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* pharmazentrum frankfurt/Center for Drug Research, Development, and Safety and
Institute of Cardiovascular Physiology, Clinic of J. W. Goethe University, Frankfurt, Germany;
Division of Immunology, Paul-Ehrlich-Institute, Langen, Germany;
Department of Host Defense, Research Institute of Microbial Diseases, Osaka University, Suita, Japan; and
¶ Section for Medical Inflammation Research, Karolinska Institutet Stockholm and Lund University, Lund, Sweden
Permanent exposure to pathogens requires decisions toward tolerance or immunity as a prime task of dendritic cells. The molecular mechanisms preventing uncontrolled immune responses are not completely clear. We investigated the regulatory function of Ncf1, an organizing protein of NADPH oxidase, in the signaling cascade of Toll-like receptors. TLR9-stimulated spleen cells from both Ncf1-deficient and B10.Q mice with a point mutation in exon 8 of Ncf1 exhibited increased IL-12p70 secretion compared with controls. This finding was restricted to stimulatory CpG2216 and not induced by CpG2088. Because only CpG/TLR9-induced IL-12p70 was regulated by Ncf1, we used TRIF–/– and MyD88–/– cells to show that TLR9/MyD88 was primarily affected. Interestingly, additional experiments revealed that spleen cells from NOX2/gp91phox-deficient mice and the blocking of electron transfer by diphenylene iodonium had no influence on CpG-induced IL-12p70, confirming an NADPH oxidase-independent function of Ncf1. Finally, proving the in vivo relevance CpG adjuvant-guided OVA immunization resulted in a strong augmentation of IL-12p70-dependent Th1 IFN-
response only in Ncf1-deficient mice. These data suggest for the first time an important role for Ncf1 in the fine tuning of the TLR9/MyD88 pathway in vitro and in vivo that is independent of its role as an activator of NOX2.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
C.R. performed most of the experiments, collected and analyzed the data, performed statistics, designed figures, and wrote the basic manuscript. M.H.S.J. performed part of the experiments. J.W. performed part of the experiments. R.P.B. provided the p47phox- and gp91phox-deficient mice and critically helped with ROS measurements and with the manuscript. M.H. supported mouse experiments with Ncf1 mutants. R.H. provided the Ncf1 mutated mice and was greatly involved in writing the manuscript. U.K. supported experiments with the TRIF- and MyD88-deficient mice. S.A. provided the MyD88–/– mice initially. J.M.P. supplied basic laboratory equipment. H.H.R. had the idea, designed and closely supervised all experiments, checked all data in detail, and wrote and finalized the manuscript.
1 This work was supported by the Deutsche Forschungsgemeinschaft (Graduate College 1172), the Dr. Hans-Schleussner-Foundation, the Swedish Research Council, the Strategic Science Foundation, and the European Union Grants Autocure (LSHB-2006-018661) and Neuropromise (LSHM-LT-018637).
2 Address correspondence and reprint requests Prof. Heinfried Radeke and Cornelia Richter, pharmazentrum frankfurt, Building 74, 2.102a, Clinic of the Goethe University, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany; E-mail addresses: radeke{at}em.uni-frankfurt.de and richter{at}med.uni-frankfurt.de
3 Abbreviations used in this paper: ROS, reactive oxygen species; DC, dendritic cell; DPI, diphenylene iodonium chloride; IRAK, IL-1R-associated kinase; IRF, IFN regulatory factor; Ncf, neutrophil cytosolic factor (Ncf, gene; Ncf, protein); pDC, plasmacytoid DC; PMA, phorbol myristate acetate; TRAF, TNFR-associated factor; TRIF, Toll/IL-1R domain-containing adapter inducing IFN-β; WT, wild type.
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