Interplay of protease-activated receptors and NOD pattern recognition receptors in epithelial innate immune responses to bacteria

Abstract

response to bacteria. Human gingival epithelial cells (GECs) were transfected with siRNA specific for PAR1 or PAR2, then stimulated with periopathogen Porphyromonas gingivalis, bridging organism between pathogens and non-pathogens Fusobacterium nucleatum, or non-pathogen Streptococcus gordonii. PAR1 or PAR2 knock-down resulted in up-regulated NOD1 and NOD2 expression with P. gingivalis or F. nucleatum stimulation (p<0.01), as well as enhanced TLR2 and TLR4 expression when cells were stimulated by bacteria that utilize TLR2 or TLR4, respectively. Involvement of PARs for induction of CC chemokine ligand 20 (CCL20), a cytokine with antimicrobial properties, was observed following stimulation of the three bacterial species. Furthermore, results from multiple cytokine ELISA array showed receptors utilized in the induction of various innate immune markers are tailored to individual bacterium tested. Our data suggest complex interplay of several receptors is required for appropriate innate immune responses to the different types of bacteria present within the oral cavity and that receptor expression itself is altered depending on which organism the cell encounters. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptImmunol Lett. Author manuscript; available in PMC 2011 July 8. Published in final edited form as:Immunol Lett. Materials and Methods Human epithelial cells and bacterial culture conditionsHealthy gingival samples were obtained from patients undergoing third-molar extraction at the Department of Oral Surgery, School of Dentistry, University of Washington. Tissue was prepared as described earlier, and subsequently isolated GECs were grown in Keratinocyte Basal Media supplemented with Keratinocyte Growth Media (Cambrex, Walkersville, MD) [21]. Cells were grown in media containing 0.15 mM calcium (Ca ++ ). P. gingivalis (ATCC 33277) cells were cultured in anaerobic conditions (85% N 2 , 10% H 2 , 5% CO 2 ) at 37°C in Trypticase soy broth (BBL, Sparks, MD) supplemented with 1 g of yeast extract, 5 mg of hemin and 1 mg of menadione per liter. Streptococcus gordonii (Challis DL1) was grown in Trypticase soy broth at 37°C under static conditions. Fusobacterium nucleatum (ATCC 25586) was grown in Todd-Hewitt broth supplemented with 1 g of yeast extract per 100 ml at 37°C in anaerobic conditions. All bacterial species used in this study are from our laboratory stock. Bacterial numbers were estimated by density in a GENios Multi-detection Reader (Phenix, Hayward, CA). Transfection of keratinocytes with siRNASpecific siRNAs were custom-synthesized...

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