Use of Short-Chain Fatty Acids for the Recovery of the Intestinal Epithelial Barrier Affected by Bacterial Toxins

1The intestinal microbiota plays a critical role in the degradation of indigestible food components, immune system function, modulation of the activity of enteric and central nervous systems, reinforcement of the epithelial barrier, protection against pathogens and their toxins, and producing several compounds that influence the nutritional and health status of the host. Low-fiber diets, stressors, or even the use of antibiotics led to an imbalanced intestinal microbiota (dysbiosis) that can trigger inflammatory bowel disease (IBD), allergic, autoimmune, and metabolic diseases, and certain types of cancer. Studies linking intestinal microbiota with nutritional status focused on the degradation of indigestible fiber by bacterial enzymes and metabolites. Several studies showed that microbial-generated short-chain fatty acids (SCFAs) in the gut might play a key role in the prevention and treatment of IBD. SCFAs are carboxylic acids with aliphatic tails of less than six carbons where acetate (C2), propionate (C3), and butyrate (C4) represent the major end-products of gut microbial fermentation. These molecules are readily absorbed in the colon where they serve as an energy source for colonocytes and aid in the repair of the wounded epithelium. SCFAs also help to regulate glucose and lipid metabolism, which has important implications for the energy homeostasis of the host. Interestingly, SCFAs are associated with changes in tight junction (TJ) proteins expression and distribution. Recent evidence points to a direct role for the GPR43, GPR41, and GPR109A receptors in mediating the protective roles of SCFAs in IBD, as well as in controlling the production of cytokines and chemokines by intestinal epithelial cells (IECs; ).

2Colonization of the proximal intestine by pathogenic microorganisms can lead to a permeable gut by the action of secreted toxins. Specifically, these toxins can alter cell physiology through various mechanisms, either being directly responsible for the pathology of the disease or favoring other bacterial processes such as manipulation of the immune response and penetration of host barriers, among others. Thus, the epithelial barrier tightly controls antigen trafficking through paracellular pathways and is essential for the maintenance of normal functioning of the intestine. SCFAs have a protective effect against bacterial pathogens by maintaining the integrity of the epithelial barrier. Therefore, the production of adequate and balanced SCFAs by healthy gut microbiota is an important factor that prevents infection by common foodborne pathogens. Here, we aim to provide an overview of microbial SCFAs production in the gut and their signaling associated with the recovery process of the intestinal barrier after being damaged by bacterial toxins.

3In physiological conditions and absence of bacterial enteric infections, the small intestine harbors a complex microbial community of approximately 103–107 microbial cells/gram. This microbiota is less diverse and abundant than the colonic microbiota with ≈1012 cells/gram. An important metabolic activity of the intestinal microbiota is the fermentation of non-digestible starch and fiber, which generates SCFAs as the principal end product. SCFAs have different production rates and their concentrations vary within the human colon: acetate is in the range of 20–43 mmol/L, while butyrate and propionate have been reported to 6–15 and 6–13 mmol/L, respectively. Butyrate is mostly produced by Gram-positive Firmicutes, while acetate and propionate are mainly produced by Gram-negative Bacteroidetes. In this sense, some investigations have shown that dysbiosis in IBD patients is associated with a reduction in the number of butyrate-producing firmicute bacteria. Additionally, mucosal immunity is greatly influenced by dietary fiber and microbial SCFAs. Acetate and butyrate improve goblet cell differentiation and mucus production, necessary for a healthfuller epithelium structure, protecting against pathogens that degrade mucus layer for invasion and/or uptake of mucus-derived nutrients.

4Short-chain fatty acids can enter IECs through passive diffusion or by carrier-mediated transport: sodium-coupled monocarboxylate transporter 1 (SMCT1) and H+-coupled monocarboxylate transporter 1 (MCT1; ). SMCT1 is mainly expressed in the distal colon, whereas MCT1 is expressed in the colon but also in monocytes, granulocytes, and lymphocytes. In addition to being transported into cells, SCFAs can act directly as signal molecules for some G-protein-coupled receptors such as GPR41 (FFAR3), GPR43 (FFAR2), and GPR109A (HCAR2;;;;;; ). GPR41 is equally activated by propionate and butyrate, whereas GPR43 is activated by acetate and propionate more than butyrate. GPR41 and GPR43 are differentially expressed in intestinal cells, adipocytes, and phagocytes and differ in their downstream signaling cascades: while GPR41 binds only to Gαi decreasing intracellular cAMP levels, GPR43 can bind to either Gαi or Gαq decreasing cAMP or increasing the generation of diacylglycerol (DAG) and inositol triphosphate, respectively, promoting the mobilization of Ca2+. When GPR43 inhibits cAMP signaling, protein kinase A (PKA) activation is prevented. On the other hand, the effect of butyrate on DAG-mediated protein kinase C (PKC) activation is not fully studied. GPR109A, which is expressed in adipose tissue and colon, is activated by butyrate and it was shown to provide a protective effect against colitis.

5Epithelial cells are the first line of defense against bacteria and their toxins. These cells are held together by different intercellular junctions: TJ, adherent junctions, gap junctions, and desmosomes (Figure 1; ). The claudins, occludins, and the zonula occludens-associated (ZO) proteins 1, 2, and 3 in the TJ are located in the most apical side of the basolateral membrane, forming the paracellular pathways that regulate the passage of ions, solutes, bacteria, and toxins across the epithelial monolayer and are also responsible for maintaining/imparting cell polarity. The integrity of the TJ structure largely depends on the expression levels of TJ proteins.

6Zonulin is the main regulator of intestinal permeability due to its action on TJ. Zonulin-induced stimulation of protease-activated receptor 2 (PAR2; ), which is located in the basolateral face, increases the intestinal permeability through the redistribution of ZO-1, occludin, and F-actin. Thereby, zonulin increases intestinal permeability to bacterial components that strongly activate the innate immune response of the intestine, and its upregulation has been linked to the development of several chronic and autoimmune inflammatory disorders such as celiac disease. Additionally, the participation of several tyrosine kinases, such as c-Src and c-Yes, serine/threonine protein kinases, such as PKC, MAPK (mitogen-activated protein kinases), and PKA, in the proper functioning of TJ has been demonstrated. This would strongly support the role of different types of kinases as mediators in the effects of activation of SCFA receptors on proper epithelial barrier function.

7In Caco-2 cells, inflammatory mediators, such as tumor necrosis factor-α (TNF-α, ) and interferon-γ (IFN-γ), dissociate TJ, mainly through changes in the expression and localization of TJ proteins and in actin cytoskeletal structure. TNF-α also plays a central role in intestinal inflammation associated with Crohn’s disease (CD) and other inflammatory disorders. In general, intestinal inflammation, commensal microbes, and dietary components are among the main factors affecting epithelial permeability.

8Butyrate has been shown to regulate proliferation, apoptosis, and cell differentiation of the gastrointestinal epithelium. Butyrate was able to recover the barrier function through the positive regulation of the expression of claudin-1, ZO-1, and occludin in Cdx2-IEC and Caco-2 cells, resulting in increased transepithelial electrical resistance (TEER;;; ); the effect of butyrate on the epithelial barrier may be mediated by upregulation of TJ proteins via activation of AMP-activated protein kinase (AMPK; ). On the other hand, showed that Caco-2 cell monolayers treated with 5 mM butyrate changed TJ proteins expression levels: claudin-2 decreased by 90%, and claudin-7 increased by 376%, suggesting that butyrate might also regulate the remodeling of the TJ for maturing the barrier function. demonstrated that butyrate reduced the negative effect of lipopolysaccharide (LPS) on epithelial integrity through an increase in the synthesis of claudin-3 and claudin-4 via activation of the Akt signaling pathway in IPEC-J2 cells.

9In colonocytes, SCFAs modulate several processes involving members of the PKC family, which require cofactor calcium and DAG to be activated. Authors suggested that PKCβ1 activation may be required for the probiotic-mediated protection of TJ from H2O2 and that the reassembly of TJ in Caco-2 cells by sodium butyrate is because of the increased activity of PKCβ2. The role of PKC mediating the effects of SCFAs were also demonstrated in studies by, which showed that acetate-mediated activation of GPR43 signals via the PLC-PKC-PTEN pathway in adipocytes. However, the signaling pathways activated by SCFAs appear to depend on the cell type and function being examined.

10Short-chain fatty acids have also shown the effects against the production of proinflammatory cytokines (e.g., IL-6, IL-1β, IL-8, and TNF-α) by decreasing NF-κB activation in TLR-induced responses, in IECs. The inhibition of histone deacetylases activity, mainly by butyrate and propionate, induces anti-inflammatory activities on intestinal cells, macrophages, and dendritic cells. Additionally, butyrate potently inhibits the production of LPS-induced IL-1β and TNF-α in peripheral blood mononuclear cells by increasing the expression of GPR43 and also inhibiting the production of IL-6 to a lesser extent. Butyrate also promotes the correct activation and functioning of the inflammasome in colonic epithelium, allowing rapid recognition of invading microbes, thus avoiding further damage to the epithelial barrier.

11The intestinal microbiota is essential for the development and functional maturation of the gut immune system. Hence, a complex interaction between the host immune system and the microbiota is required for gut homeostasis. In this sense, ulcerative colitis (UC) and CD are gastrointestinal disorders associated with dysbiosis and dysfunction of the intestinal barrier integrity induced by toxins and pathogens. It was postulated that dietary fiber-derived SCFAs promote the function of the intestinal epithelial barrier and help repair damaged epithelium. Even more, several intestinal diseases, particularly those involving mucosal inflammation, are believed to result from an imbalance in SCFAs concentrations.

12Among SCFAs, butyrate has received the most attention for its effects on colonic health. Butyrate has shown some promising effects in the treatment of UC, while propionate’s anti-inflammatory effect would increase lipogenesis and glucose absorption in omental adipose tissue and may, therefore, have therapeutic utility in the prevention of obesity. Thus, although optimal concentrations of SCFAs in the lumen of the colon would be essential for the maintenance of health, these concentrations can be reduced under various conditions such as the intake of a low-fiber diet and/or chronic use of antibiotics.

13There are different molecular mechanisms and cell targets involved in the SCFA-mediated protective effects, including the stimulation of the host’s intestinal defenses, direct and/or indirect effects on bacterial colonization and toxin production, and increased TJ function. For example, SCFAs might exert protective effects against enteric pathogen colonization inducing the production of antimicrobial peptide LL37, as demonstrated in the HT-29 cell line. On the other hand, SCFAs can also have protective effects acting on the bacteria themselves, e.g., propionate represses Salmonella Pathogenicity Island 1 (SPI-1), decreasing its virulence, and butyrate strongly inhibits virulence factor production in Listeria monocytogenes. Butyrate treatment in a Clostridium difficile infection model increased the expression of genes associated with TJ proteins, including claudin-1 and occludin. Due to this antimicrobial activity, SCFAs have promising applications in food safety and human health.

14While many bacterial pathogens produce an array of virulence factors that affect the integrity of the epithelium during infection, in this review, we focused on those that affect TJ. For example, Entamoeba histolytica produces cysteine protease EhCP-A5 that induces secretion of IFN-γ, TNF-α, and IL-13, which correlates with an increase in TJ permeability by altered expression of claudin-2, occludin, and ZO-1. Helicobacter pylori transfers the cytotoxin-associated gene A (CagA) oncoprotein into gastric epithelial cells. CagA interacts with ZO-1 and junctional adhesion molecule, disturbing the composition and function of the apical junction complex. Clostridium difficile, the major cause of antibiotic-associated diarrhea and pseudomembranous colitis, produces exotoxin A (TcdA) and exotoxin B (TcdB), which inactivate specific Rho and Ras GTPases affecting downstream pathways, including cytoskeleton organization. The disruption of the cellular actin structure leads to the death of IECs, the loss of barrier function, and, consequently, a profound inflammatory response.

15Another toxin secreted by different human pathogens is zonula occludens toxin (Zot). Zot was first discovered in Vibrio cholerae, and has been described to mimic zonulin. Zot is also used by other pathogens, such as Campylobacter concisus and Neisseria meningitidis, to increase tissue permeability. V. cholerae Zot increases ZO-1 phosphorylation via PKCα, causing a selective disconnection of ZO-1 from occludin and claudin-1, opening the TJ, and increasing intestinal permeability. Zot induces cytoskeletal rearrangement, redistributing F-actin filaments and altering TEER in Caco-2 cell monolayers.

16Zot-type toxins have also been found in Vibrio parahaemolyticus and Vibrio vulnificus, both human pathogens. According to our recent studies, Zot would be associated with the actin cytoskeletal disturbances observed in Caco-2 cells after infection with a non-toxigenic strain of V. parahaemolyticus. On the other hand, it has been hypothesized that C. concisus disrupts gastrointestinal epithelium, driving a dysregulated immune response that results in chronic inflammation in the intestine. The analysis of samples from the oral cavity of patients with IBD showed the presence of two Zot-encoding genes in the C. concisus strains, suggesting that Zot may play a critical role in the pathogenesis of these bacteria. Studies by showed that Zot from C. concisus causes prolonged damage to the intestinal epithelial barrier, produces apoptosis, and increases the production of TNF-α and IL-8 by epithelial cells and macrophages. Thus, understanding how these pathogens disrupt the epithelial barrier is essential to propose alternative treatments to improve the symptoms of the disease.

17As discussed above, SCFAs can diminish the severity and span of intestinal infections caused by different pathogens, not only regulating bacterial virulence but also improving membrane barrier function enhancing TJ proper functioning. The generation of beneficial SCFAs in the gut can be optimized with nutritional interventions such as the intake of appropriate prebiotics (complex carbohydrates that can be fermented by enteric bacteria) and probiotics (living bacteria that promote gut health); however, there is limited evidence indicating clinical improvement. Additionally, oral formulations of butyric acid have been studied, such as tributyrin (a butyric acid triglyceride;; ). Some of the beneficial effects of probiotics are listed in Table 1.

18Overall, although studies demonstrate that SCFAs serve as protective and anti-inflammatory agents in the intestine, the underlying molecular mechanisms remain poorly understood. Therefore, it is of great interest to determine the mechanisms involved in the protective function of SCFAs, to develop new therapeutic strategies against bacterial toxins-triggered intestinal damage. In this sense, we propose that SCFAs, signaling through GPCRs, activate PKCβ which phosphorylates ZO-1, allowing its reconnection with anchor proteins and actin cytoskeleton, recovering the barrier function affected by Zot-like toxins (Figure 1).

19DP-R and KG conceived the idea. DP-R, CP, EK, and KG wrote the manuscript. All the authors read, discussed, and approved the final version of this manuscript.

20The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.