MCP-1 dependent balance of inflammatory pathways and interplay of immune cells in the liver during injury, fibrosis and injury regression

  • Von der Entzündung über die Fibrose bis hin zur Wundheilung : unterschiedliche MCP-1-abhängige Entzündungsmechanismen und Interaktionen von Immunzellen in der Leber

Bäck, Christer Matthias; Conrath, Uwe (Thesis advisor); Tacke, Frank (Thesis advisor)

Aachen : Publikationsserver der RWTH Aachen University (2015)
Dissertation / PhD Thesis

Aachen, Techn. Hochsch., Diss., 2015

Abstract

Multiple potentially harmful stimuli challenge the liver, the chief metabolic and detoxifying organ of the human body. Due to its central anatomical location, continuous blood flow from the gastrointestinal tract through the hepatic sinusoids allows the metabolically active hepatocytes, the non-parenchymal cells and the various immune cell populations residing and patrolling in the liver to interact with antigens and microbiological components coming from the intestine. Cytokines are key mediators within the complex interplay of intrahepatic immune cells and hepatocytes, as they can activate effector functions of immune cells as well as hepatocytic intracellular signaling pathways controlling cellular homeostasis. Kupffer cells and liver-infiltrating monocyte-derived macrophages (iMϕ) are controlled by cytokines via cytokine-receptors and are primary sources of cytokines such as tumor necrosis factor (TNF). TNF can activate specific intracellular pathways in hepatocytes that influence cell fate in different manners, e.g. pro-apoptotic signals via the caspase cascade, but also survival pathways, namely the nuclear factor (NF)-kappaB pathway. One important cytokine-receptor on the surface of iMϕ responsible for chemotaxis and activation is the C-C motif chemokine receptor 2 (CCR2). Monocyte chemoattractant protein-1 (MCP-1, CCL2), the primary ligand of CCR2, is increased in livers patients with non-alcoholic steatohepatitis (NASH) and in murine models of steatohepatitis and fibrosis. It was recently shown that monocyte / macrophage infiltration into the liver upon injury is critically regulated by CCR2 and functionally important for perpetuating hepatic inflammation and fibrogenesis. In this study, I investigated pharmacological inhibition of MCP-1 by the L-enantiomeric RNA oligonucleotide mNOX-E36, a so-called ‘Spiegelmer’ that binds murine MCP-1 / CCL2 with high affinity and neutralizes its action, in two murine models of chronic liver diseases in vivo. Antagonizing MCP-1 by mNOX-E36 efficiently inhibited murine monocyte chemotaxis in vitro. Upon carbon tetrachloride (CCl4)- or methionine-choline-deficient (MCD) diet-induced hepatic injury in vivo, the infiltration of macrophages into the liver was significantly decreased in anti-MCP-1 treated mice (by FACS and immunohistological staining). In line with the lower level of intrahepatic macrophages, pro-inflammatory cytokines (TNFα, IFNγ, IL-6) were significantly reduced intrahepatically. In MCD diet-challenged mice a lower level of fatty liver degeneration was detected (oil red O staining, intrahepatic triglyceride measurement) in mNOX-E36-treated animals.Moreover, there is also increasing evidence that distinct monocyte and macrophage subsets exert critical functions in regression from organ fibrosis as well. Experimental mouse models of fibrosis regression have identified ‘restorative’ macrophages as Ly-6C (Gr1) low expressing, monocyte-derived cells. I aimed at elucidating molecular pathways balancing pro-inflammatory and restorative macrophages during fibrosis regression as well as pharmacologically augment beneficial macrophage functionality in fibrosis resolution. I therefore employed the above mentioned Spiegelmer-based inhibitor of the chemokine MCP-1 / CCL2 mNOX-E36 in the regression phase of the two murine models of toxic (CCl4) and metabolic (MCD diet) liver fibrosis. Although inflammation rapidly declined after cessation of injury, I observed a transient influx of Ly-6C+ infiltrating monocytes (iMΦ), which was blocked by mNOX-E36. By inhibiting the early influx of Ly-6C+ iMΦ via the CCL2-inhibitor mNOX-E36, the intra-hepatic macrophage equilibration shifted towards the ‘restorative’ Ly-6C- subset of iMΦ. Consequently, fibrosis resolution was significantly accelerated upon mNOX-E36 administration in both models. Blocking transient recruitment of infiltrating Ly-6C+ monocytes, but not direct effects of the inhibitor on the remaining macrophages, resulted in reduced pro-inflammatory cytokines like tumor necrosis factor-alpha (TNFα) in the liver. In conclusion, I demonstrate the successful pharmacological intervention using the CCL2-inhibitor mNOX-E36 in two independent experimental models of fibrosis and fibrosis-regression from toxic and metabolic liver injury.