In humans, the intestinal epithelium covers as large as 400 m² of surface area (1). Though only a single layer of cells, the intestinal epithelial cells (IECs) are the mainstay of the intestinal barrier and serve as a physical barrier (Figure 1). There are at least seven types of functional IECs—enterocytes, goblet cells, Paneth cells, microfold cells (M cells), enteroendocrine cells, cup cells, and tuft cells, although the functions of the last two cell populations are not well understood (10). Among all these cell types, enterocytes represent the absolute majority, accounting for at least 90% of crypt cells or villus cells. Enterocytes are absorptive cells and vital for nutrient uptake. However, growing evidence indicates that the functions of enterocytes are not limited to nutrient absorption. For example, enterocytes can control the abundance of Gram-positive bacteria by expressing RegIIIγ, one type of antimicrobial proteins (AMPs) (11–13). All epithelial cell types originate from Lgr5⁺ intestinal epithelial stem cells, which reside within the crypts (14). The turnover rate of IECs is high and the cells are renewed every 3–5 days in the mammalian intestine (10, 15), with the exception being the Paneth cells, which have a life span of about 2 months.

The IEC lining is continuous, and the contact between IECs is sealed by TJs (16). The paracellular pathway, in contrast to transcellular pathway, allows the transport of substances across the gut epithelium through the spaces between IECs. A large variety of molecules, mainly proteins, control the plasticity of TJs. More than 40 TJ proteins have been recognized, including occludin, claudins, junctional adhesion molecule A, and tricellulin (17). Under various pathological conditions, paracellular permeability may be increased, resulting in the entry of unwelcome, potentially harmful molecules.

On top of the gut epithelium, there are two layers of mucus, the inner and outer layers, that cover the whole intestinal epithelial lining and provide physical protection to separate luminal microorganisms from the epithelium. Organized by its major component, a highly glycosylated gel-forming mucin MUC2, the mucus contains diverse molecules including IgA as well as enzymes and proteins, such as lactoferrin (18). Goblet cells are the central cell type for the formation of mucus. They not only produce MUC2 mucin but also secret other mucus components such as ZG16, AGR2, FCGBP, CLCA1, and TFF3 (19, 20). Colitis would spontaneously develop in Muc2-deficient mice, indicating a critical role for MUC2 in mucosal protection (21). In addition to gel-forming mucins, there is another type of mucin that is in close proximity to epithelial cells, called transmembrane mucins. Enterocytes are the main producers of transmembrane mucins (20).

The gut commensal bacteria have been described as one component of the intestinal physical barrier primarily due to its two major functions (22). The first is to promote resistance to the colonization of harmful or pathogenic bacteria species by competing for nutrients, occupying attachment sites, and releasing antimicrobial substances (23, 24). Additionally, the gut microbiota regulates the digestion and absorption of nutrients to supply energy to epithelial cells, which are a major component of the physical barrier (25). A good example of the direct energy supply is the production of short-chain fatty acids by the gut microbiota, which are used by colonocytes for their development and metabolism (26). Taken together, IECs, the mucus layers, and gut microbial residents serve as the physical barrier to limit the entry of unfriendly luminal contents into host tissues.

Biochemical molecules with antimicrobial properties exist in the mucus as well as far into the lumen and include bile acids and AMPs (27, 28) (Figure 1). These diverse molecules form a complicated network to reduce the load of colonized bacteria and decrease the chance of contact between luminal antigens and host cells. They are a good supplement to the physical barrier and an essential component of the intestinal barrier function.

The proximal small intestine harbors very few microorganisms (29). But as the distance from the stomach increases, the pH rises and the number of colonized bacteria esculates (30). Facing a large number of microorganisms, which likely outnumber the number of host cells, multiple AMPs are generated to fight against invaders. These AMPs are divided into several types, including α- and β-defensins, C-type lectin, cathelicidin, lysozyme, and intestinal alkaline phosphatase (IAP) (27). Their detailed antimicrobial mechanisms are discussed elsewhere (31). As a major, but not exclusive, producer of AMPs, Paneth cells support and mediate the biochemical barrier function.

Below the intestinal epithelium, there are organized lymphoid follicles, including the Peyer’s patches and isolated lymphoid follicles. Inside the follicles, a variety of immune cells, including B cells, T cells, dendritic cells (DCs), and neutrophils, orchestrate the immune response by presenting antigens, secreting cytokines, and producing antigen-binding antibodies (Figure 1). In the intestinal epithelium where lymphoid follicles are found, M cells are present that transcytose antigens across the intestinal epithelium to the Peyer’s patches underneath (14). In addition, goblet cells present acquired luminal antigens to CD103⁺ DCs in lamina propria in small intestine by forming goblet cell-associated antigen passages (GAPs) (32, 33). Interestingly, spontaneous antigen presentation was also observed in the colon, but only when the mice were raised germ-free (GF), or housed conventionally but with oral antibiotic treatment (34). This suggests that the antigen uptake process and formation of GAPs are regulated by the colonic microbiota (35). In addition, goblet cells and GAPs are capable of sensing invasive pathogens and inhibiting the translocation of pathogenic bacteria into the host immune system (36). Furthermore, intestinal mononuclear phagocytescan sense and sample luminal contents (37, 38). CX3CR1-expressing cells are responsible for this process, and antigen sampling is dependent on structures called transepithelial dendrites (TEDs) (39, 40). The formation of TEDs is regulated by CX3CR1⁺ macrophages and the expression of CX3CL1 by certain IECs (41, 42).

Another component of the immunological barrier is secretory IgA (SIgA). As the most abundant immunoglobulin in the body, IgA resides primarily on intestinal mucosal surfaces. While some people with selective IgA deficiency appear to be healthy, SIgA is important as it presumably interacts with commensal bacteria to provide protection against pathogens. A unique feature about SIgA is that is structurally resilient in protease-rich environments allowing it to remain functionally active compared to other antibody isotypes on mucosal surfaces (43). In adult humans, about 50 mg/kg of SIgA is produced daily by plasma cells residing in the intestinal lamina propria. Finally, SIgA can be transcytosed through the epithelium and secreted into the gut lumen.

Though not mentioned here, self-modulating factors, such as nerves and diverse cytokines, are also important for maintaining the normal functions of the intestinal barrier.

Nutrients and food ingredients have been reported to contribute to the maintenance or alterations of gut microbiota and the intestinal barrier function (82). A recent review by De Santis et al. detailed many dietary factors that may modulate the intestinal barrier (83). Here, we review some recent publications and emphasize the effects of diet-induced alterations of gut microbiota on compromising the gut barrier function. Vitamin D (VD) has been recognized as an intestinal permeability protector by inducing the expression of TJ proteins ZO-1 and claudin-1. In VD receptor (VDR)-knockout mice, more severe experimental colitis has been observed, suggesting the protective effect of VD on the mucosal barrier (84). However, another group have recently found that VDR deficiency lowers, whereas VD treatment upregulates, the expression of claudin-2, a pore-forming TJ protein, which renders the intestinal epithelium leaky (85). Further analysis confirmed that VDR enhanced claudin-2 promoter activity. The exact role of VD and VDR on modulating intestinal permeability is therefore unclear and should be investigated carefully in association with gut microbiota. In a recent study by Desai et al., a low-fiber diet consumption was found to trigger the expansion of mucus-degrading bacteria, including Akkermansia muciniphila and Bacteroides caccae (45). As a result, the thickness of mucus is significantly decreased in mice fed with fiber-deficient diets, although the transcription of Muc2 gene was surprisingly heightened, possibly as a compensatory response. The thinner mucus and compromised intestinal barrier function lead to a higher susceptibility to certain colitis-causing pathogens (45). Moreover, a diet high in saturated fat has been shown to greatly decrease Lactobacillus and increase Oscillibacter, and these changes were correlated with significantly increased permeability in the proximal colon (86). Furthermore, studies revealed that the abundance of the Oscillospira genus was negatively correlated with the mRNA expression of barrier-forming TJ protein ZO-1.

Under certain circumstances, stress-induced alterations of gut microbiota and the impaired intestinal barrier would allow the occurrence of microbial translocation. Burn injury and alcohol consumption are examples of such stress. Burn injury results in increased intestinal permeability, which is mediated by increased activity of myosin light-chain (MLC) kinase (87, 88). It is known that MLC phosphorylation or kinase activation can trigger epithelial TJ opening (89–91). In burn injury, TJ proteins, including ZO-1, occluding, and claudin-1, are redistributed, which can be reversed by adding an MLC phosphorylation inhibitor (87). In addition, both humans and mice experiencing burn injury undergo similar alterations of gut microbiota, in particular, with increases of the abundance of bacteria from the Enterobacteriaceae family (88). Importantly, microbial translocation of these Gram-negative aerobic bacteria has been observed. Another research group, using a different burn injury mouse model reported increased colonic permeability together with reduced aerobic and anaerobic bacterial populations in the gut microbiota, particularly those producing butyrate (92). As a consequence, the butyrate level in the stool was significantly decreased in mice with burn injury. Interestingly, when the experimental mice received fecal microbiota transplant, their altered bacterial counts and impaired mucosal barrier function were reversed, suggesting direct involvement of microbiota in causing gut leakiness after burn injury.

Chronic alcohol consumption is responsible for intestinal barrier dysfunction, alterations on both the quality and quantity of gut microbiota, LPS translocation, and alcoholic liver disease (ALD). In both human and mouse, it has been well established that alcohol can disrupt intestinal barrier function, which is closely related to increased tumor necrosis factor (TNF) production from intestinal monocytes/macrophages and enterocytes bearing TNF-receptor 1, followed by downstream activation of MLC kinase (93). Notably, when mice given chronic alcohol also received oral antibiotic treatment, to remove the microbiota, the level of TNF production and intestinal permeability decreased to levels comparable to those in control mice (93). This indicates that the alcohol-induced, TNF-mediated gut leakiness is greatly dependent on gut microbiota. Indeed, though the mechanism is unknown, alcohol administration alters microbiota qualitatively and quantitatively in both human and mouse (94). Bacterial overgrowth has been observed with alcohol consumption, whereas antibiotics can decrease the bacterial load and attenuate ALD (53, 93, 95–97). Interestingly, probiotic Lactobacillus is significantly suppressed during alcohol consumption (53, 97). Directly supplying Lactobacillus strains or indirect stimulation of Lactobacilli with prebiotics or diets can decrease bacterial overgrowth, restore mucosal integrity of the intestine, and suppress microbial translocation (53, 94, 98, 99). Microbial translocation, especially the translocation of LPS, is involved in ALD development and progression as evidenced by the lack of ALD in mice deficient of TLR4 (100, 101). It is worth noting that some bacteria species can produce alcohol, including E. coli and Weissella confusa, and this may be the mechanism by which they compromise the intestinal barrier function (102, 103).

Infections can play a role in regulating the mucosal barrier. A good example is Helicobacter pylori, a Gram-negative bacterium infecting the human stomach (104). H. pylori is known to directly increase epithelial permeability by redistributing TJ protein ZO-1 (105, 106). In addition, bacteriophages, which are usually not considered pathogenic to mammals, can have an impact on the leaky gut. When rats were given a bacteriophage cocktail containing phages against Salmonella enterica, disruption of the intestinal barrier integrity was observed (107). The authors speculated that the gut microbiota might have been affected by bacteriophages, but sequencing data were not supplied to support their claims.

Taken together, perturbation of gut microbiota, which may be the consequence of diverse interventions, can lead to increased intestinal permeability and translocation of bacterial components and products. Such microbial translocation can subsequently trigger an abnormal immune response, causing inflammation and/or tissue damage in extraintestinal organs.

Type 1 diabetes is an organ-specific autoimmune disorder characterized by an autoimmune response against the host’s own pancreatic β cells, leading to insufficient insulin production from the pancreas (108). Some argue that the leaky gut is only an outcome of disease progression rather than an initiator or exacerbator of disease (109), but this should not be the case for T1D. This is supported by the following evidences. First, studies utilizing human subjects affected by T1D or T1D-prone animal models have indicated that impaired intestinal barrier function occurs before disease onset (110–112). Second, the pathogenic role that increased intestinal permeability plays in T1D is zonulin-dependent, and the production of zonulin relies on bacterial colonization (113). Reversion of intestinal barrier dysbiosis by adding a zonulin inhibitor ameliorated T1D manifestations in disease-prone rats (114). Third, a recent study has provided evidence that microbial translocation contributes to T1D development (115). In streptozotocin-induced T1D, mice treated with streptozotocin harbor a distinct microbiota compared to vehicle-treated controls. Importantly, gut bacteria were shown to be able to translocate into pancreatic lymph nodes (PLNs) and contribute to T1D development (115). When mice were treated with oral antibiotics, PLNs appeared to be sterile and the disease was attenuated. Further analysis revealed that the translocated bacteria in PLNs triggered NOD2 activation and exacerbated T1D. Altogether, these results suggest an essential role for the leaky gut in driving the progression of T1D.

Systemic lupus erythematosus, or lupus, is an autoimmune disorder characterized by severe and persistent inflammation that leads to tissue damage in multiple organs (116). Although SLE affects both men and women, women of childbearing age are diagnosed about nine times more often than men. LPS, a cell wall component of Gram-negative bacteria, can promote SLE development and disease progression upon penetration of the intestinal epithelium and translocation into tissues (117). In SLE patients, the higher level of soluble CD14 suggests an increase in LPS, as soluble CD14 is released from monocytes when the cells are exposed to LPS (118). Activation of TLR4 exacerbates lupus development (119–121). Mice spontaneously develop lupus when TLR4 responsiveness is increased, whereas the exacerbated disease phenotype can be significantly ameliorated when the commensal gut flora is removed by antibiotic treatment (121). This clearly indicates that TLR4 hyperresponsiveness to gut flora (which contains LPS) contributes to the pathogenesis of SLE. Moreover, the development of lupus in wild-type mice (C57BL/6 or BALB/c) immunized with phospholipid-binding proteins can be facilitated by the administration of LPS (122–124). Conversely, inhibition of TLR4 results in reduced autoantibody production and lowered renal glomerular IgG deposits in lupus-prone mice (125, 126). Taken together, these data suggest that LPS stimulation and TLR4 activation as disease-initiating factors for SLE. Lipoteichoic acid (LTA), a component of the Gram-positive bacterial cell wall, can also promote lupus disease. The expression of TLR2, the receptor of LTA, has been reported to be increased in SLE patients (127). In lupus-prone mice, TLR2 activation triggers lupus nephritis, whereas TLR2 knockout attenuates lupus-like symptoms (125, 128–130). Recently, another bacterial antigen that may mimic self-antigens has been recognized to induce autoantibody production (131).

Several downstream proteins in the TLR signaling cascade are highly relevant to the pathogenesis of SLE and are potential therapeutic targets, including MyD88, IRAKs, and IFNα (132). Deficiency of MyD88, in particular, has been shown to ameliorate lupus disease in MRL/lpr mice (133, 134), suggesting a potential role for TLRs to communicate with harmful bacteria in the gut microbiota. Conversely, there is a paucity of data pertaining to members of the NLR family. The most extensively characterized NLRs are associated with inflammasome formation (135, 136). Loss of NLRP3 and AIM2 inflammasome function was found to significantly contribute to lupus pathogenesis (137). Interestingly, both of these inflammasomes were found compromised in NZB mice, a lupus-prone model. Consistent with this finding, loss of ASC (apoptosis-associated speck-like protein containing CARD), a common adaptor protein required for inflammasome formation in B6-Fas^lpr mice led to exacerbation of lupus-like disease (138). These results suggest a potential role for NLRs to recognize protective bacteria in the gut microbiota. Therefore, it appears that TLRs and NLRs make distinct contributions to lupus pathogenesis by sensing harmful and protective bacteria, respectively. Both types of bacteria can come from gut microbiota through microbial translocation, especially in the presence of a leaky gut.