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

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-1ß). 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.

Porphyromonas gingivalis-stimulated macrophage subsets exhibit differential induction and responsiveness to interleukin-10.

OBJECTIVES: Oral mucosal macrophages (Mϕs) determine immune responses; maintaining tolerance whilst retaining the capacity to activate defences against pathogens. Mϕ responses are determined by two distinct subsets; pro-inflammatory M1- and anti-inflammatory/regulatory M2-Mϕs. Tolerance induction is driven by M2 Mϕs, whereas M1-like Mϕs predominate in inflammation, such as that exhibited in chronic Porphyromonas gingivalis (PG) periodontal infection. Mϕ responses can be suppressed to benefit either the host or the pathogen. Chronic stimulation by pathogen associated molecular patterns (PAMPs), such as LPS, is well established to induce tolerance. The aim of this study was to investigate the P. gingivalis-driven induction of and responsiveness to the suppressive, anti-inflammatory cytokine, IL-10, by Mϕ subsets. METHODS: M1- and M2-like Mϕs were generated in vitro from the THP-1 monocyte cell line by differentiation with PMA and Vitamin D3, respectively. Mϕ subsets were stimulated by PG-LPS in the presence or absence of IL-10. RESULTS: PG-LPS differentially induced IL-10 secretion and endogenous IL-10 activity in M1- and M2-like subsets. In addition, these subsets exhibited differential sensitivity to IL-10-mediated suppression of TNFα, where M2 Mϕs where sensitive to IL-10 and M1 Mϕs were refractory to suppression. In addition, this differential responsiveness to IL-10 was independent of IL-10-binding and expression of the IL-10 receptor signal transducing subunit, IL-10Rß, but was in fact dependent on activation of STAT-3. CONCLUSION: P.gingivalis selectively tolerises regulatory M2 Mϕs with little effect on pro-inflammatory M1 Mϕs; differential suppression facilitating immunopathology at the expense of immunity.