p40, which was originally isolated from the culture supernatant of Lactobacillus rhamnosus GG (LGG) (32), is the first recognized biologically active component of a Gram-positive commensal and probiotic bacterium, L. rhamnosus GG (LGG), for benefiting intestinal functional maturation and protecting IECs against inflammatory insults. Genes encoding proteins of the p40 cluster are mainly present in species related to the L. casei, L. paracasei, L. zeae and L. rhamnosus taxonomic group. In fact, p40 has been detected in culture supernatants of several strains of Lactobacillus (33–35). p40 homolog genes are also present in some species of the families Enterococcaceae and Streptococcaceae (36). C-terminal domain of p40 contains a histidine-dependent amidohydrolase/peptidase (CHAP) domain with cell wall hydrolase activity (33). Interestingly, p40 has been found to bind lipoteichoic acid (LTA) on the external surface of extracellular vesicles (EVs) released by Lactobacillus casei BL23 (37), suggesting a manner for secretion of p40 by bacteria. It is unknown that whether there is free form of p40 section.

Studies have revealed that p40 exerts immediate and long-lasting effects on IECs ( Figure 1 ) through two distinct mechanisms and these two functions are independent. The immediately effect of p40 is through transactivation of epidermal growth factor receptor (EGFR) and its downstream target, Akt, in IECs. In addition to direct ligand binding to EGFR for its activation, EGFR can be transactivated by other pathways that stimulate A Disintegrin and Metalloproteinase (ADAM)17-triggered releasing transmembrane EGFR ligands for binding to EGFR (38). Studies found that p40 up-regulated ADAM17 catalytic activity to stimulate membrane-bound heparin binding (HB)-EGF release in human and mouse intestinal epithelial cell lines and in mice (32). The biological consequences of EGFR transactivation by p40 have been unraveled, including inhibition of proinflammatory cytokine-induced apoptosis, preserving tight junctions in IECs (39, 40), and promoting mucin production by goblet cells (41). Furthermore, transactivation of EGFR by p40 not only stimulates protective roles on IECs but also induces innate immunity: p40 upregulates gene expression and protein production of proliferation inducing ligand (APRIL) gene expression in IECs, which is a cytokine involved in B cell class switching to IgA⁺ cells, thereby increasing IgA⁺ plasma cells and IgA production (42). EGFR activation contributes to multiple protective cellular effects in colitis (43). Strong evidence indicates that p40 transactivation of EGFR in IECs contributes to ameliorating colitis in mice (44).

Furthermore, EGFR signaling is required for postnatal growth (45). p40 significantly enhanced functional maturation of the intestine, including intestinal epithelial cell proliferation, differentiation, and tight junction formation, and IgA production in early life in wild-type mice, which is mediated by transactivation of EGFR in IECs (46). These results define a mechanism of p40 regulated immediate effect on protection of intestinal epithelium through induction of ADAM17-mediated EGFR ligand release, leading to transactivation of EGFR in IECs.

Colonization of the gut microbiota in a critical window in early life enables life-long health outcomes in humans and animals (47). It has been reported that p40 supplementation in early life stimulated long-lasting effects on TGF-β production by IECs. Two outcomes of this effects have been reported: promoting induction of differentiation of regulatory T cells (Tregs) in the lamina propria of the small intestine and the colon and protecting of epithelial barrier and inhibiting proinflammatory cytokine production in IECs. One ultimate result by early p40 supplementation is to prevent colitis in adulthood (44, 46).

The long-lasting effects of p40 has been related to its epigenetic modification of IECs. Epigenetic reprogramming refers to global remodeling of epigenetic marks via which the host identifies the microbial signal to convert them into the long-term specific cellular signal. In the IECs and immune cells, epigenetic modification permits the gut microbiota to regulate gene expression and cellular responses (48). The COMPASS complex contains methyltransferase and adaptors to activate target gene expression by catalyzing mono- and tri-methylation of histone 3 lysine 4 residue (H3K4me1/3) at enhancer and promoter sites (49). Setd1β, a methyltransferase in COMPASS complex, has a specific function in the assembly and regulation of H3K4 mono-, di and trimethylation. p40 has been found to upregulate Setd1β gene expression and protein production, which mediate programming the TGFβ locus into a transcriptionally permissive chromatin state and promoting TGFβ production in IECs. Interestingly, p40 supplementation in early life, but in adulthood, could induce sustained H3K4me1/3 in IECs toward TGFβ production (44). These results establish a novel mechanism involved life-long effects on maintain homeostasis by supplementation with commensal bacterium-derived factor in early life.

The gut bacteria can metabolite a compound of phytochemicals in dietary fiber-enriched meals into SCFAs, including acetate, butyrate, and propionate. In particularly, butyrate is rapidly absorbed from the intestinal lumen and is the preferred source of energy for colonic epithelial cells (50). Regulation of epithelial barrier by SCFAs has been reported through several mechanisms. Reinforcing the epithelial barrier and enhancing wound healing through promoting the expression of the actin-binding protein synaptopodin (SYNPO) has been identified as a role of a SCFA, butyrate (51). Butyrate decreases gut permeability by enhancing tight junction protein claudin-2 upregulated expression via an interleukin 10 receptor subunit alpha (IL-10RA)-dependent mechanism (52). SCFA produced by the symbiotic bacteria, Akkermansia muciniphila, Clostridium butyricum, and Faecalibacterium prausnitzii produce SCFAs in the intestinal tract function as inhibitors of histone deacetylases (HDACs) by upregulating histone acetyltransferases activity, while possessing anti-inflammatory and epithelial barrier maintenance effects in various animal models (53). As proven, the epigenetic effect of SCFAs as inhibitors of HDACs is mostly butyrate>propionate>acetate, which results in increased levels of histone acetylation, decondensation, and relaxing of chromatin (54). In vitro studies have shown that SCFAs have the potential to be the intestinal protection barrier by inhibiting the enzyme Histone Deacetylase (HDAC), which has preventative effects on DNA transcription, regulates gene expression, and increases the expression of MUC2, MUC1, MUC3, and MUC4 (55). Furthermore, both butyrate and propionate upregulated MUC2 transcript expression in LS174T cells. Analysis of the MUC2 promoter indicated that an active butyrate-responsive region comprising an AP1 (c-Fos/c-Jun) cis-element is necessary for the activation of MUC2 by acetylation and methylation of histones (56). Another study has shown that SCFAs strengthen the epithelial cell tight junctions, resulting in a robust and healthy intestinal barrier. Butyrate maintains and enhances the transepithelial electrical resistance (TEER) in Caco-2 cells that is mediated by AMP activated protein kinase (AMPK) (57).

SCFAs such as butyrate and propionate play significant roles in immunity through regulation of IECs and immune cells, such as T cells, macrophages, and dendritic cells. A report showed that SCFAs and a high-fiber diet were able to induce vitamin A metabolism in epithelial cells and CD103⁺ DCs and this was associated with enhanced Foxp3 expression in T cells (58). DCs have been found to play a significant role in the initiation of IgA production in the gut in response to SCFAs. Metabolite-sensing mammalian G protein-coupled receptor (GPR43) in DCs mediated the acetate induction of intestinal IgA response to microbiota, in that GPR43 knock out mice showed lower IgA production and decreased numbers of IgA+ B cells in the intestines (59).

In addition to normal IECs, SCFA plays roles in inhibition of colorectal cancer cell growth. Major anti-proliferative activity of SCFAs is associated with butyrate, acetate, and propionate (with acetate and propionate requiring higher concentrations to be as effective as butyrate). These SCFA compounds can promote apoptosis responses in colon cancer cells by influencing mitochondrial permeability potential, producing reactive oxygen species (ROS), and activating caspase-3 (27).

Indole and its derivatives are derived from the metabolism of tryptophan by the gut bacteria containing tryptophanase such as Lactobacillus reuteri and Clostridium sporogenes. The absorption of indole and its derivatives (Indole 3-propionic acid (IPA), indole-3-ethanol (IEA), and indole-3-acetaldehyde (IAAld) through the intestinal epithelium is due to the ability to freely diffuse through lipid membranes (60). Indole and its derivatives support intestinal immune homeostasis through activating aryl hydrocarbon receptor (AhR) to protect the intestinal tight junctional barrier. The activation of the AhR pathway in IECs is vital for protecting the stem cell niched and maintaining intestinal barrier integrity (61). The pregnane X receptor (PXR) is a physiologic regulator associated with gut permeability (62). IPA has been found as a ligand for epithelial PXR, and the administration of IPA can up-regulate tight junction protein-coding mRNAs and enhance the expression of claudins and occludins (63). Recent studies have showed that IEA, IPA, and IAAld contribute to maintaining the integrity of the intestinal tight junctional barrier in an AhR-dependent manner and alleviating dextran sodium sulfate (DSS)-induced colitis in mice (64). Indole and its derivatives enhance IL-10 expression through aryl hydrogen receptors (AhR) activation and promote IL-10 signaling which is linked with barrier function. Indole-3-aldehyde (IAld) increases epithelial cells proliferation and upregulates the differentiation of goblet cells, intestinal barrier integrity and downregulates the systemic inflammation caused by aging in geriatric mice. This effect increases the expression of the cytokine IL-10 via AhR but does not depend on the type I interferon or IL-22 signaling (65). The activation of AhRs leads to lL-22 transcription, which can further increase the expression of antimicrobial peptides and improve colonization resistance against Candida albicans in the gastrointestinal tract (66).

Overall, these studies support the feasibility of commensal bacterial metabolites as a strategy to promote mucosal barrier and intestinal homeostasis. Empirical modulation of the microbiota using probiotics can increase SCFAs and indole-producing bacteria for the maintenance of epithelial barrier integrity in inflammatory models (67). Thus, supplementation with specific probiotics for beneficial metabolites formation could provide new avenues to manage disease activity.

Bacterial surface layers contain ubiquitous proteins structures that are abundant in Gram negative and Gram-positive bacteria. In Gram-positive bacteria, the S-layer lattice is generally composed of a single protein and is attached to peptidoglycan-bound secondary cell-wall polymer by non-covalent interactions (68). As the outermost structure of the cell, the surface layer lattice is generally considered to be the first bacterial components that have a direct interaction with host cells. The effects of cell surface molecules are diverse and have been shown to play roles in many bacterial functionalities, such as adhesion to the host cells, strengthening of the gut barrier integrity, pathogen exclusion, stimulation of the host mucosal system to improve mucus production, and secretion of defense molecules such as β defensins (69).

Surface layer protein A (SPA-A) derived from Lactobacillus acidophilus NCK2187 binds to the C-type lectin SIGNR3 and initiates regulatory signals, leading to maintenance of healthy gastrointestinal microbiota, protecting gut mucosal barrier function, and prevention of colitis (70). Recent study revealed that SLP of Lactobacillus acidophilus NCFM strain led to a reduction in myeloperoxidase activity and TNF-α expression whereas significantly increased the IL-10 levels. The administration of these surface proteins significantly reversed the histopathological damages induced by the colitogens and improved the overall histological score in TNBS colitis mice model (71). Results from in vitro studies indicated that purified SLPs from L. plantarum exert a protective effect on Caco-2 cells infected with EPEC by increasing their transepithelial resistance (TEER) and down-regulating their permeability (72). Further, L. acidophilus contains three different SLPs, SLP-A, SLP-B, and SLP-X, which interact with pattern recognition receptors (PRRs) in IECs and modulate the immune response. L. acidophilus SLPs decrease interleukin (IL-8) secretion in Caco-2 cells stimulated by S. typhimurium (73).

Exopolysaccharide (EPS) are metabolic by-product of microorganisms (74). EPS consists of homopolysaccharides (HoPS) or heteropolysaccharides (HePS), depending on the main chain composition and mechanisms of synthesis. The most HePS-producing bacteria are Lactobacillus, Lactococcus, Streptococcus and Enterococcus strains frequently isolated from fermented dairy products and the human gastrointestinal tract (GIT), whereas most HoPS are produced by Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, and Weissella strains present in animal GIT, vegetables and fermented beverages (75). EPS-1 contributes to maintaining the intestinal barrier integrity against the disruption by lipopolysaccharide (LPS) in Caco-2 monolayer mediated by enhancing. the expression of tight junction. On the transcriptional level, LPS-decreased expression of several tight junction genes was inhibited by S. thermophilus-derived HePS in vivo (76). Further, the role of EPS in epithelial adhesion of commensal bacteria makes EPS of particular interest for preventing adhesion of pathogenic bacteria. It has been demonstrated that EPS produced by Lactobacillus paracasei subsp. Paracasei BGSJ2 plays an essential role in the prevention of adhesion of E. coli to Caco-2 cells (77). Further, the immunoregulatory effects of EPS on IECs are supported by the fact that EPS activates C-type lectin receptors on IECs to elicit an immunological response. Upon stimulation by EPS, IECs secrete several cytokines and chemokines, including interleukins, TNF, growth factors, and beta-defensins (78). Therefore, IECs play an essential role in the recruitment of dendritic cells, which are responsible for controlling both innate and acquired immunological responses (79). In addition, EPS has shown to mitigate experimental colitis, improving mucosal barrier function, and modulating gut microbiota composition (80–82). Moreover, recent studies have suggested that applying EPS from lactic acid bacteria to the skin enhances skin health and proven to be aid in gastrointestinal wound healing in different in-vitro and in-vivo studies (83, 84). It would be advantageous to consider EPS as a feasible prebiotic choice for therapeutic purposes due to its two feathers: EPS is indigestibility to the host cells, thus, arriving in the colon intact and consumed by specific gut microbiota in the colon (85).

Peptidoglycan (PGN) is a large polymeric molecule present in the Gram positive and Gram-negative bacterial cell wall. The basic overall structure of PGN is conserved between different organisms, but there are backbone and crosslinking modifications that increase the variability among the bacterial species. PGN in probiotic bacteria undergo variety of modifications in the sugar structure including deacetylation, O-acetylation, and N-glycosylation, which create the differences in sugar structure leading to the alteration in the properties of the cell wall (86). There are different receptors to detect the peptidoglycan or its fragments, for example, the innate immune system can detect PGN through peptidoglycan recognition proteins (PGLYRPs) (87), which are mostly expressed in eosinophils and neutrophils and could potentially act on inflammation (88). In the intestinal lumen, PGN-contained cell wall fragments can be released from commensal bacteria after digestion by Paneth cell-derived lysozyme. It has been suggested that PGN can be absorbed by crypt-based immature intestinal epithelial cells and in transported over the intestinal epithelium (89). Functional analysis has shown that PGN secreted by Lactobacillus and Bifidobacterium enhanced the expression of tight junction proteins, including claudins, occludin, and ZO-1 and improved the integrity of the gut barrier via Toll-like receptors 2 signaling (90, 91).

Lipoteichoic acid (LTA) is one of the major cell wall components of Gram-positive bacteria that can be considered the pivotal components for immunomodulating effects (92). In addition to its immunoregulatory effects, such as LTA from L. casei YIT9029 and L. fermentum YIT0159 cause TNF-α production in macrophages through TLR2 receptors (93), LTA from Lactobacillus plantarum confers anti-inflammatory responses in porcine intestinal epithelial cell line, IPEC-J2 (94).

This evidence supports the note that commensal bacterial surface components act through evolutionary-optimized mechanism that targets IECs to benefit intestinal homeostasis.