Effect of endotoxemia in mice genetically deficient in cystathionine-γ-lyase, cystathionine-β-synthase or 3-mercaptopyruvate sulfurtransferase

Abstract

Hydrogen sulfide (H2S) has been proposed to exert pro- as well as anti-inflammatory effects in various models of critical illness. In this study, we compared bacterial lipopolysaccharide (LPS)‑induced changes in inflammatory mediator production, indices of multiple organ injury and survival in wild‑type (WT) mice and in mice with reduced expression of one of the three H2S‑producing enzymes, cystathionine-γ-lyase (CSE), cystathionine-β-synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3MST). Mice were injected intraperitoneally (i.p.) with LPS (10 mg/kg). After 6 h, the animals were sacrificed, blood and organs were collected and the following parameters were evaluated: blood urea nitrogen (BUN) levels in blood, myeloperoxidase (MPO) and malondialdehyde (MDA) in the lung, cytokine levels in plasma and the expression of the three H2S‑producing enzymes (CBS, CSE and 3MST) in the spleen, lung, liver and kidney. LPS induced a tissue‑dependent upregulation of some of the H2S‑producing enzymes in WT mice (upregulation of CBS in the spleen, upregulation of 3MST in the liver and upregulation of CBS, CSE and 3MST in the lung). Moreover, LPS impaired glomerular function, as evidenced by increased BUN levels. Renal impairment was comparable in the CSE‑/‑ and Δ3MST mice after LPS challenge; however, it was attenuated in the CBS+/‑ mice. MPO levels (an index of neutrophil infiltration) and MDA levels (an index of oxidative stress) in lung homogenates were significantly increased in response to LPS; these effects were similar in the WT, CBS+/‑, CSE‑/‑ and Δ3MST mice; however, the MDA levels tended to be lower in the CBS+/‑ and CSE‑/‑ mice. LPS induced significant increases in the plasma levels of multiple cytokines [tumor necrosis factor (TNF)α, interleukin (IL)‑1β, IL‑6, IL‑10, IL‑12 and interferon (IFN)γ] in plasma; TNFα, IL‑10 and IL‑12 levels tended to be lower in all three groups of animals expressing lower levels of H2S‑producing enzymes. The survival rates after the LPS challenge did not show any significant differences between the four animal groups tested. Thus, the findings of this study indicate that a deficiency in 3MST does not significantly affect endotoxemia, while a deficiency in CBS or CSE slightly ameliorates the outcome of LPS-induced endotoxemia in vivo.

Figure 1

Baseline tissue expression of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) in the animals used in the current study. Protein levels of (A and B) CBS, (C and D) CSE and (E and F)3MST in the spleen, lung, liver and kidney of the CBS heterozygous (CBS^+/−), CSE knockout (CSE^−/−) and 3MST mutant (Δ3MST) mice are shown. Enzyme levels were normalized to those of the wild-type control, set as 100%. Please note that CBS^+/− mice show reduced CBS expression in the liver and kidney; CSE^−/− mice show reduced CSE expression in the spleen, lung, liver and kidney; and Δ3MST mice show reduced 3MST expression in the spleen and lung. Data are shown as the means ± standard error of the mean (SEM) of n=5 determinations.

Figure 2

Effect of lipopolysaccharide (LPS) [10 mg/kg, intraperitoneally (i.p.), 6 h] on protein levels of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3MST) in the spleen, lung, liver and kidney of wild-type (WT) mice. Expression levels in vehicle-treated mice for each organ are normalized as 100%. Please note that LPS induced an increase in CBS expression in the spleen and lung, an increase in CSE expression in the lung, and an increase in 3MST expression in the lung and liver. Data are shown as the means ± standard error of the mean (SEM) of n=5 determinations; ^*p<0.05 shows significant LPS-induced increase in the expression level of the indicated enzyme in the indicated organ.

Figure 3

Downregulation of cystathionine-β-synthase (CBS) attenuates the lipopolysaccharide (LPS)-induced increases in blood urea nitrogen (BUN) plasma levels in mice. BUN levels in wild-type (WT), CBS heterozygous (CBS^+/−), cystathionine-γ-lyase (CSE) knockout (CSE^−/−) and 3-mercaptopyruvate sulfurtransferase (3MST) mutant (Δ3MST) mice treated with vehicle or LPS (10 mg/kg, 6 h). LPS significantly impaired glomerular function, as evidenced by markedly increased BUN concentration; this increase was reduced in CBS^+/− mice. Data are shown as mean ± standard error of the mean (SEM) of 10 animals; ^*p<0.05 shows significant increases in response to LPS, compared to the vehicle control; ^#p<0.05 shows a significant protective effect of the CBS^+/− phenotype compared to WT.

Figure 4

Downregulation of cystathionine-β-synthase (CBS) or cystathionine-γ-lyase (CSE) attenuates the lipopolysaccharide (LPS)-induced increases in lung malondialdehyde (MDA), but not myeloperoxidase (MPO) levels in mice. Lung (A) MPO and (B) MDA levels in wild-type (WT), CBS heterozygous (CBS^+/−), CSE knockout (CSE^−/−) and 3-mercaptopyruvate sulfurtransferase (3MST) mutant (Δ3MST) mice treated with vehicle or LPS (10 mg/kg, 6 h). LPS significantly increased MPO and MDA levels; this increase was reduced in CSE^−/− and CBS^+/− mice. Data are shown as the means ± standard error of the mean (SEM) of 10 animals; ^**p<0.01 shows significant increases in response to LPS, compared to the vehicle control; ^#p<0.05 shows a reduction by the CSE^−/− and CBS^+/− phenotype compared to WT.

Figure 5

Downregulation of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3MST) attenuates the lipopolysaccharide (LPS)-induced increases in plasma tumor necrosis factor (TNF)α, but not interleukin (IL)-1β levels in mice. Plasma (A) IL-1β and (B) TNFα levels in wild-type (WT), CBS heterozygous (CBS^+/−), CSE knockout (CSE^−/−) and 3MST mutant (Δ3MST) mice treated with the vehicle or LPS (10 mg/kg, 6 h). LPS significantly increased IL-1β and TNFα levels; the increase in TNFα was reduced in CBS^+/−, CSE^−/− and Δ3MST mice. Data are shown as the means ± standard error of the mean (SEM) of 10 animals; ^**p<0.01 shows significant increases in response to LPS, compared to the vehicle control; ^#p<0.05 shows a significant reduction on TNFα plasma levels in the CSE^−/−, CBS^+/− and Δ3MST phenotype compared to WT.

Figure 6

Downregulation of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3MST) does not affect the lipopolysaccharide (LPS)-induced increases in plasma interleukin (IL)-2 or IL-4 levels in mice. Plasma (A) IL-2 and (B) IL-4 levels in wild-type (WT) mice, in CBS heterozygous (CBS^+/−), CSE knockout (CSE^−/−) and 3MST mutant (Δ3MST) mice treated with the vehicle or LPS (10 mg/kg, 6 h). LPS significantly increased IL-2 and IL-4 levels; the effect was comparable in all four groups of animals studied. Data are shown as the means ± standard error of the mean (SEM) of 10 animals; ^*p<0.05 and ^**p<0.01 show significant increases in response to LPS, compared to the vehicle control; ^#p<0.05 shows a significant reduction of IL-2 plasma levels in the Δ3MST phenotype, compared to WT.

Figure 7

Downregulation of 3-mercaptopyruvate sulfurtransferase (3MST) increases the lipopolysaccharide (LPS)-induced increases in plasma interleukin (IL)-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) levels in mice. Plasma (A) IL-5 and (B) GM-CSF levels in wild-type (WT), cystathionine-β-synthase (CBS) heterozygous (CBS^+/−), cystathionine-γ-lyase (CSE) knockout (CSE^−/−) and 3MST mutant (Δ3MST) mice treated with the vehicle or LPS (10 mg/kg, 6 h). LPS significantly increased IL-5 and GM-CSF levels; the effect was more pronounced in the Δ3MST mice than in the WT controls. Data are shown as the means ± standard error of the mean (SEM) of 10 animals; ^*p<0.05 shows significant increases in response to LPS, compared to the vehicle control; ^#p<0.05 shows significantly higher cytokine responses in the Δ3MST mice compared to the WT controls.

Figure 8

Downregulation of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3MST) attenuates the lipopolysaccharide (LPS)-induced increases in plasma interleukin (IL)-10, but not IL-6 levels in mice. Plasma (A) IL-6 and (B) IL-10 levels in wild-type (WT), CBS heterozygous (CBS^+/−), CSE knockout (CSE^−/−) and 3MST mutant (Δ3MST) mice treated with the vehicle or LPS (10 mg/kg, 6 h). LPS significantly increased IL-6 and IL-10 levels; the increase in IL-10 was reduced in CBS^+/−, CSE^−/− and Δ3MST mice. Data are shown as the means ± standard error of the mean (SEM) of 10 animals; ^*p<0.05 and ^**p<0.01 show significant increases in response to LPS, compared to the vehicle control; ^#p<0.05 shows a significant protective effect of the CSE^−/−, CBS^+/− and Δ3MST phenotype compared to WT.

Figure 9

Downregulation of cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3MST) attenuates the lipopolysaccharide (LPS)-induced increases in plasma interleukin (IL)-12, while downregulation of CSE reduces plasma interferon (IFN)γ levels in mice. Plasma (A) IL-12 and (B) IFNγ levels in wild-type (WT), CBS heterozygous (CBS^+/−), CSE knockout (CSE^−/−) and 3MST mutant (Δ3MST) mice treated with the vehicle or LPS (10 mg/kg, 6 h). LPS significantly increased IL-12 and IFNγ levels; the increase in IL-12 was reduced in CBS^+/−, CSE^−/− and Δ3MST mice while the increase in IFNγ was reduced in the CSE^−/− mice. Data are shown as the means ± standard error of the mean (SEM) of 10 animals; ^*p<0.05 and ^**p<0.01 show significant increases in response to LPS, compared to the vehicle control; ^#p<0.05 shows a significant protective effect of the CSE^−/−, CBS^+/− and Δ3MST phenotype compared to WT on IL-12 levels or the protective effect of CSE^−/− compared to WT on IFNγ levels.

Figure 10

Downregulation of, cysta thionine-β-synthase (CBS), cystathionine-γ-lyase (CSE) or 3-mercaptopyruvate sulfurtransferase (3MST) does not affect lipopolysaccharide (LPS)-induced mortality in mice. Comparison of (A) wild-type (WT) and CBS heterozygous (CBS^+/−) mice, (B) WT and CSE knockout (CSE^−/−) mice, and (C) WT and 3MST mutant (Δ3MST) mice treated with the vehicle or LPS (20 mg/kg). LPS induced a significant mortality, which was comparable in WT, CSE^−/−, CBS^+/− and Δ3MST mice at all time points. Data show survival proportions (%) of n=15 mice in each experimental group.