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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Liver fibrogenesis is a process tightly controlled by endogenous anti- and pro-fibrogenic factors. Interferon gamma (IFNγ) is a potent antifibrogenic cytokine in vitro and might therefore represent a powerful therapeutic entity. However, its poor pharmacokinetics and adverse effects, due to the presence of IFNγ receptors on nearly all cells, prevented its clinical application so far. We hypothesized that delivery of IFNγ specifically to the disease-inducing cells and concurrently avoiding its binding to nontarget cells might increase therapeutic efficacy and avoid side effects. We conjugated IFNγ to a cyclic peptide recognizing the platelet-derived growth factor beta receptor (PDGFβR) which is strongly up-regulated on activated hepatic stellate cells (HSC), the key effector cells responsible for hepatic fibrogenesis. The IFNγ conjugates were analyzed in vitro for PDGFβR-specific binding and biological effects and in vivo in acute (early) and chronic (progressive and established) carbon-tetrachloride-induced liver fibrosis in mice. The targeted-IFNγ construct showed PDGFβR-specific binding to fibroblasts and HSC and inhibited their activation in vitro. In vivo, the targeted-IFNγ construct attenuated local HSC activation in an acute liver injury model. In the established liver fibrosis model, it not only strongly inhibited fibrogenesis but also induced fibrolysis. In contrast, nontargeted IFNγ was ineffective in both models. Moreover, in contrast to unmodified IFNγ, our engineered targeted-IFNγ did not induce IFNγ-related side effects such as systemic inflammation, hyperthermia, elevated plasma triglyceride levels, and neurotropic effects. Conclusion: This study presents a novel HSC-targeted engineered-IFNγ, which in contrast to systemic IFNγ, blocked liver fibrogenesis and is devoid of side effects, by specifically acting on the key pathogenic cells within the liver. (HEPATOLOGY 2011;)

Liver cirrhosis, characterized by the extensive accumulation of an abnormal extracellular matrix, is the major cause of liver-related morbidity and mortality worldwide.1, 2 Except for an effective treatment of the underlying etiology, which is an option for some patients, there exists no clinically proven antifibrotic therapy to prevent progression of chronic liver disease to cirrhosis or to its regression.3, 4 Activated hepatic stellate cells and portal fibroblasts (collectively named activated hepatic stellate cells, HSC) are the main effector cells of liver fibrogenesis, producing most of the excessive extracellular matrix (ECM) such as fibrillar collagens.5 Following liver injury, release of several fibrogenic factors by inflammatory cells, bile duct epithelia, or hepatocytes, such as transforming growth factor-beta (TGFβ) or platelet-derived growth factor (PDGF) activate HSC to transform to contractile myofibroblastic cells characterized by enhanced proliferation and ECM production.5 There are only a few cytokines such as interferon-alpha (IFNα) and interferon-gamma (IFNγ) that can attenuate fibrogenic processes and have been explored as potential therapeutics.6 However, whereas IFNα and especially IFNγ are highly effective antifibrotic agents in vitro and in some animal models in vivo,6, 7 their antifibrotic potential in clinical trials has been disappointing, due to poor efficacy and unwanted off-target effects,8, 9 related to the ubiquitous presence of IFNγ receptor (IFNγR) on all cells except erythrocytes.10

IFNγ is a pleiotropic proinflammatory T helper 1 (Th1) cytokine produced by activated immune cells.10 It has been tested for the treatment of viral, immunological, and malignant diseases11 due to its antiviral, immunomodulatory, and antiproliferative activities. In addition, several clinical studies have explored the potential role of systemic IFNγ in renal, pulmonary, and liver fibrosis.8, 9, 12 However, its limited efficiency associated with a short circulation half-life and undesirable systemic side effects has limited its clinical utility. Many attempts to prolong the IFNγ half-life or to enhance its activity through slow release by incorporation into nanoparticles, liposomes, microspheres, or elastomers did not lead to a significant improvement.13, 14 No approach of cell-specific delivery of IFNγ has been reported, although in vivo disease activity is controlled by its local production. Experimental therapies, mimicking this local production, are therefore attractive.

In the present study we chemically engineered IFNγ by directing it to another target receptor, PDGFβR, that is abundantly expressed only on activated HSC during fibrogenesis.15, 16 IFNγ was covalently conjugated to a PDGFβR-recognizing cyclic peptide17 (PPB) either directly or indirectly using a polyethylene glycol (PEG) linker. PPB cyclic peptide (*CSRNLIDC*) has been developed by our group17 and extensively studied for PDGFβR-specific drug delivery, e.g., to tumors.18 The PPB-modified IFNγ constructs were characterized in vitro for their biological activity in fibroblasts and HSC. In vivo, the targeted constructs showed high specific binding to the target cells, inhibited HSC activation, and progression of liver fibrosis/cirrhosis in acute and chronic carbon tetrachloride (CCl4)-induced fibrosis models. Notably, the targeted IFNγ construct were devoid of unwanted IFNγ-related side effects.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Human Liver Specimens.

Human liver specimens were obtained from liver-transplanted patients suffering from liver cirrhosis and were anonymously provided by the Department of Pathology (University Medical Center Groningen UMCG, The Netherlands). Control tissue was obtained from the unaffected part of liver from transplanted patients. Necessary approvals were obtained from the hospital Medical Ethics Committee.

Cell Lines.

Mouse 3T3 fibroblasts and RAW macrophages were obtained from the American Type Culture Collection (ATCC). Human hepatic stellate cells (LX2) were kindly provided by Prof. Scott Friedman (Mount Sinai Hospital, New York). RAW macrophages and 3T3 were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS). LX2 were cultured in DMEM-Glutamax (Invitrogen) supplemented with 10% FBS.

Synthesis of IFNγ Conjugates.

IFNγ conjugates were synthesized by either direct chemical coupling of PDGFβ receptor recognizing peptide (PPB) via N-[γ-maleimidobutyryloxy] succinimide ester (GMBS; Sigma, St. Louis, MO) to generate IFNγ-PPB or by indirect conjugation using bifunctional PEG molecule (Mal-PEG-SCM, 2 kDa, Creative PEGworks, Winston Salem, NC) to synthesize IFNγ-PEG-PPB. As a control IFNγ-PEG was synthesized using monofunctional PEG (mPEG-SMB, 2 kDa, Nektar Therapeutics). The detailed syntheses and characterization using western blotting are described in the Supporting data.

Immunohistochemistry and Immunofluorescence.

The detailed protocol for immunohistochemistry and immunofluorescence is described in the Supporting data. The antibodies used are listed in Supporting Table 1.

Nitric Oxide (NO) Release Bioassay in RAW Macrophages.

The bioactivity of IFNγ and IFNγ conjugates was assessed by measuring accumulation of nitrite NO2, a stable NO metabolite produced by RAW macrophages.19 Briefly, cells seeded in 96-well plates were incubated with different concentrations of IFNγ and IFNγ conjugates. After 24 hours the secreted nitrite was measured as absorbance at 550 nm using Greiss reagent (1% sulfanilamide; 0.1% naphthylethylendiamine dihydrochloride; 3% H3PO4).

In Vitro Binding and Effects of the IFNγ Conjugates in Mouse Fibroblasts and Human HSC.

Cells were seeded in Lab-Tek (Nunc, Roskilde, Denmark) or in 24-well plates and cultured overnight. For binding study, cells were incubated with IFNγ or IFNγ conjugates (1 μg/mL). To block the PDGFβR-mediated binding, anti-PDGFβR IgG (Santa Cruz Biotechnology, Santa Cruz, CA) was added 1 hour before IFNγ conjugates. After 2 hours, cells were fixed and immunofluorescent staining for PPB and IFNγ was performed.

To assess effects on fibrotic parameters, cells were starved for 24 hours and incubated with IFNγ and IFNγ conjugates with 5 ng/mL of human recombinant TGFβ1 (Roche, Mannheim, Germany) for 48 hours. Subsequently, cells were fixed and stained for collagen I and III.

To assess effects on proliferation, cells were starved for 24 hours and incubated with IFNγ and IFNγ conjugates plus 50 ng/mL of human recombinant PDGF (Peprotech) for 48 hours. Subsequently, cells were incubated with 0.25 μCi/mL of thymidine 3[H] for 6 hours, after which the cells were washed thoroughly, fixed with 5% TCA, lysed with 1 mL of 1M NaOH, mixed with 4 mL of scintillation fluid, and measured using a scintillation counter. All measurements were performed in duplicate in three independent experiments.

CCl4-Induced Acute Liver Injury Mouse Model.

Male C57BL/6 mice (20-22 g) were treated with a single intraperitoneal injection of olive oil or CCl4 (1 mL/kg in olive oil) at day 1. At day 2 and day 3, CCl4-treated mice intravenously received different treatments or phosphate-buffered saline (PBS) (n = 6 per group). At day 4, all mice were sacrificed; blood and different organs were collected for subsequent analysis.

For in vivo biodistribution of the conjugates (n = 6 per group), mice were treated with different constructs 10 minutes prior to sacrifice on day 4 after CCl4 injection.

CCl4-Induced Advanced Liver Fibrosis Mouse Model.

Male balb/c mice (20-22 g) were treated with olive oil or increasing doses of CCl4 (week 1: 0.5 mL/kg; week 2: 0.8 mL/kg and week 3-8: 1 mL/kg prepared in olive oil) twice weekly by intraperitoneal injections for 8 weeks as described.20 At weeks 7 and 8, mice were treated intravenously with PBS, IFNγ, IFNγ-PEG, or IFNγ-PEG-PPB (2.5 μg/mice, thrice per week, n = 6 per group). All mice were sacrificed at week 8; blood and different organs were collected for subsequent measurements. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and plasma triglycerides levels were measured by standard automated laboratory methods. Plasma levels of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 were analyzed using a cytometric bead array (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions. IFNγ-induced fever was determined21 by measuring the rectal temperature after 30 minutes of treatments using a digital thermometer with lubricated thermocouple inserted 1.5 cm into the rectum of mice. Hepatic collagen content was determined by liver hydroxyproline assay as reported, with minor modifications.22 The relative hydroxyproline (mg/g liver) was calculated based on individual liver weights. All the experimental protocols for animal studies were approved by the Animal Ethical Committee of the University of Groningen.

Quantitative Real-Time Polymerase Chain Reaction (PCR).

The detailed protocol for quantitative real-time PCR is described in the Supporting data. The primers used are listed in Supporting Table 2.

Statistical Analyses.

Data are presented as mean ± standard error of the mean (SEM). Multiple comparisons between different groups were performed by one-way analysis of variance (ANOVA) with Bonferroni post-test.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

PDGFβR Expression on Fibroblasts and Hepatic Stellate Cells.

We first examined the expression of PDGFβR in mouse and human fibrotic livers. PDGFβR was highly up-regulated in areas of active fibrogenesis (Fig. 1A,B) and specifically colocalized with desmin-positive HSC (Fig. 1C). Conversely, PDGFβR was virtually absent in normal livers and other organs (Fig. 1D). High expression of PDGFβR on activated HSC in fibrotic livers relative to other tissues and cells known to express PDGFβR indicates that this receptor is an appropriate target for HSC-specific delivery in active liver fibrogenesis.

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Figure 1. PDGFβ receptor expression in human and mouse livers and other mouse organs (lung, kidney, heart, and spleen), PDGFβR-targeted IFNγ constructs, and their in vitro biological activity. (A) Representative photomicrographs depicting up-regulation of PDGFβR in cirrhotic human and in CCl4-fibrotic mouse liver sections as compared to normal livers. Scale bars = 100 μm. (B) PDGFβR transcripts were significantly elevated in the fibrotic livers. #P < 0.05 and ##P < 0.01 versus PBS-treated olive oil (normal) group. (C) Fluorescent pictures of liver sections showing colocalization of HSC and PDGFβR in mouse (×100) and human (×200) fibrotic livers stained for desmin (HSC marker, green), PDGFβR (red), and nuclei (blue). (D) PDGFβR-stained tissue sections from CCl4-fibrotic mice showing negligible expression of PDGFβR in other tissues. Scale bars = 100 μm. (E) Schematic representation of the chemically engineered IFNγ conjugates. (F) Nitrogen oxide (NOx) release in mouse RAW macrophages (n = 3) after incubation with unmodified native IFNγ and IFNγ conjugates showing that IFNγ conjugates retain their biological activity relative to unmodified IFNγ.

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Syntheses and In Vitro Characterization of Targeted-IFNγ Constructs.

To target IFNγ to HSC, we modified IFNγ with PDGFβR-recognizing cyclic peptide (PPB) using different conjugation strategies as illustrated in Fig. 1E. PPB was directly conjugated to IFNγ (IFNγ-PPB) or by way of a 2 kDa hydrophilic hetero-bifunctional PEG linker (IFNγ-PEG-PPB). In addition, we synthesized IFNγ-PEG as a control. The synthesis details are illustrated in Supporting Fig. 1. The synthesized conjugates were characterized by western blot analyses with anti-IFNγ and anti-PPB antibodies (Supporting Fig. 2).

Because chemical modifications of cytokines can diminish their biological activity, we examined the activity of the IFNγ conjugates compared to unmodified IFNγ in mouse RAW macrophages. These cells express the IFNγR but lack PDGFβR. IFNγ and its constructs IFNγ-PPB, IFNγ-PEG, and IFNγ-PEG-PPB all induced a similar dose-dependent increase in nitric oxide (NOx) release in RAW cells (Fig. 1F). There was no significant difference in dose-response slopes, demonstrating that all IFNγ conjugates retained full biological activity.

In Vitro Binding and Effects of the Targeted-IFNγ Conjugates in Mouse 3T3 Fibroblasts and Human LX2 Hepatic Stellate Cells.

IFNγ binds to its receptor, which is strictly species-specific, whereas PDGFβR binding is not. In order to discriminate between IFNγR- and PDGFβR-mediated bindings, we used mouse NIH3T3 fibroblasts, primary rat HSC, and human LX2 hepatic stellate cells. The results confirmed the species specificity of IFNγ; mouse IFNγ and mouse derived IFNγ-PEG showed binding to mouse 3T3 fibroblasts (Fig. 2A) but not to rat HSC and human HSC (Fig. 2B; Supporting Fig. 3). However, PPB-modified mouse IFNγ conjugates showed high binding to mouse, rat, and human cells (Fig. 2A,B; Supporting Fig. 3), which was almost completely blocked with anti-PDGFβR IgG (Fig. 2B). This demonstrates the specific binding of PPB-modified IFNγ constructs to PDGFβR, which is species-nonspecific.

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Figure 2. In vitro PDGFβR-specific binding and antifibrotic effects of targeted-IFNγ conjugates in mouse 3T3 fibroblasts and human hepatic stellate cells. (A) Fluorescent photographs (×400) showing binding of IFNγ-PEG, IFNγ-PPB, or IFNγ-PEG-PPB to mouse NIH3T3 fibroblasts. (B) Fluorescent photographs (×200) showing binding of targeted mouse-derived IFNγ conjugates to human HSC (LX2) and blocking of PDGFβR-specific binding by PDGFβR antibody. Mouse IFNγ-PEG did not show any binding to human LX2 cells due to species differences. (C,D) Representative pictures (C) and quantitative analysis (D) of collagen I/III stained LX2 and 3T3 cells, incubated with TGFβ (5 ng/mL) in combination with IFNγ or IFNγ conjugates (1 μg/mL). (E) Bars represent the cell proliferation (thymidine incorporation) after incubation with PDGFBB (50 ng/mL) in combination with IFNγ or IFNγ conjugates (1 μg/mL) in 3T3 fibroblasts. The groups were normalized to control cells (untreated). In 3T3 cells, unmodified mouse IFNγ and IFNγ conjugates caused a significant reduction of TGFβ-induced collagen expression and PDGFBB-induced proliferation. However, in human LX2 cells, only PPB-modified IFNγ conjugates attenuated collagen expression, whereas unmodified mouse IFNγ and IFNγ-PEG did not cause any reduction due to species restriction. Scale bars = 200 μm. ##P < 0.01 versus untreated cells; *P < 0.05 and **P < 0.01 versus TGFβ-treated or PDGFBB-treated cells (n = 3).

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Subsequently, we investigated the antifibrotic effects of the constructs in mouse 3T3 fibroblasts and in human HSC after their activation with TGFβ. Both mouse IFNγ and IFNγ conjugates induced significant reduction in collagen expression in mouse cells (Fig. 2C,D). In addition, mouse IFNγ and IFNγ conjugates inhibited PDGF-induced cell proliferation in 3T3 fibroblasts as assessed by thymidine incorporation assays (Fig. 2E). Interestingly, in human LX2 cells, TGFβ1-induced collagen expression was strongly inhibited by treatment with the PDGFR-specific IFNγ constructs (Fig. 2C,D), whereas unmodified mouse IFNγ and IFNγ-PEG did not induce any effect in human cells due to species differences. These results clearly demonstrate that mouse IFNγ, which is inactive in human cells, can become biologically active in other species by directing it to the PDGFβR.

In Vivo Localization of HSC Targeted-IFNγ Constructs.

The tissue biodistribution of human serum albumin (HSA)-modified PPB has been previously extensively investigated by our group using radiolabeled and imaging studies showing high distribution of PPB-HSA to fibrotic livers and to PDGFβR-expressing activated hepatic stellate cells.17 To determine whether our PPB-modified IFNγ constructs specifically accumulate in HSC in vivo, IFNγ and IFNγ conjugates (5 μg/mouse) were administered to mice that had received a single intraperitoneal injection of CCl4 and their localization was analyzed after 10 minutes (Fig. 3A). Liver uptake and cellular distribution were determined by double staining for desmin (HSC marker) and peptide PPB. IFNγ-PPB and IFNγ-PEG-PPB largely colocalized with desmin-positive cells, whereas they were absent in nondamaged areas depicted by arrows (Fig. 3A). No costaining studies could be performed for exogenously administered IFNγ due to endogenous IFNγ.

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Figure 3. HSC-specific delivery IFNγ conjugates in the acute CCl4-induced liver injury model in mice. (A) Representative immunofluorescent images showing HSC-specific accumulation of IFNγ-PPB and IFNγ-PEG-PPB in the damaged areas in livers, whereas staining was absent in nondamaged areas as indicated by arrows (n = 6 per group). PPB was visualized with a specific antibody (red); HSC with antidesmin IgG (green) and nuclei by counterstaining with DAPI (blue). 400× magnification and further enlargement in the inserts. Representative photomicrographs (B) and quantitative image analysis (C) of MHC-II-stained liver sections of acutely CCl4-treated mice following treatment with IFNγ or IFNγ constructs. Scale bar = 200 μm. IFNγ-PEG-PPB conjugate induced significant up-regulation of MHC-II expression in livers relative to all other groups. (D) Staining for MHC-II (red), desmin (green) and nuclei (blue) showing enhanced expression of MHC-II in HSC-containing damaged areas. Original magnification, 200×. #P < 0.05 versus PBS-treated olive oil (normal) group; **P < 0.001 versus PBS-treated CCl4 animals. Bars represent mean ± SEM of six mice per group.

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We also assessed major histocompatibility class II (MHC-II) expression, which is known to be up-regulated by IFNγ,23 to assess the biological activity of the conjugates in livers. IFNγ-PEG-PPB treatment induced a remarkable up-regulation in MHC-II expression (P < 0.001) (Fig. 3B,C) within the damaged areas that were characterized by accumulation of activated HSC (Fig. 3D).

Effects of Targeted-IFNγ in Acute CCl4-Induced Liver Injury.

IFNγ, IFNγ-PEG, and targeted-IFNγ conjugates (IFNγ-PPB and IFNγ-PEG-PPB) were subsequently evaluated for their antifibrotic effects in the acute CCl4 liver-injury model. Only IFNγ-PEG-PPB conjugate significantly attenuated collagen I and alpha smooth muscle actin (α-SMA) expression (P < 0.05; Fig. 4A,B). Apart from collagen expression and deposition, the balance between collagen degrading matrix metalloproteinases-13 (MMP-13) and their major endogenous inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1), is also an important determinant of fibrosis progression. IFNγ-PEG-PPB induced a significant increase of the MMP-13/TIMP-1 transcript ratio (P < 0.01), suggesting fibrolytic activation (Fig. 4C). As the IFNγ-PEG-PPB construct was found to be the most effective, it was further investigated in an established CCl4-induced liver fibrosis model.

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Figure 4. Effects of targeted-IFNγ on fibrosis-related parameters in the acute CCl4-induced model in mice. Representative pictures (A) and quantitative analysis (B) of collagen I and α-SMA-stained liver sections from CCl4-treated mice (acute model) that received IFNγ or IFNγ constructs. Scale bars = 200 μm. For quantitative analysis, the groups were normalized to vehicle group (PBS treated-CCl4 mice). IFNγ-PEG-PPB induced a dramatic reduction in collagen I expression (*P < 0.05) and α-SMA expression (**P < 0.01). #P < 0.05 denotes significance versus PBS-treated olive oil (normal) group. (C) Effect of IFNγ constructs on fibrolytic activation as determined by the ratio of MMP13 and TIMP-1 transcripts. (#P < 0.05 compared to PBS-treated CCl4 animals). Bars represent mean ± SEM of six mice per group.

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Resolution of Liver Fibrosis by IFNγ-PEG-PPB in Advanced CCl4-Induced Fibrosis.

Mice received CCl4 for 8 weeks to induce advanced liver fibrosis/cirrhosis. During the last 2 weeks, six doses of IFNγ or IFNγ conjugate (2.5 μg/dose/mouse) or PBS were administered intravenously (Fig. 5A). Control CCl4 mice developed extensive bridging fibrosis, substantial deposition of collagen, and increased expression of the HSC markers α-SMA and desmin (Fig. 5B). ALT and AST levels were strongly up-regulated in all CCl4-treated animals. Treatment with IFNγ, IFNγ-PEG, or IFNγ-PEG-PPB induced a 20%-30% reduction in these levels (P < 0.05, Supporting Fig. 4). However, only treatment with IFNγ-PEG-PPB significantly inhibited bridging and reduced stainable collagen I by >70% (P < 0.001), accompanied by a substantial reduction in α-SMA and desmin-positive HSC and relative hydroxyproline content (Fig. 5B-D). These reductions were paralleled by a significant decrease in respective transcript levels (Fig. 5E). Untargeted IFNγ (IFNγ and IFNγ-PEG) only induced a moderate decrease in collagen expression and there was no significant decrease in the HSC activation markers α-SMA and desmin, indicating that only IFNγ-PEG-PPB specifically affected fibrogenic HSC activation and proliferation (Fig. 5B-E). The reduction in desmin-positive HSC was due to decreased proliferation but not due to killing of the cells, as confirmed by absence of apoptotic cells using caspase staining (data not shown), whereas significant reduction in proliferating nuclei (stained with Ki67 antibody) was observed with targeted IFNγ construct (Supporting Fig. 5). In addition, the HSC-targeted conjugate but not IFNγ and IFNγ-PEG significantly enhanced the MMP-13/TIMP-1 transcript ratio, implying activation of fibrolysis (Fig. 5E). Finally, the chemokine receptor CXCR4 and its ligand CXCL12/SDF1α, which were recently implicated in HSC activation,24 were significantly down-regulated by IFNγ-PEG-PPB (Fig. 5F), whereas IFNγ and IFNγ-PEG had no effect.

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Figure 5. Effects of targeted-IFNγ on fibrosis-related parameters in CCl4-treated cirrhotic mice. (A) Regimen of liver fibrosis induction. After 6 weeks of CCl4 injections, animals received IFNγ, IFNγ-PEG, IFNγ-PEG-PPB (2.5 μg/mice, 3 times a week), or PBS alone (n = 6 per group) intravenously for 2 weeks, while CCl4 injections were continued. (B) Representative photomicrographs and (C) computerized quantitative analysis of collagen I, α-SMA-, and desmin-stained liver sections of olive oil-treated animals (normal) and fibrotic animals treated with PBS, IFNγ, or modified IFNγ. Scale bars = 200 μm. For quantitative analysis, the groups were normalized to vehicle group (PBS treated-CCl4 mice). (D) Relative hepatic hydroxyproline content. (E) Quantitative real-time PCR analysis of procollagen α1(I), α-SMA, desmin, MMP13/TIMP-1, and (F) SDF1α and CXCR4 in olive oil-treated mice (normal) and CCl4 animals treated with PBS, IFNγ, IFNγ-PEG, IFNγ-PEG-PPB. Expression levels were normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Bars represent mean ± SEM of six mice per group. #P < 0.05 versus olive oil; *P < 0.05, **P < 0.01 versus PBS treated-CCl4 mice. These data show reduction in fibrotic parameters with targeted-IFNγ construct containing the PEG spacer and PDGFβ-receptor homing peptide PPB (IFNγ-PEG-PPB), while native IFNγ and IFNγ-PEG showed limited effectiveness.

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IFNγ-PEG-PPB Inhibits Angiogenesis and Liver Inflammation in Chronic CCl4-Induced Liver Fibrosis.

Angiogenesis that is induced by hypoxia within an injured liver and appears to aggravate hepatic fibrogenesis.25 Accordingly, using CD31 immunostaining, we noted significant neovascularization that was paralleled by an increased angiopoietin-1 and fibronectin expression26, 27 in livers of mice chronically treated with CCl4. All these parameters were ameliorated by IFNγ and IFNγ-PEG treatment, but most dramatically by IFNγ-PEG-PPB (Fig. 6A,B). Because PDGF receptor blockade can also lead to antiangiogenic effects we administered higher doses of PPB coupled to a nonbioactive protein (albumin) and did not observe any reduction in CD31 staining in a chronic CCl4 model (Supporting Fig. 6).

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Figure 6. Effect of targeted-IFNγ on angiogenesis and liver inflammation in CCl4 fibrotic mice. (A) Representative photomicrographs of sections stained for CD31, angiopoietin-1, and fibronectin from olive oil-treated mice (normal) and advanced fibrotic mice treated with IFNγ, IFNγ-PEG, IFNγ-PEG-PPB, or PBS alone (n = 6). Scale bars = 200 μm. (B) Quantitative real-time PCR analysis of CD31 and angiopoietin1 messenger RNA (mRNA) expression, normalized with GAPDH. #P < 0.05 versus olive oil; *P < 0.05, **P < 0.01 versus PBS treated-CCl4 mice. Results show significant inhibition of angiogenesis by IFNγ-PEG-PPB. (C) Representative photomicrographs showing liver sections stained for the macrophage marker F4/80. The pictures show effective inhibition of liver inflammation with HSC-specific targeting of IFNγ. (D) Transcripts levels for CD68, F4/80, and MIP-2. Significant reduction of CD68, F4/80, and MIP-2 transcripts with IFNγ-PEG-PPB was observed. Bars represent mean ± SEM of six mice per group. #P < 0.05 versus PBS-treated olive oil group; *P < 0.05, **P < 0.01 versus PBS treated-CCl4 group.

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Liver fibrosis is also an inflammation-driven process28 and HSC can modulate the recruitment of inflammatory cells during fibrogenesis.5 Compared to PBS, IFNγ, and IFNγ-PEG, IFNγ-PEG-PPB showed a significant decrease in MIP2 (macrophage inflammatory protein 2) expression and lower numbers of macrophages as evidenced by staining for F4/80, and CD68 and F4/80 RNA transcript levels (Fig. 6C,D). Additional effects on other inflammatory cells (neutrophils, CD4, CD8, and dendritic cells) were investigated but no significant differences were observed (Supporting Fig. 7).

Reduction in IFNγ-Related Side Effects with IFNγ-PEG-PPB Conjugate.

The main hurdles in IFNγ-based therapies are the adverse effects due to the proinflammatory activity of IFNγ, one reason for its failures in clinical trials. To investigate whether targeting of IFNγ could ameliorate these IFNγ-mediated side effects, we focused on clinically relevant side effects such as fever, elevated plasma triglycerides, endothelial cell activation, proinflammatory cytokine release, and central nervous system (CNS) effects.29-31 Although IFNγ-PEG treatment induced a significant rise in body temperature, endothelial cell activation (eNOS), plasma TNF-α, and IL-6 levels (Fig. 7A-D), these side effects were completely absent in animals treated with HSC-targeted IFNγ-PEG-PPB (Fig. 7A-D). We also observed increased plasma triglycerides induced by IFNγ, which remained unaltered after IFNγ-PEG-PPB treatment (Fig. 7E). Neurotropic effects were analyzed by examining the expression of MHC-II, the norepinephrine transporter (NET), and indoleamine-pyrrole 2, 3-dioxygenase-I (IDO-I) in the brain. Although IFNγ or IFNγ-PEG induced significant up-regulation of MHC-II, NET, and IDO-I expression, these parameters were unchanged in the IFNγ-PEG-PPB treated animals (Fig. 7F and Supporting Fig. 8).

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Figure 7. Effect of targeted-IFNγ on IFNγ-related side effects in CCl4-fibrotic mice. Analysis of adverse effects after 2 weeks of treatment with PBS, IFNγ, IFNγ-PEG, or IFNγ-PEG-PPB (2.5 μg/mouse). (A) Rectal temperature of CCl4-treated cirrhotic mice receiving PBS or the different IFNγ constructs. #P < 0.05 versus PBS-treated CCl4 group, *P < 0.05 versus IFNγ-PEG-treated group. (B,C) Plasma levels of TNF-α and IL-6 in IFNγ- and IFNγ-construct-treated cirrhotic mice. #P < 0.05 versus PBS-treated CCl4group. (D) mRNA expression of eNOS in the livers from treated animals as analyzed by qRT-PCR (normalized with GAPDH). #P < 0.05 versus PBS-treated CCl4 group; *P < 0.05 versus IFNγ-PEG-treated group. (E) Triglyceride levels in plasma of CCl4-fibrotic mice treated with different compounds. #P < 0.05 represents significance versus PBS-treated CCl4 group; *P < 0.05 versus IFNγ treated group. (F) MHC-II mRNA expression in brain tissue from PBS, IFNγ, and IFNγ constructs-treated cirrhotic mice as determined by qPCR. #P < 0.05 versus PBS-treated CCl4 group; *P < 0.05 versus IFNγ-PEG-treated group and **P < 0.01 versus IFNγ-treated group. Bars represent mean ± SEM of n = 6 mice per group. These data show that IFNγ and IFNγ-PEG induce clinically relevant adverse effects but IFNγ-PEG delivered through the homing peptide PPB is devoid of these side effects.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Liver cirrhosis is a slowly progressive process tightly controlled by endogenous mediators. Although certain local mediators might provide novel therapeutic opportunities, their systemic use is fraught with tremendous hurdles such as insufficient access to the fibrotic liver and adverse reactions, as is particularly true for IFNγ. To date, no proven antifibrotic pharmacotherapy is available to inhibit the progression or induce regression of human liver fibrosis and cirrhosis. Our strategic approach was therefore to chemically engineer the IFNγ molecule to redirect it to the fibrogenic target cells, whereas avoiding undesired off-target effects. We could show that specific targeting of IFNγ to activated HSC, key effector cells of liver fibrogenesis, increased its therapeutic efficacy but strikingly reduced unwanted side effects by avoiding its interaction with nontarget cells in the liver and other tissues. We used the PDGFβ receptor as the target receptor for delivery of IFNγ due to its specific and high induction on activated HSC during liver injury.15, 16 To date, such an approach has not been described.

Due to their potency, cytokines have been the focus of several new biological therapeutics.32 However, only very few cytokines have made their way to the clinic, mainly due to their short half-life and adverse side effects. Their plasma stability and circulation life-span can be increased through PEGylation (e.g., PEGASYS and PEGIntron), liposomal encapsulation, or coupling to carriers.33, 34 However, although these approaches improved the biological's half-life, they still fail to prevent their interaction with nontarget cells and concomitant side effects. Here we demonstrate for the first time that redirecting a cytokine from its ubiquitously expressed receptor to another target cell-specific receptor (PDGFβR) can both lead to enhanced therapeutic efficiency and reduced side effects.

PDGFβR is highly induced on activated HSC (and portal myofibroblasts) in rodent and human liver fibrogenesis but it is also expressed to a minor extent on vascular smooth muscle cells. Keeping in mind the hurdles of pharmacokinetics and in vivo instability, we designed different strategies to conjugate the cyclic PDGFβR-binding peptide PPB to mouse IFNγ using either direct coupling (IFNγ-PPB) or via a PEG linker (IFNγ-PEG-PPB), in order to provide hydrophilicity, stability, and conformational flexibility for appropriate receptor interaction.

Modification of cytokines is always a challenging task, as this can result in a complete or partial loss of biological activity.35 However, using mild conditions during synthesis, PPB-modified IFNγ constructs completely retained their biological activity as analyzed in mouse RAW macrophages. PDGFβR-specific binding was subsequently demonstrated in mouse fibroblasts, primary rat HSC, and human hepatic stellate cell line LX2 using specific antibodies. Moreover, whereas mouse-derived IFNγ was only effective in murine cells, PPB containing IFNγ constructs were also effective in human cells, further demonstrating that the biological activity of IFNγ-PEG-PPB is mediated through PDGFβ receptor.

It is known that IFNγ has a receptor binding sequence and a nuclear signaling sequence (NLS) that mediates its biological effects.36 IFNγ-mediated effects occur through intracellular uptake of the nuclear signaling sequence which subsequently binds to its intracellular target through the JAK-STAT pathway and modulates IFNγ-responsive genes. Although our construct is taken up via the PDGFR, the internalized construct is apparently able to bind to its intracellular target. Most likely, the internalized construct releases IFNγ or its metabolite intracellularly, but this needs to be further explored. Of note, to test whether the antifibrotic and antiangiogenic effects were not due to a blockade of the PDGFβR induced by PPB, we coupled PPB to human serum albumin (PPB-HSA) and no significant effects were observed in vitro or in vivo.

Our targeted-IFNγ constructs quite rapidly accumulated in HSC and HSC-specific accumulation was confirmed also by local up-regulation of MHC-II expression, a well-known activity of IFNγ.23 In these experiments we found that IFNγ-PEG-PPB was more potent than IFNγ-PPB, illustrating the relevance of the PEG linker. Furthermore, improved antifibrotic effect of targeted-IFNγ constructs such as reduction of α-SMA and collagen I expression and significant increase of the MMP-13/TIMP-1 ratio demonstrated the rationale and efficiency of cell-specific targeting. In all experimental settings, IFNγ-PEG-PPB induced the most prominent antifibrotic effects.

Advanced liver fibrosis/cirrhosis develops during many years in patients with chronic liver disease and to induce regression of fibrosis represents a major challenge. In our study we induced advanced liver cirrhosis in mice by chronic treatment with CCl4 for 6 weeks and then examined the therapeutic efficacy of the targeting construct for another 2 weeks, while continuing the CCl4 injections mimicking the ongoing liver damage that occurs in humans. IFNγ-PEG-PPB treatment strikingly diminished fibrosis by a strong inhibition of HSC activation and proliferation, a significant reduction in collagen deposition, and an increase in the MMP-13/TIMP-1 ratio, whereas untargeted IFNγ was only modestly effective (nonsignificant) due to the relatively low dose used in the present study (2.5 μg/dose/mice) compared to other studies (5 μg/dose/mice).37

Recent reports highlight the relevance of angiogenesis in the pathogenesis of liver fibrosis,25-27 whereas the role of chronic inflammation was recognized earlier.28 The significant inhibition of these processes by the targeted construct and not by native IFNγ further highlights the potency of our targeting approach.

Adverse effects have been the main reason for patient withdrawal from clinical trials with IFNγ. IFNγ is a very potent proinflammatory cytokine with a ubiquitous receptor expression, and therefore IFNγ-based experimental therapies are associated with side effects like severe flu-like symptoms, systemic endothelial and immune cell activation, neurotropic effects, and hyperlipidemia. In our study we observed that both free IFNγ and PEGylated IFNγ induced hyperthermia, elevated plasma triglyceride levels, endothelial cell activation, serum TNF-α and IL-6 levels, and CNS inflammation. Notably, these adverse reactions were completely absent following IFNγ-PEG-PPB administration.

In conclusion, we report here a novel approach of chemically engineering a cytokine to shift its receptor specificity by directing it to the myofibroblast-like cells (activated HSC) in liver. Using this approach, targeting of IFNγ ameliorated advanced liver fibrosis and eliminated the IFNγ-related adverse effects. We believe that this illustrates new opportunities to utilize cytokines more effectively for therapy of hepatic fibrosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank C. Reker-Smit, A. de Jager-Krikken, and M. de Ruiter for their technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

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HEP_24395_sm_suppinfo.doc39007KSupporting Information.

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