Figure 1.
Txnip−/− mice are hypersusceptible to endotoxic shock as compared to WT mice.
(A) Left panel, LPS (10 mg/kg body weight) was injected i.p. into WT mice (n = 17) and Txnip−/− mice (n = 17). Right panel, E. coli (108 CFU of live E. coli (DH5α)) was injected i.p. into WT mice (n = 9) and Txnip−/− mice (n = 9). (B and C) After LPS injection, the blood glucose and body temperature were measured in WT and Txnip−/− mice at the indicated times. Values represent the mean ± SD from 4 mice at each time point. Significant differences between WT and Txnip−/− mice at the indicated time points are denoted with *P<0.05 and **P<0.01. Data are representative of 3 independent experiments. (D) Enhanced cell infiltration in the lungs of LPS-challenged TXNIP−/− mice (right). BAL fluid was collected 18 h after LPS administration from WT or Txnip−/− mice, and total cells counts in the BAL fluid were determined by optical microscopy after cytocentrifugation and H&E staining. Original magnification (400×). The arrows indicate immune cells in the blood vessel. (E) Size of the spleens from WT and Txnip−/− mice at 2, 4, 8, and 18 h after injection of LPS. Quantitation of the spleen size is shown. (F) Spleen histology with H&E staining, original magnification (100×). TUNEL assays were performed on sections of spleens from mice at 18 h post-LPS injection. The arrows denote apoptotic cells (WP, white pulp; RP, red pulp). Quantitation of the TUNEL staining is shown. We counted apoptotic cells within 6 randomly selected fields. The data shown in (A) are presented as Kaplan-Meyer curves from 3 independent experiments. The data shown in D-F are representative of 3 independent experiments with 3 mice at each time point (**P<0.01).
Figure 2.
TXNIP does not affect the production of inflammatory cytokines by macrophages or neutrophils.
(A) Peritoneal macrophages from WT or Txnip−/− mice were treated with 100 ng/ml LPS or E. coli at a multiplicity of infection (MOI) of 10. The data shown are representative of 3 independent experiments with 3 mice at each time point. (B) BM neutrophils from WT or Txnip−/− mice were treated with 100 ng/ml LPS, and then their supernatants were harvested at the indicated times. TNF-α and IL-6 were measured in the culture supernatants by ELISA. A representative experiment using neutrophils pooled from 2 WT or Txnip−/− mice and performed in triplicate is shown. Three additional experiments with separate groups of mice demonstrated similar results. WT mice (n = 5 per group) and Txnip−/− mice (n = 5 per group) were injected with LPS (10 mg/kg body weight) or E. coli (108 CFU). Following these injections, TNF-α (C) and IL-6 (D) were measured in the sera or tissue lysates (liver, lung, and spleen) from WT and Txnip−/− mice at the indicated time points. The data shown in (C) and (D) are representative of 3 independent experiments with 3 mice at each time point.
Figure 3.
The expression of iNOS and the production of NO are significantly elevated in LPS-treated Txnip−/− mice.
Peritoneal Macrophages from WT and Txnip−/− mice on the C57BL/6 background were treated with 100 ng/ml LPS or E. coli at a MOI of 10, and cell lysates and supernatants were harvested at the indicated times. (A) NO production in response to LPS or E. coli was determined by measuring the amount of nitrite in the culture media using Griess reagents. (B) iNOS mRNA expression levels were assessed by real-time PCR, and (C) iNOS protein levels were determined by immunoblotting with an anti-iNOS antibody. One representative experiment using macrophages pooled from 3 WT and Txnip−/− is shown. Three additional experiments with separate groups of mice provided similar results (data shown in A-C). (D) Expression levels of iNOS in macrophages from WT and Txnip−/− mice were determined by performing immunofluorescence with an antibody specific for iNOS (conjugated to the red fluorescent dye Alexa Fluor 546 (Alexa 546)). Then, LPS-treated cells were fixed, permeabilized, and analyzed by confocal microscopy (Carl Zeiss, LSM510). Confocal images were processed using the program Metamorph 6.1 (Universal Imaging, Media, PA). The graph represents 3 independent experiments in which 10 cells were analyzed for each condition. Confocal images were processed using the program Metamorph 6.1 (Universal Imaging, Media, PA). **P<0.01 versus WT. Scale bar, 20 µm. (E) After LPS or E. coli injection in WT (n = 5 per group) and Txnip−/− mice (n = 5 per group), serum NO levels were determined. (**P<0.01, ***P<0.001). (F) Liver, lung, and spleen samples were obtained from WT (n = 5 per group) and Txnip−/− mice (n = 5 per group) at 0, 4, and 18 h after LPS administration. Tissue lysates were prepared by homogenization. Nitrite plus nitrate (NOx) was measured in the serum, and tissue lysates were measured using a nitrate reductase-based colorimetric assay kit. These data are representative of at least 3 independent experiments (*P<0.05, **P<0.01, ***P<0.001). (G) Tissue lysate samples from (F) were analyzed by SDS-PAGE and immunoblotting with an anti-iNOS antibody. (H) Real-time PCR analysis of iNOS mRNA expression in the liver, lung, and spleen from WT and Txnip−/− mice. Both types of mice were injected with LPS (10 mg/kg) for the indicated time periods. Data are presented as the mean ± SD of 3 independent experiments (*P<0.05; **P<0.01).
Figure 4.
An iNOS inhibitor (1400W) rescues Txnip−/− mice injected with LPS in vivo.
(A) 1400W (10 mg/kg body weight) was used to treat mice at 1 h prior to LPS (10 mg/kg body weight; i.p.) injection. Mice were divided into a 1400W group (n = 7), an LPS-alone group, and an LPS+1400W group (n = 8). Viability was assessed every 5 h. (B) The concentrations of serum NO in Txnip−/− mice (n = 5 per group) were determined. After LPS injection, the serum concentrations of NO were significantly affected by 1400W treatment. These data represent repeated results from at least 3 independent experiments (**P<0.01). (C) WT or Txnip−/− mice were BM-transplanted as indicated, and survival rates were determined after LPS (10 mg/kg; i.p.) treatment (**P<0.01). (D) Lung fibroblasts from WT or Txnip−/− mice were treated with 100 ng/ml LPS, and the cell lysates were harvested at the indicated times. Western blot analysis was performed using iNOS, phospho-p65, HIF-1α, and TXNIP antibodies. The detection of β-actin in each sample served as a loading control. The data are representative of at least 3 repeated experiments.
Figure 5.
TXNIP negatively regulates the production of NO and iNOS expression in response to LPS.
(A) RAW264.7 cells were transfected with control siRNA and TXNIP siRNA. At 48 h post-transfection, cells were treated with 100 ng/ml LPS for 6 or 12 h and then harvested. The expression of iNOS and TXNIP in RAW264.7 cells was measured by western blot. (B) NO production in response to LPS was determined by measuring the amount of nitrite in the culture media using Griess reagents. Data are presented as the mean ± SD of 3 independent experiments (**P<0.01). (C) Txnip−/− macrophages were infected with TXNIP retrovirus or control retrovirus at 200 plaque-forming units (PFU) per cell, and the cells were incubated in the presence or absence of 100 ng/ml LPS at the indicated times. The expression of TXNIP and iNOS was measured by western blot (top). NO production from the macrophages was determined by measuring the amount of nitrite in the culture media using Griess reagents (bottom). Data are presented as the mean ± SD of 3 independent experiments (**P<0.01). (D) For the overexpression of TXNIP, RAW264.7 cells were infected with TXNIP retrovirus or control retrovirus at 200 PFU/cell, and the cells were incubated in the presence or absence of 100 ng/ml LPS at the indicated times. The expression of TXNIP and iNOS was measured by western blot. Data are representative of at least 3 repeated experiments. (E) Macrophages from WT or Txnip−/− mice were incubated with or without LPS overnight, washed, (F) replenished with fresh medium with (+) or without (−) 10 µM of the proteasomal inhibitor MG-132, and then lysed at the indicated time points after washing. Western blot analysis was conducted using an anti-iNOS antibody, and the detection of actin in each sample serves as a loading control. Data are representative of at least 3 repeated experiments.
Figure 6.
TXNIP deficiency promotes NF-kB activation mediated by LPS or E. coli stimulation.
Peritoneal macrophages from WT or Txnip−/− mice were treated with 100 ng/ml LPS or E. coli at a MOI of 10, and cell lysates were harvested at the indicated times. (A) The levels of phosphorylated ERK, JNK, and p38 were determined by western blotting. The same blots were stripped and reprobed with an anti–β-actin antibody. (B) The experimental conditions followed the pattern outlined in (A). Western blot analysis was performed using anti-IκBα, anti–phospho-IκBα, and anti–phospho-p65 antibodies. The detection of β-actin in each sample serves as a loading control. The data are representative of at least 3 repeated experiments. (C) Cytosolic fractions and nuclear extracts were prepared from macrophages treated with 100 ng/ml LPS at the time points indicated. The NF-κB p65 levels were determined by western blot analysis. The detection of β-actin in cytosolic fractions and histone H1 in nuclear extracts served as a loading control. The data are representative of at least 3 repeated experiments. (D) The nuclear translocation of NF-κB was evaluated by immunohistochemistry using an antibody for the NF-κB p65 subunit (stained with DAPI (blue) and conjugated to the red fluorescent dye Alexa Fluor 546 (Alexa546)). LPS-treated cells were fixed, permeabilized, and analyzed by confocal microscopy (Carl Zeiss, LSM510). Scale bar, 20 µm. (E) Nuclear extracts (10 µg) of isolated macrophages from WT and Txnip−/− mice were analyzed by EMSA. The specificity of NF-κB binding was assessed by incubating the nuclear extracts with a 100-fold excess of unlabeled specific probe. (F) Peritoneal macrophages from WT or Txnip−/− mice were stimulated with LPS, and the cells were processed for ChIP assays at the indicated time points. The antibodies used for the ChIP assays are shown on the right. The input indicates the control.
Figure 7.
TXNIP deficiency-induced NO inhibits activation of the NLRP3 inflammasome via S-nitrosylation.
(A) Peritoneal macrophages from WT or Txnip−/− mice were treated with 100 ng/ml LPS and E. coli at a MOI of 10. The supernatants were harvested at the indicated times. The levels of IL-1β in the culture supernatants were measured by ELISA. (B and C) After LPS or E. coli injection in WT (n = 5 per) and Txnip−/− mice (n = 5 per), the levels of serum IL-1β (B) and IL-18 (C) were measured by ELISA. Bacterial peritonitis was induced by the i.p. injection of 108 CFU of live E. coli (DH5α). (D) Concentrations of serum IL-1β in Txnip−/− mice (n = 5 per group) were determined. After LPS injection, the serum concentrations of NO were significantly altered by 1400W treatment. These data represent at least 3 independent experiments (*P<0.05, **P<0.01). (E) Immunoblot analysis of inflammasome components, including caspase-1, NLRP3, and ASC, in WT and Txnip−/− macrophages at the indicated time points. (F) The total level of S-nitrosylation of inflammasome components was determined by immunoblot analysis. LPS was used to treat macrophages from WT and Txnip−/− mice at the indicated time points. Below (lysate), immunoblot analysis of total lysate fractions. Data are representative of at least 3 repeated experiments. Peritoneal macrophages (G) and BMDMs (H) from WT and Txnip−/− mice were primed with LPS for 12 h and then stimulated with inflammasome activators such as ATP (5 mM) for 30 min, nigericin (10 µM) for 30 min, or MSU (200 µg/ml) for 6 h. IL-1β secretion was measured in the culture supernatants by ELISA. Data are presented as the mean ± SD of 3 independent experiments (**P<0.01).
Figure 8.
A schematic model showing TXNIP-mediated regulatory pathways for LPS-mediated inflammatory signaling.
TXNIP-iNOS-NO is a main pathway involved in the generation of septic shock. The excessive NO production in Txnip−/− mice reduced the production of IL-1β by inhibiting the NLRP3 inflammasome.