Abstract

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.

Keywords

Modulation of macrophages, Probiotics Lactobacillus spp., Live bacteria, SOCS3, Endotoxin tolerance, Caco-2, THP-1, IL-23, IL-10, IL-6, IL-16, TRAIL, TOLLIP, SAT3, NLRP3, IL-1beta, TNFalfa, Tolerisation

Document Type

Thesis

Publication Date

2021

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