Lacticaseibacillus casei Strain Shirota Modulates Macrophage-Intestinal Epithelial Cell Co-Culture Barrier Integrity, Bacterial Sensing and Inflammatory Cytokines

Abstract

Probiotic bacteria modulate macrophage immune inflammatory responses, with functional cytokine responses determined by macrophage subset polarisation, stimulation and probiotic strain. Mucosal macrophages exhibit subset functional heterogeneity but are organised in a 3-dimensional tissue, over-laid by barrier epithelial cells. This study aimed to investigate the effects of the probiotic Lacticaseibacillus casei strain Shirota (LcS) on macrophage-epithelial cell cytokine responses, pattern recognition receptor (PRR) expression and LPS responses and the impacts on barrier integrity. THP-1-derived M1 and M2 subset macrophages were co-cultured in a transwell system with differentiated Caco-2 epithelial cells in the presence or absence of enteropathogenic LPS. Both Caco-2 cells in monoculture and macrophage co-culture were assayed for cytokines, PRR expression and barrier integrity (TEER and ZO-1) by RT-PCR, ELISA, IHC and electrical resistance. Caco-2 monocultures expressed distinct cytokine profiles (IL-6, IL-8, TNFα, endogenous IL-10), PRRs and barrier integrity, determined by inflammatory context (TNFα or IL-1β). In co-culture, LcS rescued ZO-1 and TEER in M2/Caco-2, but not M1/Caco-2. LcS suppressed TLR2, TLR4, MD2 expression in both co-cultures and differentially regulated NOD2, TLR9, Tollip and cytokine secretion. In conclusion, LcS selectively modulates epithelial barrier integrity, pathogen sensing and inflammatory cytokine profile; determined by macrophage subset and activation status.

Discovery of Lactomodulin, a Unique Microbiome-Derived Peptide That Exhibits Dual Anti-Inflammatory and Antimicrobial Activity against Multidrug-Resistant Pathogens

“…Inoculation with five Lactobacillus strains resulted in a one- to five-fold increase in IL-8 compared to the untreated cells, except for L4. These data are consistent with previous reports showing that some Lactobacillus strains can result in initial stimulation of the immune response through the induction of IL-8 and IL-1β secretion and the generation of reactive oxygen species [ 33 , 34 ]. Cells incubated with L1-L8 strains prior to the pathogen challenge showed a 50–90% drop in IL-8 secretion.…”

Development of an Inflammation-Triggered In Vitro “Leaky Gut” Model Using Caco-2/HT29-MTX-E12 Combined with Macrophage-like THP-1 Cells or Primary Human-Derived Macrophages

“…IFN-γ priming of Caco-2 cells together with the stimulation of differentiated THP-1 cells by LPS and IFN-γ, induced an inflammation-like response in their diseased intestine model, evident by intestinal barrier disruption and pro-inflammatory cytokine release. Based on this model of Kämpfer, co-culture models with differentiated Caco-2 cells and PMA-differentiated THP-1 cells in the presence of inflammatory stimulators (e.g., LPS with/without IFN-γ) have been used to evaluate the potential immunomodulatory and anti-inflammatory effects of phytochemicals [ 43 , 72 , 73 ], marine natural products [ 74 , 75 , 76 ], bacterial β-glucans [ 77 ], siRNA-based nanomedicine [ 42 ], bovine milk-derived extracellular vesicles [ 78 ], and probiotic bacteria [ 41 ].…”

“…Thus, some researchers have started to combine intestinal cell lines with human macrophage-like cells (e.g., differentiated monocytic THP-1 cells), or monocyte-derived macrophages from peripheral blood mononuclear cells (PBMCs) [ 38 , 39 , 40 ]. However, each of these IBD models has its disadvantages, because of either (1) the lack of mucus-producing cells [ 41 , 42 , 43 , 44 ], (2) a spatial distance between the intestinal cells and macrophages [ 45 , 46 , 47 ], or (3) being specifically adapted for buoyant particles [ 48 ].…”

Mechanistic insights into the role of probiotics in modulating immune cells in ulcerative colitis

“…LcS exhibited differential effects on the co‐culture system depending on the macrophage subset and activation status. Specifically, LcS restored the integrity of the tight junction protein ZO‐1 and the trans‐epithelial electrical resistance (TEER) in the M2/Caco‐2 co‐culture but not in the M1/Caco‐2 co‐culture 62 . Moreover, LcS modulated the expression of PRRs, such as TLR2, TLR4, and myeloid differentiation factor 2, in both co‐cultures, indicating its influence on pathogen sensing.…”

“…Additionally, the interaction between macrophages and IEC is crucial for maintaining the integrity of the gut barrier. This study aimed to explore how LcS affects these interactions and subsequent immune responses 62 . Using a co‐culture system consisting of M1 and M2 subset macrophages derived from THP‐1 cells and differentiated Caco‐2 epithelial cells, the researchers assessed cytokine responses, PRR expression, and barrier integrity in the presence or absence of enteropathogenic LPS, a bacterial component known to induce inflammation 62 .…”