Probiotic Lactobacillus reuteri suppress proinflammatory cytokines via c-Jun

Abstract

Differential immunoregulatory capabilities of probiotic Lactobacillus were explored in the context of pediatric Crohn's disease. Experimental strategies addressed molecular mechanisms of tumor necrosis factor (TNF) suppression in activated macrophages by transcriptional regulation. Secreted factors produced by probiotic Lactobacillus reuteri strains were harvested and tested with human monocytes and macrophages. Quantitative immunoassays and real-time reverse-transcriptase polymerase chain reaction (RT-PCR) were used to examine relative quantities of human cytokines and TNF mRNA, respectively, and reporter assays assessed transcriptional regulation of TNF by probiotics. DNA-protein macroarrays interrogated probiotic-mediated effects on transcription factor activation. Finally, enzyme-linked immunosorbent assays (ELISAs) and immunoblots examined the involvement of the specific transcription factor AP-1 and its components. Probiotic L. reuteri strain ATCC PTA 6475 demonstrated the ability to potently suppress human TNF production by lipopolysaccharide-activated monocytes and primary monocyte-derived macrophages from children with Crohn's disease. Quantities of the chemokine MCP-1/CCL2 were also reduced by probiotic L. reuteri strain ATCC PTA 6475 in macrophages of children in remission. Quantitative real-time RT-PCR and luciferase reporter assays showed that transcriptional regulation of human TNF was a primary mechanism of probiotic-mediated immunomodulation. Probiotic L. reuteri suppressed TNF transcription by inhibiting activation of MAP kinase-regulated c-Jun and the transcription factor, AP-1. Human TNF and MCP-1 suppression by probiotic L. reuteri was strain-dependent, and the activation of c-Jun and AP-1 represent primary targets for probiotic-mediated suppression of TNF transcription. This report emphasizes the clonal nature of immunoprobiosis and delineation of a specific immunomodulatory mechanism for probiotic strain selection in future inflammatory bowel disease-oriented clinical trials.