Myeloperoxidase-Hepatocyte-Stellate Cell Cross Talk Promotes Hepatocyte Injury and Fibrosis in Experimental Nonalcoholic Steatohepatitis - PubMed (original) (raw)
Myeloperoxidase-Hepatocyte-Stellate Cell Cross Talk Promotes Hepatocyte Injury and Fibrosis in Experimental Nonalcoholic Steatohepatitis
Benjamin Pulli et al. Antioxid Redox Signal. 2015.
Abstract
Aims: Myeloperoxidase (MPO), a highly oxidative enzyme secreted by leukocytes has been implicated in human and experimental nonalcoholic steatohepatitis (NASH), but the underlying mechanisms remain unknown. In this study, we investigated how MPO contributes to progression from steatosis to NASH.
Results: In C57Bl/6J mice fed a diet deficient in methionine and choline to induce NASH, neutrophils and to a lesser extent inflammatory monocytes are markedly increased compared with sham mice and secrete abundant amounts of MPO. Through generation of HOCl, MPO directly causes hepatocyte death in vivo. In vitro experiments demonstrate mitochondrial permeability transition pore induction via activation of SAPK/JNK and PARP. MPO also contributes to activation of hepatic stellate cells (HSCs), the most important source of collagen in the liver. In vitro MPO-activated HSCs have an activation signature (MAPK and PI3K-AKT phosphorylation) and upregulate COL1A1, α-SMA, and CXCL1. MPO-derived oxidative stress also activates transforming growth factor β (TGF-β) in vitro, and TGF-β signaling inhibition with SB-431542 decreased steatosis and fibrosis in vivo. Conversely, congenital absence of MPO results in reduced hepatocyte injury, decreased levels of TGF-β, fewer activated HSCs, and less severe fibrosis in vivo.
Innovation and conclusion: Cumulatively, these findings demonstrate important cross talk between inflammatory myeloid cells, hepatocytes, and HSCs via MPO and establish MPO as part of a proapoptotic and profibrotic pathway of progression in NASH, as well as a potential therapeutic target to ameliorate this disease.
Figures
**FIG. 1.
Myeloperoxidase (MPO)-expressing cells, MPO protein, and MPO activity are increased in nonalcoholic steatohepatitis (NASH). (a) Flow cytometric analysis of liver myeloid cells in NASH: neutrophils (red gate) and inflammatory Ly-6Chigh monocytes (blue gate) (_n_=5 per group). (b) Relative fold increase of different myeloid cell populations in NASH over sham (_n_=5 per group). (c) Immunohistochemistry for MPO in NASH compared with sham mice (arrows indicate MPO positive cells, bar=50 μm, _n_=3 per group). (d) Liver MPO protein and MPO activity (_n_=5–6 per group). (e) Pie chart of MPO-expressing cells as quantified by flow cytometry. Overall pie chart size reflects the absolute numbers of MPO-positive cells per liver, while numbers in pie chart reflect the percentages of cell types (_n_=4–5 per group). (f) Extracellular fluid MPO activity in wild-type (WT) and MPO knockout (MPO−/−) NASH mice compared with sham mice (_n_=3–4 per group). All data are mean±SEM. Lin=CD90/CD49.2/B220/NK1.1/Ly-6G. *p<0.05, **p<0.01, ***p<0.001. KC, Kupffer cells; DC, dendritic cells; N, neutrophils; M, monocytes. To see this illustration in color, the reader is referred to the web version of this article at
**FIG. 2.
MPO deficiency attenuates the severity of fibrosis and hepatocyte injury. (a) Masson's trichrome (top row) and hematoxylin and eosin (H&E, bottom row) staining of liver sections of wild-type (WT) and MPO knockout (MPO−/−) NASH mice. In the top row, collagen is stained in blue; in the bottom row, hepatocyte ballooning is marked with arrows (bars=50 μm). (b) Quantification of fibrosis on histology (_n_=4 per group) and with hydroxyproline assay (_n_=6 per group). (c) Quantification of steatosis on histology and on Oil Red O assay (_n_=4–6 per group). (d) Quantification of hepatocyte ballooning and NAFLD activity score (NAS, _n_=4 per group) (e) real time (RT)-polymerase chain reaction mRNA quantification of COL1A1, TIMP-1, TNF, _IL-1_β, and IL-10 relative to sham mice (_n_=6 per group). (f) Adiponectin in serum and visceral fat, as well as endotoxin in serum and liver as determined by ELISA as well as biochemical assay, respectively. All data are mean±SEM. *p<0.05, **p<0.01. To see this illustration in color, the reader is referred to the web version of this article at
**FIG. 3.
MPO directly contributes to hepatocyte death in vivo and in vitro . Liver immunofluorescence (a) and flow cytometry (b) of Sytox Red-labeled hepatocytes in wild-type (WT) and MPO knockout (MPO−/−) NASH and sham mice (_n_=4 per group, arrowheads indicate Sytox Red positive cells, bar in low magnification represents 1 mm, bar in high magnification represents 50 μm, blue counterstain is DAPI). (c) Liver immunofluorescence in NASH mice for MPO (anti-MPO, green) and injured hepatocytes (Sytox Red, red, counterstain is DAPI, bar=50 μm). (d) Cell death (propidium iodide, PI) in primary hepatocytes (bright-field panel) cultured in the presence of MPO and glucose oxidase (GOX) (_n_=4 per group, bar=100 μm). *p<0.05, **p<0.01, ***p<0.001. To see this illustration in color, the reader is referred to the web version of this article at
**FIG. 4.
MPO-derived oxidative stress causes hepatocyte injury via induction of mitochondrial permeability transition. (a) Cell death (propidium iodide, PI) in primary hepatocytes preincubated with ABAH, taurine, marimastat, SB-431542, or a protease inhibitor cocktail and incubated with MPO (_n_=3 per group, bar=50 μm). (b) Heat map with hierarchical clustering of signaling pathways in primary hepatocytes stimulated with MPO. Molecules in bold are statistically significantly changed from baseline (_n_=4 per group). (c) Western blots of phosphorylated AMPK, SAPK/JNK, and p38, as well as caspase 3/7 activity in MPO- or vehicle-stimulated hepatocytes (_n_=4 per group). Camptothecin (CPT) was used as a positive control for the caspase assay. (d) Cell death in hepatocytes preincubated with the mitochondrial permeability transitioning pore inhibitor cyclosporine A (_n_=4 per group). (e) Cell death (propidium iodide, PI) in primary hepatocytes isolated from NASH or control mice and incubated with MPO (_n_=4 per group, bar=50 μm). All data are mean±SEM. For MPO, += 0.1 U/ml, ++=1.0 U/ml. *p<0.05, **p<0.01, or ***p<0.001, #p<0.05 compared with vehicle. To see this illustration in color, the reader is referred to the web version of this article at
**FIG. 5.
MPO-derived oxidative stress activates hepatic stellate cells. (a) α-smooth muscle actin (α-SMA) and COL1A1 expression of primary HSCs (bright-field and vitamin A fluorescence overlay, left panel) stimulated with MPO (_n_=3–5 per group). Primary hepatic stellate cells (HSCs) were also preincubated with ABAH, taurine, marimastat, or SB-431542 and stimulated with MPO (_n_=3 per group). (b) Heat map with hierarchical clustering of signaling pathways in primary HSCs stimulated with MPO. Molecules in bold are statistically significantly changed from baseline (_n_=4 per group). (c) Immunohistochemistry for α-SMA (bar=50 μm) and (d) flow cytometric analysis of vitamin A fluorescent quiescent HSCs (_n_=4–5 per group). (e) CXCL1 protein secretion in primary HSCs (_n_=3–4 per group). CXCL1 and CXCL2 mRNA in HSCs stimulated with MPO in vitro. (f) CXCL1 protein and mRNA levels in wild-type (WT) and MPO knockout (MPO−/−) NASH mice, as well as sham mice (_n_=6 per group). All data are mean±SEM. +=0.05 U/ml, ++=0.5 U/ml, *p<0.05, **p<0.01, ***p<0.001, #p<0.05 compared to vehicle. To see this illustration in color, the reader is referred to the web version of this article at
**FIG. 6.
MPO activates TGF-β, and TGF-β signaling inhibition ameliorates steatosis and fibrosis in NASH. (a) Activation of TGF-β in vitro. TGF-β was incubated with glucose oxidase (GOX) and MPO (_n_=3 per group). +=0.01 U/ml. ++=0.1 U. +++=1 U/ml. (b) TGF-β in wild-type (WT) and MPO knockout (MPO−/−) mice with NASH and sham mice (_n_=6 per group). (c) Hematoxylin and eosin (H&E, bars=100 μm) and Masson's trichrome (collagen in blue, bars=500 μm) stains of liver sections from NASH mice treated with the TGF-β signaling inhibitor SB-431542 or vehicle for 4 weeks. (d) Quantification of steatosis, fibrosis, and NAFLD activity. All data are mean±SEM. +=0.05 U/ml, ++=0.5 U/ml, *p<0.05, **p<0.01, ***p<0.001. To see this illustration in color, the reader is referred to the web version of this article at
**FIG. 7.
Myeloid cell subsets and cytokine expression are similar in MPO knockout and wild-type mice with NASH. (a) Liver myeloid cell populations in wild-type (WT) and MPO knockout (MPO−/−) with NASH and sham mice. Representative analysis of neutrophils (red gate) and Ly-6Chigh monocytes (blue gate) is shown, and relative numbers of neutrophils, monocytes, and Kupffer cells are presented (_n_=4–5 per group). (b) TNF secretion of Ly-6Chigh monocytes (_n_=4–5 per group). (c) Total liver TNF levels as determined by ELISA (_n_=5 per group). (d) TGF-β secretion of Kupffer cells (_n_=4–5 per group). All data are mean±SEM. *p<0.05, ***p<0.001. To see this illustration in color, the reader is referred to the web version of this article at
**FIG. 8.
Pathophysiology of NASH relevant to MPO. MPO catalyzes the formation of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2). This induces hepatocyte (HC) injury. HS apoptosis leads to TGF-β secretion and HSC activation. HOCl also activates TGF-β and matrix metalloproteinases (MMPs), resulting in hepatic stellate cell (HSC) activation. Activated HSCs deposit collagen and thus contribute to fibrosis. Activated HSCs secrete chemokines to attract inflammatory myeloid cells CXCL1. HC necrosis releases damage-associated molecular pattern molecules (DAMPs), among other factors, which also recruit inflammatory myeloid cells to the site of injury. Solid lines represent findings from the present article, and dashed lines are included to link these findings to established knowledge.
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