Developmental and Comparative Immunology 34 (2010) 797–804 Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci Flagellin expression enhances Salmonella accumulation in TLR5-positive macrophages H.J. Metcalfe a , A. Best b , T. Kanellos c , R.M. La Ragione b,d , D. Werling a,∗ a Molecular Immunology Group, Department for Pathology and Infectious Diseases, Royal Veterinary College, Hawkshead Campus, Hawkshead Lane, North Mymms, Hertfordshire AL9 7TA, United Kingdom b Department for Food and Environmental Safety, Veterinary Laboritories Agency (Weybridge), Woodham Lane, Addlestone, Surrey KT15 3NB, United Kingdom c Pfizer R&D, Sandwich, Kent, United Kingdom d Faculty of health and Medical sciences, University of Surrey, Guildford, Surrey, GU2 7XH, United Kingdom article info Article history: Received 4 January 2010 Received in revised form 8 February 2010 Accepted 8 February 2010 Available online 4 March 2010 Keywords: Salmonella Flagellin TLR5 Macrophage Enteritidis Typhimurium Bovine abstract Recently, it has been reported that Salmonella secrete flagellin in response to host produced lysophos- pholipids. However, this monomer of the bacterial flagella activates Toll-like receptor 5 (TLR5) in the innate immune system. The objective of this study was to examine the role of flagellin expression during infection of species-specific macrophages (M) which either expressed or lacked TLR5. Initially, TLR5- activity was confirmed in bovine M using Salmonella typhimurium derived-flagellin. Within these cells, recombinant FliC induced a potent CXCL8 response when compared to the heterogeneous (FliC/FljB) form of purified flagellin. Furthermore, neither form of flagellin induced nitrite secretion which was subsequently detected after exposing bovine M to LPS in the presence of IFN-. Flagellin enhanced the accumulation of Salmonella enteritidis in TLR5-positive bovine and human M which was independent of adhesion in bovine M. In contrast, murine Ms which lacked TLR5 were equally susceptible to hosting S. enteritidis, with or without flagellin. However, lack of flagellin in S. typhimurium marginally inhibited bacterial accumulation in bovine M, where FljB and FliC compensated for the lack of each other. This study suggests that flagellin may be inducing TLR5-dependent internalisation mechanisms in MФ which vary qualitatively between different species and Salmonella serotypes. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Salmonella enterica (S.) is a world-wide, serologically diverse bacterium with the potential of causing zoonotic diseases rang- ing from mild gastroenteritis to systemic infection in animals and humans [1]. The increasing prevalence of multi-drug resis- tant strains such as Salmonella serotype Typhimurium definitive type 104 is a current concern, adding complications to pri- mary and acquired immunosuppression [2–4]. This present study contributes towards our knowledge of differences in immunopathology between vertebrates which may influence the development of new immune strategies for sustainable disease control. Macrophages (M), like dendritic cells (DCs), act as sentries to infection in the innate immune system, continuously sampling antigens below the physical barriers. These cells express germ-line encoded receptors, such as Toll-like receptors (TLRs), which utilise microbial-associated molecular patterns to distinguish self from ∗ Corresponding author. Tel.: +44 1707 666573; fax: +44 1707 652090. E-mail address: dwerling@rvc.ac.uk (D. Werling). non-self [5]. TLRs are signalling receptors conserved throughout most eukaryotes and can sense a diverse array of structures from different types of pathogen [6–8]. Over the last 10 years, a highly conserved region of flagellin which is a monomer of the bacterial flagellum, was described to induce a pro-inflammatory response by binding to TLR5 [9–11]. Similar to other TLRs, TLR5 can induce very complex pleiotrophic, parallel and cross regulatory mechanisms [12–15]. In addition, there is a significant degree of TLR5 sequence variation and tissue-specific TLR variation between species [7,16]. This diverse selective pressure can lead to varied immunopathol- ogy such as systemic infection of mice by Salmonella typhimurium in contrast to localised S. typhimurium infection in cattle and humans [17]. Bacterial flagella are crucial for bacterial motility, colonisation, penetration of the mucosal layer, adhesion/invasion of epithe- lial cells and exploiting inflammation to compete with intestinal microbiota in vivo [18–21]. However, the role of monomeric flag- ellin must be multifaceted because Salmonella increase flagellin synthesis and secretion, independent of flagella, after sensing host- produced lysophospholipids from epithelial cells [22]. Another role for flagellin, as a virulence factor, may include recruiting M/DCs, through TLR5 induced chemokine secretion, for intracel- 0145-305X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2010.02.008