Naegleria fowleri induces MUC5AC and pro- inflammatory cytokines in human epithelial cells via ROS production and EGFR activation Isaac Cervantes-Sandoval, 1,3 Jose ´ de Jesu ´ s Serrano-Luna, 2 Patricia Meza-Cervantez, 1 Rossana Arroyo, 1 Vı ´ctor Tsutsumi 1 and Mineko Shibayama 1 Correspondence Mineko Shibayama mineko@cinvestav.mx 1 Department of Infectomic and Molecular Pathogenesis, Center for Research and Advanced Studies, National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico 2 Department of Cell Biology, Center for Research and Advanced Studies, National Polytechnic Institute, Av. IPN 2508, Mexico City 07360, Mexico 3 Department of Immunology, National School of Biological Sciences, National Polytechnic Institute, Av. Manuel M. Carpio and Plan de Ayala, Mexico City 11340, Mexico Received 7 May 2009 Revised 9 July 2009 Accepted 30 July 2009 Naegleria fowleri is an amoeboflagellate responsible for the fatal central nervous system (CNS) disease primary amoebic meningoencephalitis (PAM). This amoeba gains access to the CNS by invading the olfactory mucosa and crossing the cribriform plate. Studies using a mouse model of infection have shown that the host secretes mucus during the very early stages of infection, and this event is followed by an infiltration of neutrophils into the nasal cavity. In this study, we investigated the role of N. fowleri trophozoites in inducing the expression and secretion of airway mucin and pro-inflammatory mediators. Using the human mucoepidermal cell line NCI-H292, we demonstrated that N. fowleri induced the expression of the MUC5AC gene and protein and the pro-inflammatory mediators interleukin-8 (IL-8) and interleukin-1b (IL-1b), but not tumour necrosis factor-a or chemokine c-c motif ligand 11 (eotaxin). Since the production of reactive oxygen species (ROS) is a common phenomenon involved in the signalling pathways of these molecules, we analysed if trophozoites were capable of causing ROS production in NCI-H292 cells by detecting oxidation of the fluorescent probe 2,7-dichlorofluorescein diacetate. NCI-H292 cells generated ROS after 15–30 min of trophozoite stimulation. Furthermore, the expression of MUC5AC, IL-8 and IL-1b was inhibited in the presence of the ROS scavenger DMSO. In addition, the use of an epidermal growth factor receptor inhibitor decreased the expression of MUC5AC and IL-8, but not IL-1b. We conclude that N. fowleri induces the expression of some host innate defence mechanisms, such as mucin secretion (MUC5AC) and local inflammation (IL-8 and IL-1b) in respiratory epithelial cells via ROS production and suggest that these innate immune mechanisms probably prevent most PAM infections. INTRODUCTION Naegleria fowleri is an ubiquitous, free-living amoeba that has been isolated from diverse water and soil sources. Under certain conditions, which are not fully understood, these amoebae may infect the human central nervous system (CNS), giving rise to primary amoebic meningoen- cephalitis (PAM). Infection usually occurs in young healthy people with a history of recent recreational water activities (Marciano-Cabral & Cabral, 2007). Despite the ubiquity and worldwide distribution of these amoebae, only a little over 200 cases have been reported worldwide. Trophozoites invade the host by penetrating the olfactory neuroepithe- lium and migrating through the cribriform plate, before reaching the olfactory bulbs (Carter, 1970; John & Nussbaum, 1983; Jarolim et al., 2000; Rojas-Herna ´ndez et al., 2004). Once in the CNS, the amoebae induce a strong inflammatory reaction, haemorrhage and lytic necrosis (Carter, 1970; Cervantes-Sandoval et al., 2008a). Abbreviations: CCL11, chemokine c-c motif ligand 11; CNS, central nervous system; EGFR, epidermal growth factor receptor; FDA, 2,7- dichlorofluorescein diacetate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL-1b, interleukin-1b; IL-8, interleukin-8; MUC5AC, mucin 5AC; PAM, primary amoebic meningoencephalitis; PMA, phorbol 12-myristate 13-acetate; ROS, reactive oxygen species; TGF-a, transforming growth factor-a; TNF-a, tumour necrosis factor-a. Microbiology (2009), 155, 3739–3747 DOI 10.1099/mic.0.030635-0 030635 G 2009 SGM Printed in Great Britain 3739