Flagellin/TLR5 signalling activates renal collecting duct cells and facilitates invasion and cellular translocation of uropathogenic Escherichia coli Marcelle Bens, 1,2 Sophie Vimont, 3 Sanae Ben Mkaddem, 1 Cécilia Chassin, 1,2 Jean-Michel Goujon, 4 Viviane Balloy, 5,6 Michel Chignard, 5,6 Catherine Werts 7 and Alain Vandewalle 1,2 * 1 Centre de Recherche sur l’Inflammation (CRI), UMRS 1149, Université Denis Diderot – Paris 7, Paris, France. 2 Groupe ATIP-AVENIR INSERM, Université Denis Diderot – Paris 7, Paris, France. 3 Service de Bactériologie, Hôpital Tenon, Université Paris 6 Pierre et Marie Curie, Paris, France. 4 Service d’Anatomie et Cytologie Pathologiques, CHU de Poitiers, Université de Poitiers, Poitiers, France. 5 Institut Pasteur, Unité de Défense Innée et Inflammation, Paris, France. 6 INSERM U874, Paris, France. 7 Institut Pasteur, Unité Biologie et Génétique des parois bactériennes, Paris, France. Summary Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysac- charide, stimulated the production of the chemo- attractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild-type (WT) and Tlr4 −/− MCDs, but not in Tlr5 −/− MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strik- ingly, the invasion capacity and transcellular trans- location of the UPEC strain HT7 were significantly lower in Tlr5 −/− than in WT MCDs. The non-motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5 −/− kidneys exhibited less infil- trating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 post- infected Tlr5 −/− kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers. Introduction Urinary tract infection (UTI) and acute pyelonephritis (APN), which are mainly due to flagellated uropathogenic Escherichia coli (UPEC), are the most frequent renal bac- terial infections causing significant morbidity and mortality in humans (Brown et al., 2005; Freedman, 2005). Early recognition of bacterial motifs by a number of pattern recognition receptors, including Toll-like receptors (TLRs), is essential for the removal of bacterial pathogens (Takeuchi and Akira, 2010). UPEC colonizing the urinary tract are recognized by several TLRs (Song and Abraham, 2008), including TLR4, which senses lipopolysaccharide (LPS) and also TLR11, which is expressed in murine bladder epithelial cells and renal tubule cells, but is only expressed as a pseudo-gene in humans (Zhang et al., 2004). TLR5 that senses flagellin, the most abundant protein in the tails of most flagellated bacteria, has also been shown to be involved in host defence against UPEC. TLR5 recognizes a conserved site on flagellin, which is encoded by fliC (Hayashi et al., 2001; Smith et al., 2003). Flagellum-mediated motility facilitates the spread of UPEC infection and contributes to the retrograde ascent of UPEC from the bladder to the kidneys (Lane et al., 2005; 2007; Wright et al., 2005). Andersen-Nissen et al. (2007) evidenced lower inflam- matory response in the bladder and renal pelvis from Tlr5 knockout (Tlr5 −/− ) mice than from wild-type (WT) mice, two days after the transurethral inoculation of the UPEC strain CFT073. These authors also showed that Tlr5 −/− mice had significantly more bacteria in the bladder and kidneys, 5 days after UPEC inoculation. These findings indicated that TLR5 plays a role in the control of the innate immune Received 13 December, 2013; revised 3 April, 2014; accepted 18 April, 2014. *For correspondence. E-mail alain.vandewalle@ inserm.fr; Tel. (+33) 1 57 27 75 50; Fax (+33) 1 57 27 74 85. Cellular Microbiology (2014) doi:10.1111/cmi.12306 © 2014 John Wiley & Sons Ltd cellular microbiology