Dok-1 and Dok-2 are negative regulators of lipopolysaccharide-induced signaling

Endotoxin, a bacterial lipopolysaccharide (LPS), causes fatal septic shock via Toll-like receptor (TLR)4 on effector cells of innate immunity like macrophages, where it activates nuclear factor κB (NF-κB) and mitogen-activated protein (MAP) kinases to induce proinflammatory cytokines such as tumor necrosis factor (TNF)-α. Dok-1 and Dok-2 are adaptor proteins that negatively regulate Ras–Erk signaling downstream of protein tyrosine kinases (PTKs). Here, we demonstrate that LPS rapidly induced the tyrosine phosphorylation and adaptor function of these proteins. The stimulation with LPS of macrophages from mice lacking Dok-1 or Dok-2 induced elevated Erk activation, but not the other MAP kinases or NF-κB, resulting in hyperproduction of TNF-α and nitric oxide. Furthermore, the mutant mice showed hyperproduction of TNF-α and hypersensitivity to LPS. However, macrophages from these mutant mice reacted normally to other pathogenic molecules, CpG oligodeoxynucleotides, poly(I:C) ribonucleotides, or Pam3CSK4 lipopeptide, which activated cognate TLRs but induced no tyrosine phosphorylation of Dok-1 or Dok-2. Forced expression of either adaptor, but not a mutant having a Tyr/Phe substitution, in macrophages inhibited LPS-induced Erk activation and TNF-α production. Thus, Dok-1 and Dok-2 are essential negative regulators downstream of TLR4, implying a novel PTK-dependent pathway in innate immunity

The Assembly of EDC4 and Dcp1a into Processing Bodies Is Critical for the Translational Regulation of IL-6

<div><p>Macrophages play critical roles in the onset of various diseases and in maintaining homeostasis. There are several functional subsets, of which M1 and M2 macrophages are of particular interest because they are differentially involved in inflammation and its resolution. Here, we investigated the differences in regulatory mechanisms between M1- and M2-polarized macrophages by examining mRNA metabolic machineries such as stress granules (SGs) and processing bodies (P-bodies). Human monocytic leukemia THP-1 cells cultured under M1-polarizing conditions (M1-THPs) had less ability to assemble oxidative-stress-induced SGs than those cultured under M2-polarizing conditions (M2-THPs). In contrast, P-body assembly in response to oxidative stress or TLR4 stimulation was increased in M1-THPs as compared to M2-THPs. These results suggest that mRNA metabolism is controlled differently in M1-THPs and M2-THPs. Interestingly, knocking down EDC4 or Dcp1a, which are components of P-bodies, severely reduced the production of IL-6, but not TNF-α in M1-THPs without decreasing the amount of IL-6 mRNA. This is the first report to demonstrate that the assembly of EDC4 and Dcp1a into P-bodies is critical in the posttranscriptional regulation of IL-6. Thus, improving our understanding of the mechanisms governing mRNA metabolism by examining macrophage subtypes may lead to new therapeutic targets.</p></div

SG and P-body components are expressed in M1-THPs and M2-THPs.

<p>(A) M1-THPs and M2-THPs with no treatment (NT) or stimulated with LPS for 1, 4, 8, or 24 hours were harvested and subjected to semi-quantitative RT-PCR (A) and immunoblot analyses using antibodies against the SG or P-body components indicated; (B) β-actin served as a loading control.</p

Oxidative stress does not induce P-body accumulation in M1-THPs or M2-THPs.

<p>(A) M1- and M2-polarized THP-1 cells, either untreated (NT) or treated with 0.5 mM arsenite (Ars) for 30 minutes, were fixed and stained with a goat polyclonal antibody specific for EDC4 and a mouse monoclonal antibody specific for Dcp1a, followed by staining with Alexa 488-conjugated donkey anti-goat IgG and Alexa 568-conjugated donkey anti-mouse secondary antibodies. (B) Bar graph showing the average number of EDC4- or Dcp1a- positive foci per cell. The fluorescent dots were counted as described in Methods, and the error bars indicate SD. **p<0.01, ***p<0.001</p

SG accumulation is induced by oxidative stress in M1- and M2-polarized macrophages.

<p>(A) The upregulation of M1 and M2 biomarkers in polarized PMA-differentiated THP-1 cells was confirmed by RT-PCR analysis. PMA-differentiated THP-1 cells were either left untreated (M0) or treated with LPS and IFN-γ (M1 activation) or IL-4 and IL-13 (M2 activation); RNA was isolated 8 and 24 hours after treatment. RT-PCR was performed to determine quantitative changes in mRNAs serving as activation biomarkers. W: no template control for PCR analysis. (B-F) SG formation in response to arsenite- or H₂O₂-induced oxidative stress was stronger in M2-THPs than in M1-THPs. (B) M1-THPs and M2-THPs, either untreated (NT) or treated with 0.5 mM arsenite (Ars) for 30 minutes, were fixed, stained with a mouse monoclonal antibody specific for G3BP1 and goat polyclonal antibody specific for TIA-1, and then stained with Alexa 488-conjugated donkey anti-goat IgG and Alexa 568-conjugated donkey anti-mouse secondary antibodies. The cells were stained with Hoechst 33342 to visualize nuclei, and the images were merged digitally. (C,D) Cells were left untreated or treated with arsenite, and the number of distinct cytoplasmic puncta positive for TIA1, G3BP1, TIA1, or G3BP1 (total), and both TIA1 and G3BP1 (merge) was counted for each cell type. ***p<0.001 (C) The number of fluorescent dots per cell was counted in 30 cells for each cell type, and the cells were categorized as shown in the bar graph. (D) The average number of dots positive for TIA1, G3BP1, TIA1 or G3BP1, and TIA1 and G3BP1 is shown for each cell type; dots were counted in 30 individual cells of each type. Error bars indicate SD. (E) SG formation in M1-THPs and M2-THPs, either untreated (NT) or treated with 1 mM H₂O₂ for 4 hours, was examined by immunofluorescence as in B. (F) Bar graph showing the average number of foci positive for both TIA1 and G3BP1 per cell. Error bars indicate SD. ***p<0.001</p