ORIGINAL ARTICLE Staphylococcus aureus biofilm activates the nucleotide-binding oligomerization domain containing 2 (Nod2) pathway and proinflammatory factors on a human sinonasal explant model Daniel Cantero, MD, Clare Cooksley, PhD, Ahmed Bassiouni, MD, Peter-John Wormald, MD and Sarah Vreugde, MD, PhD Background: The presence of Staphylococcus aureus biofilms on sinonasal mucosal surfaces is associated with recalcitrant chronic rhinosinusitis (CRS), but lile is known about the innate immune response they trigger. We aimed to study the human paern recognition receptor (PRR) nucleotide-binding oligomerization domain containing 2 (Nod2) receptor and downstream pathway in response to initial S. aureus biofilm infection. Methods: Using a validated protocol, sinonasal mucosae from 4 non-CRS donors were cultured with and without S. aureus biofilms and planktonic cells. Aſter 24 hours, RNA was extracted and gene expression was analyzed using a human antibacterial response polymerase chain reaction (PCR) array. Immunohistochemistry was performed to con- firm the presence and determine the immunolocalization of selected proteins. Results: C-X-C motif (CXC) chemokine ligands 1 and 2, interleukin-6 (IL-6), and genes related to the Nod2 pathway were significantly upregulated in biofilm-treated tissues compared with control samples. Nod2 pathway–specific gene expression was increased in biofilm-treated tissues compared with planktonic S. aureus–treated explants. En- hanced expression of Nod2 and nuclear factor kappa B1 (NF-κ B1) was also detected with immunohistochemistry in control and biofilm-treated tissues. Conclusion: S. aureus biofilms exerted a proinflammatory response in the mucosa and activation of the Nod2 pathway, indicating this receptor to be involved in the innate immune response to S. aureus biofilms. Further studies are required to elucidate the role of this pathway in CRS. C  2013 ARS- AAOA, LLC. Key Words: Staphylococcus aureus; biofilms; Nod2 protein; Nod2 re- ceptor; organ culture; nasal mucosa; sinusitis; confocal; innate immunity; paranasal sinuses How to Cite this Article: Cantero D, Cooksley C, Bassiouni A, Wormald P-J, Vreugde S. Staphylococcus aureus biofilm activates the nucleotide-binding oligomerization domain containing 2 (Nod2) pathway and proinflammatory factors on a hu- man sinonasal explant model. Int Forum Allergy Rhinol. 2013;3:877–884. S taphylococcus aureus is one of the most prevalent bac- teria recovered from chronic rhinosinusitis (CRS) pa- Department of Otorhinolaryngology–Head and Neck Surgery, Division of Surgery, The Queen Elizabeth Hospital, and the University of Adelaide, Adelaide, South Australia, Australia Correspondence to: Peter-John Wormald, MD, Department of Otorhinolaryngology Head and Neck Surgery, The Queen Elizabeth Hospital, 28 Woodville Rd, Woodville South, South Australia 5011, Australia; e-mail: peterj.wormald@adelaide.edu.au Funding sources for the study: The University of Adelaide, Faculty of Health Sciences, Adelaide, South Australia. Potential conflict of interest: P.J.W. receives royalties from Medtronic for instruments designed and is a consultant for Neilmed Pty Ltd.; these are not relevant to this study. Received: 3 June 2013; Revised: 28 June 2013; Accepted: 5 July 2013 DOI: 10.1002/alr.21213 View this article online at wileyonlinelibrary.com. tients using traditional and/or molecular methods. 1, 2 This bacterium possesses a broad armamentarium of virulence factors such as the production of adhesion proteins and the secretion of toxins and enzymes. 3 Features like biofilm formation and surviving intracellularly 4, 5 may increase the ability of S. aureus to avoid antimicrobial treatments and host defenses in CRS. The host immune response to this bacterium is complex and only partially understood. Although some researchers have looked at the acquired immune response to S. aureus biofilms in CRS, 6 little has been studied regarding the innate immune response to S. aureus biofilms in paranasal cavities. The innate immune system of the airway represents the first line of defense against invading pathogens and orchestrates adaptive immune responses. Epithelial and immune cells express pattern recognition receptors 877 International Forum of Allergy & Rhinology, Vol. 3, No. 11, November 2013