Ncf1 Provides a Reactive Oxygen Species-Independent Negative Feedback Regulation of TLR9-Induced IL-12p70 in Murine Dendritic Cells

Abstract

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-γ 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.

PP2A-dependent control of transcriptionally active FOXO3a in CD8+ central memory lymphocyte survival requires p47phox

“…Consistent with these observations, a recent report by Purushothaman and Sarin showed short hairpin RNA inhibition of gp91 phox , but not p47 phox inhibited neglect-induced death in activated T cells, and that activated T-cell survival, as well as antigen-specific T-cell persistence and recall, were improved in gp91 phox−/− mice following super-antigen challenge. 31 Also consistent with our observations, Richter et al 32 reported that independent of its role in generating Nox2-dependent ROS, p47 phox is an important protein for integrating signaling downstream of TLR9 in mouse dendritic cells, thus supporting the idea of distinct and divergent functions for gp91 phox and p47 phox .…”

NADPH Oxidase–Derived Superoxide Provides a Third Signal for CD4 T Cell Effector Responses

“…In the presence of the ROS donor xanthine oxidase, pro-inflammatory cytokines could be rescued, but Type I interferon synthesis remained decreased (21). Our studies and others (21, 37, 38) provide evidence that NOX subunits are also directly involved in physically transducing signals independent of superoxide generation. The p47 phox subunit has been shown to physically interact with TNF-associated factor 4 (TRAF) 4 to inhibit TLR9 signaling and IFN-β synthesis (38).…”

“…The p47 phox subunit has been shown to physically interact with TNF-associated factor 4 (TRAF) 4 to inhibit TLR9 signaling and IFN-β synthesis (38). Additionally, TLR9/MyD88 signaling and dendritic cell expression of IL-12p70 was shown to be negatively regulated via p47 phox -independent mechanisms (37). Future studies will seek to examine these physical interactions by the p47 phox subunit to propagate adaptive immune responses necessary for Th1 cytokine responses.…”

NADPH Oxidase-2 Derived ROS Dictates Murine DC Cytokine-Mediated Cell Fate Decisions during CD4 T Helper-Cell Commitment

“…However, an important question raised by these data is the relative roles of the p47 phox component of the Nox2 complex, and the ROS produced by the fully assembled and activated Nox2 holoenzyme. A recent report showed that p47 phox , independent of Nox2, negatively regulates TLR-9 induced IL-12p70 expression in splenic DC [30]. To address this, we used ELISA to assess IFNγ/LPS stimulated IL-12p70 secretion in Nox2 catalytic subunit Nox2 −/− DC.…”