Identification of Two Forms of TNF Tolerance in Human Monocytes: Differential Inhibition of NF-κB/AP-1– and PP1-Associated Signaling

Abstract

The molecular basis of TNF tolerance is poorly understood. In human monocytes we detected two forms of TNF refractoriness, as follows: absolute tolerance was selective, dose dependently affecting a small group of powerful effector molecules; induction tolerance represented a more general phenomenon. Preincubation with a high TNF dose induces both absolute and induction tolerance, whereas low-dose preincubation predominantly mediates absolute tolerance. In cells preincubated with the high TNF dose, we observed blockade of IκBα phosphorylation/proteolysis and nuclear p65 translocation. More prominent in cells preincubated with the high dose, reduced basal IκBα levels were found, accompanied by increased IκBα degradation, suggesting an increased IκBα turnover. In addition, a nuclear elevation of p50 was detected in tolerant cells, which was more visible following high-dose preincubation. TNF-induced phosphorylation of p65-Ser536, p38, and c-jun was inhibited, and basal inhibitory p65-Ser468 phosphorylation was increased in tolerant cells. TNF tolerance induced by the low preincubation dose is mediated by glycogen synthesis kinase-3, whereas high-dose preincubation-mediated tolerance is regulated by A20/glycogen synthesis kinase-3 and protein phosphatase 1–dependent mechanisms. To our knowledge, we present the first genome-wide analysis of TNF tolerance in monocytic cells, which differentially inhibits NF-κB/AP-1–associated signaling and shifts the kinase/phosphatase balance. These forms of refractoriness may provide a cellular paradigm for resolution of inflammation and may be involved in immune paralysis.

RNAi Screening Identifies that TEX10 Promotes the Proliferation of Colorectal Cancer Cells by Increasing NF‐κB Activation

“…[29] We next investigated whether TEX10 could affect this process. Consistent with a previous report, [30] TNF treatment increased the nuclear accumulation of RELA, while overexpression of TEX10 further increased the nuclear localization of RELA in HT29 cells ( Figure 5A). Additionally, TEX10 knockdown significantly impaired RELA nuclear translocation in HCT116 and DLD1 cells ( Figure 5B,C).…”

C/EBPβ-LAP*/LAP Expression Is Mediated by RSK/eIF4B-Dependent Signalling and Boosted by Increased Protein Stability in Models of Monocytic Differentiation

“…After blocking with 5% skim milk (Merck Millipore) or bovine serum albumin (BSA, Roche) in TBST or PBST, membranes were incubated with the specific primary antibodies at 4°C overnight. Following incubation with HRP-coupled secondary antibodies, protein bands were visualised using HRP substrates enhanced chemoluminescence (ECL) or SuperSignal West Femto (Thermo Fisher, Bonn, Germany) and densitometric analyses were performed using the Bio-imaging system MF-ChemiBis 3.2 and the TotalLab TL100 analysis software (Nonlinear Dynamics, Newcastle, UK) [ 15 , 17 , 20 ].…”

“…RNA concentrations were determined using a Nanodrop ND-1000 (Peqlab, Erlangen, Germany). cDNA synthesis and qRT-PCR were performed as previously described [ 15 , 17 , 20 ]. For mRNA expression analysis, two specific primer pairs were used for C/EBPβ cDNA detection (5’ mRNA section at pos.…”

GSK3: A Kinase Balancing Promotion and Resolution of Inflammation

“…TNF tolerance may occur in two forms, i.e., absolute tolerance (in which gene expression is blocked) and induction tolerance (in which gene expression is not further inducible). Low-dose preincubation predominantly results in absolute tolerance, whereas high-dose preincubation induces both absolute and induction tolerance [289]. Amongst other mechanisms, the development of low-dose TNF tolerance as well as TNF-induced cross-tolerance towards LPS depends on the activity of GSK3 [286], since GSK3 remained in an active state during TNF preincubation [288], while pharmacological inhibition or genetic deletion of GSK3 are able to reverse the formation of these forms of tolerance [288,289].…”

“…Amongst other mechanisms, the development of low-dose TNF tolerance as well as TNF-induced cross-tolerance towards LPS depends on the activity of GSK3 [286], since GSK3 remained in an active state during TNF preincubation [288], while pharmacological inhibition or genetic deletion of GSK3 are able to reverse the formation of these forms of tolerance [288,289]. Mechanistically, GSK3 inhibition appears to counteract several molecular effects observed in low-dose tolerized cells, especially the increased phosphorylation of NF-κB-p65 at the inhibiting site Ser468 and the reduced phosphorylation at the activating site Ser536 [289]. The latter can be traced to direct association of C/EBPβ with p65 in tolerant cells [290].…”