Neutrophil-derived myeloperoxidase aggravates non-alcoholic steatohepatitis in low-density lipoprotein receptor-deficient mice - PubMed (original) (raw)
Neutrophil-derived myeloperoxidase aggravates non-alcoholic steatohepatitis in low-density lipoprotein receptor-deficient mice
Sander S Rensen et al. PLoS One. 2012.
Abstract
Background: Chronic inflammation and oxidative stress play fundamental roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). Previously, we reported that myeloperoxidase (MPO), an aggressive oxidant-generating neutrophil enzyme, is associated with NASH severity in man. We now investigated the hypothesis that MPO contributes to the development and progression of NASH.
Methodology: Low-density lipoprotein receptor-deficient mice with an MPO-deficient hematopoietic system (LDLR(-/-/)MPO(-/-tp) mice) were generated and compared with LDLR(-/-/)MPO(+/+tp) mice after induction of NASH by high-fat feeding.
Results: High-fat feeding caused a ~4-fold induction of liver MPO in LDLR(-/-/)MPO(+/+) mice which was associated with hepatic sequestration of MPO-positive neutrophils and high levels of nitrotyrosine, a marker of MPO activity. Importantly, LDLR(-/-/)MPO(-/-tp) mice displayed markedly reduced hepatic neutrophil and T-lymphocyte infiltration (p<0.05), and strong down regulation of pro-inflammatory genes such as TNF-α and IL-6 (p<0.05, p<0.01) in comparison with LDLR(-/-/)MPO(+/+tp) mice. Next to the generalized reduction of inflammation, liver cholesterol accumulation was significantly diminished in LDLR(-/-/)MPO(-/-tp) mice (p = 0.01). Moreover, MPO deficiency appeared to attenuate the development of hepatic fibrosis as evident from reduced hydroxyproline levels (p<0.01). Interestingly, visceral adipose tissue inflammation was markedly reduced in LDLR(-/-/)MPO(-/-tp) mice, with a complete lack of macrophage crown-like structures. In conclusion, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in response to a high fat diet, supporting an important role for neutrophils in the pathogenesis of metabolic disease.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Strong high-fat diet-induced induction of MPO in the liver.
A) Total liver MPO content of LDLR −/− mice as assessed by ELISA was almost four-fold increased by three weeks of high-fat feeding (19±1 vs. 68±10 ng/µg protein, p<0.01; n = 6 in both groups). B) MPO immunostaining reveals infiltration of neutrophils into the liver of LDLR −/− mice after three weeks of high-fat feeding (see arrows; 100× magnification). Many neutrophils are organized into aggregates predominantly surrounding steatotic hepatocytes (right panel; 200× magnification). MPO was not detected in Kupffer cells.
Figure 2. Reduced hepatic MPO and MPO-derived nitrated proteins in LDLR −/−/ MPO −/−tp mice after 8 weeks of high-fat feeding.
A) Plasma MPO levels of LDLR −/−/ MPO −/−tp and LDLR −/−/ MPO +/+tp mice (22±7 vs. 324±52 ng/ml, p<0.01). B) The MPO-positive cell number is strongly reduced in the liver of LDLR −/−/ MPO −/−tp vs. LDLR −/−/ MPO +/+tp mice (49.5±7.6 vs. 4.0±1.6 cells/mm2, p<0.01), and much lower than those observed in LDLR −/−/ MPO +/+ mice on chow. C) Hepatic levels of nitrotyrosine, a marker of MPO activity, are reduced in LDLR −/−/ MPO −/−tp animals in comparison with LDLR −/−/ MPO +/+tp mice (126±7 vs. 149±9 mmol/µg protein, p = 0.02).
Figure 3. Liver histology of LDLR −/−/ MPO −/−tp animals in comparison with LDLR −/−/ MPO +/+tp mice after 8 weeks of high-fat feeding.
Representative pictures of HE-stained liver sections of LDLR −/−/ MPO −/−tp and LDLR −/−/ MPO +/+tp mice indicating steatosis and inflammation (arrows).
Figure 4. Decreased cholesterol accumulation in the liver of LDLR −/−/ MPO −/−tp mice.
A) Representative Oil red O stainings of liver sections of LDLR −/−/ MPO −/−tp and LDLR −/−/ MPO +/+tp mice fed a high-fat diet for 8 weeks, showing comparable lipid accumulation (100× magnification). B) Similar hepatic triglyceride levels in LDLR −/−/ MPO −/−tp and LDLR −/−/ MPO +/+tp mice after high-fat feeding (0.31±0.02 vs. 0.35±0.02 µg/µg protein, p = 0.24). Chow-fed LDLR −/−/ MPO +/+ mice show a lower level of liver triglycerides. C) Plasma triglyceride levels are similar in LDLR −/−/ MPO −/−tp and LDLR −/−/ MPO +/+tp animals after high-fat feeding (1.50±0.09 vs. 1.64±0.09 mmol/l, p = 0.42). D) High-fat feeding results in higher plasma cholesterol levels in LDLR −/−/ MPO +/+tp animals as compared with LDLR −/−/ MPO −/−tp mice (33.5±0.1 vs. 39.5±2.0 mmol/l, p = 0.02). E) Diet-induced liver cholesterol accumulation is reduced in LDLR −/−/ MPO −/−tp mice compared with LDLR −/−/ MPO +/+tp animals (0.072±0.004 vs. 0.090±0.004 µg/µg protein, p = 0.01), but does not reach the level observed in chow-fed LDLR −/−/ MPO +/+ mice. F) Hepatic mRNA expression of key enzymes in cholesterol metabolism is not altered in LDLR −/−/ MPO −/−tp mice, whereas scavenger receptor expression is reduced (SR-B1 1.7-fold, p<0.01; CD36 1.4-fold, p<0.01, SR-A 1.2-fold, p = 0.63).
Figure 5. General reduction of diet-induced hepatic inflammation in LDLR −/−/ MPO −/−tp mice.
A) Significantly lower number of hepatic Ly-6G+ neutrophils and CD3+ T-lymphocytes in LDLR −/−/ MPO −/−tp mice as compared with LDLR −/−/ MPO +/+tp mice after 8 weeks of high-fat feeding (Ly-6G: 36.7±2.6 vs. 47.8±3.1 cells/mm2, p = 0.03; CD3: 49.1±4.2 vs. 62.9±5.0 cells/mm2, p = 0.04). Pictures represent examples of the stainings (200× magnification). B) Hepatic pro-inflammatory cytokine/chemokine expression is substantially reduced in LDLR −/−/ MPO −/−tp mice after 8 weeks high-fat diet (TNF-α 1.8-fold, p = 0.03, IL-1α 1.6-fold, p<0.01, IL-6 1.3-fold, p = 0.67, Mcp-1 2.5-fold, p<0.01), in parallel with a reduction of CD68 expression (1.3-fold, p<0.05).
Figure 6. Reduced diet-induced adipose tissue inflammation in LDLR −/−/ MPO −/−tp mice.
A) Lack of high-fat diet-associated macrophage ‘crown-like structures’ in visceral adipose tissue of LDLR −/−/ MPO −/−tp mice as revealed by F4/80 immunostaining (200× magnification). B) Adipose tissue mRNA expression of the macrophage marker Mac-1 and the macrophage chemokine Mcp-1 is significantly lower in LDLR −/−/ MPO −/−tp mice fed a high-fat diet for 8 weeks (p<0.05). C) Reduced adipose tissue expression of the adipokines leptin and TNF-α in LDLR −/−/ MPO −/−tp mice after 8 weeks of high-fat feeding, whereas expression of adiponectin is increased.
Figure 7. Attenuation of liver fibrosis in LDLR −/−/ MPO −/−tp mice after 8 weeks high-fat diet.
A) Sirius red staining of liver sections of LDLR −/−/ MPO −/−tp,LDLR −/−/ MPO +/+tp, and chow-fed LDLR −/−/ MPO +/+ mice (200× magnification). B) The amount of hydroxyproline, a protein modification specifically present in collagen and elastin, is significantly lower in the liver of LDLR −/−/ MPO −/−tp mice in comparison with LDLR −/−/ MPO +/+tp mice (3.3±0.2 vs. 4.6±0.7 nmol/mg tissue, p<0.01). C) LDLR −/−/ MPO −/−tp mice display reduced expression of collagen type I, PAI-1, TIMP1, α-SMA, MMP-13, TGF-β1, and BAMBI, genes associated with hepatic fibrosis.
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