Alpha-Melanocyte Stimulating Hormone Protects against Cytokine-Induced Barrier Damage in Caco-2 Intestinal Epithelial Monolayers.

Judit Váradi,András Harazin,Katalin Réti-Nagy,Zoltán Ujhelyi,Péter Gogolák,Pálma Fehér,Miklós Vecsernyés,Gábor Vasvári,David Haines,Ferenc Fenyvesi,György Vámosi,Mária A Deli,Ildikó Bácskay,Eszter Róka

doi:10.1371/journal.pone.0170537

Abstract

Alpha-melanocyte-stimulating hormone (α-MSH) is a potent anti-inflammatory peptide with cytoprotective effect in various tissues. The present investigation demonstrates the ability of α-MSH to interact with intestinal epithelial cell monolayers and mitigate inflammatory processes of the epithelial barrier. The protective effect of α-MSH was studied on Caco-2 human intestinal epithelial monolayers, which were disrupted by exposure to tumor necrosis factor-α and interleukin-1β. The barrier integrity was assessed by measuring transepithelial electric resistance (TEER) and permeability for marker molecules. Caco-2 monolayers were evaluated by immunohistochemistry for expression of melanocortin-1 receptor and tight junction proteins ZO-1 and claudin-4. The activation of nuclear factor kappa beta (NF-κB) was detected by fluorescence microscopy and inflammatory cytokine expression was assessed by flow cytometric bead array cytokine assay. Exposure of Caco-2 monolayers to proinflammatory cytokines lowered TEER and increased permeability for fluorescein and albumin, which was accompanied by changes in ZO-1 and claudin-4 immunostaining. α-MSH was able to prevent inflammation-associated decrease of TEER in a dose-dependent manner and reduce the increased permeability for paracellular marker fluorescein. Further immunohistochemistry analysis revealed proinflammatory cytokine induced translocation of the NF-κB p65 subunit into Caco-2 cell nuclei, which was inhibited by α-MSH. As a result the IL-6 and IL-8 production of Caco-2 monolayers were also decreased with different patterns by the addition of α-MSH to the culture medium. In conclusion, Caco-2 cells showed a positive immunostaining for melanocortin-1 receptor and α-MSH protected Caco-2 cells against inflammatory barrier dysfunction and inflammatory activation induced by tumor necrosis factor-α and interleukin-1β cytokines.

Highlights

Epithelial cells are key components of the intestinal barrier by forming tight junctions (TJ) sealing the paracellular cleft, restricting free flux of cells and molecules from the gut to the blood
The goal of the present study was to evaluate the protective effect of α-MSH on proinflammatory cytokine-induced Caco-2 monolayer activation and barrier injury
We demonstrated the polarized expression of MC1R on human Caco-2 epithelial cells with a predominant apical localization (Fig 1) for the first time

Summary

Introduction

Epithelial cells are key components of the intestinal barrier by forming tight junctions (TJ) sealing the paracellular cleft, restricting free flux of cells and molecules from the gut to the blood. Dysfunction of the epithelial barrier is a common feature in inflammatory diseases of the gastrointestinal system [1]. The damage of the protective epithelial barrier contributes to the pathomechanism and both local and systemic inflammation. Proinflammatory cytokines tumor necrosis factor-α (TNF- α) and interleukin-1β (IL-1 β) are overexpressed in inflammatory bowel diseases and directly damage the intestinal barrier including the interepithelial TJs [1]. Cell culture models of intestinal epithelium are widely used in the characterization of gut disease pathomechanisms, and to evaluate selected pharmacotherapies. The Caco-2 human intestinal epithelial cell line is a well-characterized model to study intestinal absorption processes [2], and is used to investigate intestinal inflammation [3,4,5,6]

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