recommended by The World Health bacteria (4). There are few studies related to Arg and protection Organization for the treatment of diarrhea (135). Zn also has a of the integrity of the epithelial barrier. In heat-stress conditions, beneficial effect on infectious diseases like shigellosis improving pre-treatment with L-Arg partly reverted the decrease on TEER IEB, nitrogen absorption, and symptoms (135–137). Finally, and increased paracellular permeability (117). In a model of zinc carnosine, a health food supplement, stabilizes small bowel hypoxia in jejunal IPEC-J2 cells, Arg prevented the reduction integrity and stimulates gut repair processes after indomethacin of TEER and increased inulin paracellular permeability (118). A treatment, as shown in a placebo-controlled trial (138). great compilation of the last 30 years of clinical trials performed with Gln and Arg is also available (119), highlighting a reduction Microbiota-Based Factors of the infection rate and mortality by Gln and a decrease of Microbiota exerts many crucial functions (thoroughly reviewed complications by Arg in surgical patients. Nevertheless, some of in other papers in this monography) including IEB maintenance these trials have methodologic flaws and many do not evaluate (139, 140). intestinal permeability. Hence, further and well-designed trials are needed for justifying the use of these amino acids. Antibiotics Tryptophan is an essential amino acid also studied by its Antibiotics are recommended to treat bacterial infections. potential link between imbalanced gut microbiota, impairment Independently of the origin of the infection, antibiotic of intestinal immunity and disease development. Recent evidence administration has adverse effects on the gut indigenous underlines that the enzyme indoleamine 2,3-dioxygenase 1 microbial community leading to mid to long term dysbiosis expressed by the host is relevant to generate indole metabolites (141) and mycobyosis (142), with some compositional effects (120), which are involved in the re-establishment of IEB lasting for 6 months (143), to ease colonization by pathogens such integrity in the context of intestinal inflammatory diseases and as Salmonella or Cl. Difficile, and to increase antibiotic resistance metabolic syndrome. (144, 145). In addition, many antibiotics induce changes in A recent review on amino acid supplementation in weaned intestinal permeability that may be linked to alterations in piglets, disclosed that several of them (Arg, Gln, tryptophan, α-diversity and relative abundance of specific bacteria within sulfur-containing amino acids, and branched-chain amino acids) the gut microbiota as shown in rats (146). Moreover, changes in may have a role in the maintenance and improvement of intestinal permeability are accompanied by reduction of SCFAs, intestinal morphology and function, increasing proliferation of and increased activity of NLRP3 inflammasome and autophagy epithelial cells and preserving intestinal mucosal integrity (121). (147). Therefore, the use of some antibiotics in disorders In mice, radiation-induced intestinal barrier disruption was associated with barrier dysfunction may lead to additional ameliorated by an amino acid-based oral rehydration solution, complications, though these findings should be translated to enhancing TJ protein expression and improving paracellular the clinic. In addition, bioavailability of antibiotics seems to permeability (122). depend also on the composition of microbiota and on intestinal permeability as well (148). Minerals However, some antibiotics may have a better profile for the Zinc (Zn) is an essential trace element [10% of the human microbiota. Rifaximin is a poorly-absorbed broad spectrum oral genome encodes Zn-binding proteins (123) that plays an antibiotic prescribed for GI disorders such as IBS, IBD, small important role in diarrheal diseases and GI infections and intestine bacterial overgrowth, traveler’s diarrhea or diverticular it is closely linked to mucosal integrity and IEB (124). Zn disease (DD) (149, 150). Rifaximin seems to exert eubiotic effects deficiency leads to reduced expression of occludin (OCLN) and on the microbiota, increasing Bifidobacterium, Faecalibacterium Frontiers in Nutrition | www.frontiersin.org 9 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction prausnitzii, and Lactobacillus abundance, with no major change before DSS-colitis induction improved intestinal barrier integrity in the overall gut microbiota composition, what may represent involving transcriptional modulations of TJ genes (ZO-1, OCLN, a therapeutic advantage in specific clinical settings (150, 151). In CLDN-1) (167). addition, Xu et al. (152), showed how oral rifaximin prevented Stress clearly affects intestinal barrier function and mucosal inflammation, impairment to intestinal barrier function, probiotics have been shown to prevent some of changes. and visceral hyperalgesia by altering the composition of bacterial Weissella paramesenteroides WpK4 ingestion reduced intestinal communities in the ileum while other antibiotics were not permeability and reduced anxiety-like and depressive-like as effective. behaviors in stressed mice submitted to DSS (168). Lactobacillus farciminis prevented stress-induced gut hyperpermeability Probiotics and mucus alterations in different animal models (169, 170). Probiotics are live microorganisms which, when consumed in In rats subjected to partial restraint stress fermented milk adequate amounts, confer a health benefit on the host (153). containing Bifidobacterium lactis CNCM I-2494 prevented This benefit relates in part to the ability of probiotics to stress-induced increase in intestinal permeability and restored modulate the IEB. A large amount of evidence has accumulated OCLN and JAM-A expressions to control levels (171). More to support the efficacy of probiotics to enhance IEB tightness recently, Wang et al. have shown that Lactobacillus casei and integrity, and to modulate intestinal inflammation (154). Zhang significantly increased jejunum villus height, villus We will only review here the most representative evidence. height-crypt depth ratio, muscle thickness, and expression For instance, Bifidobacterium was able to adhere to mucus, to of proliferating cell nuclear antigen and TJ proteins ZO-1 inhibit and displace the adhesion of pathogenic bacteria (155) and OCLN in early-weaned piglets, and prevented E. coli and to increase TJ integrity, protecting them from Escherichia K88-induced jejunum damage (172). Similarly, Lactobacillus coli O157:H7 (156). Incubation of T84 cell monolayers fermentum CECT 5716 prevented maternal separation and with multispecies probiotic completely prevented LPS-induced WAS-induced intestinal barrier dysfunction in newborn rats, increase in paracellular permeability in a dose dependent reducing small intestine permeability and increasing ZO-1 manner. This multispecies probiotic also prevented the epithelial expression (173). disruption induced either by intracolonic infusion of fecal In humans, Lactobacillus plantarum WCFS1 administration supernatant from IBS patients or by water avoidance stress into the duodenum was associated with an increase in of (WAS) in C57/Bl6 mice. In addition, these formula increased ZO-1 and OCLN in healthy subjects (174). In contrast, the expression of OCLN and decreased TNF-α secretion in Lactobacillus GG significantly reduced the alteration of gastric response to LPS (157). Similarly, Lactobacillus rhamnosus CNCM (but not intestinal permeability induced by indomethacin I-3690 prevented changes in intestinal permeability in Caco-2 administration in healthy subjects suggesting that probiotics are cells stimulated with TNF-α and in a mouse model of increased useful to enhance barrier function possibly in a location-specific colonic permeability, to a similar degree that Faecalibacterium manner (175). prauznitzii A2-165 in the last (158). In a post-infectious IBS Bifidobacterium lactis CNCM I-3446 induced a significant mouse model, probiotic treatment promoted the expression of decrease of intestinal permeability in infants with NEC major TJ proteins CLDN-1 and OCLN in the mouse ileon (176). In IBD patients, particularly in those with severe (159). Similarly, Lactobacillus rhamnosus GG improves intestinal pouchitis, administration of a probiotic combination effectively barrier function in the immature murine gut through the prevented flare-ups (177), combination that has been shown induction of CLDN 3 expression (160). In obese and type2 to promote recovery from IFN-γ-induced intestinal barrier DM mice, Akkermansia. muciniphila treatment increased the dysfunction (178). intestinal levels of endocannabinoids that control inflammation Recently, beneficial effects of probiotics have also been and gut barrier (161). shown to occur through the release of extracellular vesicles Probiotics can also prevent intestinal barrier damage in IBD (EV). EV contain a vast number of active compounds conditions. Both LGG and a probiotic formulation containing and bacterial mediators that play a key role in bacteria- Lactobacillus acidophilus, Bifidobacterium lactis, Lactobacillus host interactions, but also between probiotics and other plantarum, and Bifidobacterium breve reduced the disruption bacteria. In vitro, pretreatment with Akkermansia muciniphila- of barrier function in DSS-induced colitis in mice (162, 163). derived EV decreased IL-6 production from colon epithelial In a similar way, the administration of a probiotic mixture cells stimulated by pathogenic Escherichia coli EV (179). prevented not only the decrease in TJ proteins expression, Akkermansia muciniphila-derived EV (179) also protected from but also the increase of epithelial apoptotic ratio induced by DSS-induced colitis in C57BL/6 mice. Moreover, in high-fat diet- acute colitis (164). Oral Bifidobacterium infantis conditioned induced diabetic mice, Akkermansia muciniphila-derived EV medium administration reduced colonic permeability in IL-10- administration enhanced tight junction function, reduced body deficient mice in part through enhanced protein expression weight gain and improve glucose tolerance in association with of CLDN-4, ZO-1, and OCLN, and decreased expression of an increase in the expression of occludin, zonula occludens, and CLDN-2 (165). Similarly, Escherichia coli Nissle 1917 has been claudin-5 (180). In fact, in the same study, more Akkermansia shown to inhibit leaky gut by enhancing mucosal integrity muciniphila-derived EV were found in the fecal samples of through up-regulation of ZO-1 expression in murine DSS colitis healthy controls when compared with type 2 diabetes patients (166). Lactobacillus rhamnosus MTCC-5897 administration and Akkermansia muciniphila-derived EV treatment improved Frontiers in Nutrition | www.frontiersin.org 10 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction intestinal permeability in LPS-treated Caco-2 cells, by increasing Bioactive Pharmaceutical Molecules and the expression of occludin. Signaling Peptide-Based Therapeutic Vesicles secreted by Bacteroides fragilis have been Strategies shown to contain capsular polysaccharide, which induces Glucagon-Like Peptide 2 immunomodulatory effects on dendritic cells and prevents Glucagon-like peptide 2 (GLP-2) (Figure 3) is an intestinal experimental colitis through TLR2-signaling pathways peptide derived from proglucagon that exerts its function (181). In Caco2 cells, B. fragilis EV significantly decreased through the GLP-2 receptor (GLP-2R), expressed predominantly TLR2 and slightly increased TLR4 mRNA levels along in the intestinal tract (191, 192). Endogenous GLP-2 promotes with an increase in anti-inflammatory cytokines and intestinal growth after a fasting period or in response to enteritis the inhibition of interferon (IFN)-γ (182). Stimulation (193). Exogenous GLP-2 is exerts profound effects expanding of bone marrow derived dendritic cells (BMDCs) with the crypt-villus epithelium through enhanced proliferation and EV originated from another Gram-negative probiotic, survival, to increase nutrient digestion, absorption, and blood Bacteroides vulgatus mpk, contributes to immune response flow (192, 194, 195). silencing through induction of a tolerant BMDCs GLP-2 also improves intestinal barrier function in both phenotype (183). health conditions and disease models (59, 196, 197), reducing Escherichia coli Nissle 1917 (EcN) has also been shown to paracellular transport of ions and small molecules, and restore barrier function in experimental models of increased dramatically inhibiting endocytic macromolecules uptake gut barrier permeability. Prophylactic administration of EcN in mice (197). GLP-2 chronic administration enhances gut resulted in reduced inflammation, and preservation of intestinal barrier function and decreases epithelial barrier permeability. permeability in a DSS murine model of colitis (184). EcN In fact, GLP2 in mice model decreases the transcellular treatment significantly upregulated the colonic expression of passage of ions, 51Cr-EDTA and fluorescein-isothiocyanate the tight junction proteins ZO-1 and occludin, preserving as well as the endocytosis of horseradish peroxidase (HRP), the mucus- layer and restoring intestinal permeability. a marker of transcellular permeability (198–200). Following Oral administration of purified EcN EV before DSS intake, studies have demonstrated that this ability is GLP-2R- significantly reduced clinical symptoms and histological scores dependent, in association with increased TJ expression, in a DSS-induced colitis mouse model (185). Similarly, in most notably CLDN-3 and−7 (196, 199). In addition, rats colonic cell lines, EcN EV promoted upregulation of ZO-1 receiving subcutaneous exogenous GLP-2 exhibited less and claudin-14, and induced IL-22 expression reinforcing the intestinal structural damage, longer intestinal villi, and increased intestinal barrier (186). immunoglobulin (Ig)A expression, in a model of obstructive However, few studies have specifically addressed intestinal jaundice (201). permeability in vivo in humans. One study assessed the It has been widely reported the GLP-2 effect on increasing efficacy of a probiotic mix in intestinal permeability, immune microvillus length, however, how this is achieved is poorly function and in the prevention of multiple organ dysfunction understood. Recently, Markovic et al. (202) demonstrated that syndrome in critically ill patients. They found that patients the increase in microvillus length with GLP-2 treatment requires responded with a significantly larger increase in systemic the intestinal epithelial insulin-like growth factor-1 receptor (IE- IgA and IgG concentrations and in most of them, intestinal IGF-1R) in mice. Villin, an actin-binding protein, is regulated permeability decreased, compared to placebo or sonicates (187). by the GLP-2-IE-IGF-1R pathway. Villin has a well-established Mujagic et al. evaluated the effects of Lactobacillus plantarum role in epithelial wound repair, with both insulin growth factor on small intestinal barrier function through the lactulose- 1 and villin levels decreased in Crohn’s disease (203, 204). rhamnose ratio after intake of indomethacin, but there was These findings suggest a new mechanism by which GLP- no significant effect. However, in small intestinal biopsies, L. 2 may attenuate Crohn’s disease and/or other inflammatory plantarum TIFN101 modulated gene transcription pathways pathologies. These results are consistent with previous research related to cell-cell adhesion with high turnover of genes which has already shown the effects of GLP-2 through the IE- involved in tight- and adhesion junction protein synthesis and IGF-1R modulating intestinal TJ proteins (199). In this regard, degradation (188). A recent meta-analysis, evaluated the effect of a study in pediatric patients with acute ileal CD showed that probiotics/synbiotics on serum levels of zonulin, as a measure of these patients have decreased post-prandial GLP-2 release, and intestinal permeability, showing favorable effects although results increased intestinal permeability. Healing of CD was associated should be interpreted with caution due to high heterogeneity with the normalization of post-prandial GLP-2 release and (189). Another recent meta-analysis highlights also the potential intestinal permeability (205). More recently, an abnormal post- beneficial role of probiotics in GI mucositis and the reduction of prandial glucagon-like peptide 2 release has also been described intestinal permeability and maintenance of the mucus layer (190). in adult patients with Crohn’s disease (206). Taken together, all these data suggest that probiotics enhance Finally, GLP-2 analog teduglutide has been successfully intestinal barrier tightness and integrity by several mechanisms, introduced in clinical practice as a new treatment for parenteral and that mucosal restoration positively impacts the clinical nutrition-dependent short bowel syndrome (207). It can course of disease. However, specific studies measuring intestinal effectively increase the residual intestinal absorption capacity permeability through a validated method are needed to achieve through the induction of intestinal mucosa hypertrophy and more robust conclusions. Frontiers in Nutrition | www.frontiersin.org 11 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction FIGURE 3 | Molecules intended to regulate intestinal permeability and/or mucosal inflammation. Representation of a transversal section of the small intestine, including epithelial and goblet cells. The mucus layer and the lumen are found inside; the lamina propria is outside. (A) Dexmedetomidine reduces epithelial cell death, TJ damage and inflammation. (B) Larazotide is a zonulin antagonist, able to bind zonulin receptor and block its toxic effects. (C) Mucoprotectants cover the epithelial cells surface forming a film barrier that helps to preserve intestinal permeability. (D) CRF antagonist binds to CRF receptors blocking the binding of stress-released CRF and decreasing subsequent proinflammatory responses. (E) MC stabilizers are responsible for MC cell membrane stabilization inhibiting degranulation. (F) Lubiprostone increases water flux to the intestinal lumen and bowel transit, enhancing the expression of TJ proteins. (G) Glucocorticoids inhibit the activation of the immune system via transcription of anti-inflammatory genes. (H) miRNA exert different roles within the epithelial barrier regulation, being able to enhance the intestinal permeability or reducing inflammation (I) Anti-IL-13 treatment inhibits IL-13 effects in the IEB, increasing cell survival and decreasing the expression of the pore-forming TJ claudin 2. (J) Anti-TNF-α increases cell survival and TJ expression while decreases claudin 2 expression through TNF-α antagonism. (K) Anti-INF-γ treatment inhibits INF-γ effects in the IEB, increasing the expression of TJs. (L) GLP-2 binds to GLP-2R, predominantly expressed in the intestinal tract, resulting in an increase of cell survival and villi length, restoring Th1/Th2 balance. CRF, Corticotropin-releasing factor; CRFR, Corticotropin releasing factor receptor; GC, Glucocorticoids; GLP-2, Glucagon-like peptide 2; GLP-2R, Glucagon-like peptide 2 receptor; IEB, Intestinal epithelial barrier; IL-13, Interleukin 13; INF-y, Interferon gamma; LBP, Lubiprostone; MC, Mast cell; TJ, Tight junction; TNF-α, Tumor necrosis factor alpha. hyperplasia, the increase of intestinal perfusion and the reduction therapy in severely active CD since its fluctuating inflammatory of intestinal motility and gastric acid secretion, achieving a activity can be considered at greater risk (213). reduction of parenteral nutrition (208, 209). Several cases of All these findings support GLP-2 treatment as a possible active Crohn’s disease and short bowel syndrome successfully effective therapy for enhancing, maintaining, or recovering treated with teduglutide have been recently described (210–212). normal barrier function in intestinal disorders. However, to Yet, there are no systematic data about off-label teduglutide date, human studies evaluating the effect of GLP-2 on intestinal Frontiers in Nutrition | www.frontiersin.org 12 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction permeability do not exist. Nevertheless, teduglutide must be used translated in clinical practice for the management of stress- with caution and discontinued in case of intestinal neoplasia induced IBS. because of its effect on intestinal epithelial proliferation. Corticotropin-Releasing Factor Humanized Antibodies Against Tumor Necrosis Corticotropin-releasing factor (CRF) is a signaling peptide Factor-α (Figure 3), secreted both in the central nervous system and in the Tumor necrosis factor-α (TNF-α) (Figure 3) and myosin- periphery, including the GI tract, which stimulates the secretion light chain kinase (MLCK) are the main regulators of the of adrenocorticotropic hormone from the pituitary gland in leak paracellular pathway (102). TNF-α is a multifunctional response to stress. CRF and related molecules such as urocortins pro-inflammatory cytokine that has wide effects on cells 1, 2, and 3 have been extensively involved in the regulation of and structures related to the intestinal barrier function. stress-mediated motor, sensory and permeability changes in the One of the barrier-deteriorating effects likely comes from GI tract, in animal models and humans, acting via the G-Protein TNF-α’s ability to induce apoptosis (241). However, TNF- coupled CRF receptors (CRF-R) 1 and CRF-R2 (214–217). α was found to deteriorate paracellular integrity even in Many authors have described the effects of stress on the presence of an apoptosis blocker, suggesting additional gut permeability. Studies in rats and pigs have shown mechanisms involved. Indeed, in vitro studies found that that CRF-induced changes over the barrier function were TNF-α modulates TJ structure by breaking strands of ZO- equivalent to those triggered by stress. Among other alterations, 1 and thus modifying the structure of the epithelial barrier CRF induces mucus layer thickening, enhanced conductance (242). TNF-α stimulation could also increase permeability by and transepithelial and paracellular macromolecular flux, TJ inducing the expression of the pore-forming TJ protein CLDN- reorganization and activation of the immune system in the small 2 (243). There is also evidence for TNF-α increasing paracellular intestine and colon (218–224). Consistent evidence indicates that permeability by activating long MLCK transcription, expression, many of these mechanisms are predominantly driven by the enzymatic activity, and recruitment to the actomyosin ring activation and degranulation of MCs (221, 223–225), although (244–247). MLCK activation triggers perijunctional actomyosin recent studies lay stress on eosinophils as potential contributors ring contraction that leads to molecular reorganization of TJ to the stress-mediated gut dysfunction, specifically in IBS-D structure, including OCLN endocytosis. patients (226). Opposite effects of CRF-R1 and CRF-R2 are The use of monoclonal antibodies against TNF-α has shown observed on stress-mediated intestinal mucosal barrier function mixed results, some proven successful in inducing remission in pigs, with CRF-R2 preventing permeability changes and CRF- in cases of IBD (248), but also at the cost of adverse events R1 enhancing them (227). and high number of non-responders over time (249, 250). Interestingly, chronic stress has shown how barrier Few but promising evidence suggest targeting TNF-α can impairment could be persistent if the stressor is repeated improve specific parts of the intestinal barrier function and (222, 228–230). Vicario et al. reported increased gut epithelial endoscopic signs on the mucosal tissue, collectively termed permeability, hyperactivation of the hypothalamic-pituitary- mucosal healing (251). adrenal axis and reversible inflammation in rats submitted to Adalimumab is a monoclonal antibody against TNF-α shown a repeated stress or CRF, developing visceral hypersensitivity to improve both IEB and clinical features in IBD patients (252– afterwards (230). Similarly, CRF and sauvagine, a stress-like 256). Human colonic cell culture showed that simultaneous peptide, enhanced intestinal ion, and macromolecular flux, use of both TNF-α and interferon (IFN-γ) disrupted the which could be inhibited by astressin, a potent non-specific CRF epithelial barrier, leading to a significant drop in TEER (257), inhibitor, and doxantrazole, a MC stabilizer. The alterations appearance of irregularities in the TJ structures, disruption of intestinal permeability evoked by various stressors or CRF of OCLN and increase phosphorylation of MLC. All of these are inhibited by peptide CRF receptor antagonists and selective effects were reversed upon administration of Adalimumab. When CRF-R1 antagonists (219, 221–224, 226, 228–232) [for further subjecting a 3D Caco-2 cell model to plasma from patients with review, see Taché et al. (217)]. Moreover, Nozu et al. have recently active Crohn’s disease, paracellular permeability increased via reported in a rat IBS model that imipramine dose-dependently breakdown of ZO-1 and OCLN (258). inhibited visceral hypersensitivity, colonic hyperpermeability, Infliximab, a chimeric monoclonal antibody against both and other GI effects of CRF or repeated stress through membrane bound and soluble TNF-α, has shown successful α2-adrenoceptors, dopamine and opioid receptors (233). results in both patients with Crohn’s disease and UC (256, 259– In healthy humans and IBS patients, functional studies 261). Crohn’s disease patients display an increased baseline also demonstrated that peripheral CRF largely reproduces the permeability compared to healthy controls, that was normalized increased colonic motility, intestinal permeability, MC activation after 7-days course of infliximab, for a final 10-fold decrease of and visceral hypersensitivity observed in animals (224, 234). the lactulose/mannitol ratio. The effect of Infliximab on barrier Changes in intestinal and colonic permeability were mediated by function was also investigated by mounting non-inflamed colonic MC activation and reversed by disodium cromoglycate, another biopsies from Crohn’s disease patients in Ussing chambers and MC stabilizer (235). Nonetheless, despite raising high expectation in Caco-2 cells. The results showed a significant decrease in early on, and several clinical assays performed with several CRF paracellular permeability and normalization of transmucosal antagonists (236–240), unfortunately, to date, this has not been permeability to near control levels for adherent invasive Frontiers in Nutrition | www.frontiersin.org 13 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction Escherichia coli (262). Efficacy of infliximab has not been well- natural killer cells (277) (Figure 3). The effect of IL-13 on barrier studied for other conditions characterized by intestinal barrier function has not been widely studied but in vitro experiments dysfunction. However, anti-TNF-α therapy is a common rescue using colonic epithelial cell lines have shown upregulation of the medication for diarrheal conditions refractory to steroid therapy, pore-forming TJ protein CLDN-2 together with an increase in including immune-related diarrhea after immune checkpoint paracellular permeability (278, 279). Activation of MLCK can inhibitor therapy (263) or microscopic colitis (264). A case-study lead to an increased production of mucosal IL-13 together with of a refractory CD patient showed improvement in symptoms an upregulation of CLDN-2 in mice (279). Further on, IL-13 also and intestinal histology after Infliximab treatment, suggesting a shares with TNF-α the ability of inducing epithelial apoptosis and possible effect on the barrier function (265). this effect can be enhanced by the stimulation of both cytokines Inhibition of MLCK expression or enzymatic activity results simultaneously, hinting at a possible synergistic effect (280). in systemic toxicity making these molecules unsuitable as An increased expression of IL-13 has been found in the lamina therapeutic targets for barrier control. However, recently a new propria mononuclear cells from Crohn’s disease and ulcerative molecule, termed divertin prevents MLCK1 recruitment to the colitis (UC) patients (281). In the same study, stimulation with acto-myosin ring without inhibiting enzymatic function. In this IL-13 displayed a decreased TEER in a cell culture model of HT- way, divertin restores TNF-induced barrier dysfunction and 29/B6 cells and by an increase in the pore-forming TJ CLDN- prevents disease progression in experimental chronic IBD (266). 2, while levels of both ZO-1 and OCLN were unaffected. In Collectively, these results indicate that mucosal healing and addition, they also found significantly increased permeability clinical remission in IBD patients may be strongly related of the sugar probes lactulose and mannitol, and higher rate to the immunomodulatory effects from blocking TNF-α, with of apoptosis in vitro (281). Although information on mucosal improvements to the intestinal barrier function occurring as a IL-13 levels in patients with IBS is scarce, there are results secondary effects that synergistically improve the outcome. showing serum levels of IL-13 being significantly increased in UC patients with IBS-like symptoms (282). However, the role of IL- Interferon-Gamma (IFN-γ) 13 in the pathophysiology of inflammatory intestinal disorders is Interferon type II (IFN-γ) is widely known as a pro-inflammatory controversial as later clinical studies with monoclonal antibodies cytokine with potent effects on intestinal barrier function against IL-13 (tralokinumab, anrukinzumab) in UC fail to report (267) (Figure 3). Studies performed in vitro have found IFN- convincing results (277, 283). These studies did not investigate γ to influence paracellular permeability by affecting structural any direct parameters of intestinal barrier function such as properties of the epithelial barrier. It has been shown in colonic permeability or TJ gene/protein expression, thus it’s possible that T84 cell lines that IFN-γ can internalize the TJ proteins OCLN, the anti-IL-13 agents could have had affected such parameters, CLDN-1, CLDN-4 and junctional adhesion molecule A, thereby mimicking the in vitro studies, but to an ineffective degree. The decreasing TEER and increasing the passage of paracellular anti-IL-13 agent lebrikizumab seems to have positive effects on markers (268, 269). The internalization process was found to patients with atopic dermatitis, a chronic inflammatory skin involve cytoskeletal contraction in a MLCK-independent manner condition characterized by skin-barrier defects. Even though (269), which separates it from the mechanisms of TNF-α through the mechanisms behind improvement could speculatively be more direct effects on barrier integrity, even though the end linked to restoration of skin barrier function, to our knowledge, result is similar. Modern in vitro techniques using intestinal no studies have yet elucidated such mechanisms (284, 285). organoids (3D cell culture models) have further verified the It is likely the anti-inflammatory effects from inhibiting IL- ability of IFN-γ to disrupt the epithelial barrier function through 13 indirectly also could help to maintain barrier integrity to TJ protein degradation and delocalization (270). A number of some extent. However, these results do not seem to suggest studies show a synergistic deleterious effect on intestinal barrier targeting only IL-13 is an effective option in treating conditions of function from the combination of IFN-γ and TNF-α (267, 271). intestinal barrier dysfunction. Since multiple cytokines can have One of the mechanisms behind this synergistic effect could come deleterious effects on the barrier function, it’s possible inhibiting from IFN-γ’s ability to increase the expression of TNF receptor-2, several cytokines at the same time would have stronger effects. as shown by restoration of barrier function when blocking TNF receptor-2 but not TNF receptor-1 (272). Viceversa, TNF-α has Larazotide also been demonstrated to increase the IFN- receptor expression Larazotide acetate, also known as AT-1001, is a synthetic peptide in vitro (16426148). IFN-γ is seen increased in many intestinal derived from the Vibrio cholerae zonula occludens toxin (ZO-T conditions that also are characterized by gut barrier dysfunction, or zonulin) which behaves as a zonulin antagonist and proposed such as IBD and IBS (273, 274). Despite several attempts to create as permeability regulator (286) (Figure 3). Zonulin is released antibodies for IFN-γ or its receptors (275), clinical applicability is by intestinal epithelial cells after diet or microbiota stimuli. difficult due to its ubiquity in cells and organs and its pleiotropic Zonulin-mediated detachment of the ZO-1 protein from the effects. Anyhow, a monoclonal antibody against IFN-γ, AMG TJ protein complex has a direct effect in increasing intestinal 811, is under development (276). permeability (287). Larazotide prevents TJ opening, being able to block zonulin receptors locally, by joining the receptors Humanized Antibodies Against Interleukin-13 itself, decreasing TJ detachment and promoting TJ assembly IL-13 is a cytokine extensively involved in inflammatory reactions and structural filaments rearrangement (288). Larazotide was and mainly produced by T helper-2 cells, MCs, eosinophils, and developed for the treatment of CD (289) and later tested in Frontiers in Nutrition | www.frontiersin.org 14 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction type 1 diabetes, inflammatory bowel disease, Kawasaki disease, reduced intestinal injury in a rat model of intestinal ischemia respiratory diseases (290), collagen-induced arthritis (291) and (310), and also improved intestinal microcirculatory dysfunction intestinal ischemic injury (292, 293). and barrier dysfunction in endotoxemic rats (311) in association Four clinical trials using larazotide acetate have been with a reduction of OCLN cleavage and bacterial influx into published, all in CD (289, 294–296). These studies confirmed its the spleen. After traumatic brain injury, GI system dysfunction safety and efficacy for reducing gluten-induced symptoms as well and impairment of barrier function are common features (312). as an interesting inverse dose effect, that is, greater reduction DMM was able to reduce systemic inflammatory cytokines and of symptoms with lower doses. Nevertheless, positive results barrier dysfunction, and to improve villus structure in a rat model cannot be linked to a reduction of small bowel permeability, of brain injury (313). DMM has also proved to protect against measured by the lactulose-mannitol test, due to huge variability, heat stroke-induced inflammatory response and multi-organ leading to controversial results. A phase 3 trial is ongoing dysfunction (314). DMM also demonstrated capacity to reverse (ClinicalTrials.gov Identifier: NCT03569007) to test larazotide burn-induced intestinal epithelial hyperpermeability by reducing in lower doses in CD patients on a gluten-free diet. Hence, inflammation and enhancing the expression and distribution of although some clinical benefit has been observed, a more the TJ proteins ZO-1 and OCLN (315). accurate evaluation of larazotide effect on intestinal permeability In humans, a randomized, double-blinded trial using either is needed, not only in CD but also for other pathologies with perioperative DMM or placebo in patients who underwent an paracellular intestinal barrier dysfunction. hepatectomy not only showed a decrease in clinical relevant biomarkers of intestinal injury but also a reduction of intestinal Lubiprostone failure scores at 72 h after surgery (316). In another randomized, Lubiprostone (LBP) is a prostaglandin E1-derivative able to double-blinded prospective study, DMM enhanced the recovery bind and activate the chloride channel type 2 (ClC-2) located of GI and reduced intestinal injuries and permeability, reflected in the luminal side of the epithelium, improving bowel by decreased serum diamine oxidase and intestinal fatty acid- frequency and stool consistency in constipated-IBS patients binding protein expression (317). A recent randomized double- (297–301) (Figure 3). Moreover, LBP has been reported to blinded prospective study, suggests DMM as a more suitable enhance intestinal barrier function, reversing IFNγ-induced anesthetic for patients undergoing GI surgery as it is associated decrease in TEER and the increase in fluorescein labeled- with a decrease in TNF-α, and D-Lactate along with an increase dextran permeability and enhancing the expression of CLDN-1 in the activation of α7nAChR (318). in vitro (302). LBP reduced the severity of colitis as well as intestinal Mast Cell Stabilizers and Flavonoids permeability in both DSS and TNBS-induced colitis in murine Intestinal mast cells (MCs) (Figure 3) play an essential role in models (303). Alternatively, when LBP was administered to barrier function regulation and gut homeostasis as shown both ClC-2 knockout mice, the protective effect against DSS colitis in vitro, in animal models and in humans as reviewed elsewhere was limited, suggesting a central role of chloride channels in (216). MC activation leads to the release of a wide variety of the restoration of barrier function and TJ architecture driven proinflammatory and regulatory mediators, and many of them all by LBP (303). LBP also reduced mannitol flux in ischemia- have an effect on intestinal barrier as well as modulating immune injured intestine in ex vivo porcine models (304), and decreased response and enteric nervous system. Though the inhibition chronic water avoidance stress-induced visceral hyperalgesia in of MC activation has been extensively investigated, and many rats, partly by down-regulation of OCLN and also up-regulation different approaches are possible (319–321), the use of MC of CLDN-2 in rat colon crypts (305, 306). The potential of LBP to stabilizers has gained some consideration in the management prevent small intestinal injury and increased permeability related of several intestinal disorders in humans, mainly because its to non-steroidal anti-inflammatory drugs has been reported in a beneficial role in the regulation of IEB is based on a solid and rat model (307). vast literature in preclinical models (228, 322–332). Among MC One study assessed the effect of LBP on human intestinal stabilizers, only ketotifen and disodium cromoglycate (DSCG), barrier function after administration of diclofenac, showing a have been translated to the clinic. significant reduction of lactulose-mannitol ratio compared to the There are few studies exploring the effect of MC stabilizers control group (308). Unfortunately, the three randomized trials in modulating IEB in humans. In one study, ketotifen was that support the use of LBP in IBS with constipation did not able to reestablish GI permeability in a small group of food evaluate intestinal permeability as an endpoint. allergy patients (333). In a trial with IBS patients, ketotifen reduced several IBS symptoms, although barrier function was Dexmedetomidine not explored (334). Although preliminary, ketotifen has also Dexmedetomidine (DMM) (Figure 3) is a highly selective 2- shown significant benefits for the treatment of post-operative adrenoreceptor agonist, used as a sedative and anesthetic ileus (335), a condition that seems to be also related with adjuvant. Interestingly, it also shows a protector role against dysfunction of IEB (336). DSCG reverted the increase of barrier dysfunction and intestinal injury. However, the exact intestinal permeability triggered by CRF or stress in healthy mechanisms are not completely elucidated, although it is able volunteers (224). Previously, DSCG pretreatment reduced milk- to accelerate intestinal wound healing by increasing intestinal induced in intestinal permeability, in children with cow’s milk epithelial cell proliferation (309). Pretreatment with DMM allergy (337, 338) or food allergy (339), and in patients with Frontiers in Nutrition | www.frontiersin.org 15 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction dyshidrotic eczema (340) but nor in atopic eczema (341), how LRH-1 restoration reestablished epithelial integrity in mouse although these studies were performed in small groups. Several and human organoids as well as its overexpression protected mice other studies have shown the potential utility of DSCG for IBS from developing colitis (377). treatment (342–344), but again, little clinical evidence is available to support its use as a possible modulator of the IEB. Flavonoids are natural substances with variable phenolic Mucoprotectors structures commonly present in fruits, vegetables, tea, wine, Mucoprotectants are compounds of different nature (insoluble grains, bark, roots, stems, and flowers (345). Flavonoids present salts, hemicellulose, tannic acid, gelatins. . . ) with the ability a natural antioxidant, antimicrobial, cytoprotective, and anti- of enhancing the intestinal barrier by creating a film-forming inflammatory activity (346). Multiple in vitro studies show barrier over the intestinal mucosa (Figure 3), helping to reduce the ability of several flavonoids, including 8-prenylnaringenin, the effect of pathogens and to improve the function of the anthocyanins, berberine, puerarin, genistein, kaempferol, intestinal barrier (378). These compounds work intraluminally naringenin, quercetin, and luteolin, among others, to restore to modify enteric contents and may represent an alternative barrier dysfunction, predominantly in a Caco-2 cells (347– or complementary therapy for dealing with acute and chronic 354). Moreover, it has been described their ability to increase diarrheal disorders (379, 380). the expression, assembling and production of different TJ proteins such as ZO-1 and 2, OCLN and CLDN 1, 3, and Xyloglucan 4 through the activation of AMPK and the inhibition of Xyloglucan (XG) is a water-soluble, high molecular weight NAPDH oxidase /NF-γB and MLCK and MLC phosphorylation branched polysaccharide hemicellulose. XG helps to reduce (244, 352, 353, 355–357). In vivo studies in rat, highlight the effect permeability changes, preserving TJ, and invasion by E. coli of flavonoids in the upregulation of several pathways involved in in Caco2/Goblet cells (381), and binding to MUC1, in mice the expression of several TJ proteins 1 (351, 357, 358) In humans, exposed to DSS (382). XG is non-toxic and resistant to digestive we are awaiting for ongoing clinical trials to determine the role of enzymes, reaching the colon unaltered, where it is partially natural flavonoids in the management of IEB dysfunction (346). broken down to oligosaccharides by bacterial endo-ß-glucanases, followed by bacterial fermentation of oligosaccharides (383, 384). Glucocorticoids The molecular structure of XG is known to possess mucomimetic Glucocorticoids (GCs) (Figure 3) play an important role and mucoadhesive properties (382). XG is often combined with in maintaining homeostasis through anti-inflammatory and gelatin or gelose to prolong its availability within the intestine, immunosuppressive actions (359), mediated mostly through but showing similar protective effects as XG alone on barrier GC receptors (360) GCs synthetic derivatives are essential function in rats after Salmonella enterica and Enterococcus in the clinical treatment of inflammatory and autoimmune hirae infections (385). The combination of XG, pea proteins, diseases (361). tannins from grape xylo-oligosaccharides also offered protection GCs are released after barrier disruption, in part to neutralize against stress-induced visceral hypersensitivity and intestinal the effect of TNF, via inhibition of MLCK activity (362). In the hyperpermeability in rats (380). same in vitro model, GCs triggered a time and dose-dependent In humans, several clinical trials have shown the efficacy of increase in TEER in a GC receptor-dependent manner although XG in the treatment of acute diarrhea in children (386, 387) no changes were observed in TJ architecture (362). GCs also and adults (388), and also in chronic diarrhea in IBS patients, regulated CLDN expression via MKP-1 in cell lines (363), but also improving the majority of symptoms (389, 390). So far, these in human and rat colon mucosal crypts (364). In addition, it has findings have not been linked to its ability to regulate IEB, and been described that GCs modulate the expression of several other additional trials are needed to support this concept. molecules related with TJ polarization and development (365). An interaction between GCs and IL-10 p38 MAPK improved Gelatine Tannate barrier integrity after TNF-α challenge in a Caco-2 model (366). Gelatine tannate (GT) is a complex of tannic acid and gelatin GC receptor deficiency aggravated barrier integrity in an animal which forms electrostatic bonds with mucin to create a protein- model of colitis (367). based biofilm over the intestinal mucosa (391, 392). Gelatin is GCs reduced intestinal permeability in a large number of a collagen derivate, which is ingested as an insoluble powder patients, mostly in Crohn’s disease (368, 369), but also in the at acidic pH, that becomes a gelatin at pH > 5.5 (393). In rectum of collagenous colitis patients (370). In addition, the the intestine, this complex increases the epithelial resistance effect of GCs on permeability is not restricted to IEB because against E. coli, contributing to restore the normal physiology similar modulatory effects have been shown in lung epithelia of barrier function in Caco and Goblet cellsTM (394). GT also (371) and the blood brain barrier (372). In addition, the use helps to restore the mucus layer and to modulate the intestinal of GCs for treating intestinal inflammation during sepsis has microbiota in the DSS model of murine colitis (395), and in been proposed to reduce intestinal barrier dysfunction (373). Caco-2 cells, where it prevents the release of TNF-α induced by Finally, UC patients display decreased levels of the liver receptor LPS (396). Furthermore, tannins allow the precipitation of pro- homolog-1 (LRH-1) in the colon (374, 375). LRH-1 is involved inflammatory molecules from the intestinal mucus and their fecal in the replacement of the adrenal steroidogenic factor 1 and GC elimination (396). Together, these observations may explain the synthesis in the adrenal medulla (376). A recent study has shown protective effect of GT on intestinal barrier function. Frontiers in Nutrition | www.frontiersin.org 16 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction Several clinical trials have been performed with GT for acute increasing barrier permeability through the degradation of diarrhea in children, and adults, with mixed results (379, 397, OCLN mRNA (412). 398). The combination of GT and tyndallized probiotics has By inactivating the endonuclease RNase Dicer, enzyme been claimed as highly effective in the treatment of moderate responsible of the pre-miRNAs cleavage and the following and prolonged diarrhea, but clinical evidence awaits the results maturation to functional miRNAs (413), McKeena et al. showed of an ongoing clinical trial (ISRCTN63068134). Similar to XG, altered expression of Cadherin 1 and Cathepsin B. Dicer1 additional evidence is needed to link the positive effects to the mutants also displayed impaired epithelial barrier function, most protective effect on IEB. probably due to the disorganization of the epithelial layer and the junctional complexes (414). Hence, the role of miR-144 Diosmectite in downregulating OCLN and ZO-1 expression and enhancing Diosmectite is a medicinal clay (aluminum and magnesium intestinal permeability has been reported in a rat model od silicate) frequently used as an adjuvant therapy in children and IBS-D (415). adults with acute diarrhea (399, 400), to reduce stool output, to Other miRNAs, such as miR-93 and miR-29a/b seem to offer provide symptomatic relief and to prevent dehydration (398). protective effects on barrier function. miRNA-93 is responsible The mechanism of action is complex, but partly related to of PTK6 downregulation in YAMC cells, reversing the effects modifications of the rheological characteristics of the GI mucus in permeability caused by TNF-α and IFN-γ (416). miR-29a barrier, to reduce penetration of toxins, adsorptive properties, and miR-29b prevented inflammation in mice after DSS-induced reduction of intestinal permeability by increasing the expression colitis when delivered with supercarbonate apatite nanoparticle of OCLN, CLDN-1, and ZO-1, and increased MUC2 expression. (417). Increased levels of miR-29a have also been found in These mechanisms have been replicated mainly in Caco-2 and blood, small bowel and colon in IBS and colon in IBD patients HT-29 cell lines, and in rodent and piglets animal models in (113, 418, 419), but, as a matter of fact, its role is sometimes response to TNF, acetic acid or TNBS (401–405). contradictory (420). miR-29a overexpression increased epithelial Diosmectite has been shown to improve acute and chronic permeability by targeting glutamine synthetase gene (GLUL), diarrhea, based on a number of open and randomized double- alteration that has been previously associated with increased blind, placebo-controlled clinical trials, performed mostly in membrane permeability (113). Moreover, in IBS-D patients, children with acute diarrhea, and highlighted in a recent increased levels of miRNA-29a and miRNA-29b have been Cochrane review (398). Nonetheless, the clinical benefit has not described together with a reduction of CLDN1, ZO-1 and nuclear been associated with its barrier protective characteristics. factor-kB repressing factor, while increased levels have also been observed in mice models of IBS or colitis (113, 419). Epigenetic and Exosome-Mediated After showing a correlation between differential mRNA Regulation of Intestinal Barrier Function expression and ultrastructural changes in the epithelium of IBS- In the last decade, exosomes (food and host-derived) and D patients (421), the same group reported the ability of miR-16 enclosed micro-RNA (miRNA)s’ role as modulators of immune and miR-125b to modulate barrier function in the jejunum of responses and IEB function has been widely reported. miRNAs these patients through the regulation of claudin-2 and cingulin are small (21–23 bp) non-coding RNAs that regulate gene expression, respectively (422). In a recent study, Martínez et al. expression either by binding to the 3′ untranslated region of reported several regulated miRNAS in the rectal mucosa of post- their target mRNAs or via endonucleolytic mRNA cleavage, infectious-IBS patients along with downregulation of their target promoting post-transcriptional repression and influencing mRNAs involved in barrier function (423). Finally, Mahurkar- intestinal homeostasis (406, 407). miRNAs have been implicated Joshi et al. (424) have reported decreased levels of miR-219a-5p in several GI physiologic and pathophysiologic mechanisms and and miR-338-3p in sigmoid biopsies in IBS, particularly in IBS- studied widely in intestinal immune and inflammatory diseases, C. Inhibition of miR-219a-5p resulted in altered expression of including IBS and IBD, though studies are highly heterogeneous. proteasome/barrier function genes and enhanced permeability of Both in vitro and in vivo assays have recently shown that intestinal epithelial cells. Additionally, inhibition of miR-338-3p after IL-1β administration both Caco-2 cells and enterocytes in cells caused alterations in the mitogen-activated protein kinase from mice with colitis display increased small intestinal TJ (MAPK) signaling pathway genes (424). permeability, a rapid increase in miR200C-3p and reduced levels Not only host miRNA but also plant and bacterial-derived of OCLN mRNA and protein, meanwhile the antagomiR-200c miRNAS have gained special attention for their potential role, prevented OCLN and permeability changes (408). Moreover, yet disputed, as cross-kingdom gene expression regulators, colon tissues and organoids from patients with UC had increased influencing plant interactions with animals and microorganisms levels of IL-1β mRNA and miR200C-3p compared with healthy to regulate a number of physiological functions (425). In controls. In other studies, transfection of miR-21 in Caco-2 cells humans, diet would be a primary source of plant miRNA also resulted in the loss of TJ as well as ultrastructural changes uptake, whereby have been termed as xeno-miRNAs (xenomiRs), enhancing intestinal permeability through the degradation of moreover, effective detection and quantification of dietetically RhoB and PTEN (409, 410). An increase of miR-21 and miR- absorbed plant microRNAs in human plasma (426). Several 126 has been also observed in colon, feces and blood of UC, and mechanisms, including transmembrane miRNA carriers, CD patients (409, 411). In addition, increased expression of miR- receptor-facilitated endocytosis, phagocytosis, macropinocytosis, 122a has been also noticed in Caco-2 cells after TNF-α exposure, clathrin-mediated, caveolin-mediated, and clathrin and Frontiers in Nutrition | www.frontiersin.org 17 October 2021 | Volume 8 | Article 718093 Fortea et al. Therapeutic Approaches to Barrier Dysfunction caveolin-independent endocytosis, paracellular diffusion, Probably, the truth lies somewhere between these two extremes. and luminal immune cell capture have been proposed to What is certain is that pathophysiological, functional and explain xenomiRs uptake by intestinal epithelial cells, though, molecular knowledge has advanced enormously in that period to date, still unresolved (425, 427). Later, xenomiRs could of time, and with it, the search for better therapeutic options to be packaged into microvesicles (426) or associated with manage intestinal permeability disorders. In this review, we have proteinase K-resistant complexes (428) to be transported discussed the most relevant therapeutic approaches to improve and released into the bloodstream to reach out target intestinal permeability and barrier dysfunction. To move tissues. It has been shown that absorption of more stable forward, more clinical trials, new molecules intended directly xenomiRs, such as miR-2911, could be promoted when intestinal to fine tuning of intestinal permeability, differential treatments permeability is enhanced, which may be of use for engineering according to the affected intestinal segment, according to sex, delivery of dietary miRNA (429). Moreover, gut microbiota according to age, and to many other variants are needed. In could be responsible for enhancing xenomiRs absorption, addition, it is important to recognize the role of other factors, increasing their bioavailability by degrading the exosomes such as immune activation and microbiota-related regulation of components (430). barrier defects in order to deal better with chronic inflammation Despite the well-known role of xenomiRs in shaping in the gastrointestinal tract because presumably targeting only microbiota and modulating inflammation and immune the barrier may not be sufficient to change the natural history of activation (410, 430, 431), including the down-regulation of TNF many of these conditions. The advancement in our knowledge receptor (432), direct regulation of the epithelial barrier has not of the intimate mechanisms and the inducing and predictive been reported (430). However, given TNF-α ability to modulate factors of changes in permeability will lead to the development barrier permeability, xenomiRs could be indirectly involved in of a better therapeutic approach in several digestive and also the regulation of IEB. In addition, several studies have shown the extradigestive diseases. ability of plant-derived exosomes, enriched for diverse miRNAs, to contribute to intestinal barrier integrity (410, 433–435), AUTHOR CONTRIBUTIONS though, again, a direct link could not be established and the effect on barrier function could be dependent on other non-xenomiR- MF, MA-B, MA-G, JP-GM, XS-R, AH-P, EE, AG-C, DG, BL, related mechanisms. Because some plant xenomiRs modulate the and CA-C reviewed scientific literature and collected data. MF, expression of enterocyte transporters (436), cytokines involved MA-B, MA-G, JP-GM, XS-R, AH-P, CA-C, and JS wrote the in barrier function such as TNF-α or IL-1β, and activate Wnt/β- paper. MF, MA-B, and MA-G prepared the tables and figures. All catenin pathway (437), it remains plausible that xenomiRs could authors critically reviewed and edited the manuscript in its final act as regulators of intestinal barrier permeability. Furthermore, version and approved the final draft of the manuscript. gut microbiota, by enhancing xenomiRs absorption, may also regulate the expression of different miRNAs in IECs (438). FUNDING Therefore, considering miRNAs promising role on modulating intestinal barrier function, miRNA based therapeutic This work was supported in part by Fondo Europeo de strategies are moving from bench to the bedside. Although with Desarrollo Regional (FEDER), Fondo de Investigación Sanitaria several limitations, regulation of miRNAs expression can be and Centro de Investigación Biomédica en Red de Enfermedades achieved by administering synthetic miRNAs or miRNAs Hepáticas y Digestivas (CIBEREHD), Instituto de Salud Carlos expressing vectors or by anti-sense nucleotides (439). Their III, Subdirección General de Investigación Sanitaria, Ministerio therapeutic application for barrier function and intestinal de Economía y Competitividad, Ajuts per a la contractació de inflammation and cancer is under development though further personal investigador FI–Agència de Gestió d’Ajuts Universitaris research is needed (440–442). i de Recerca (AGAUR), Generalitat de Catalunya; The Swedish Research Council, and the European Commision. PI19/0164 SUMMARY AND CONCLUSIONS (BL), FI20/00256 and 2020FI_B1 00127, 2019FI_B 00817 (MA-B); PI17/0190 (JS); CIBERehd CB06/04/0021 (JS and This is an exciting time for intestinal permeability. We have gone CA-C); dnr 2019-00653 (JP-GM); GA no:848228 (JS, CA-C, from not recognizing the importance of intestinal permeability, EE, AG-C, and BL). MF received support from FWO (Fonds no more than 30 years ago, to express now that most digestive Wetenschappelijk Onderzoek) grant G.093818 to P. Vanden and extradigestive disorders have something to do with it. Berghe, KU Leuven. REFERENCES 3. Anderson JL, Hedin CR, Benjamin JL, Koutsoumpas A, Ng SC, Hart AL, et al. Dietary intake of inulin-type fructans in active and inactive Crohn’s 1. Turner JR. Intestinal mucosal barrier function in health and disease. Nat Rev disease and healthy controls: a case-control study. J Crohn’s Colitis. (2015) Immunol. (2009) 9:799–809. doi: 10.1038/nri2653 9:1024–31. doi: 10.1093/ecco-jcc/jjv136 2. 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Hossian AKMN, Mackenzie GG, Mattheolabakis G. miRNAs in gastrointestinal diseases: can we effectively deliver RNA-based therapeutics Citation: Fortea M, Albert-Bayo M, Abril-Gil M, Ganda Mall J-P, Serra-Ruiz X, orally? Nanomedicine. (2019) 14:2873–89. doi: 10.2217/nnm-2019-0180 Henao-Paez A, Expósito E, González-Castro AM, Guagnozzi D, Lobo B, Alonso- 442. Zhang L, Cheng J, Fan XM. MicroRNAs: New therapeutic targets for Cotoner C and Santos J (2021) Present and Future Therapeutic Approaches to intestinal barrier dysfunction. World J Gastroenterol. (2014) 20:5818– Barrier Dysfunction. Front. Nutr. 8:718093. doi: 10.3389/fnut.2021.718093 25. doi: 10.3748/wjg.v20.i19.5818 Conflict of Interest: CA-C discloses past scientific collaboration with Noventure Copyright © 2021 Fortea, Albert-Bayo, Abril-Gil, Ganda Mall, Serra-Ruiz, S.L. JS has served as consultant for Noventure and discloses present and past Henao-Paez, Expósito, González-Castro, Guagnozzi, Lobo, Alonso-Cotoner and recent scientific collaborations with Salvat, Norgine, Alfa-Sigma, Cosmo, Adare, Santos. This is an open-access article distributed under the terms of the Devintecpharma, Pileje and Danone that do not constitute a conflict of interest in Creative Commons Attribution License (CC BY). The use, distribution or developing the content of the present manuscript. reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in The remaining authors declare that the research was conducted in the absence of this journal is cited, in accordance with accepted academic practice. No use, any commercial or financial relationships that could be construed as a potential distribution or reproduction is permitted which does not comply with these conflict of interest. terms. Frontiers in Nutrition | www.frontiersin.org 31 October 2021 | Volume 8 | Article 718093