A Co-Culture Model of the Developing Small Intestine Offers New Insight in the Early Immunomodulation of Enterocytes and Macrophages by Lactobacillus spp. through STAT1 and NF-kB p65 Translocation

Martin Trapecar,Ales Goropevsek,Mario Gorenjak,Lidija Gradisnik,Marjan Slak Rupnik,Markus M Heimesaat

doi:10.1371/journal.pone.0086297

Martin Trapecar, Ales Goropevsek + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0086297

Copy DOI

Abstract

The early establishment of a complete microbiome has been shown to play an integral part in the development and maintenance of an intact intestine and its immune system, although much remains unknown about the specific mechanisms of immune modulation in newborns. In our study we show in a co-culture model of the undeveloped small intestine that members of Lactobacillus spp. influence STAT1 and NF-kB p65 nuclear translocation in both intestinal epithelial cells as well as underlying macrophages. Moreover, by using imaging flow cytometry we were able to monitor each individual cell and create a framework of the percentage of cells in which translocation occurred in challenged versus control cell populations. We also observed a significant difference in baseline translocation in intestinal cells when cultured alone versus those in a co-culture model, underpinning the importance of 3D models over monolayer set-ups in epithelial in vitro research. In conclusion, our work offers new insights into the potential routes by which the commensal microbiome primes the early immune system to fight pathogens, and shows how strain-specific these mechanisms really are.

Highlights

An established microbiome is prerequisite to the early development of the intestine in newborns acquiring tolerance and immunity [1]
Lactobacillus spp. trigger STAT1 and NF-kB p65 translocation in H4-1 small intestinal epithelial cells cultured in a human 3D model of the gut Untransformed H4-1 IEC co-cultured with TLT macrophages on microporous membranes and treated with different bacterial strains were stained with antibodies against STAT1 as well as NFkB p65 and further analyzed with imaging flow cytometry (Figure 2)
Addition of Lactobacillus spp. increased the number of cells in which nuclearization of STAT1 occurred as shown in Figure 3a,b no significant differences could be detected among individual strains LGG, PCS 20 and PCS 26

Summary

Introduction

An established microbiome is prerequisite to the early development of the intestine in newborns acquiring tolerance and immunity [1]. The role of priming is to potentiate early responses and to raise the level of alertness of mononuclear cells against pathogens without actual activation of the cells. In this process bacteria-derived extracellular stimuli such as lipopolysaccharides and lipoteichoic acids induce TLR-dependent activation of NF-kB signaling as well as secretion of type I interferons, extended to the activation of STAT1, in eneterocytes, followed by a translational cascade reaching macrophages and other cells of the innate immune system [6,7,8]. A dysregulation in those signaling cascades in early intestinal developmental often results in necrotizing enterocolitis (NEC), an inflammatory bowel necrosis of premature infants [9]. NEC in newborns is marked with a high infiltrate of macrophage leukocytes in affected areas and a very low count of lymphocytes, which underpins macrophages as the first line of defense, especially in the premature gut [10]

Objectives

Methods

Results

Discussion

Conclusion