The human microbiome assumes a pivotal role in influencing human health and disease states (1). An increasing number of studies suggest that the beneficial effects associated with the establishment of a symbiotic gut microbiota are predominantly mediated by the metabolic by-products generated by microbial activity.

In 2012, the term “postbiotics” was coined to refer to “any soluble factor resulting from the metabolic processes of live bacteria or any released molecule capable of conferring health benefits through both direct and indirect mechanisms” (2). Since then, extensive research has been conducted on postbiotics and their effects, primarily dissecting their impact within the gut, but also on the skin. It is well-established that bacterial metabolites can cross the gut vascular barrier (3) and have been detected in the bloodstream (4) . Indeed, short-chain fatty acids (SCFAs), commonly produced by bacteria, have been detected in the blood (5) and their influence on inflammatory signaling pathways has been thoroughly investigated (4). Consequently, postbiotics hold promise as a therapeutic strategy for maintaining gut homeostasis.

The gastrointestinal tract serves as the primary interface between the body's internal environment and external surroundings (6). It comprises specialized barriers, such as the mucus layer, intestinal epithelial layer, and the lamina propria housing mucosal immune cells. These barriers dynamically receive and respond to various stimuli (7) .

Among them, the intestinal epithelial barrier (IEB) plays a crucial role in preventing harmful antigens and microorganisms from entering while facilitating the absorption of nutrients and water. Maintaining this delicate balance is essential for human well-being and is tightly regulated. The functionality of the IEB relies on intercellular junctions, namely the apical junctional complex, which includes tight junctions (TJs), adheres junctions (AJs), and desmosomes (8), (9).

TJs are a multi-protein complex, that are responsible for controlling the passage of antigens through the IEB and have a key role in maintaining barrier integrity. Various factors such as cytokines, gut microbiota, and dietary components influence intestinal TJs (10), (11).

Finally, the gut vascular barrier (GVB) is the deepest layer of the intestinal barrier. The GVB refers to a specialized structure within the gastrointestinal tract that regulates the passage of molecules, ions, and cells between the intestinal tissue and the bloodstream (12). When this barrier is compromised, endothelial cells can become damaged, and intestinal permeability can increase, allowing toxins, pathogens, and other harmful molecules to easily cross the intestinal mucosa and reach the bloodstream.

Recent researches have extensively demonstrated the IEB dysregulation can facilitate the passage of harmful substances through the intestine, which can then interact with the GVB favoring the occurrence of numerous intestinal and extra-intestinal disorders (13). In these conditions, dysfunction of the IEB leads to increased intestinal permeability, allowing antigens and microorganisms to enter the circulation and disseminate to distant organs (14). This process activates the immune system, triggering and sustaining chronic low-grade inflammation, typical of these disorders (15).

Given the link between intestinal permeability and numerous conditions, postbiotics can represent an innovative strategy to prevent or reduce intestinal permeability (Figure 1).

Description of the leaky gut syndrome and consequent pathologic conditions

The preservation of both epithelial (IEB) and endothelial (GVB) barrier function is essential to prevent various pathologic conditions, such as LGS, characterized by the passage of harmful agents, such as bacteria, toxins, and viruses, into the bloodstream (17). However, factors like stress, an unhealthy diet, alcohol, antibiotics, and certain medications can disrupt the balance of intestinal microbiota and compromise the integrity of the intestinal barrier, leading to increased intestinal permeability. LGS is characterized by chronic inflammation and altered tight junctions (TJs) in the intestine increasing intestinal hyperpermeability allowing harmful agents to cross the intestinal epithelium and enter the bloodstream, affecting various organs and systems. LGS and intestinal barrier dysfunction are associated with both intestinal diseases, such as inflammatory bowel disease (IBD) (17), (18), (19), encompassing Crohn’s disease (CD) and ulcerative colitis (UC), and irritable bowel syndrome (IBS) (20), celiac disease (21), (22) and extra-intestinal pathological conditions, including heart disease and metabolic disorders (obesity, type 2 diabetes and NAFLD/NASH (23), (24), (25)). Recently, also neurodegenerative disorders have been associated to the LGS (26), (27), (28), (29).

Consistently, IBS patients exhibit reduced levels of TJ proteins and elevated cytokine levels (20); patients affected by metabolic disorders (Obesity, type 2 Diabetes and NAFLD) display gut microbiota dysbiosis, microbial translocation and intestinal barrier disfunctions associated with changes in TJs integrity, and gut permeability (30) (31). Chronic heart failure (CHF) patients exhibit increased bowel permeability, leading to bacterial translocation and inflammation (32).

These findings underscore the critical role of intestinal permeability in various pathological conditions and highlight potential therapeutic targets for managing these diseases.

Microbiota-modulating therapeutic strategies to counteract the leaky gut

Reinforcing the intestinal barrier, in particular restoring the paracellular pathways stringency, mainly targeting TJs, has been suggested as a therapeutic strategy to restore intestinal homeostasis and prevent related-disorders.

Two major regulators of intestinal barrier have been identified in diet - prebiotics, and secondly within the intestinal microbiota – probiotics. Both players are related to lifestyle, suggesting that environmental factors might have an impact on intestinal barrier and gut health (33).

Nutritional interventions play a significant role in managing the leaky gut syndrome, with strategies focusing on avoiding sugar and fat-rich diets while incorporating elements like prebiotics and probiotics.

High-fiber intake has shown beneficial in various chronic diseases as fiber fermentation by gut microbiota produces valuable molecules like SCFAs, glutamine and vitamins among others, which play a crucial role in protecting intestinal barrier integrity (34), (35), (36). However, assumption of fibers is often associated with production of gas, leading to abdominal pain in susceptible individuals such as IBS patients.

Regarding intestinal bacteria and probiotics, various strains have been found to directly or indirectly influence intestinal barrier function (37). Probiotic supplementation, like Bifidobacteria and Lactobacillae species, has shown promise in increasing the expression of TJ proteins and improving transepithelial resistance (38) (39). These probiotics can protect against acute colitis and revert inflammatory dysfunctions induced by cytokines such as TNF-alpha and IFN-gamma, thus improving barrier function in both humans and mice (40). Research into leaky gut treatments emphasizes the potential of functional foods and ingredients supporting an improvement of intestinal barrier function and reducing permeability.

Focus on the importance of finding innovative therapeutic strategies to prevent and revert intestinal barrier damage

Postbiotics are considered safe as they do not contain live bacteria, mitigating concerns associated with administering live microorganisms that might translocate across a damaged intestine (41) This innovative safety aspect offers a targeted approach to address LGS without the risks associated with live bacteria on fragile populations. Differently from probiotics that need to find the right environment to release beneficial metabolites, postbiotics provide an immediate and reparative action on both the impaired intestinal barrier and microbiota diversification (42) (43).

For instance, recent research has underscored the significant beneficial role of a specific bacterial strain, Akkermansia muciniphila (44), in reinforcing gut barrier (45) function across various conditions marked by heightened inflammation, such as obesity (46) (47), metabolic disorders (48) and autoimmune diseases (49). Researchers have later on demonstrated that this microorganism mainly exhorts its beneficial functions through the production and release of diverse metabolites, including proteins, short-chain fatty acids like acetate and propionate (50), but also through cell envelope components like phosphatidylethanolamine and peptidoglycans (47). These observations were further confirmed by a clinical study in obese patients reporting how daily administration of the pasteurized form of this microorganism markedly improves patients’ metabolic parameters, indicating its potential as a therapeutic intervention (46).

Hence, microorganism derived metabolites are identified as the true contributors to the beneficial effects associated with probiotics, and their ability to be administered at higher concentrations without the necessity for colonizing the gut provides significant flexibility in dosage and delivery. Further, as they derive from microbial fermentation, differently from prebiotics are devoid of side effects such as gas production. The selectivity of postbiotics can be tailored for specific applications, enhancing their efficacy in targeted treatment for disorders characterized by an impairment of the intestinal barrier integrity.

Effects of postbiotic in the treatment of Chron disease associated intestinal barrier damage in ex-vivo models

IBD is characterized by excessive inflammation in various parts of the intestinal wall, leading to mucosal damage, ulcerations, and fibrosis.

Probiotics have garnered attention for their potential therapeutic role in IBD, but caution is advised, as the efficacy of probiotics varies depending on the bacterial strain and clinical context. Clinical studies have highlighted potential risks associated with administering probiotics to patients with acute inflammation (51).­­­

In our laboratory, we tested the immunomodulatory properties of three different probiotic strains, Lactobacillus paracasei B21060, Lactobacillus rhamnosus GG (LGG) and Lactobacillus plantarum NCIMB8826, on tissues derived from Chron patients using a polarized organ culture system (2).

In healthy tissues, inoculation with L. paracasei B21060 or L. rhamnosus GG (LGG) did not result in significant changes, with the cytokine secretion profile remaining similar to negative control samples. Surprisingly, stimulation with L. plantarum NCIMB8826 led to tissue deterioration, characterized by immune cell homing towards the epithelial layer, along with an upregulation of key inflammatory molecules such as CCL4 and Interleukin (IL)-1 beta. This suggests that in inflamed tissues with potentially increased intestinal permeability and bacterial translocation, otherwise harmless bacteria may exacerbate inflammation. By contrast, strains that were harmless on healthy tissues exacerbated the inflammation on IBD tissues. Therefore, caution is warranted when considering probiotic use in patients during the acute phase of IBD.

To overcome this challenge, Lactobacillus paracasei B21060 was used to produce postbiotics and the preparation was tested to explore its activity in ex-vivo organ culture after Salmonella thyphimurim (S. thyphimurium) infection of healthy tissues or on Crohn’s disease samples. Treatment with postbiotic resulted in reduction of TNF secretion paired by a reduction in ulceration of the epithelium and glandular destruction, histological features altered by S.thyphimurium infection.

When tested on tissue from CD patients, evaluation of NF-kB activation, a pathway associated with TNF production, showed reduced p65 translocation to the nucleus in samples treated with postbiotic, indicating a release of the inflammatory condition. Overall, these findings highlight the potential of postbiotics, (derived from L. paracasei B21060), to modulate inflammatory responses without compromising protecting anti-inflammatory mechanisms.

Effects of postbiotic in the prevention of Salmonella thyphimurium induced intestinal barrier disruption

To test the direct effect of postbiotics on IEB upon colonic epithelium injury induced by the pathogenic agent (S. thyphimurium) we performed bot​​​​​​​h in vitro and in vivo studies (52).

Caco-2 cells were used to mimic the intestinal epithelial barrier in vitro and transepithelial electrical resistance (TEER) was used as an indicator of intestinal epithelial integrity. Treatment with postbiotic-derived from Lactobacillus paracasei CNCM I-5220 prevented S. thyphimurium barrier disruption, maintaining TEER values similar to those of non-infected cells.

Further analysis focused on TJs highlighted the ability of postbiotic-derived from L. paracasei CNCM I-5220 to protect TJ structures, altered by S. typhimurium infection. Importantly, the postbiotic did not exhibit direct antibacterial effects, but was able to restore the altered morphology of tight junctions after exposure to pathogen.

Intestinal barrier protective effect of postbiotic-derived from L. paracasei CNCM I-5220 was also evaluated in vivo. Mice treated orally for 10 days with the postbiotic showed reduced bacterial translocation to the colon and liver upon infection with S. typhimurium compared to controls. Moreover, the postbiotic preserved the integrity of the GVB and restored mRNA expression levels of α-Defensin and TGF-beta, markers of antimicrobial peptide release and anti-inflammatory response, respectively. Additionally, the postbiotic maintained gut microbiota composition preserving the abundance of the Ruminococcaceae family which was reduced upon S. typhimurium infection. These findings suggest that the postbiotic protects the intestinal barrier both in vitro and in vivo preventing pathogenic bacterial translocation.

Furthermore, the postbiotic was shown to directly interact with S. typhimurium, blocking biofilm formation and reducing bacterial pathogenicity. Additionally, biosurfactants isolated from the postbiotic, replicated the inhibitory effect on S. typhimurium biofilm formation exerted by the whole postbiotic, suggesting a direct role of these molecules in antibiofilm activity.

These findings provide evidence that postbiotics can have a protective role in preserving the integrity of IEB.

Dysregulation of the IEB has been linked to numerous intestinal and extra-intestinal disorders, characterized by increased intestinal permeability and chronic inflammation. These findings together suggest that postbiotics hold promise as a non-pharmacologic strategy for preventing and mitigating the leaky gut syndrome and its associated disorders, offering a novel approach to maintaining gut homeostasis and overall health.

Further studies are needed to dissect the full potential and molecular mechanisms at the base of protective effects of postbiotics on gut health.

Clinical studies should be performed to assess both the efficacy of postbiotics in the treatment of patients affected by LGS and as a possible combined therapeutic strategy to current therapies for pathologies characterized by mucosal barrier disruption extending their possible use in extra-intestinal disorders.