Targeting of macrophages and hepatic stellate cells for the treatment of liver diseases
The PhD defence of Dhadhang Kurniawan will take place (partly) online and can be followed by a live stream.
Dhadhang Kurniawan is a PhD student in the research group Advanced Organ bioengineering and Therapeutics (AOT). Supervisors are prof.dr. J. Prakash and prof.dr. G. Storm, co-supervisor is dr. R. Bansal, all from the Faculty of Science & Technology (S&T).
Liver fibrosis and its progression to liver cirrhosis and hepatocellular carcinoma (HCC) is a growing health problem affecting millions of people worldwide. This growing global health burden is attributed to hepatitis viral infections (viral hepatitis), metabolic disorders such as obesity and diabetes (non-alcoholic fatty liver disease, NAFLD) and alcohol abuse (alcohol-associated liver disease, ALD), among others. Upon persistent liver injury, chronic inflammation ensues that develops into fibrosis, that further develops into cirrhosis (end-stage liver failure) and/or hepatocellular carcinoma (HCC).
Following hepatocellular damage, liver inflammation is triggered by the resident immune cells followed by activation and infiltration of immune cells, mainly monocytes/macrophages, that lead to chronic liver inflammation. Liver inflammation instigates proliferation, activation and trans-differentiation of quiescent hepatic stellate cells (HSCs) to myofibroblasts that secretes excessive amounts of extracellular matrix proteins, mainly collagen, resulting in tissue stiffness and distortion of liver architecture referred to liver fibrosis that further develops into liver cirrhosis (liver dysfunction) and cancer development.
Despite the increasing prevalence of such liver diseases, unfortunately no clinically approved drugs are available for their treatment. Removal of the underlying cause e.g. by alcohol abstinence, lifestyle modifications i.e. healthy diet and exercise (weight loss), and regular monitoring of liver function is recommended for patients with acute/early liver disease (liver fibrosis). Liver transplantation is the only option available in case of end-stage liver failure (liver cirrhosis). Due to the lack of sufficient liver donors, lifelong immunosuppressive drugs, high costs and other associated problems, liver transplantation is used for only a minority of patients. Therefore, there is an urgent need to develop a drug-based treatment for fatty liver and fibrotic liver diseases.
Macrophages and hepatic stellate cells (HSCs) are known to play a crucial role in the development of liver diseases, both during acute and chronic liver injury. Therefore, in this thesis, we developed and investigated novel therapeutic approaches targeting macrophages and HSCs. As summarized in chapter 1, we investigated the utility of novel compounds and nanoparticle-based approaches to achieve this goal.
In an initial preliminary study, we used a profiler array tool, applied to murine RAW 264.7 cells. Unstimulated M0 macrophages, lipopolysaccharide (LPS)/interferon gamma (IFNγ)-stimulated pro-inflammatory M1 macrophages and interleukin (IL)-4/IL-13-stimulated pro-resolving M2 macrophages were analyzed for differential gene expression. In the profiler array, we found that spleen tyrosine kinase (SYK) was highly upregulated in M1 macrophages, among other genes that were upregulated in M1 macrophages (17 in total). This phenomenon is also highlighted in our literature review (chapter 2), in which we describe and discuss the role of the SYK signaling pathway in liver diseases. This review points out that SYK is highly expressed in liver diseases, and that SYK expression and activation of its signaling pathway correlates with the disease severity in different etiological liver diseases including viral hepatitis (B and C), NASH, ALD, liver cirrhosis and HCC. SYK is known to be widely expressed in hepatocytes, hepatic macrophages and activated HSCs. We further provide an overview of different SYK inhibitors that have/can be explored for the treatment of liver diseases. We have also highlighted the limitations of using SYK inhibitors e.g., poor pharmacokinetics (small molecule inhibitors) and adverse effects due to involvement of SYK in normal cellular functions, and future applications of targeting SYK inhibitors using nanoparticles.
In chapter 3, motivated with the results obtained from the profiler array, we explored the effects of a small molecule SYK inhibitor, R406 (without and with nanoparticles) in vitro and in vivo. As shown previously by others, we found a positive correlation of SYK expression with the pathogenesis of NASH and alcoholic hepatitis in patients. But not shown previously, we observed that SYK expression was particularly induced in M1-differentiated pro-inflammatory macrophages. Following this observation, we assessed the implication of the SYK pathway in macrophages and found that inhibition of the SYK pathway using R406 resulted in the inhibition of inflammation markers i.e., nitric oxide (NO) release, and enhanced gene expression of IL-1β, FcγR1, iNOS, CCL2, IL-6, and CCR2, in a dose-dependent manner.
Based on the significant inhibition of the SYK pathway by R406 in M1 (pro-inflammatory) macrophages and to overcome the limitations as mentioned above, we encapsulated this drug into poly lactic-co-glycolic acid (PLGA) nanoparticles. PLGA was chosen considering its biodegradability, biocompatibility as well as the fact that it is a FDA approved polymer. After extensive characterization of the R406-PLGA nanoparticles, we examined the efficacy of R406-PLGA nanoparticles in in vitro cultured RAW macrophages and BMDMs polarized towards the M1 phenotype. We found that both R406-PLGA nanoparticles as well as R406 itself could significantly inhibit the gene expression of several inflammatory markers in vitro. In a subsequent in vivo study, using the MCD-diet NASH mouse model, we found that R406-PLGA nanoparticles significantly ameliorated liver inflammation, fibrosis, and steatosis as compared to free R406. We concluded that R406-PLGA nanoparticles could be a promising approach for the treatment of NASH. However, further studies in mouse models i.e. western diet models and/or the Stelic Animal Model (STAM) model, that more closely mimic the clinical NASH situation, are needed.
Since SYK pathway is closely associated with SRC kinase pathway, we then explored the involvement and role of SRC kinase pathway in liver diseases in chapter 4. We confirmed that the Src gene was highly upregulated in human liver tissues obtained from patients with NASH, alcoholic hepatitis, cirrhosis, and biliary atresia. We also observed that the Src gene was highly upregulated in RAW 264.7 macrophages, murine BMDMs, and human MDMs (marrow-derived macrophages), when polarized to their respective M1 phenotype. For a further investigation of the role of the SRC kinase pathway and potential therapeutic benefit of inhibition of this pathway, we used the selective small molecule SRC kinase inhibitor, KX2-391.
KX2-391 inhibited the phosphorylation of SRC, SYK and significantly reduced the NO release in RAW macrophages. Moreover, gene expression analysis in RAW macrophages and BMDMs evidenced that SRC pathway inhibition mediated by KX2-391 attenuated the expression of different inflammatory markers including iNOS, CCL2, and FcγR1. Additionally, in a precision-cut liver slices (PCLS) model, KX2-391 decreased the gene expression of SRC, iNOS and CCL2 without affecting the cell/tissue viability. Moreover, KX2-391 attenuated hepatocytic lipid accumulation; TGFβ-induced HSCs activation, contractility and collagen expression.
Next, we investigated the relevance of the SRC pathway in NASH and ASH using a MCD-diet NASH mouse model and a Lieber-De Carli ALD mouse model, respectively. The results indicated that KX2-391 attenuated inflammation, fibrosis, and steatosis in both NASH and ASH mouse models. Mechanistic studies further revealed that SRC mediated the effects through the FAK/PI3K/AKT pathway.
Accordingly, we proposed that inhibiting the SRC kinase pathway using inhibitors such as KX2-391 represents a potential treatment for liver diseases like NASH and ALD. The SRC kinase inhibitor KX2-391 is also very promising to be studied as a potential therapy for other chronic diseases including cancer.
In chapter 5, we focused on targeting of HSCs to inhibit fibrosis. Since the fibroblast growth factor receptor 1 (FGFR1) is highly overexpressed on activated HSCs, we decided to use basic FGF (FGF2) to inhibit HSCs activation. We observed that FGF2 was able to inhibit TGFβ-induced HSCs activation (confirmed by reduced collagen-I and α-SMA expression), migration of HSCs (confirmed by wound healing assays) and contraction of HSCs (confirmed by 3D-gel contraction assays). These results laid the foundation for our further studies in which we conjugated FGF2 to superparamagnetic iron oxide nanoparticles (FGF2-SPIONs) to improve the stability and half-life of FGF2 and thereby to improve the therapeutic efficacy of FGF2 for the treatment of liver fibrosis. In addition to enhancing therapeutic efficacy, conjugation of FGF2 with SPIONs is also expected to be applied for diagnostic-related purposes due to its paramagnetic properties, providing theranostic potential. After performing several in vitro and in vivo experiments, the results revealed that the potency of FGF2-SPIONs conjugate is significantly improved as compared to free FGF2. Besides improved therapeutic efficacy, we also concluded that the conjugation of FGF2 with SPIONs may provide a personalized theranostic approach with combined therapy and diagnosis for personalized disease management. However, more studies are required to validate our findings and theranostic potential of FGF2-SPIONs in more advanced and representative liver fibrosis animal models.
In conclusion, the approaches described in this thesis are promising to be explored further for the treatment of liver diseases like NASH or ALD-based liver fibrosis. With nanotechnological approaches such as polymeric nanoparticles and SPIONs, the efficacy of active compounds with poor pharmacokinetic profile can be significantly increased. In addition to increasing efficacy, nanoparticulate delivery systems can also reduce the adverse effects of these active compounds. Since, the majority of administered nanoparticles will accumulate and be sequestered in the liver after intravenous administration into the body, this thesis work confirms our initial hypothesis that the design of nano-delivery systems can effectively improve drug delivery to the liver and therefore have great potential to ameliorate the therapy of liver diseases as presented in this thesis.
