Surface Layer of Lactobacillus helveticus MIMLh5 Promotes Endocytosis by Dendritic Cells

Abstract

Surface layers (S-layers) are proteinaceous arrays covering the cell walls of numerous bacteria. Their suggested properties, such as interactions with the host immune system, have been only poorly described. Here, we aimed to elucidate the role of the S-layer from the probiotic bacterial strain Lactobacillus helveticus MIMLh5 in the stimulation of murine bone-marrow-derived dendritic cells (DCs). MIMLh5 induced greater production of interferon beta (IFN-β), interleukin 10 (IL-10), and IL-12p70, compared to S-layer-depleted MIMLh5 (naked MIMLh5 [n-MIMLh5]), whereas the isolated S-layer was a poor immunostimulator. No differences in the production of tumor necrosis factor alpha (TNF-α) or IL-1β were found. Inhibition of the mitogen-activated protein kinases JNK1/2, p38, and ERK1/2 modified IL-12p70 production similarly in MIMLh5 and n-MIMLh5, suggesting the induction of the same signaling pathways by the two bacterial preparations. Treatment of DCs with cytochalasin D to inhibit endocytosis before the addition of fluorescently labeled MIMLh5 cells led to a dramatic reduction in the proportion of fluorescence-positive DCs and decreased IL-12 production. Endocytosis and IL-12 production were only marginally affected by cytochalasin D pretreatment when fluorescently labeled n-MIMLh5 was used. Treatment of DCs with fluorescently labeled S-layer-coated polystyrene beads (Sl-beads) resulted in much greater uptake of beads, compared to noncoated beads. Prestimulation of DCs with cytochalasin D reduced the uptake of Sl-beads more than plain beads. These findings indicate that the S-layer plays a role in the endocytosis of MIMLh5 by DCs. In conclusion, this study provides evidence that the S-layer of L. helveticus MIMLh5 is involved in endocytosis of the bacterium, which is important for strong Th1-inducing cytokine production. IMPORTANCE Beneficial microbes may positively affect host physiology at various levels, e.g., by participating in immune system maturation and modulation, boosting defenses and dampening reactions, thus affecting the whole homeostasis. As a consequence, the use of probiotics is increasingly regarded as suitable for more extended applications for health maintenance, not only microbiota balancing. This implies a deep knowledge of the mechanisms and molecules involved in host-microbe interactions, for the final purpose of fine tuning the choice of a probiotic strain for a specific outcome. With this aim, studies targeted to the description of strain-related immunomodulatory effects and the identification of bacterial molecules responsible for specific responses are indispensable. This study provides new insights in the characterization of the food-origin probiotic bacterium L. helveticus MIMLh5 and its S-layer protein as a driver for the cross-talk with DCs.

Probiotics Modulate Mouse Gut Microbiota and Influence Intestinal Immune and Serotonergic Gene Expression in a Site-Specific Fashion

“…Other significant modulations of the expression of genes involved in intestinal immune responses were observed. L. helveticus MIMLh5 induced an increase of TNF-α in the cecum, which partly confirms the immunostimulatory attitude of this bacterium observed in vitro ( Taverniti et al, 2012 , 2013 , 2019 ). In addition, B. bifidum MIMBb23sg and L. paracasei DG impacted on the gene expression of iNOS in the ileum, an effect already observed for DG in ex vivo investigations on colonic biopsies ( Compare et al, 2017 ).…”

“…Specifically, we gavaged wild type mice with the viable cells of three bacterial strains representative of the taxonomic groups most commonly used as probiotics, including L. paracasei DG, a strain employed in commercial preparations available in Europe, America, and Asia, which has been demonstrated to efficiently survive the gastrointestinal (GI) transit in adult and pediatric populations ( Arioli et al, 2018 ; Radicioni et al, 2019 ), affect the gut microbiota taxonomic composition and butyrate levels in healthy adults and irritable bowel syndrome (IBS) patients ( Ferrario et al, 2014 ; Cremon et al, 2018 ), modulate the immune response in IBS and ulcerative colitis patients ( D’Inca et al, 2011 ; Cremon et al, 2018 ), and maintain remission of diverticular disease ( Tursi et al, 2008 , 2013 ). In addition, we tested Lactobacillus helveticus MIMLh5, a bacterial strain of dairy origin used in commercial paraprobiotic formulations available in Europe, which was shown in vitro to display antagonistic activity against pathogens and immunomodulatory properties on epithelial and antigen presenting cells ( Guglielmetti et al, 2010 ; Taverniti et al, 2012 , 2013 , 2019 ); in addition, MIMLh5 was shown during a clinical trial to contribute to the prevention of recurrent upper respiratory tract infections in adults and children with recurrent rhinopharyngitis ( Cavaliere et al, 2020 ). Finally, B. bifidum strain MIMBb23sg has been studied here as a representative member of its species, which possesses marked in vitro ability to efficiently adhere to Caco-2 enterocytes, utilize mucin as its sole carbon source, catabolize human milk oligosaccharides, and modulate cytokine expression in bone marrow derived dendritic cells and Caco-2 enterocytes.…”

Dairy starters and fermented dairy products modulate gut mucosal immunity

“…In L. helveticus MIMLh5, S-layer protein SlpA mediates the stimulatory effect on innate immunity [138]. In murine bone-marrow-derived dendritic cells, L. helveticus MIMLh5 induced greater production of IFN-β, IL-10, and IL-12p70, compared to S-layer-depleted MIMLh5, and S-layer promotes endocytosis by these dendritic cells [137]. In stimulated human monocyte derived dendritic cells, different strains of L. helveticus, isolated from Italian hard cheeses, induced release of both the pro-inflammatory cytokine IL-12p70 and the anti-inflammatory cytokine IL-10, while corresponding cell-free culture supernatants triggered a dose-dependent decrease of IL-12p70 and an increase of IL-10 [149].…”

The effect of microbial metabolites from colonic protein fermentation on bacteria-induced cytokine production in dendritic cells

“…BMDCs were generated from C57BL/6NTac mice (Taconic, Lille Skensved, Denmark) as described previously. [57,58] Briefly, cells were isolated from the tibia and femur by flushing the bones with cold PBS, after which cells were seeded at 3 * 10 5 cells/mL in RPMI 1640 medium containing 10 % (v/v) heat-inactivated FBS, L-glutamine (4 mM), penicillin (100 U/ mL), streptomycin (100 U/mL), and 2-mercaptoethanol (50 μM). On days 3 and 6, culture medium containing 15 ng/ml granulocyte-macrophage colony stimulating factors (GM-CSF) was added to Petri dishes for DC differentiation.…”