Molecular Immunology 47 (2010) 1450–1457 Contents lists available at ScienceDirect Molecular Immunology journal homepage: www.elsevier.com/locate/molimm Peptidoglycan, not endotoxin, is the key mediator of cytokine gene expression induced in rainbow trout macrophages by crude LPS  Simon A. MacKenzie a,1 , Nerea Roher a,1 , Sebastian Bolta ˜ na a , Frederick W. Goetz b,∗ a Institute of Biotechnology and Biomedicine, Dep. Biologia Cellular, Immunologia i Fisiologia Animal, Universitat Autónoma de Barcelona, 08193 Barcelona, Spain b Great Lakes WATER Institute, University of Wisconsin-Milwaukee, 600 E. Greenfield Avenue, Milwaukee, WI 53204, USA article info Article history: Received 13 January 2010 Received in revised form 6 February 2010 Accepted 16 February 2010 Keywords: Macrophages Lipopolysaccharide Peptidoglycans Endotoxin Rainbow trout Inflammatory gene expression abstract In rainbow trout macrophages, phenol-extracted lipopolysaccharide (LPS) preparations stimulate proin- flammatory cytokine gene expression but ultrapure preparations of LPS are inactive. Crude LPS preparations could potentially have a number of contaminants including peptidoglycans (PGNs), nucleic acids and lipoproteins. Thus, in the current study we individually tested potentially contaminating pathogen associated molecular patterns (PAMPs) on rainbow trout (Oncorhynchus mykiss) macrophages to determine which ones could induce proinflammatory cytokine expression. We found that PGNs derived from Gram-negative bacteria (Escherichia coli 0111:B4 and K12), are potent inducers of IL-1 and IL-6 gene expression and were equal to, or more potent than, crude LPS. On the other hand, PGNs of Gram-positive bacteria, DNA, RNA and lipoteichoic acid were weak stimulators, and lipid A, lipoprotein (Pam3CSK4) and ultrapure LPS were nonstimulatory. More importantly, crude LPS treated with lysozyme to degrade PGNs, exhibited greatly reduced activity in stimulating IL-1 and IL-6 gene expression, indicating that PGNs in the crude LPS are responsible for a significant amount of the proinflammatory activity. Finally, we showed that PGN treatment induces expression of COX-2 and the subsequent synthesis and release of prostaglandin E 2 (PGE 2 ), an important mediator of inflammatory processes. The strong stimulatory effect of E. coli PGNs by themselves on trout macrophages suggests that the recognition of Gram-negative bac- teria in trout is through PGNs in the bacterial wall, and indicates that the systems responsible for bacterial recognition in invertebrates (e.g., Drosophila) may also be conserved in some vertebrates. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction Pathogens are recognized by the immune system through spe- cific components referred to as pathogen associated molecular patterns (PAMPs), including lipopolysaccharides (LPSs) of Gram- negative bacteria, peptidoglycans (PGNs) found in Gram-positive and Gram-negative bacteria, -glucans of fungi, and viral nucleic acids. The study of LPS and the mechanism by which it stimu- lates an immune response has been a central focus in vertebrate immunology, particularly in view of the potential lethal effects of LPS over-stimulation. LPS is the major constituent of the exter- nal layer of the outer membrane of Gram-negative bacteria. It is composed of a polysaccharide portion consisting of a carbohydrate O-antigen and an oligosaccharide core region, and a lipid portion  This study was supported by the Consolider-Ingenio Programme 2010, project CSD2007-0002 funded by the Spanish Ministry of Science and Education, Spain to S.M. N.R. and S.B. are funded by Consolider-Ingenio 2010. ∗ Corresponding author. Tel.: +1 414 382 1742; fax: +1 414 382 1705. E-mail address: rick@uwm.edu (F.W. Goetz). 1 These authors contributed equally. termed “lipid A” that is responsible for the innate immune response in mammals and confers the endotoxic properties of LPS (Bishop, 2005; Raetz and Whitfield, 2002). Mammalian cells are extremely sensitive to the effects of LPS, in part because of the facilitatory action of a serum protein called lipopolysaccharide binding protein (LBP) (Gallay et al., 1993). In mammals, LPS aggregates are initially recognized by LBP (Mathison et al., 1992) that facilitates the trans- fer of LPS to the co-stimulatory molecule CD14 (Tobias et al., 1995) and then in monomeric form to LY96 (Gioannini et al., 2005). LY96 is associated with Toll-like receptor 4 (TLR4) and specifically binds the endotoxin moiety of LPS (Akashi et al., 2003). The activation of TLR4 by LPS/LY96 is followed by the recruitment of intracellu- lar adaptor molecules including a pathway involving MyD88 and TIRAP (Burns et al., 1998; Horng et al., 2001), and another pathway including TICAM1 and TICAM2. The MyD88 pathway leads to the early activation of NFB while the TICAM1/2 pathway leads to the later activation of NFB and also the induction of antiviral genes (Seya et al., 2005). It has been known for some time that nonmammalian verte- brates, and particularly fish, are immune to the toxic effects of LPS that cause septic shock in mammals (Berczi et al., 1966). Fur- ther, fish leukocytes are orders of magnitude less sensitive than 0161-5890/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2010.02.009