Ncf1 provides a reactive oxygen species-independent negative feedback regulation of TLR9-induced IL-12p70 in murine dendritic cells.
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/gp91(phox)-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-gamma 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.
Richter
Cornelia
C
pharmazentrum frankfurt/Center for Drug Research, Development, and Safety, Clinic of J. W. Goethe University, Frankfurt, Germany. richter@med.uni-frankfurt.de
Juan
Martina Herrero San
MH
Will
Jutta
J
Brandes
Ralf P
RP
Kalinke
Ulrich
U
Akira
Shizuo
S
Pfeilschifter
Josef M
JM
Hultqvist
Malin
M
Holmdahl
Rikard
R
Radeke
Heinfried H
HH
D000818:Animals; D003713:Dendritic Cells / Q000276:immunology* / Q000378:metabolism; D025461:Feedback, Physiological / Q000276:immunology*; D005434:Flow Cytometry; D018664:Interleukin-12 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D053594:Myeloid Differentiation Factor 88 / Q000276:immunology / Q000378:metabolism; D019255:NADPH Oxidases / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D017382:Reactive Oxygen Species / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000276:immunology*; D051217:Toll-Like Receptor 9 / Q000276:immunology* / Q000378:metabolism
C507780:Myd88 protein, mouse; D053594:Myeloid Differentiation Factor 88; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D051217:Toll-Like Receptor 9; D018664:Interleukin-12; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1