Differential NF-kappaB pathways induction by Lactobacillus plantarum in the duodenum of healthy humans correlating with immune tolerance

Abstract

How do we acquire immune tolerance against food microorganisms and commensal bacteria that constitute the intestinal microbiota? We investigated this by stimulating the immune system of adults with commensal Lactobacillus plantarum bacteria. We studied the in vivo human responses to L. plantarum in a randomized double-blind placebo-controlled cross-over study. Healthy adults ingested preparations of living and heat-killed L. plantarum bacteria. Biopsies were taken from the intestinal duodenal mucosa and altered expression profiles were analyzed using whole-genome microarrays and by biological pathway reconstructions. Expression profiles of human mucosa displayed striking differences in modulation of NF-kappaB-dependent pathways, notably after consumption of living L. plantarum bacteria in different growth phases. Our in vivo study identified mucosal gene expression patterns and cellular pathways that correlated with the establishment of immune tolerance in healthy adults.

Fig. 1.

Validation of microarray results by QPCR, using total RNA from biopsies (pooled data from 8 persons). After consumption of midlog and dead bacteria, amounts of mRNA for the genes CD55 and SLPI were low. In all figures and text, down-regulated genes are indicated with negative fold-changes for ease of interpretation and comparison, although this is mathematically incorrect.

Fig. 2.

Cellular pathways significantly modulated after bacterial consumption. Statistical significance of pathway modulation was calculated via a right-tailed Fisher's Exact test in Ingenuity Pathway Analysis and represented as −log(P value); −log values exceeding 1.30 were significant (P < 0.05).

Fig. 3.

Ingenuity protein–protein interaction networks. The interaction map was derived by plotting interacting proteins involved in specific cellular functions: immune response, immune system development and function, cell signaling, cell cycle, cell growth, cell death and proliferation. Transcriptional information was projected onto the interaction map; up-regulated genes are depicted in shades of red, down-regulated genes in shades of green. Relevant pathways that feature modulated genes were indicated as well. (A) transcriptional networks modulated after consumption of midlog bacteria. From this interaction map, it can be seen that the up-regulated PARP1, MYC, and CCND1 are important nodes associated with the mentioned pathways. (B) transcriptional networks modulated after consumption of dead bacteria. Major nodes are proteins from the CEBP, JUN, and NFKB family but also proinflammatory TNF and IFN regulatory factor, IRF1.

Fig. 4.

Proposed model of cellular proteins and processes associated with immune tolerance induced in the proximal small intestine of healthy adults after consumption of L. plantarum. (A and B) Because of up-regulation of factors such as A20 and IκBα and lack of p65 (RelA), no continuous NF-κB driven proinflammatory gene transcription is expected. Interleukins (blue arrows) are exclusively up-regulated during the interaction with dead bacteria (A) and are proposed to stimulate the maturation of CD4⁺ cells (B); overt stimulation by ILs is probably balanced by the suppressor SOCS3. (C) The crossed-out red arrows between IECs and dendritic, T and B cells indicate that no stimulatory or activating contact takes place between these cells through lack of expression of necessary genes such as BAFF, AID, IL-1, and TSLP.

Fig. 5.

Histological examination of human biopsies does not show evidence for infiltration of immune cells after consumption of L. plantarum. The bright-field images (A–C) are from representative areas of biopsy sections; the FITC fluorescence image (D Bottom Right) is from the same area of the section labeled “stationary” (C) and shows autofluorescent granules. G, goblet cell; M, macrophage; N, neutrophil; g, granules. (Magnifications: A and B, 500×; C and D, 1,000×. Scale bars: A, 50 μm; C, 25 μm.) Images are representative for at least 12 sections from biopsies taken after consumption of the 3 bacterial preparations and placebo control by 3 individuals.