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. 2009 Oct;86(4):863-75.
doi: 10.1189/jlb.0309189. Epub 2009 Aug 5.

Endotoxin tolerance dysregulates MyD88- and Toll/IL-1R domain-containing adapter inducing IFN-beta-dependent pathways and increases expression of negative regulators of TLR signaling

Wenji Piao  1 , Chang Song, Haiyan Chen, Marco A Quevedo Diaz, Larry M Wahl, Katherine A Fitzgerald, Liwu Li, Andrei E Medvedev
Affiliations

Endotoxin tolerance dysregulates MyD88- and Toll/IL-1R domain-containing adapter inducing IFN-beta-dependent pathways and increases expression of negative regulators of TLR signaling

Wenji Piao et al. J Leukoc Biol. 2009 Oct.

Abstract

Endotoxin tolerance reprograms cell responses to LPS by repressing expression of proinflammatory cytokines, while not inhibiting production of anti-inflammatory cytokines and antimicrobial effectors. Molecular mechanisms of induction and maintenance of endotoxin tolerance are incompletely understood, particularly with regard to the impact of endotoxin tolerization on signalosome assembly, activation of adaptor-kinase modules, and expression of negative regulators of TLR signaling in human cells. In this study, we examined LPS-mediated activation of MyD88-dependent and Toll-IL-1R-containing adaptor inducing IFN-beta (TRIF)-dependent pathways emanating from TLR4 and expression of negative regulators of TLR signaling in control and endotoxin-tolerant human monocytes. Endotoxin tolerization suppressed LPS-inducible TLR4-TRIF and TRIF-TANK binding kinase (TBK)1 associations, induction of TBK1 kinase activity, activation of IFN regulatory factor (IRF)-3, and expression of RANTES and IFN-beta. Tolerance-mediated dysregulation of the TLR4-TRIF-TBK1 signaling module was accompanied by increased levels of suppressor of IkappaB kinase-epsilon (SIKE) and sterile alpha and Armadillo motif-containing molecule (SARM). LPS-tolerant cells showed increased expression of negative regulators Toll-interacting protein (Tollip), suppressor of cytokine signaling (SOCS)-1, IL-1R-associated kinase-M, and SHIP-1, which correlated with reduced p38 phosphorylation, IkappaB-alpha degradation, and inhibited expression of TNF-alpha, IL-6, and IL-8. To examine functional consequences of increased expression of Tollip in LPS-tolerized cells, we overexpressed Tollip in 293/TLR4/MD-2 transfectants and observed blunted LPS-inducible activation of NF-kappaB and RANTES, while TNF-alpha responses were not affected. These data demonstrate dysregulation of TLR4-triggered MyD88- and TRIF-dependent signaling pathways and increased expression of negative regulators of TLR signaling in endotoxin-tolerant human monocytes.

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Figures

Figure 1.
Figure 1.
Endotoxin tolerization dysregulates LPS-mediated TLR4-TRIF, TRIF-TBK1 associations and inhibits TBK-1 activation in human monocytes. After prior exposure to medium or 10 ng/ml LPS for 20 h, human monocytes were washed and treated with medium or challenged with 100 ng/ml LPS. (A) Whole cell lysates were prepared, immunoprecipitated with α-TLR4 (H80) or α-TRIF Abs, and immune complexes were analyzed by immunoblotting with the indicated antibodies. Shown are data of a representative (n=3) experiments. (B) HEK293T cells were transfected with either empty vector (pCDNA3) or pCDNA3-Flag-TBK1 and recovered for 24 h; whole lysates were prepared and immunoprecipitated with α-Flag or α-TBK1 Abs. Samples of whole cell lysates and Flag- or TBK1-containing immune complexes were fractionated by SDS-PAGE; proteins were transferred onto PVDF membrane and immunoblotted with α-Flag or α-TBK1 Abs. Numbers on the left indicate molecular mass standards (kDa). The results of a representative (n=2) experiments are shown. (C) Cells were lysed and whole cell lysates were immunoprecipitated with α-TBK1 Ab or isotype control IgG. Immunoprecipitates were subjected to in vitro kinase assay, using GST-IRF3 and GST recombinant proteins as substrates (top two panels) or to Western blot analyses with α-TBK1 Ab to control total expression of endogenous TBK1 proteins. (A and C): Expression levels of p-p38, IκB-α and total p38 were analyzed in whole cell lysates by immunoblotting with the respective Abs to control for LPS inducibility/endotoxin tolerization. Shown are data of a representative experiment (n=3).
Figure 2.
Figure 2.
Endotoxin tolerance inhibits LPS-mediated activation of IRF3. Human monocytes were pretreated for 20 h with medium or 10 ng/ml LPS, washed and challenged with 100 ng/ml LPS for the indicated time points (A) or for 60 min (B). (A) Whole cell lysates were fractionated by native gel electrophoresis (top panel) or SDS-PAGE (other panels), transferred onto PVDF membranes and immunoblotted with the indicated Abs. β-actin immunoblots were used to control for protein loading. Data of a representative experiment (n=4) are presented. (B) Confocal analysis of IRF3 nuclear translocation. Monocytes were stained with mouse α-HLA/α-mouse IgG-Texas Red Abs before fixation (to stain cell surface HLA molecules), cells were fixed with 2% paraformaldehyde, permeablized with 100% ice-cold methanol, and stained with rabbit α-IRF3 /α-rabbit IgG-FITC Abs (to detect intracellular IRF3); nuclei were costained with DAPI. Cells were seeded on glass slides and analyzed by confocal microscopy, using a LSM510 META laser scanning microscope. Shown are results of a representative (n=3) experiment. (C) 293/TLR4/MD-2 cells were pretreated for 20 h with medium or 10 ng/ml LPS, washed, and transfected with expression plasmids encoding Gal4-IRF3 (DBD-IRF3 in control cultures), UAS-luciferase and Renilla luciferase. Following LPS stimulation for 5 h, firefly and Renilla luciferase activities were measured in cell lysates. Data were calculated as firefly luciferase/Renilla luciferase activities in Gal4-IRF3-expressing cells divided by those values obtained in Gal4-DBD-expressing cultures. Results from a representative experiment (n=3) are depicted.
Figure 3.
Figure 3.
Increased expression of SARM and SIKE mRNA in endotoxin-tolerant human monocytes. Human monocytes were pretreated for 20 h with medium or 10 ng/ml LPS, as shown. RNA was isolated, reverse-transcribed, and analyzed by real-time PCR to determine expression levels of SARM (A) and SIKE (B) mRNA. Results of three (SARM) and two (SIKE) independent experiments (mean ± se) are depicted.
Figure 4.
Figure 4.
Inhibited LPS-inducible expression of RANTES and IFN-β in endotoxin-tolerant human monocytes. After prior exposure to medium or 10 ng/ml LPS for 20 h, human monocytes were washed and treated with medium or challenged with 100 ng/ml LPS for 1h (A) or with the indicated concentrations of LPS for 24 h (B). (A): Following stimulation, RNA was isolated, reverse-transcribed and analyzed by real-time PCR with gene-specific primers for RANTES, IFN-β and HPRT. Results of a representative experiment (n=5) are depicted. (B) RANTES secretion levels were assayed in cell-free culture supernatants by ELISA. Shown are data (mean ± sd) of 5 independent experiments. *, P < 0.05; **, P < 0.01.
Figure 5.
Figure 5.
Effect of endotoxin tolerance on expression of Tollip, SOCS-1, IRAK-M, and SHIP-1. Human monocytes were pretreated for 20 h with medium or 10 ng/ml LPS, washed and treated with medium, or restimulated with 100 ng/ml LPS as shown. RNA was isolated, reverse-transcribed, and analyzed by real-time PCR to determine expression levels of Tollip, SOCS-1, IRAK-M (A), and SHIP-1 (B). The results of five independent experiments (mean ± sd) are shown. (C) and (D) Whole cell lysates were examined by Western blot analyses to determine protein expression of Tollip, IRAK-M (C) and SOCS-1 (D), using the corresponding Abs. Total p38 and β-actin immunoblots were run to control for protein loading, and IκB-α and p-p38 proteins were immunoblotted to monitor for LPS inducibility/tolerization. Results of a representative experiment (n=3) are shown.
Figure 6.
Figure 6.
Deficient expression of proinflammatory cytokines and chemokines in endotoxin-tolerant human monocytes. Human monocytes were pretreated for 20 h with medium or 10 ng/ml LPS, washed three times, and treated with medium or restimulated with 100 ng/ml LPS for 3h (A and B), or for the indicated time points (C and D). (A and B) Total RNA was isolated, reverse-transcribed and subjected to real-time PCR analyses for the indicated cytokine genes. Data were processed according to 2−ΔΔCT method [49] and expressed as fold induction compared with values detected in medium-treated control monocytes taken as 1. (C, D) Cell-free supernatants were collected and secretion levels of the indicated cytokines and chemokines were analyzed by ELISA. Data (mean ± sd) of 5 experiments are shown. *, P < 0.05; **, P < 0.01.
Figure 7.
Figure 7.
The effect of Tollip overexpression on LPS-mediated activation of NF-κB- and RANTES-driven luciferase reporters. HEK293T cells were plated in 24-well tissue culture plates (105 cells per well), grown overnight, and transiently transfected with pCDNA3-TLR4 (50 ng per well), pCDNA3-CD14 (20 ng per well), pEFBOS-MD2 (2 ng per well), pRL-TK (50 ng per well), pELAM-luciferase or pGL3-RANTES-luciferase (200 ng per well each), and the indicated doses of pCDNA3-Flag-Tollip. Total level of plasmid DNA was adjusted to 1 μg per well with empty (pCDNA3) vector. After recovery, cells were stimulated for 5 h with medium (open bars), 100 ng/ml LPS (hatched bars, A and B) or 20 ng/ml TNF-α (hatched bars, C). Cell lysates were assayed for firefly vs. Renilla luciferase activities. Shown are data of a representative (n=3) experiment.
Figure 8.
Figure 8.
Comparable LPS inducibility of TNFRII, FpRI, BPIL2, and PtGes mRNA in control and endotoxin-tolerant human monocytes. Cells were incubated with medium or 10 ng/ml LPS for 20 h, washed and treated with medium or restimulated with 100 ng/ml LPS for the indicated time periods. RNA was isolated and reverse transcribed; and expression levels of HPRT, TNFRII, FpRI, BPIL2, and PtGes mRNA were examined by real-time PCR. Data were processed according to 2−ΔΔCT method [49] and expressed as fold induction compared with values detected in medium-treated control monocytes taken as 1. Results of three (TNFRII, PtGes, and BPIL2) and six (FpRI) independent experiments (mean ± sd) are shown.
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