The Role of Chemokines in Acute Liver Injury

Chemokines are small molecular weight proteins primarily known to drive migration of immune cell populations. In both acute and chronic liver injury, hepatic chemokine expression is induced resulting in inflammatory cell infiltration, angiogenesis, and cell activation and survival. During acute injury, massive parenchymal cell death due to apoptosis and/or necrosis leads to chemokine production by hepatocytes, cholangiocytes, Kupffer cells, hepatic stellate cells, and sinusoidal endothelial cells. The specific chemokine profile expressed during injury is dependent on both the type and course of injury. Hepatotoxicity by acetaminophen for example leads to cellular necrosis and activation of Toll-like receptors while the inciting insult in ischemia reperfusion injury produces reactive oxygen species and subsequent production of pro-inflammatory chemokines. Chemokine expression by these cells generates a chemoattractant gradient promoting infiltration by monocytes/macrophages, NK cells, NKT cells, neutrophils, B cells, and T cells whose activity are highly regulated by the specific chemokine profiles within the liver. Additionally, resident hepatic cells express chemokine receptors both in the normal and injured liver. While the role of these receptors in normal liver has not been well described, during injury, receptor up-regulation, and chemokine engagement leads to cellular survival, proliferation, apoptosis, fibrogenesis, and expression of additional chemokines and growth factors. Hepatic-derived chemokines can therefore function in both paracrine and autocrine fashions further expanding their role in liver disease. More recently it has been appreciated that chemokines can have diverging effects depending on their temporal expression pattern and the type of injury. A better understanding of chemokine/chemokine receptor axes will therefore pave the way for development of novel targeted therapies for the treatment of liver disease

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

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

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 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

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