SELECTIVE USE OF MEMBRANE CD14, BUT NOT SOLUBLE CD14 ANDINHIBITION BY SERUM IN THE INFLAMMATORY CYTOKINE RESPONSE INDUCED BY A NOVELTLR LIGAND (P. AERUGINOSA EXOENZYME S)

Background/Purpose: Soluble andmembrane CD14 (sCD14 and mCD14) bind microbial products, are toll-like receptor(TLR) coreceptors, and induce inflammation. Clinical relevance is demonstrated by a correlation of sCD14 levels with morbidity and mortality in inflammatory diseases. Moreover, P. aeruginosa induces damaging inflammation in the lungs of cysticfibrosis patients and its virulence is often attributed to the TLR2 and TLR4ligand exoenzyme S (ExoS). Because of the importance of the response of CD14 toTLR ligands and its potential for therapeutic manipulation, its role inExoS-induced inflammatory cytokines was investigated. Methods: The ability of ExoS to induce TNFand IL-6 cytokine production in cell lines that express TLR and either CD14 positive or negative was assessed (THP-1 or U373 cells, respectively). Recombinant CD14 or CD14 blocking agents were used to test the contribution ofmCD14 and sCD14. Results: Enhancing expression of mCD14 on THP-1 cells increased TNF production, which was abrogated by blocking or removing mCD14. Transfecting mCD14 into U373 cells demonstrated that mCD14 was required for binding ExoS and subsequent IL-6 production. Unlike TLR ligands that only stimulate one TLR, such as lipopolysaccharide, neither sCD14 nor serum enhanced production of cytokine in response to ExoS. Uniquely, serum inhibited ExoS induced TNF. Conclusion: This work demonstrates a fundamental difference in the requirement of ExoS and other TLR ligands for CD14, which must be considered when designing therapies to block microbe-induced inflammation. The presence of a potential therapeutic molecule in serum could help in the development of an ExoS neutralizing agent.