The role of probiotic and commensal bacteria in the physiology of the gastrointestinal tract is incompletely understood, mostly because of the complex and intricate array of cellular and molecular partners involved. In particular, the mechanisms underlying the beneficial effects of probiotics, as well as the impact on epithelial maturation and communication with the mucosal immune system are still in need of investigation. Using polarized Caco-2 cell monolayers, we found that incubation with the probiotics LPR and BL led to modifications of several features including adhesion, permeability, and signaling events involved in NF-κB nuclear translocation, production of pIgR, and induction of immune mediators. The further novelty of our data resides in the demonstration that binding of nonspecific SIgA to bacteria potentiates their effect on selected events associated with adhesion and cell signaling, reflecting that different sensing pathways could be identified in the in vitro setting used inhere.
Preferential adhesion of LPR, BL, and Nissle 1917 probiotics strains to Caco-2 cell monolayer in comparison with E. coli TG1 is in agreement with previous studies using similar in vitro systems. Our data allowed us to demonstrate that SIgA increased adhesion to Caco-2 epithelial cells grown as polarized monolayers. Such an effect can find an explanation in the surface expression of epithelial CD71 (the transferrin receptor), which exhibits SIgA binding properties (29), as anchoring of the Ab through mucin cannot occur in Caco-2 cells unable to produce mucus. Human SIgA purified from colostrum, when combined with bacteria, displayed the same potentiating effect on adhesion to polarized CaCo-2 cells monolayers.3 Unexpectedly, enhanced adhesion did not translate into further increased TER, which ranged in between 18–25% for all conditions tested. This supports the hypothesis that SIgA-mediated enhanced adhesion of bacteria to Caco-2 cells does not necessarily improve permeability, as confirmed by the stability in TER observed upon incubation of Caco-2 cells with 5 times less or 5 times more bacteria. Consistent with this, phosphorylation of ZO-1 and occludin linked to epithelial tightness was the same for bacteria and SIgA-based complexes, suggesting that improved adhesion to Caco-2 cells might he consequences in other pathways, pathways we identified in this work.
Infection of epithelial cells with pathogenic microbes including S. flexneri induces rapid degradation of IκBα, resulting in the release of the NF-κB complex that translocates to the nucleus where it triggers transcription of a variety of genes required for immune responses (30). In contrast, exposure to LPR or BL only partially induced nuclear translocation of the transcription factor, an effect potentiated by SIgA. This indicates that exposure of epithelial cells to certain probiotic microorganisms such as LPR and BL maintains a low, if not basal, degree of NF-κB activation that may be instrumental for the maintenance of homeostasis (31). This is with keeping in mind that although a central regulator in gene expression, NF-κB acts in concert with a plethora of other transcription factors to promote optimal and cell-specific transcriptional control of multiple target genes. Consistent with this, induction of NF-κB after exposure to BL or SIgA-probiotic complexes did not lead to the production by Caco-2 cells of chemokines involved in the recruitment of pro-inflammatory cells. However, production of TSLP involved in maintaining an intestinal noninflammatory environment (25) was promoted by Caco-2 cells incubated with either LPR or BL, and significantly further enhanced when complexed with SIgA.
Efficient transport of IgA from the lamina propria into mucosal secretions is mediated by pIgR produced by epithelial cells (23). Up-regulation of intestinal pIgR mRNA expression has been reported in formely germ-free mice colonized with the commensal Bacteroides thetaiotamicron (32). In comparison with LPR or BL alone, we found that exposure of Caco-2 cell monolayers to SIgA-probiotic complexes triggered production of the pIgR protein. Increased production of pIgR that in turn would augment IgA translocation can be seen as an additional step in controlling the microbiota through a regulatory loop mechanism implying both innate and adaptive immunity.
Moreover, the data presented provide a mechanistic explanation to the underestimated function of natural SIgA in the regulation of the endogenous microbiota, both preweaning through maternal Ab, and after weaning through early, low affinity, Ab responses induced in the neonates (7). Indeed, by intervening in the complex interactions governing host-microbe cross-talk, SIgA may contribute to regulate epithelial cell responses to their environment. Maternal SIgA, by coating gut commensal bacteria in the neonatal intestine, would control initial exposure to the developing immune system and thus subsequent maturation. This may keep the level of gut-colonizing bacteria at bay until sufficient amounts of endogeneous SIgA can be produced by the neonate at the time of weaning.
“Natural” existence of bacterium-SIgA complexes anytime may contribute to maintain commensals in close association with the epithelium, and guarantee self-limiting control of microorganisms permanently colonizing the gut (33). We postulate that such a monitoring mechanism would promote optimal gut microbial colonization and ensures a dynamic and plastic interplay with epithelial cells. This adds to the already documented role of SIgA in limiting dissemination of microorganisms in the gastrointestinal lymphoid tissue (17, 34), and the immunomodulatory properties of SIgA in the intestine (35–37). Together, this will result in the onset of modulatory pathways crucial to maturation of both the epithelium and innate and adaptive immunity.
Our results further unrel that intimate association of microorganisms with SIgA potentiates the communication with epithelial monolayers to different degrees, as reflected by the observation that not all features examined were subject to changes, and that differences between Lactobacillus LPR and Bifidobacterium BL were indeed identified. The observation that the mucus-binding protein of another Lactobacillus species exhibits IgG and IgA binding activity might suggest a track to explore to better understand the complexity of interactions that ultimately lead to gut and mucus adhesion (38). Maintenance of intestinal integrity and proper functioning requires that harmonious microbial-epithelial interactions occur, and our novel data reveal the functional importance of SIgA in participating to this complex homeostatic balance.