Abstract
Hepatic stellate cells (HSCs), following transdifferentiation to myofibroblasts plays a key role in liver fibrosis. Therefore, attempts to attenuate this myofibroblastic phenotype would be a promising therapeutic approach. Interferon gamma (IFNγ) is a potent anti-fibrotic cytokine, but its pleiotropic receptor expression leading to severe adverse effects has limited its clinical application. Since, activated HSC express high-level of platelet derived growth factor beta receptor (PDGFβR), we investigated the potential of PDGFβR-specific targeting of IFNγ and its signaling peptide that lacks IFNγR binding site (mimetic IFNγ or mimIFNγ) in liver fibrosis. We prepared DNA constructs expressing IFNγ, mimIFNγ or BiPPB (PDGFβR-specific bicyclic peptide)-IFNγ, BiPPB-mimIFNγ fusion proteins. Both chimeric proteins alongwith IFNγ and mimIFNγ were produced in E.coli. The expressed proteins were purified and analyzed for PDGFβR-specific binding and in vitro effects. Subsequently, these recombinant proteins were investigated for the liver uptake (pSTAT1α signaling pathway), for anti-fibrotic effects and adverse effects (platelet counts) in CCl4-induced liver fibrogenesis in mice. The purified HSC-targeted IFNγ and mimIFNγ fusion proteins showed PDGFβR-specific binding and significantly reduced TGFβ-induced collagen-I expression in human HSC (LX2 cells), while mouse IFNγ and mimIFNγ did not show any effect. Conversely, mouse IFNγ and BiPPB-IFNγ induced activation and dose-dependent nitric oxide release in mouse macrophages (express IFNγR while lack PDGFβR), which was not observed with mimIFNγ and BiPPB-mimIFNγ, due to the lack of IFNγR binding sites. In vivo, targeted BiPPB-IFNγ and BiPPB-mimIFNγ significantly activated intrahepatic IFNγ-signaling pathway compared to IFNγ and mimIFNγ suggesting increased liver accumulation. Furthermore, the targeted fusion proteins ameliorated liver fibrogenesis in mice by significantly reducing collagen and α-SMA expression and potentiating collagen degradation. IFNγ also induced reduction in fibrogenesis but showed significant decrease in platelet counts, which was restored with targeted proteins. These results suggest that these rationally designed proteins can be further developed as novel anti-fibrotic therapeutics.
Highlights
IFNc is a pleiotropic homodimeric Th1 cytokine mainly produced by activated inflammatory cells and has been documented to be highly effective in viral, immunological and malignant diseases [1,2]
Others and we have shown that activated Hepatic stellate cells (HSCs) abundantly express the platelet derived growth factor receptor (PDGFbR) during liver fibrosis, while its expression is relatively weak on other cells and normal tissues [20,21,22]
Hepatocellular damage, hepatic inflammatory cell infiltration and extensive tissue remodeling culminate into the development of progressive fibrosis and cirrhosis [31]
Summary
IFNc is a pleiotropic homodimeric Th1 cytokine mainly produced by activated inflammatory cells and has been documented to be highly effective in viral, immunological and malignant diseases [1,2]. Many attempts have been made to prolong the half-life of IFNc by PEGylation or by increasing its activity through slow release by incorporation in nanoparticles, elastomers, microspheres or liposomes [9,10,11]. These approaches have shown to be beneficial, but adverse effects due to the longer exposure of IFNc to non-target tissues can still be detrimental. Targeted approaches leading to an increased therapeutic efficacy without eliciting adverse effects would be ideal to treat slowly progressing chronic diseases [12]
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