Transplanted adult human hepatic stem/progenitor cells prevent histogenesis of advanced hepatic fibrosis in mice induced by carbon tetrachloride

Transplantation of adult human hepatic stem/progenitor cells (hHSPCs) has been considered as an alternative therapy, replacing donor liver transplantation to treat liver cirrhosis. This study assessed the antifibrotic effects of hHSPCs in mice with fibrosis induced by carbon tetrachloride (CCl4) and examined the actions of hHSPCs on the fibrogenic activity of human hepatic stellate cells (HSCs) in a coculture system. Isolated hHSPCs expressed stem/progenitor cell phenotypic markers. Mice were given CCl4 (twice weekly for 7 weeks) and hHSPC transplantation weekly. CCl4 induced advanced fibrosis (bridging fibrosis and cirrhosis) in mice, which was prevented by hHSPC transplantation. The liver of hHSPC-transplanted mice showed only occasional short septa and focal parenchymal fibrosis, and a 50% reduction in hepatic collagen, assessed by Sirius red stain histomorphometry. Moreover, the proteins for α-smooth muscle actin (α-SMA) and collagen I were decreased. While α-SMA, collagen α1(I), and tissue inhibitor of metalloproproteinase-1 mRNAs were decreased, matrix metalloproteinase (MMP)-1 mRNA was increased, consistent with decreased fibrogenesis. MMP-2 and transforming growth factor-β were not affected. Alanine aminotransferase and aspartate aminotransferase were lower, suggesting improvement of liver function/damage. In coculture, hHSPCs elicited changes of α-SMA and fibrogenic molecules in HSCs similar to those observed in vivo, providing evidence for a functional link between hHSPCs and HSCs. A decreased HSC proliferation was noted. Thus, transplantation of hHSPCs prevents histogenesis of advanced liver fibrosis caused by CCl4. hHSPCs mediate downregulation of HSC activation coincident with modulation of fibrogenic molecule expression, leading to suppression of fibrogenesis both in vivo and in vitro

Transplanted adult human hepatic stem/progenitor cells prevent histogenesis of advanced hepatic fibrosis in mice induced by carbon tetrachloride

Transplantation of adult human hepatic stem/progenitor cells (hHSPCs) has been considered as an alternative therapy, replacing donor liver transplantation to treat liver cirrhosis. This study assessed the antifibrotic effects of hHSPCs in mice with fibrosis induced by carbon tetrachloride (CCI4) and examined the actions of hHSPCs on the fibrogenic activity of human hepatic stellate cells (HSCs) in a coculture system. Isolated hHSPCs expressed stem/progenitor cell phenotypic markers. Mice were given CCl4 (twice weekly for 7 weeks) and hHSPC transplantation weekly. CCl4 induced advanced fibrosis (bridging fibrosis and cirrhosis) in mice, which was prevented by hHSPC transplantation. The liver of hHSPC-transplanted mice showed only occasional short septa and focal parenchymal fibrosis, and a 50% reduction in hepatic collagen, assessed by Sirius red stain histomorphometry. Moreover, the proteins for a-smooth muscle actin (alpha-SMA) and collagen I were decreased. While alpha-SMA, collagen alpha 1(I), and tissue inhibitor of metalloproproteinase-1 mRNAs were decreased, matrix metalloproteinase (MMP)-1 mRNA was increased, consistent with decreased fibrogenesis. MMP-2 and transforming growth factor-beta were not affected. Alanine aminotransferase and aspartate aminotransferase were lower, suggesting improvement of liver function/damage. In coculture, hHSPCs elicited changes of alpha-SMA and fibrogenic molecules in HSCs similar to those observed in vivo, providing evidence for a functional link between hHSPCs and HSCs. A decreased HSC proliferation was noted. Thus, transplantation of hHSPCs prevents histogenesis of advanced liver fibrosis caused by CCl4. hHSPCs mediate down-regulation of HSC activation coincident with modulation of fibrogenic molecule expression, leading to suppression of fibrogenesis both in vivo and in vitro

Leukadherin-1-Mediated Activation of CD11b Inhibits LPS-Induced Pro-inflammatory Response in Macrophages and Protects Mice Against Endotoxic Shock by Blocking LPS-TLR4 Interaction

Dysregulation of macrophage has been demonstrated to contribute to aberrant immune responses and inflammatory diseases. CD11b, expressed on macrophages, plays a critical role in regulating pathogen recognition, phagocytosis, and cell survival. In the present study, we explored the effect of leukadherin-1 (LA1), an agonist of CD11b, on regulating LPS-induced pro-inflammatory response in macrophages and endotoxic shock. Intriguingly, we found that LA1 could significantly reduce mortalities of mice and alleviated pathological injury of liver and lung in endotoxic shock. In vivo studies showed that LA1-induced activation of CD11b significantly inhibited the LPS-induced pro-inflammatory response in macrophages of mice. Moreover, LA1-induced activation of CD11b significantly inhibited LPS/IFN-γ-induced pro-inflammatory response in macrophages by inhibiting MAPKs and NF-κB signaling pathways in vitro. Furthermore, the mice injected with LA1-treated BMDMs showed fewer pathological lesions than those injected with vehicle-treated BMDMs in endotoxic shock. In addition, we found that activation of TLR4 by LPS could endocytose CD11b and activation of CD11b by LA1 could endocytose TLR4 in vitro and in vivo, subsequently blocking the binding of LPS with TLR4. Based on these findings, we concluded that LA1-induced activation of CD11b negatively regulates LPS-induced pro-inflammatory response in macrophages and subsequently protects mice from endotoxin shock by partially blocking LPS-TLR4 interaction. Our study provides a new insight into the role of CD11b in the pathogenesis of inflammatory diseases

X4 Human Immunodeficiency Virus Type 1 gp120 Promotes Human Hepatic Stellate Cell Activation and Collagen I Expression through Interactions with CXCR4

<div><h3>Background & Aims</h3><p>Patients coinfected with HIV-1 and HCV develop more rapid liver fibrosis than patients monoinfected with HCV. HIV RNA levels correlate with fibrosis progression implicating HIV directly in the fibrotic process. While activated hepatic stellate cells (HSCs) express the 2 major HIV chemokine coreceptors, CXCR4 and CCR5, little is known about the pro-fibrogenic effects of the HIV-1 envelope protein, gp120, on HSCs. We therefore examined the <em>in vitro</em> impact of X4 gp120 on HSC activation, collagen I expression, and underlying signaling pathways and examined the <em>in vivo</em> expression of gp120 in HIV/HCV coinfected livers.</p> <h3>Methods</h3><p>Primary human HSCs and LX-2 cells, a human HSC line, were challenged with X4 gp120 and expression of fibrogenic markers assessed by qRT-PCR and Western blot +/− either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Downstream intracellular signaling pathways were evaluated with Western blot and pre-treatment with specific pathway inhibitors. Gp120 immunostaining was performed on HIV/HCV coinfected liver biopsies.</p> <h3>Results</h3><p>X4 gp 120 significantly increased expression of alpha-smooth muscle actin (a-SMA) and collagen I in HSCs which was blocked by pre-incubation with either CXCR4-targeted shRNA or anti-CXCR4 neutralizing antibody. Furthermore, X4 gp120 promoted Extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and pretreatment with an ERK inhibitor attenuated HSC activation and collagen I expression. Sinusoidal staining for gp120 was evident in HIV/HCV coinfected livers.</p> <h3>Conclusions</h3><p>X4 HIV-1 gp120 is pro-fibrogenic through its interactions with CXCR4 on activated HSCs. The availability of small molecule inhibitors to CXCR4 make this a potential anti-fibrotic target in HIV/HCV coinfected patients.</p> </div

The role of CYP2A5 in liver injury and fibrosis: chemical-specific difference

Liver injuries induced by carbon tetrachloride (CCL4) or thioacetamide (TAA) are dependent on cytochrome P450 2E1 (CYP2E1). CYP2A5 can be induced by TAA but not by CCL4. In this study, liver injury including fibrosis induced by CCL4 or TAA were investigated in wild-type (WT) mice and CYP2A5 knockout (cyp2a5−/− ) mice as well as in CYP2E1 knockout (cyp2e1−/− ) mice as a comparison. Acute and subchronic liver injuries including fibrosis were induced by CCL4 and TAA in WT mice but not in cyp2e1−/ − mice, confirming the indispensable role of CYP2E1 in CCL4 and TAA hepatotoxicity. WT mice and cyp2a5−/− mice developed comparable acute liver injury induced by a single injection of CCL4 as well as subchronic liver injury including fibrosis induced by 1 month of repeated administration of CCL4, suggesting that CYP2A5 does not affect CCL4- induced liver injury and fibrosis. However, while 200 mg/kg TAA-induced acute liver injury was comparable in WT mice and cyp2a5−/− mice, 75 and 100 mg/kg TAA-induced liver injury were more severe in cyp2a5−/− mice than those found in WT mice. After multiple injections with 200 mg/kg TAA for 1 month, while subchronic liver injury as indicated by serum aminotransferases was comparable in WT mice and cyp2a5−/− mice, liver fibrosis was more severe in cyp2a5−/− mice than that found in WT mice. These results suggest that while both CCL4- and TAA-induced liver injuries and fibrosis are CYP2E1 dependent, under some conditions, CYP2A5 may protect against TAA-induced liver injury and fibrosis, but it does not affect CCL4 hepatotoxicity

Gp120 is present in liver tissue from HIV/HCV infected patients.

<p>Paraffin-embedded liver biopsies from HIV/HCV coinfected patients prior to initiation of ART were immunostained with monoclonal anti-gp120 or isotype control. Sinusoidal staining was appreciated in all 3 specimens. Representative images shown (Magnification 100×, 630×). Viral load ranges: HIV RNA levels ranged from 514–2,455 copies/ml; HCV RNA levels ranged from 1.2 million–1.6 million IU/ml.</p

X4 gp120 induction of collagen I occurs via the ERK 1/2 pathway.

<p>(A, B) To determine whether X4 gp120 activates PI3K-Akt and/or ERK 1/2 pathways, primary HSCs were incubated with 500 ng/ml of X4 gp120 and levels of phospho-Akt and phospho-ERK 1/2 assessed by Western blot. An increase in phospho-ERK 1/2 was seen while no increase in phospho-Akt was seen in response to X4 gp120. (C) Pretreatment of cells with ERK inhibitor (UO126; 10 nM) 30 minutes prior to gp120 treatment resulted in a marked decrease in X4 gp120-induced collagen I protein expression in HSCs. ß-tubulin and total ERK1/2 were used as loading controls. Representative Western blots of three independent experiments with normalized densitometric units shown.</p

X4 gp120 induction of a-SMA and collagen I expression is CXCR4-dependent.

<p>(A) Passage #3 primary HSCs were plated at a density of 2×10⁴ cells/well in 6-well plates, serum-starved for 24 hours, and then transfected with shCXCR4 or shControl. A 75% reduction in CXCR4 protein expression was noted 72 hours after transfection by Western blot. (B) 72 hours after shControl or sh CXCR4 transfection, primary HSCs were then challenged with X4 gp120 for 2 hours, RNA harvested, reverse transcribed and qRT-PCR performed for CXCR4, a-SMA, and coll I (a1). A 40–50% reduction in gp120-induced collagen (a1) and a-SMA mRNA levels was observed with CXCR4 knockdown. Data are expressed as the mean ± standard deviation of three independent experiments. (C) 72 hours after shCXCR4 knockdown, HSCs were challenged with HIV-IIIB for 8 hours and cell lysates used for Western blot where a 70% reduction in the protein expression of both a-SMA and collagen I was observed. (D) Human primary HSCs were pretreated with anti-CXCR4 antibody for 30 min (20 µg/ml) followed by treatment with gp120 (500 ng/mL) for 8 hours. Both X4 gp120-induced a-SMA and coll I protein expression were attenuated by anti-CXCR4 neutralizing antibody. ß-actin, a-tubulin, and GAPDH were used as loading controls. Representative Western blots with normalized densitometric arbitary units shown.</p

X4 HIV-1 gp120 induces fibrogenic gene expression in human stellate cells.

<p>(A) LX-2 cells were serum-starved for 24 hrs, treated with X4 HIV-1 gp120 at a final concentration of 500 ng/ml for 2 hours, RNA harvested, reverse transcribed, and qRT-PCR performed for TGF-ß1, type I TGF-ß receptor, a-SMA and coll I (a1) mRNA levels. To confirm X4 gp120 effects were CD4-independent, cells were pre-incubated with 25 µg/mL anti-CD4 30 minutes prior to challenge with X4 gp120. (B, C) Effect on collagen I (a1) was confirmed in primary HSCs with both X4 gp120 as well as AT-2 treated X4-tropic HIV-IIIB, which presents gp120 in its oligomeric confirmation. All data are expressed as means +/− standard deviation of at least three independent experiments.</p

X4 HIV-1 gp120 promotes a-SMA and collagen I protein expression in human HSCs.

<p>(A, B) LX-2 cells were incubated with 500 ng/ml X4 gp120 for 0–12 hours, protein harvested, and expression of a-SMA and collagen I examined by Western blot analysis. (C) The effect of X4 gp120 versus vehicle control on collagen I protein expression was further confirmed in passage #3 primary HSCs. Densitometry was performed and normalized arbitrary units represented numerically.</p