Please cite this article in press as: Bertuccini L, et al. Lactoferrin prevents invasion and inflammatory response following E. coli strain LF82 infection in experimental model of Crohn’s disease. Dig Liver Dis (2014), http://dx.doi.org/10.1016/j.dld.2014.02.009 ARTICLE IN PRESS G Model YDLD-2586; No. of Pages 9 Digestive and Liver Disease xxx (2014) xxx–xxx Contents lists available at ScienceDirect Digestive and Liver Disease jou rnal h om epage: www.elsevier.com/locate/dld Alimentary Tract Lactoferrin prevents invasion and inflammatory response following E. coli strain LF82 infection in experimental model of Crohn’s disease Lucia Bertuccini a , Manuela Costanzo b , Francesca Iosi a , Antonella Tinari a , Fabio Terruzzi c , Laura Stronati b , Marina Aloi d , Salvatore Cucchiara d , Fabiana Superti a,∗ a Department of Technology and Health, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy b ENEA, Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Department of Radiobiology and Human Health, Via Anguillarese 301, 00123 Rome, Italy c Giellepi S.p.A., Via G. Verdi, 41/Q, 20831 Seregno (MB), Italy d Department of Pediatrics, Sapienza University of Rome, Viale Regina Elena, 324, 00161 Rome, Italy a r t i c l e i n f o Article history: Received 29 November 2013 Accepted 9 February 2014 Available online xxx Keywords: Adherent-invasive Escherichia coli Bovine lactoferrin Crohn’s disease Inflammatory bowel diseases a b s t r a c t Background: Crohn’s disease is a multifactorial disease in which an aberrant immune response to com- mensal intestinal microbiota leads to chronic inflammation. The small intestine of patients with Crohn’s disease is colonized by a group of adherent-invasive Escherichia coli strongly able to adhere and invade intestinal epithelial cells lactoferrin is an iron-binding glycoprotein known to have anti-bacterial and anti-inflammatory activities. Aims: We explore the ability of bovine lactoferrin to modulate the interactions between the adherent- invasive E. coli strain LF82 and intestinal epithelial cells as well as the inflammatory response. Methods: Bacterial adhesion and invasion assays were used to assess the antimicrobial activity of lacto- ferrin. Electron microscopy was used to characterize bacteria–cell interactions. The mRNA expression of pro-inflammatory cytokines was measured both in cultured cells and in biopsies taken from intestine of patients affected by Crohn’s disease. Results: Lactoferrin inhibited bacterial invasion through minimally affecting adhesion. This divergence was due to a mannose-dependent lactoferrin binding to the bacterial type 1 pili and consequent bacterial aggregation on the intestinal epithelial cell surface. Expression of pro-inflammatory cytokines, such as TNF-alpha, IL-8, and IL-6, was markedly inhibited by lactoferrin both in cultured and Crohn-derived intestinal cells. Conclusions: Bovine lactoferrin might function via an antibacterial and/or anti-inflammatory mechanism in the treatment of Crohn’s disease. © 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved. 1. Introduction Crohn’s disease (CD) is an inflammatory bowel disease (IBD) characterized by a chronic transmural, segmental, and typically granulomatous inflammation of the intestine in humans with unknown aetiology. Deregulation of the innate and adaptive immune responses directed against luminal bacteria or their prod- ucts, which are normally under tight immunological tolerance, are immune factors characteristic of CD and other IBDs [1]. Among the bacteria that may play a role as target of de-regulated immunity in the pathogenesis of CD, a pathovar of Escherichia coli called AIEC (adherent-invasive E. coli) has been strongly implicated ∗ Corresponding author. Tel.: +39 064990 3149; fax: +39 064990 3563. E-mail address: fabiana.superti@iss.it (F. Superti). [2]. AIEC encompass a subgroup of E. coli spp. named from their characteristic capability to adhere to intestinal cells, to invade the infected eukaryotic cells, and to replicate in epithelial cells and macrophages, ultimately causing intestinal diseases in humans [3–7]. LF82, an E. coli strain (serotype O83:H1) originally isolated from a patient with CD [3], represents the prototype AIEC strain. Lactoferrin (Lf), a member of the transferrin family [8] presents in milk and other exocrine secretions, constitutes one of the major antimicrobial components of the innate immune system [9]. Biolog- ical properties reported for Lf include antimicrobial activity against a wide range of pathogenic bacteria, fungi, protozoa and virus, as well as anti-inflammatory, antitumor and immunomodulatory activities [10,11]. Antimicrobial functions attributed to this protein or its peptides include iron sequestration [12], destabilization of Gram-negative bacteria outer membrane through binding of bacte- rial lipopolysaccharides (LPS) [13–16], selective penetration of ions http://dx.doi.org/10.1016/j.dld.2014.02.009 1590-8658/© 2014 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.