Human neutrophil peptide-1 promotes alcohol-induced hepatic fibrosis and hepatocyte apoptosis

<div><p>Background and aims</p><p>Neutrophil infiltration of the liver is a typical feature of alcoholic liver injury. Human neutrophil peptide (HNP)-1 is an antimicrobial peptide secreted by neutrophils. The aim of this study was to determine if HNP-1 affects ethanol-induced liver injury and to examine the mechanism of liver injury induced by HNP-1.</p><p>Methods</p><p>Transgenic (TG) mice expressing HNP-1 under the control of a β-actin-based promoter were established. Ethanol was orally administered to HNP-1 TG or wild-type C57BL/6N (WT) mice. SK-Hep1 hepatocellular carcinoma cells were used to investigate the effect of HNP-1 on hepatocytes <i>in vitro</i>.</p><p>Results</p><p>After 24 weeks of ethanol intake, hepatic fibrosis and hepatocyte apoptosis were significantly more severe in TG mice than in WT mice. Levels of CD14, TLR4, and IL-6 in liver tissues were higher in TG mice than in WT mice. Apoptosis was accompanied by higher protein levels of caspase-3, caspase-8, and cleaved PARP in liver tissue. In addition, phosphorylated ASK1, ASK1, phosphorylated JNK, JNK1, JNK2, Bax, Bak and Bim were all more abundant in TG mice than in WT mice. In contrast, the level of anti-apoptotic Bcl2 in the liver was significantly lower in TG mice than in WT mice. Analysis of microRNAs in liver tissue showed that miR-34a-5p expression was significantly higher in TG mice than in WT mice. Furthermore, in the presence of ethanol, HNP-1 increased the apoptosis with the decreased level of Bcl2 in a concentration-dependent manner <i>in vitro</i>.</p><p>Conclusions</p><p>HNP-1 secreted by neutrophils may exacerbate alcohol-induced hepatic fibrosis and hepatocyte apoptosis with a decrease in Bcl2 expression and an increase in miR-34a-5p expression.</p></div

Glycoprotein Nonmetastatic Melanoma B (Gpnmb)-Positive Macrophages Contribute to the Balance between Fibrosis and Fibrolysis during the Repair of Acute Liver Injury in Mice

<div><p>Background and aims</p><p>Glycoprotein nonmetastatic melanoma B (Gpnmb), a transmembrane glycoprotein that is expressed in macrophages, negatively regulates inflammation. We have reported that Gpnmb is strongly expressed in the livers of rats fed a choline-deficient, L-amino acid-defined (CDAA) diet. However, the role of macrophage-expressed Gpnmb in liver injury is still unknown. This study aimed to clarify the characteristics of infiltrating macrophages that express Gpnmb, and the involvement of Gpnmb in the repair process in response to liver injury.</p><p>Methods</p><p>C57BL/6J, DBA/2J [DBA] and DBA/2J-Gpnmb⁺ [DBA-g+] mice were treated with a single intraperitoneal injection of carbon tetrachloride (CCl₄) at a dose of 1.0 mL/kg body weight. Mice were sacrificed at predetermined time points, followed by measurement of serum alanine aminotransferase (ALT) levels and histological examination. Expression of Gpnmb, pro-/anti-inflammatory cytokines, and profibrotic/antifibrotic factors were examined by quantitative RT-PCR and/or Western blotting. Immunohistochemistry, fluorescent immunostaining and flow cytometry were used to determine the expression of Gpnmb, CD68, CD11b and α-SMA, phagocytic activity, and the presence of apoptotic bodies. We used quantitative RT-PCR and ELISA to examine TGF-β and MMP-13 expression and the concentrations and supernatants of isolated infiltrating hepatic macrophages transfected with siGpnmb.</p><p>Results</p><p>In C57BL/6J mice, serum ALT levels increased at two days after CCl₄ injection and decreased at four days. Gpnmb expression in the liver was stimulated four days after CCl₄ injection. Histological examination and flow cytometry showed that Gpnmb-positive cells were almost positive for CD68-positive macrophages, contained engulfed apoptotic bodies and exhibited enhanced phagocytic activity. Isolated infiltrating hepatic macrophages transfected with siGpnmb showed high MMP-13 secretion. There was no significant difference in the magnitude of CCl₄-induced liver injury between DBA-g+ and DBA mice. However, hepatic MMP-13 expression, as well as α-SMA expression and collagen production, increased significantly in DBA-g+ compared with DBA mice.</p><p>Conclusions</p><p>Gpnmb-positive macrophages infiltrate the liver during the recovery phase of CCl₄–induced acute liver injury and contribute to the balance between fibrosis and fibrolysis in the repair process following acute liver injury.</p></div

Features of DBA-g+ and DBA mice.

<p>(A) Expression of Gpnmb is observed in DBA-g+ but not DBA mice following a single injection of CCl₄. However, (B) sequential changes in serum ALT levels and the degree of liver injury are not affected by the lack of Gpnmb-positive macrophages (original magnification, x100). Conversely, (C) the areas of fibrosis and (D) the number of α-SMA-positive cells are significantly decreased in the liver tissues of DBA mice compared to DBA-g+ mice (original magnification, x200). Additionally, (E) although lack of Gpnmb-positive macrophages does not affect expression of TGF-β or Col1α1, expression of MMP-9, MMP-13, and TIMP-1 are significantly decreased in mice lacking Gpnmb expression at six or eight days after single injection of CCl₄. Values are mean ± SEM (<i>n</i> = 4). * <i>P</i> < 0.05 (Mann-Whitney U test).</p