Glycogen synthase kinase-3β (GSK-3β)-modulated IFN-γ-induced inflammation has been reported; however, the mechanism that activates GSK-3β and the effects of activation remain unclear. Inhibiting GSK-3β decreased IFN-γ-induced inflammation. IFN-γ treatment rapidly activated GSK-3β via neutral sphingomyelinase- and okadaic acid-sensitive phosphatase-regulated dephosphorylation at Ser9, and proline-rich tyrosine kinase 2 (Pyk2)-regulated phosphorylation at Tyr216. Pyk2 was activated through phosphatidylcholine-specific phospholipase C (PC-PLC)-, protein kinase C (PKC)-, and Src-regulated pathways. The activation of PC-PLC, Pyk2, and GSK-3β was potentially regulated by IFN-γ receptor 2-associated Jak2, but it was independent of IFN-γ receptor 1. Furthermore, Jak2/PC-PLC/PKC/cytosolic phospholipase A2 positively regulated neutral sphingomyelinase. Inhibiting GSK-3β activated Src homology-2 domain-containing phosphatase 2 (SHP2), thereby preventing STAT1 activation in the late stage of IFN-γ stimulation. All these results showed that activated GSK-3β synergistically affected IFN-γ-induced STAT1 activation by inhibiting SHP2.

BackgroundAnesthetic propofol has immunomodulatory effects, particularly in the area of anti-inflammation. Bacterial endotoxin lipopolysaccharide (LPS) induces inflammation through toll-like receptor (TLR) 4 signaling. We investigated the molecular actions of propofol against LPS/TLR4-induced inflammatory activation in murine RAW264.7 macrophages.Methodology/Principal FindingsNon-cytotoxic levels of propofol reduced LPS-induced inducible nitric oxide synthase (iNOS) and NO as determined by western blotting and the Griess reaction, respectively. Propofol also reduced the production of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 as detected by enzyme-linked immunosorbent assays. Western blot analysis showed propofol inhibited LPS-induced activation and phosphorylation of IKKβ (Ser180) and nuclear factor (NF)-κB (Ser536); the subsequent nuclear translocation of NF-κB p65 was also reduced. Additionally, propofol inhibited LPS-induced Akt activation and phosphorylation (Ser473) partly by reducing reactive oxygen species (ROS) generation; inter-regulation that ROS regulated Akt followed by NF-κB activation was found to be crucial for LPS-induced inflammatory responses in macrophages. An in vivo study using C57BL/6 mice also demonstrated the anti-inflammatory properties against LPS in peritoneal macrophages.Conclusions/SignificanceThese results suggest that propofol reduces LPS-induced inflammatory responses in macrophages by inhibiting the interconnected ROS/Akt/IKKβ/NF-κB signaling pathways.

there is a link between autophagy and both IFN-␥ signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.Autophagy, or autophagocytosis, is required for cellular regulation in response to a variety of stimuli, including starvation, pathogen-associated molecular patterns that are recognized by pattern-recognition receptors such as Toll-like receptors, and cytokines such as tumor necrosis factor (TNF)-␣ and interferon (IFN)-␥ (1, 2). In addition to maintaining cell survival and metabolic homeostasis (3), autophagy provides a cell-autonomous defense system for recognizing viral infections (4, 5) and eliminating intracellular pathogens via the autophagosome-lysosome pathway (6 -8).Proinflammatory cytokine IFN-␥, a type II IFN produced by T cells and natural killer cells, is involved in promoting diverse bioactivities, including antigen processing, intracellular microbial killing, and proinflammation (9). After binding with IFN-␥ receptors (IFNGRs), 2 IFN-␥ typically activates Jak2-STAT1 signaling and then regulates its bioactivities. For Jak2-STAT1 activation, Jak2 is first autophosphorylated at its tyrosine residues (Tyr 1007 /Tyr 1008 ) and then leads to Jak1 transphosphorylation (Tyr 1022 /Tyr 1023 ). The activation of Jak1 then phosphorylates IFNGR1 (Tyr 440 ), which induces the recruitment and activation of STAT1 through Jak2-mediated phosphorylation (Tyr 701 ). SOCS1 (suppressor of cytokine signaling-1), SOCS3, and SHP2 (dual-phosphatase Src homology-2 domain-containing phosphatase) provide feedback regulation by suppressing Jak2-STAT1 signaling (9, 10). SOCS1 and SOCS3 interact with IFNGRs, and SHP2 causes the dephosphorylation of Jak2 and STAT1. IFN-␥ induces, STAT1-dependently, SOCS1 and SOCS3 expression; however, the mechanisms for SHP2 activation remain undocumented.IFN-␥ uses a process that involves autophagy to increase the eradication of intracellular mycobacteria and chlamydia (6, 11). IFN-inducible immunity-related GTPases (Irgs (immunoreactive glucagons)), such as Irgm1 and Irga6 (6, 11), and IFN-inducible eukaryotic initiation factor (eIF)-2␣ kinase, protein kinase R (12), are potential autophagic regulators; however, the mechanisms for IFN-␥-induced autophagy are currently undocumented. In addition, the functions of autophagic machinery for IFN-␥-activated Jak2-STAT1 signaling and bioactivities require further investigation. In this study, we examined the role of autophagy and its molecular actions in the IFN-␥-induced Jak2-STAT1 activation and cellular inflammation. EXPERIMENTAL PROCEDURES

Interferon-gamma (IFN-gamma) plays a crucial role in innate immunity and inflammation. It causes the synergistic effect on endotoxin lipopolysaccharide (LPS)-stimulated inducible nitric oxide synthase (iNOS)/NO biosynthesis; however, the mechanism remains unclear. In the present study, we investigated the effects of glycogen synthase kinase-3 (GSK-3)-mediated inhibition of anti-inflammatory interleukin-10 (IL-10). We found, in LPS-stimulated macrophages, that IFN-gamma increased iNOS expression and NO production in a time-dependent manner. In addition, ELISA analysis showed the upregulation of tumor necrosis factor-alpha and regulated on activation, normal T expressed and secreted, and the downregulation of IL-10. RT-PCR further showed changes in the IL-10 mRNA level as well. Treating cells with recombinant IL-10 showed a decrease in IFN-gamma/LPS-induced iNOS/NO biosynthesis, whereas anti-IL-10 neutralizing antibodies enhanced this effect, suggesting that IL-10 acts in an anti-inflammatory role. GSK-3-inhibitor treatment blocked IFN-gamma/LPS-induced iNOS/NO biosynthesis but upregulated IL-10 production. Inhibiting GSK-3 using short-interference RNA showed similar results. Additionally, treating cells with anti-IL-10 neutralizing antibodies blocked these effects. We further showed that inhibiting GSK-3 increased phosphorylation of transcription factor cyclic AMP response element binding protein. Inhibiting protein tyrosine kinase Pyk2, an upstream regulator of GSK-3beta, caused inhibition on IFN-gamma/LPS-induced GSK-3beta phosphorylation at tyrosine 216 and iNOS/NO biosynthesis. Taken together, these findings reveal the involvement of GSK-3-inhibited IL-10 on the induction of iNOS/NO biosynthesis by IFN-gamma synergized with LPS.