cells Review The Intestinal Barrier and Current Techniques for the Assessment of Gut Permeability Ida Schoultz 1 and Åsa V. Keita 2,* 1 Faculty of Medicine and Health, School of Medical Sciences, Örebro University, 703 62 Örebro, Sweden; [email protected] 2 Department of Biomedical and Clinical Sciences, Linköping University, 581 85 Linköping, Sweden * Correspondence: [email protected]; Tel.: +46-101-038-919 Received: 2 July 2020; Accepted: 14 August 2020; Published: 17 August 2020 Abstract: The intestinal barrier is essential in human health and constitutes the interface between the outside and the internal milieu of the body. A functional intestinal barrier allows absorption of nutrients and fluids but simultaneously prevents harmful substances like toxins and bacteria from crossing the intestinal epithelium and reaching the body. An altered intestinal permeability, a sign of a perturbed barrier function, has during the last decade been associated with several chronic conditions, including diseases originating in the gastrointestinal tract but also diseases such as Alzheimer and Parkinson disease. This has led to an intensified interest from researchers with diverse backgrounds to perform functional studies of the intestinal barrier in different conditions. Intestinal permeability is defined as the passage of a solute through a simple membrane and can be measured by recording the passage of permeability markers over the epithelium via the paracellular or the transcellular route. The methodological tools to investigate the gut barrier function are rapidly expanding and new methodological approaches are being developed. Here we outline and discuss, in vivo, in vitro and ex vivo techniques and how these methods can be utilized for thorough investigation of the intestinal barrier. Keywords: intestinal barrier; gut permeability; paracellular route; techniques; transcellular route; paracellular probes 1. Introduction The increased attention of the concept “leaky gut” and its association with numerous gastrointestinal (GI) disorders has led to an intensified interest from researchers with diverse backgrounds to perform functional studies of the intestinal barrier in different conditions. The methodological tools to investigate the intestinal barrier function are rapidly expanding and new methodological approaches are being developed continuously. For research groups new to the field of intestinal functional studies the most appropriate technique might therefore be difficult to identify. Gut barrier function involves the regulation of translocating luminal content such as antigens and bacteria that pass through the epithelial cell layer either between the epithelial cells (paracellular route) or through the cells (transcellular route) into the underlying mucosa. Intestinal permeability can be measured by recording the passage of permeability markers over the epithelium via these passage routes [1]. An increased intestinal permeability and signs of a dysfunctional barrier have been observed in several different conditions, such as Parkinson disease [2], obesity [3] and diabetes type 2 [4], as summarized in Table1. Cells 2020, 9, 1909; doi:10.3390/cells9081909 www.mdpi.com/journal/cells Cells 2020, 9, 1909 2 of 30 Table 1. Major diseases and conditions associated with an increased intestinal permeability. Transcellular Uncategorized Permeability Disease/Condition Paracellular Permeability Permeability Changes in vivo; altered expression and ex vivo; augmented Inflammatory bowel distribution of tight junction mucosal passage of in vivo; increased urinary disease proteins [5]; ex vivo, increased bacteria and horseradish secretion of probes [9] passage of paracellular probes [6,7] peroxidase (HRP) [6–8] altered expression of tight junction ex vivo; increased in vivo; increased urinary Irritable bowel syndrome proteins [10]; ex vivo; increased transepithelial passage of secretion of probes [12] passage of paracellular probes [1,2]. bacteria and HRP [11] in vivo: altered structure of tight ex vivo; augmented in vivo; increased urinary junction proteins [13,14] ex vivo; internalization of secretion of probes [15,19], Celiac disease increased passage of paracellular bacteria [17]; increased increased levels of zonulin in probes [15]; alteration in transcellular uptake of blood [20] electrophysiological parameters [16] gliadin [18] ex vivo; increased in vivo; increased levels of in vivo; altered expression of tight lipid-induced Obesity zonulin and lipopolysaccharide junction proteins [3] transcellular (LPS) in blood [3] permeability [3] in vivo; increased urinary secretion of probes [4]; increased Diabetes type 2 - - levels of LPS [21] and zonulin [22] in blood in vivo: increased LPS [23] and Alzheimer’s disease - - zonulin levels in blood [24] in vivo: increased urinary ex vivo: augmented Parkinson disease - secretion of probes [2]; increased uptake of bacteria [2] blood zonulin levels [25] in vivo: increased permeability Major depression -- markers in blood, I-FABP and disorder zonulin [26] Autism spectrum in vivo: altered expression of tight in vivo: increased levels of - disorders junction proteins [27] zonulin [28,29] Many of the conditions associated with a leaky gut show signs of both increased paracellular and transcellular permeability. Moreover, new exciting findings connecting different conditions to a dysfunctional barrier by primarily in vivo biomarkers could potentially be complemented with other techniques to strengthen these findings. To guide researchers new to functional studies of the intestine we outline how paracellular and transcellular permeability can be assessed in vivo, in vitro and ex vivo. Although, markers of intestinal inflammation or gut microbiota composition are not the focus of this review it is important to acknowledge that these factors play an essential role in gut barrier homeostasis and are important complements in studies of intestinal barrier function, particularly when investigating the underlying mechanisms of a perturbed intestinal barrier. 2. The Intestinal Barrier The intestinal barrier constitutes the interface between the outside and the internal milieu. A functional intestinal barrier allows absorption of nutrients and fluids but simultaneously prevents harmful substances like toxins and bacteria from passing through the intestinal epithelium to the underlying tissue [30]. There is a delicate balance to keep a functional barrier and it is maintained by physical defense mechanisms including both the junctional complexes linking adjacent epithelial cells and the mucosal surface of the epithelial cell lining (Figure1). Cells 2020, 9, 1909 3 of 30 Cells 2020, 9, x 3 of 31 FigureFigure 1. A1. schematicA schematic drawing drawing of the of intestinalthe intestinal barrier barrier and passageand passage routes routes across across the epithelium. the epithelium. Solutes canSolutes pass the can intestinal pass the epitheliumintestinal epithelium via either via the either (A) paracellular the (A) paracellular route (larger route hydrophilic(larger hydrophilic solutes); (B)solutes) transcellular; (B) transcellular route (small route hydrophilic (small hydrophilic and lipophilic and lipophilic solutes) (solutes)C); transcellular (C); transcellular route via route aqueous via poresaqueous (small pores hydrophilic (small hydrophilic solutes) or activesolutes) carrier-mediated or active carrier absorption-mediated absorption (nutrients); (nutrients) or (D) endocytosis,; or (D) followedendocytosis, by transcytosis followed andby transcytosis exocytosis (larger and exocytosis particles, (larger peptides particles, and proteins). peptides The and barrier proteins). constitutes The of (1)barrier the lumen,constitutes bacteria of (1) and the antigenslumen, bacteria are degraded and antigens by biliary are degraded juices, gastric by bil andiary pancreatic juices, gastric acids and and thepancreatic colonization acids of pathogensand the colonization is inhibited of by pathogens commensal is inhibited bacteria producingby commensal antimicrobial bacteria producing substances; (2)antimicrobial the microclimate; substances unstirred; (2) waterthe microclimate; layer, glycocalyx, unstirred bacterial water adhesionlayer, glycocalyx, is prevented bacterial by mucusadhesion and IgAis secretion; prevented (3) by the mucus epithelial and IgA cells; secretion luminal; (3) content the epithelial is transported cells; luminal while content noxious is transported stimuli is impeded while by chloridenoxious stimuli secretion is impeded and production by chloride of antimicrobial secretion and peptides production (AMP), of antimicrobial junctional complexes peptides (AMP), between junctional complexes between the cells regulate permeability, for details see right panel; 4) the lamina the cells regulate permeability, for details see right panel; (4) the lamina propria; immunoglobulins propria; immunoglobulins and cytokines are secreted from cells of both the innate and acquired and cytokines are secreted from cells of both the innate and acquired immunity with direct or indirect immunity with direct or indirect effects on permeability, interactions with the endocrine and enteric effects on permeability, interactions with the endocrine and enteric nervous system. TAMP: tight nervous system. TAMP: tight junction-associated-MARVEL proteins including occludin, tricellulin junction-associated-MARVEL proteins including occludin, tricellulin and Marvel D3; JAM: junctional and Marvel D3; JAM: junctional adhesion molecule; MLC: myosin light