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Shedding of the tumor necrosis factor (TNF) receptor from the surface of hepatocytes during sepsis limits inflammation through cGMP signaling

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Science Signaling  27 Jan 2015:
Vol. 8, Issue 361, pp. ra11
DOI: 10.1126/scisignal.2005548
  • Fig. 1 Multiple TLR ligands and cytokines stimulate HC-TNFR1 shedding in vitro and in vivo.

    (A) Wild-type (WT) and Tlr4−/− mice were subjected to CLP. In the control mice, laparotomy and cecal manipulation were performed without ligation and puncture. Eight hours later, plasma sTNFR1 concentrations were measured by ELISA. Data are means ± SD from 8 to 10 mice per group from two separate experiments. (B) Mouse hepatocytes were left untreated (Control) or were stimulated for 24 hours with the TLR ligands HKLM (1 × 108 cells/ml), LPS (100 ng/ml), HMGB1 (5 μg/ml), FLA-ST (1 μg/ml), and ODN1669 (5 μg/ml). The concentrations of TNFR1 in the culture media were measured by ELISA. Data are means ± SD from three experiments. *P < 0.05 versus control by two-tailed unpaired t test. (C and D) Mouse hepatocytes were left untreated or were incubated with TNF (500 U/ml) or IL-1β (100 U/ml) for the indicated times. At each time point, (C) cell viability and (D) the concentration of TNFR1 in the culture media were determined. Data are means ± SD from three experiments. *P < 0.05 versus control by two-tailed unpaired t test. (E) Mice of the indicated genotypes were subjected to CLP. Eight hours later, the plasma concentrations of TNFR1 were determined by ELISA. Data are means ± SD from six mice per group of each genotype from two experiments. *P < 0.05 versus control by two-tailed unpaired t test.

  • Fig. 2 TNFR1 shedding from hepatocytes and myeloid cells during sepsis depends on Myd88.

    (A and B) WT, Myd88−/−, and Trif−/− mice were subjected to CLP. Eight hours later, (A) plasma sTNFR1 concentrations were determined by ELISA, and (B) Tnfr1 mRNA abundance in the liver was determined by real-time reverse transcription polymerase chain reaction (RT-PCR) analysis. Data are means ± SD from 8 to 10 mice per group from two experiments. *P < 0.05 versus control by two-tailed unpaired t test. (C and D) Hepatocytes (C) and NPCs (D) from the indicated mice were left untreated (zero-hour time point) or were treated with LPS for the indicated times. At each time point, the concentration of TNFR1 in the culture media was determined by ELISA. Data are means ± SD from three experiments. *P < 0.05 by one-way analysis of variance (ANOVA). (E and F) Myd88flox, HC-Myd88−/−, and LysM-Myd88−/− mice were subjected to CLP. Eight hours later, plasma concentrations of (E) TNFR1 and (F) IL-1β were determined by ELISA. Data are means ± SD from 8 to 10 mice per group from two experiments. *P < 0.05 versus Myd88flox by two-tailed unpaired t test. N.D., not detectable.

  • Fig. 3 Shedding of HC-TNFR1 during polymicrobial sepsis is mediated through a MyD88- and iNOS-dependent pathway.

    (A and B) Mice of the indicated genotypes were subjected to CLP for 8 hours. (A) The relative expression of Nos2 was determined by real-time RT-PCR analysis. Data are means ± SD from six to eight mice of each genotype from two experiments. *P < 0.05 by one-way ANOVA. (B) The production of iNOS in the liver of the indicated mice was analyzed by immunofluorescence microscopy. Nuclear staining is in blue, whereas iNOS is shown in green. Scale bar, 50 μm. (C) Hepatocytes isolated from the indicated mice were left untreated (zero time point) or were treated with LPS (100 ng/ml) for the indicated times. Whole-cell lysates were then prepared and analyzed by Western blotting with antibodies specific for iNOS and TNFR1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) abundance was analyzed to control for loading. Western blots are representative of three independent experiments. (D) WT and Nos2−/− mice were subjected to CLP. Eight hours later, plasma concentrations of TNFR1 were determined by ELISA. Data are means ± SD from six mice per group from two experiments. *P < 0.05 by one-way ANOVA. (E) WT mice were subjected to CLP and then were left untreated or were treated with 1400W (5 mg/kg, administered subcutaneously). Eight hours after CLP, plasma concentrations of TNFR1 were determined by ELISA. Data are means ± SD from six mice per group from two experiments. *P < 0.05 by one-way ANOVA.

  • Fig. 4 iNOS-stimulated HC-TNFR1 shedding during polymicrobial sepsis is mediated by cGMP and TACE.

    (A to D) WT mice were subjected to CLP and then were immediately treated subcutaneously with saline, 1400W (5 mg/kg), 8-CPT-cGMP (5 mg/kg), both 1400W and 8-CPT-cGMP, or ODQ (20 mg/kg). Eight hours after CLP, the concentrations of (A) cGMP in liver homogenates and (B) TNFR1 in plasma were determined. Data are means ± SD from six to eight mice per group from two experiments. *P < 0.05 by one-way ANOVA. (C) Some of the same mice were analyzed by immunofluorescence microscopy to detect active TACE in the liver. TACE is shown in green, nuclei are in blue, and actin is shown in white. Scale bar, 50 μm. (D) Liver homogenates from the same mice were analyzed by Western blotting to detect TACE. GAPDH was used as a loading control. Western blots are representative of two independent experiments. (E) Hepatocytes isolated from WT mice were left untreated (zero-hour time point) or were treated in vitro with LPS (100 ng/ml) in the absence or presence of 400 nM TAPI-2 for the indicated times. The concentration of TNFR1 in the culture media was determined by ELISA. Data are means ± SD from three independent experiments. *P < 0.05 by one-way ANOVA.

  • Fig. 5 Regulation of hepatic TNFR1 shedding through the iNOS-cGMP pathway modulates inflammatory responses and liver injury during sepsis.

    (A to F) WT mice were subjected to CLP and then were immediately treated subcutaneously with saline, 1400W (5 mg/kg), 8-CPT-cGMP (5 mg/kg), both 1400W and 8-CPT-cGMP, or ODQ (20 mg/kg). Eight hours after CLP, plasma concentrations of (A) IL-6, (B) TNF, (C) KC, (D) MIP-1α, (E) HMGB1, and (F) ALT were measured by ELISA. Data are means ± SD from 8 to 10 mice per group from two experiments. *P < 0.05 by one-way ANOVA. (G to I) WT mice were subjected to CLP and then were immediately treated subcutaneously with saline or sildenafil (50 mg/kg). Eight hours after CLP, the concentrations of (G) cGMP in liver homogenates and of (H) TNFR1 and (I) IL-6 in the plasma were determined. Data are means ± SD from six mice per group. *P < 0.05 by one-way ANOVA.

  • Fig. 6 TNFR1 shedding by human hepatocytes in vitro is mediated through an iNOS-cGMP-TACE pathway.

    (A) Human hepatocytes were left untreated (Control) or treated with LPS (100 ng/ml) or 400 nM IL-1β for the indicated times. The concentration of TNFR1 in the culture media was determined by ELISA. Data are means ± SD from three independent experiments. *P < 0.05 by one-way ANOVA. (B) Human hepatocytes were left untreated (Control) or were treated with LPS (100 ng/ml). Cells were then analyzed by immunofluorescence microscopy to detect iNOS (red) and TACE (green). Arrows indicate the colocalization of iNOS and TACE. Scale bar, 50 μm. Images are representative of two independent experiments. (C to F) Human hepatocytes were left untreated (zero-hour time point) or were treated with LPS (100 ng/ml) in the absence (Cont) or presence of 400 nM TAPI-2 for the indicated times. (C) The concentration of TNFR1 in the culture media was determined by ELISA. Data are means ± SD from three independent experiments. *P < 0.05 by one-way ANOVA. (D) The hepatocytes were analyzed by Western blotting with antibodies specific for TACE and TNFR1. Densitometric analysis was performed to determine the relative abundances of (E) TACE and (F) TNFR1 proteins normalized to the abundance of actin. Data are means ± SD from three independent experiments. *P < 0.05 by two-tailed unpaired t test. (G and H) Human hepatocytes were left untreated (zero-hour time point) or were treated with LPS (100 mg/ml) in the absence (Cont) or presence of 400 nM TAPI-2 for the indicated times. (G) Samples were then subjected to immunoprecipitation (IP) with an anti-TACE antibody and were analyzed by Western blotting (IB) with antibodies specific for TACE and iNOS. Bands corresponding to iNOS in the TAPI-2–treated samples were only detectable when Western blots were separately developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). (H) Densitometric analysis was performed to determine the relative abundance of iNOS protein normalized to that of TACE. Data are means ± SD from three independent experiments. *P < 0.05 by two-tailed unpaired t test. (I) Human hepatocytes were treated with vehicle (0.01% DMSO), 20 μM 1400W, 100 μM 8-CPT-cGMP, or 20 μM ODQ, and then were stimulated with LPS (100 ng/ml) for the indicated times. The concentration of TNFR1 in the culture media was determined by ELISA. Data are means ± SD from three experiments. *P < 0.05 by one-way ANOVA.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/361/ra11/DC1

    Fig. S1. The kinetics of TNFR1 and TNF release after CLP.

    Fig. S2. Analysis of plasma IL-1β concentrations at 8 hours after CLP.

    Fig. S3. The responses to LPS of hepatocytes from Alb-Cre mice are similar to those of hepatocytes from wild-type mice.

    Fig. S4. The responses to LPS of cells from Lyz-Cre mice are similar to those of cells from wild-type mice.

  • Supplementary Materials for:

    Shedding of the tumor necrosis factor (TNF) receptor from the surface of hepatocytes during sepsis limits inflammation through cGMP signaling

    Meihong Deng, Patricia A. Loughran, Liyong Zhang, Melanie J. Scott, Timothy R. Billiar*

    *Corresponding author. E-mail: billiartr{at}upmc.edu

    This PDF file includes:

    • Fig. S1. The kinetics of TNFR1 and TNF release after CLP.
    • Fig. S2. Analysis of plasma IL-1β concentrations at 8 hours after CLP.
    • Fig. S3. The responses to LPS of hepatocytes from Alb-Cre mice are similar to those of hepatocytes from wild-type mice.
    • Fig. S4. The responses to LPS of cells from Lyz-Cre mice are similar to those of cells from wild-type mice.

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    Citation: M. Deng, P. A. Loughran, L. Zhang, M. J. Scott, T. R. Billiar, Shedding of the tumor necrosis factor (TNF) receptor from the surface of hepatocytes during sepsis limits inflammation through cGMP signaling. Sci. Signal. 8, ra11 (2015).

    © 2014 American Association for the Advancement of Science

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