Mucin Depleted Foci, Colonic Preneoplastic Lesions Lacking Muc2, Show Up-Regulation of Tlr2 but Not Bacterial Infiltration

Angelo Pietro Femia,Piero Dolara,Maddalena Salvadori,Alexander Swidsinski,Giovanna Caderni,Amedeo Amedei,Yohannes Tesfaigzi

doi:10.1371/journal.pone.0029918

Abstract

Mucin depleted foci (MDF) are precancerous lesions of the colon in carcinogen-treated rodents and humans at high risk. Since MDF show signs of inflammation we hypothesized that the defective mucous production would expose them to the risk of being penetrated by intestinal bacteria, which can be sensed by Toll-like receptors (Tlrs) and activate inflammatory pathways. To verify this hypothesis we tested the expression of 84 genes coding for Tlrs and associated pathways using RT-qPCR in MDF (n = 7) from 1,2-dimethylhydrazine (DMH)-treated rats. Among the 84 tested genes, 26 were differentially expressed in MDF with 5 genes significantly up-regulated and 21 down-regulated when compared to the normal mucosa. Tlr2, as well as other downstream genes (Map4k4, Hspd1, Irak1, Ube2n), was significantly up-regulated. Among the genes regulating the NFkB pathway, only Map4k4 was significantly up-regulated, while 19 genes were not varied and 6 were down-regulated. Tlr2 protein was weakly expressed both in normal mucosa and MDF. To determine whether inflammation observed in MDF could be caused by bacteria contacting or infiltrating crypts, we performed fluorescence in situ hybridization (FISH) experiments with a rRNA universal bacterial probe. None of the 21 MDF tested, showed bacteria inside the crypts, while among the colonic tumors (n = 15), only one had very few bacteria on the surface and on the surrounding normal mucosa. In conclusion, the up-regulation of Tlr2 in MDF, suggests a link between this receptor and carcinogenesis, possibly related to a defective barrier function of these lesions. The data of FISH experiments do not support the hypothesis that inflammation in MDF and tumors is stimulated by bacterial infiltration.

Highlights

Mucin depleted foci (MDF) are precancerous lesions of the colon identified in carcinogen-treated rodents [1] and high-risk humans [2]
Toll-like receptors (Tlrs) pathway expression in MDF The expression of 84 genes coding for Tlrs and associated pathways was studied by RT-qPCR with commercial arrays in MDF (n = 7) and their corresponding normal mucosa
The aim of this study was to understand whether the increased expression of inflammatory markers observed in MDF [6] could be related to variations in the expression of Tlr and pathways related to these receptors and/or to a direct contact of bacteria with these lesions

Summary

Introduction

Mucin depleted foci (MDF) are precancerous lesions of the colon identified in carcinogen-treated rodents [1] and high-risk humans [2]. While in normal mice the intestinal flora is separated from the colon mucosa by a densely packed layer of mucus [10,11], in Muc2-/- mice bacteria are in direct contact with the mucosa and even penetrate the crypts [11], an observation possibly explaining the proneness of this strain to develop intestinal inflammation and, eventually, cancer [11,12] This rodent genetic model proves the principle that Muc deficiency causes a certain degree of inflammation in the colon, it does not imply that focal mucin depletion, as observed in the preneoplastic lesions MDF, may trigger and sustain inflammation through a closer contact with luminal bacteria

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