[Frontiers in Bioscience 16, 2106-2127, June 1, 2011]

Functional characterization of MIMP for its adhesion to the intestinal epithelium

Zhihua Liu1, Tongyi Shen2, Hongqi Chen1, Yukun Zhou1, Peng Zhang1, Yanlei Ma1, Mary Pat Moyer3, Ming Zhang1, Zhaoxin Chu1, Huanlong Qin1

1Department of Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, 200233, China, 2Department of Surgery, Shanghai, 200072, Shanghai Tenth People's Hospital, China, 3INCELL Corporation, San Antonio Texas, 78249, United States of America

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. MIMP, antibodies and major reagents
3.2. Bacterial strains and culture conditions
3.3. NCM460 cells, iDCs and mice
3.4. Determination of MIMP effect on the intestinal permeability in vitro, ex vivo and in vivo
3.4.1. Measurement of transepithelial electrical resistance (TER) in NCM460 cell monolayers
3.4.2. Determination of dextran permeability in NCM460 monolayers
3.4.3. Ussing chamber assay for determining the intestinal permeability measurement in isolated mouse colons
3.4.4. Measurement of the intestinal permeability and colonic damage in mice
3.5. Determination of MIMP effects on TJ proteins and TJ ultrastructure in vitro and in vivo
3.5.1. Fluorescence staining and Western blotting for determining the distribution and expression of TJ proteins in NCM460 cell monolayers and mice
3.5.2. Quantitative real time PCR for detecting the mRNA expression of TJ proteins in NCM460 cells and mice
3.5.3. Transmission electron microscopy for the observation of tight junctions in NCM460 cell monolayers and mice
3.6. Determination of MIMP effects on DC-mediated immune responses
3.6.1. Flow cytometry for determining DC adhesion, maturation and Th1/Th2 differentiation
3.6.2. Western blotting, fluorescence staining, and enzyme-linked immunosorbent assays (ELISA) for detecting MIMP binding to DC-SIGN of iDCs
3.6.3. ELISA for the determination of cytokine production by DCs
3.7. Determination of MIMP effects on colonic inflammation in IL-10-/- mice
3.7.1. Histological examination of colonic inflammation
3.7.2. Determination of mucosal cytokine and myeloperoxidase (MPO) concentrations
3.8. Statistical analysis
4. Results
4.1. MIMP effects on intestinal permeability in vitro and in vivo
4.1.1. MIMP prevents EIEC/EPEC-induced decrease of TER in NCM460 cells
4.1.2. MIMP inhibits increased macromolecular permeability of NCM460 cell monolayers in response to EIEC/EPEC
4.1.3. MIMP prevents the increase in intestinal permeability in IL-10-/- mice
4.2. MIMP effects on TJ proteins and TJ ultrastructure in vitro
4.2.1. MIMP prevents decrease in the expression and rearrangement of TJ proteins and F-actin as detected by fluorescence staining
4.2.2. MIMP prevents the decrease in the expression of TJ proteins as detected by Western blotting
4.2.3. MIMP prevents the decrease in the expression of TJ proteins as detected by quantitative RT-PCR
4.2.4.MIMP prevents the destruction of the TJ structure as detected by TEM
4.3. MIMP effects on DC-mediated immune responses
4.3.1. MIMP adheres to iDCs as detected by flow cytometry
4.3.2. MIMP adheres to iDCs by binding to DC-SIGN as detected by Western blotting, fluorescence staining, and ELISA
4.3.3. MIMP promotes the production of anti-inflammatory cytokines in DCs as detected by ELISA
4.3.4. MIMP induces DCs to mediate Th2 predominant differentiation as detected by flow cytometry
4.4. MIMP effects on colonic inflammation in IL-10-/- mice
4.4.1. MIMP affects the expression of cytokines and myeloperoxidase (MPO) and ameliorates colonic inflammation
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

The micro integral membrane protein (MIMP), the domain within the integral membrane protein of Lactobacillus plantarum CGMCC 1258, has been shown to adhere to mucin and antagonize the adhesion of enteroinvasive E. coli and enteropathogenic E. coli. To further characterize the functions of MIMP, we investigated its effects on the intestinal permeability, expression of tight junction (TJ) proteins and TJ ultrastructure in vitro and in vivo. We also determined the interaction between MIMP and dendritic cells (DCs). We observed that MIMP reduced intestinal permeability and restored the expression and distribution of TJ proteins in both NCM460 cell monolayers and in IL-10-/- mice. MIMP adhered to immature (i) DCs by binding to DC-SIGN, and induced DCs to produce anti-inflammatory cytokines and to mediate Th2 differentiation. Moreover, MIMP stimulated the expression of anti-inflammatory cytokines in colonic mucosa and attenuated colitis in IL-10-/- mice. In conclusion, MIMP is the main functional component of L. plantarum that contributes to its protective effects, and thus may be a potential therapeutic agent for intestinal diseases.