Two mechanistically and temporally distinct NF-kappaB activation pathways in IL-1 signaling.

The cytokine interleukin-1 (IL-1) mediates immune and inflammatory responses by activating the transcription factor nuclear factor kappaB (NF-kappaB). Although transforming growth factor-beta-activated kinase 1 (TAK1) and mitogen-activated protein kinase (MAPK) kinase kinase 3 (MEKK3) are both crucial for IL-1-dependent activation of NF-kappaB, their potential functional and physical interactions remain unclear. Here, we showed that TAK1-mediated activation of NF-kappaB required the transient formation of a signaling complex that included tumor necrosis factor receptor-associated factor 6 (TRAF6), MEKK3, and TAK1. Site-specific, lysine 63-linked polyubiquitination of TAK1 at lysine 209, likely catalyzed by TRAF6 and Ubc13, was required for the formation of this complex. After TAK1-mediated activation of NF-kappaB, TRAF6 subsequently activated NF-kappaB through MEKK3 independently of TAK1, thereby establishing continuous activation of NF-kappaB, which was required for the production of sufficient cytokines. Therefore, we propose that the cooperative activation of NF-kappaB by two mechanistically and temporally distinct MEKK3-dependent pathways that diverge at TRAF6 critically contributes to immune and inflammatory systems.

TRAF6 establishes innate immune responses by activating NF-kappaB and IRF7 upon sensing cytosolic viral RNA and DNA.

BACKGROUND: In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor kappaB (NF-kappaB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-kappaB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed. PRINCIPAL FINDINGS: Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-kappaB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFbeta-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-kappaB activation, were not essential for RLH-mediated NF-kappaB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-kappaB and IRF7. CONCLUSIONS/SIGNIFICANCE: Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection.

Limitations in the use of rifampicin-gelatin grafts against virulent organisms.

OBJECTIVE: Efficacy and duration of antibacterial activity of rifampicin-gelatin grafts against virulent organisms were evaluated in an animal model. MATERIALS AND METHODS: Rifampicin-gelatin grafts were prepared with impregnation of Gelseal (Vascutek Ltd, Scotland) graft in 1 mg/mL rifampicin solution. Rifampicin-gelatin grafts (6 cm long; n = 24) and plain Gelseal grafts as controls (n = 4) were implanted into the canine abdominal aorta with inoculation of Staphylococcus epidermidis, Escherichia coli, or methicillin-resistant Staphylococcus aureus (MRSA), and the rifampicin-gelatin grafts were retrieved after 1 to 4 weeks. Disks cut from the retrieved rifampicin-gelatin grafts were placed on agar plates streaked with one of the organisms, and the graft antibacterial activity was assessed with the width of the inhibition zone. RESULTS: In in vitro tests, initial inhibition zones (inhibition zone of 24 hours after incubation) of rifampicin-gelatin grafts against S epidermidis, MRSA, and E coli were 40.0 +/- 0.3 mm, 36.0 +/- 0.2 mm, and 11.8 +/- 0.1 mm, respectively. In the implantation, S epidermidis -inoculated rifampicin-gelatin grafts had no findings of graft infection, and no colony growth was recognized on the plates streaked with the perigraft fluids. Initial inhibition zones of S epidermidis -inoculated rifampicin-gelatin grafts retrieved at 1 or 2 weeks were 20.1 +/- 1.1 mm and 7.6 +/- 1.0 mm, respectively. In E coli -inoculated and MRSA-inoculated rifampicin-gelatin grafts, all of the eight animals had perigraft abscess, and blood culture test results probed septicemia in five animals with patent grafts at death. Inhibition zones against E coli or MRSA were not formed on the plates streaked with the same organism, whereas initial inhibition zones of E coli -inoculated and MRSA-inoculated rifampicin-gelatin grafts on S epidermidis -streaked plates were 8.0 +/- 0.2 mm and 18.5 +/- 0.5 mm, respectively. In the MRSA group, however, recolonization of high minimal inhibitory concentration strains developed within the inhibition zones as early as 24 hours. Histologically, neither organisms nor inflammatory cells were found in S epidermidis -inoculated rifampicin-gelatin grafts and tissue ingrowth was recognized at 2 to 4 weeks, whereas E coli -inoculated and MRSA-inoculated rifampicin-gelatin grafts had aggressive neutrophil infiltration into the graft interstices, revealing establishment of uncontrollable graft infection. CONCLUSION: These results suggested that rifampicin-gelatin grafts are clearly valid for S epidermidis infection, whereas no efficacy was recognized against either MRSA or E coli graft infection because of early development of high minimal inhibitory concentration MRSA strains or poor susceptibility.