In this study, we compared two probiotic strains of L. gasseri, OLL2809 and L13-Ia, and found that these strains had distinctive abilities to modulate in vitro mechanisms of innate immunity and antioxidant/detoxifying defenses.
OLL2809 was isolated from human feces . The beneficial activity of this strain on mucosal inflammation has been previously shown in mice, where administration of OLL2809 was effective in reducing endometriotic lesions . L13-Ia was isolated from raw whole bovine milk and was considered a potential probiotic strain  as it survived a selective in vitro digestion protocol. Another probiotic property of these strains has been confirmed in this study (Table 1).
The intestinal microbiota interacts with the local immune system promoting mechanisms of intestinal homeostasis . Harnessing the contribution of probiotics to this physiological function has been proposed as a potential beneficial treatment for inflammatory bowel disease . The activity of these probiotic organisms is thought to be mediated by the interaction of microbe-associated molecular patterns (MAMPs) with pattern recognition receptors (PRRs) on antigen-presenting cells. In particular, the immune response against lactobacilli is dictated by conserved MAMPs . As a result of these interactions, some L. gasseri strains induce DCs to produce high levels of IL-10, IL-6, IL-12, and TNF-α . In line with these data, herein we showed that direct exposure of L. gasseri strains to DCs resulted in strong cytokine responses with no deviation toward a specific phenotype. Notably, the reported pro-inflammatory phenotype of mDCs derived from this mouse strain  was abrogated after challenge with both L. gasseri strains as IL-10 was also induced. Nevertheless, all of these cytokines may contribute to innate immunity by inducing the proliferation and differentiation of natural killer cells in vivo.
In functional experiments, we set the bacteria: eukaryotic cell ratio to 30:1 on the basis of a study showing that this proportion was optimal to stimulate cells . Using this protocol, a differential activity of the two L. gasseri strains was shown following bacteria challenge of mature DCs. This in vitro condition resembles the physiologic interaction occurring between bacteria and DC protrusions across the intestinal epithelium that reflects an active response to local commensal flora and bacterial products . In our experiments, the percentage of CD11b+CD11c+ DCs and the expression of co-stimulatory markers (CD40 and CD80) were increased following maturation. Intestinal lamina propria (LP) DCs are classified into CD11chiCD11bhi and CD11chiCD11blo DCs , which were found to be equivalent to CD103+CD8α- and CD103+CD8α+ LPDCs subsets, respectively . Interestingly, only OLL2809 sustained maturation of DCs in our experiments, leaving unchanged the percentage of CD11b+CD11c+ DCs and by increasing the expression of co-stimulatory markers.
We also examined the interaction of L. gasseri strains with intestinal epithelial cells (IECs). IECs, in addition to their metabolic functions, play a major role in the generation of innate immunity. To explore this function of IECs, we used a murine epithelial cell line (MODE-K) derived from the small intestine . We found that the two L. gasseri strains differentially influenced MODE-K cells. In particular, OLL2809 was more effective than L13-Ia in stimulating IL-6 secretion without inducing surface expression of MHC class II molecules. However, L13-Ia induced the expression of MHC class II, a phenomenon that allows IECs to stimulate CD4+ T cells during inflammation or in response to infection. Moreover, only SupOLL2809 induced IL-6 secretion in MODE-K cells, thus further highlighting the existence of distinctive responses elicited by these strains. The biological significance of the IL-6 increase remains controversial because this cytokine has both pro-and anti-inflammatory activities. Its receptor, IL-6R, is expressed on the surface of only a few cell types including hepatocytes and some leukocytes. The IL-6/IL-6R complex associates with gp130, which dimerizes and initiates intracellular signaling that triggers anti-inflammatory activities, such as inhibition of apoptosis and a parallel induction of proliferation in IECs . However, IL-6 trans-signaling appears to mediate the pro-inflammatory activity of this cytokine, a process involving the binding of the soluble form of IL-6R to gp130 on cells that do not express IL-6R . Our findings suggest that OLL2809 might contribute to gut immune homeostasis better than L13-Ia. Moreover, our results strengthen the concept that a probiotic activity can be induced not only from whole microorganisms and cell wall components but also from secreted metabolites. Stabilization of the enterocyte cytoskeleton was found to be mediated by a protease-sensitive metabolite secreted by the probiotic mixture VSL#3 . More recently, exposure to probiotic-conditioned media was shown to attenuate the inflammatory responses induced in different enterocyte models .
In the intestinal lamina propria, DCs are classically immature DCs that, following antigen encounter, migrate into mesenteric lymph nodes where they are primed. The existence of IEC-DC crosstalk has been suggested by observations showing that IECs can drive differentiation of Treg cell-promoting DCs. This differentiation is mediated by IEC-secreted transforming growth factor-β and retinoic acid . In agreement with these findings, we confirmed that medium conditioned by unstimulated MODE-K cells induced a regulatory phenotype in DCs, as shown by the reduced surface expression of co-stimulatory markers and, most importantly, reversal of the IL-12/IL-10 ratio. In the presence of a pro-inflammatory stimulus (i.e., treatment with TNF-α), this regulatory phenotype was abrogated, confirming that IEC-DC crosstalk is highly regulated. We further addressed this issue by evaluating the ability of L. gasseri strains to modulate the IEC-DC interaction. Our data indicate that both strains influenced IEC-DC crosstalk with distinct outcomes compared to those induced by SupMODE. In particular, these strains markedly enhanced the expression of co-stimulatory markers and downregulated IL-12, TNF-α and IL-10 secretions by mDCs. In addition, similar alterations were induced by SupOLL2809 and SupL13-Ia, thus excluding a synergistic effect of IECs. However, our model does not allow us to further elucidate this probiotic activity because MODE-K cells do not form a confluent monolayer, instrumental to analyze the different roles played by paracellular and transcytosis pathways . Taken together, our data suggest that the L. gasseri influence on IEC-DC crosstalk is dominant over IEC activity. Importantly, MODE-K cells and L. gasseri are able to produce different outcomes, regulatory mDCs and “low-responsive” mDCs, respectively.
Another beneficial effect on host immunity arising from the interaction between epithelia and commensal bacteria is the generation of reactive oxygen species that may activate the Nrf2 pathway and lead to improved antioxidant/detoxifying defenses . The Nrf2-Keap1 complex serves as an intracellular oxidative stress sensor, and Nrf2 release, triggered by mild ROS production, activates the synthesis of a battery of cytoprotective/defensive proteins including GSH, GST and NQO1 that protect cells against oxidative stress and promote cell survival . GSH plays a key role in the maintenance and regulation of the cell’s redox status. Our data showing opposing effects of bacterial strains on MODE-K cells’ and DCs’ intracellular GSH content are consistent with the reported pro-oxidant activity exhibited by probiotic strains  and with the improved ability of DCs to survive in an oxidant-rich environment . Under normal conditions, intracellular GSH levels depend upon the rates of GSH synthesis/oxidation and on GSH export from cells, and the GSH/GSSG pair is widely used as an indicator of redox status. Data from this study on MODE-K cells, according to the literature, indicates that the lack of intracellular GSSG accumulation is associated with depletion and increased export of intracellular GSH . In contrast, the increased intracellular GSH concentration accompanied by the increase in GSHtot export from the DCs without any measurable raise of intracellular GSSG concentration indicates the ability of DCs to respond to L. gasseri-induced oxidative stress by increasing GSH synthesis. These results, along with the results showing the improvement of GST and NQO1 activities in DCs directly exposed to L. gasseri strains or to conditioned supernatants from MODE-K cells, along with in vivo studies, further support the ability of bacterial strains to activate the Nrf-2 pathway [8, 9].