Abstract Probiotic bacteria are live organisms, if consumed in adequate amounts might confer health benefits. These bacteria, such as Lactic acid bacteria (LAB), include a number of strains that have specific health promoting activi-ties, attributed to their immunomodulatory and anti-inflammatory properties. Gut mucosal macrophage subsets play a fundamental role in driving muco-sal immune responses. These include, tolerance, associated with an M2-, regulatory macrophage phenotype and inflammatory activation with an M1-like phenotype. The cross-link between mucosal tolerance and inflammatory cytokine suppression, and augmentation of IL-10 production in the gut relate to endotoxin tolerance. Endotoxin tolerance is a context; it could present an example for cell drive through a hypo-responsive state. An example is mu-cosal inflammatory pathologies, such as Crohn’s disease. When tolerance is broken, causing the destruction of gut mucosal tissue. This is where the macrophage phenotype, has been transformed from a regulatory M2- to an inflammatory M1-like phenotype. This is seen as a reaction to both, patho-genic and commensal bacteria. This investigation was aimed at assessing the activities of live probiotic bacteria; Lactobacillus salivarius strain MS13 and Lactobacillus plantarum strain C28 in the immunomodulation of macro-phage subsets in health, inflammation, and endotoxin tolerance. M1- and M2-like macrophages were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and Vitamin D3, respectively. Additionally, differentiated epithelial cells (Caco-2) were obtained by long term culturing for 21 days. The role of Lactobacillus strains C28 and MS13 to modulate epi-thelial barrier integrity and macrophage-epithelial cell inflammation was in-vestigated. TNFα, IL-1β, IL-18, IL-23, IL-12, IL-6, IL-8, and IL-10 were quanti-fied by ELISA and RT-PCR, whereas TLR-2, TLR-4, Tollip, SOCS3, STAT3 and TRAIL by RT-PCR. This study revealed that, first, live C28 and MS13 stimulated the proinflammatory cytokine by M2-like macrophages as well as the anti-inflammatory cytokine in a homeostatic status; whereas in an in-flammatory environment, C28 and MS13 differentially upregulated TNFα and IL-1β by M1 and M2-like macrophages induced by E.coli K12-LPS. Both strains downregulated K12-LPS induced IL-10 by M2-like macrophages. The response of stimulated M1 and M2 macrophages to C28 and MS13, was to differentially induce the gene expression of TLR-2, TLR-4, Tollip, NLRP3, SOCS-3, STAT3 and TRAIL. Second, the repeat-stimulation/tolerisation of M1 and M2 macrophages by live probiotic bacteria revealed, TNFα, IL-1β, IL-23, IL-18, IL-6 and IL-10 were upregulated in M1-like macrophages by C28, whereas MS13 upregulated TNFα, IL-1β, IL-18, and downregulated IL-12, IL-6, and IL-10. On the other hand, the tolerisation of M2-like macrophages by C28 and MS13 resulted in the downregulation of TNFα and IL-12p35 and upregulation of IL-1β, IL-18, IL-23, IL-12, IL-6, and IL-10. These findings were linked with the differential macrophage subset upregulation of TLR-4, NLRP3, STAT-3 and TRAIL gene expression. On the other hand, TLR-2, Tol-lip and SOCS-3 were downregulated in tolerised macrophage subsets by C28 and MS13. Furthermore, the role of lactobacilli strains C28 and MS13 in the modulation of endotoxin tolerance was to; upregulate TNF-α, IL-18, IL-23 and IL-10 by M1 and M2-like macrophages. This investigation also focused on the induction of the zona-occludin-1 (Zo-1), human β defensin-2 (hBD-2), and cytokine production IL-8 by Caco-2 cells. Trans epithelial electrical re-sistance (TEER) and RT-PCR measured the main cytokines studied pro-duced by Caco-2, were IL-8, also the epithelial barrier function. Live probiotic C28 and MS13 suppressed the production of IL-8 (in the presence or ab-sence TNFα and IL-1β). Moreover, in the co-culture of Caco-2 with macro-phage subsets, MS13 enhanced the expression of hBD-2 and ZO-1. These findings allow for the better understanding of live probiotic roles on macro-phage subsets functions and endotoxin tolerisation mechanisms, which may be beneficial for the development of in vivo models of probiotic bacteria and therapeutic targeting of inflammatory bowel disease.

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