Strong tight junctions and curtailed inflammatory responses under stressful conditions are key for optimal digestive health. Bacillus -based probiotics are increasingly being used to maintain broilers' health, but their mode of action is often not well-defined. In the present study we used Caco-2 cells as a model for intestinal epithelia and assessed the effect of three Bacillus -based probiotics on intestinal barrier function and intestinal inflammation. Experimental results showed that one of the three tested strains, Bs 29784, significantly reinforced intestinal barrier integrity under basal conditions through an up-regulation of the expression of tight junction's proteins, whereas the others had no or detrimental effects. When Caco-2 cells were pre-treated with Bacillus subtilis strains, the subsequent IL-8 release to various pro-inflammatory signals (IL-1β, deoxynivalenol, or flagellin) was blunted compared to cells that had not been pretreated, but to a different extent depending on the strain of Bacillus used. Bs 29784, was able to significantly decrease IL-8 production in all stressed conditions tested. Mechanistically, Bs 29784 appeared to limit nuclear translocation of NF-κB during IL-1β exposure by preventing IκB degradation. The effects of Bs 29784 were observed independently with supernatant and cells but in a lesser extent than with the combination, indicating that they can thus likely be attributed to both secreted metabolites and cell-associated compounds. Moreover, under inflammatory conditions, Bs 29784 significantly reduced the upregulation of iNOS protein levels further underlining its intestinal anti-inflammatory potential. Our data show that Bacillus -based probiotics may indeed improve digestive health by strengthening intestinal barrier and limiting inflammatory responses and that these properties are strain-dependent.

B cells that bind antigens displayed on antigen-presenting cells (APCs) form an immune synapse, a polarized cellular structure that optimizes the dual functions of the B cell receptor (BCR), signal transduction and antigen internalization. Immune synapse formation involves polarization of the microtubule-organizing center (MTOC) towards the APC. We now show that BCR-induced MTOC polarization requires the Rap1 GTPase (which has two isoforms, Rap1a and Rap1b), an evolutionarily conserved regulator of cell polarity, as well as cofilin-1, an actin-severing protein that is regulated by Rap1. MTOC reorientation towards the antigen contact site correlated strongly with cofilin-1-dependent actin reorganization and cell spreading. We also show that BCR-induced MTOC polarization requires the dynein motor protein as well as IQGAP1, a scaffolding protein that can link the actin and microtubule cytoskeletons. At the periphery of the immune synapse, IQGAP1 associates closely with Factin structures and with the microtubule plus-end-binding protein CLIP-170 (also known as CLIP1). Moreover, the accumulation of IQGAP1 at the antigen contact site depends on F-actin reorganization that is controlled by Rap1 and cofilin-1. Thus the Rap1-cofilin-1 pathway coordinates actin and microtubule organization at the immune synapse.

Although it is known that a stiffening of the stroma and the rearrangement of collagen fibers within the extracellular matrix facilitate the movement of tumor cells away from the primary lesion, the underlying mechanisms responsible are not fully understood. We now show that this invasion, which can be initiated by applying tensional loads to a three-dimensional collagen gel matrix in culture, is dependent on the Rap1 GTPases (Rap1a and Rap1b, referred to collectively as Rap1). Under these conditions Rap1 activity stimulates the formation of focal adhesion structures that align with the tensional axis as single tumor cells move into the matrix. These effects are mediated by the ability of Rap1 to induce the polarized polymerization and retrograde flow of actin, which stabilizes integrins and recruits vinculin to preformed adhesions, particularly those near the leading edge of invasive cells. Rap1 activity also contributes to the tensioninduced collective invasive elongation of tumor cell clusters and it enhances tumor cell growth in vivo. Thus, Rap1 mediates the effects of increased extracellular tension in multiple ways that are capable of contributing to tumor progression when dysregulated.

The intestinal epithelium plays a variety of roles including providing an effective physical barrier and innate immune protection against infection. Two-dimensional models of the intestinal epithelium, 2D enteroids, are a valuable resource to investigate intestinal cell biology and innate immune functions and are suitable for high throughput studies of paracellular transport and epithelial integrity. We have developed a chicken 2D enteroid model that recapitulates all major differentiated cell lineages, including enterocytes, Paneth cells, Goblet cells, enteroendocrine cells and leukocytes, and self-organises into an epithelial and mesenchymal sub-layer. Functional studies demonstrated the 2D enteroids formed a tight cell layer with minimal paracellular flux and a robust epithelial integrity, which was maintained or rescued following damage. The 2D enteroids were also able to demonstrate appropriate innate immune responses following exposure to bacterial endotoxins, from Salmonella enterica serotype Typhimurium and Bacillus subtilis. Frozen 2D enteroids cells when thawed were comparable to freshly isolated cells. The chicken 2D enteroids provide a useful ex vivo model to study intestinal cell biology and innate immune function, and have potential uses in screening of nutritional supplements, pharmaceuticals, and bioactive compounds.

Bacterial debris in the gastrointestinal tract (GIT) are continuously being produced by the microbiota present upon bacterial division and death. One of the most abundant components in bacterial debris are peptidoglycans (PGN), a structural cell wall component in gram- positive and negative bacteria. The objective of this work was to investigate if addition of a novel microbial muramidase (Muramidase 007; MUR) that hydrolyzes PGN, would reduce PGN adhesion to porcine intestinal cells in-vitro and hydrolyze PGN in the GIT of swine. Adhesion efficacy of intact and MUR hydrolyzed fluorescein-isothiocyanate (FITC)-labelled PGN were compared using fluorescence-microscopy (3 wells/condition). Catalytic performance of MUR on intact FITC-labelled PGN adhered to intestinal cells were also tested. In-vivo MUR-supplementation at 50,000 LSU/kg diet to gestating and lactating sows and/or their subsequent offspring for 42-d post-weaning was investigated. Mass-spectroscopy was used to quantify soluble and total muramic acid, which is only found in PGN, in the ileal and cecal digesta (8 piglets/treatment) to calculate percentage soluble-PGN. Cell-culture data were analyzed using GraphPad-Prism 8.0 and in-vivo data using mixed-models in JMP 14.0. MUR hydrolyzed PGN adhered 10x less to the IPEC-J2 cell line culture compared to intact-PGN (P< 0.05). In addition, data show that MUR hydrolyzed PGN attached to cell surfaces by 2x, as attached PGN were also reduced by 50% following MUR incubation (P< 0.05). Offering sows MUR-diets had no carryover effect on the percentage soluble PGN in their piglets’ digesta, and there were no interactions observed for sow x piglet x tract neither (P >0.05). Percentage soluble-PGN increased in piglets fed MUR compared to control-piglets (47.18 vs. 29.84% SEM:1.624; P< 0.001) and was higher in cecal digesta compared to ileal digesta (49.91 vs. 27.11%; SEM:1.634; P< 0.001), irrespective of MUR-supplementation. These results suggest that MUR may reduce PGN adhesion to intestinal cells and may hydrolyze PGN found in the GIT.