STAT1/3 Signaling COntributes To The Effects Of Interleukin 10/22 And NKT Cells On Liver Regeneration After Partial Hepatectomy

The liver is the only solid organ in mammals with remarkable regenerative capabilities. The differentiated hepatocytes that are normally quiescent can reenter the cell cycle in response to tissue loss or injury and divide until the original liver mass is restored. Two-thirds partial hepatectomy (PHx) represents the most commonly used model for the study of liver regeneration. Liver regeneration involves a sequence of signaling events with highly synchronized cell-cycle profile to restore liver mass and function. After 2/3 PHx, hepatocytes are the first type of liver cells to start proliferating and undergo 1–2 rounds of cell division with DNA synthesis starting within 24 hours and proliferation peaks (S phase) at around 36-42 hours after surgery. Restoration of liver mass is nearly complete by 7-10 days in rodents and by 3-4 months in humans.Several lines of evidence suggest that liver regeneration is controlled by a wide variety of cytokines, growth factors, hormones, and their downstream signaling pathways. It is well established that after PHx, the ability of the remnant liver to detoxify endotoxin (LPS) decreases, leading to elevation of hepatic LPS levels. LPS stimulates Kupffer cells to produce inflammatory cytokines including tumor necrosis factorα(TNF-α) and interleukin 6 (IL-6) that subsequently initiate liver regeneration. This priming phase usually takes place shortly after PHx with a transient increase in low inflammation. The proinflammatory cytokine TNF-αand its downstream signaling molecule NF-κB have been shown to play a particularly important role in the earliest step of priming of hepatocytes and stimulation of hepatocyte proliferation via induction of IL-6 during liver regeneration. The action of IL-6 is mediated via binding the IL-6 receptor complex (gp80/gp130) on hepatocytes, followed by activation of STAT3 and promoting hepatocyte survival and proliferation. The findings from these literatures suggest that the inflammatory response and cytokine production are particularly active during the first 24h after PHx, a time period when hepatocyte transition from a quiescent state into the cell cycle occurs. However, how these inflammatory responses are controlled during liver regeneration remains largely unknown. In the current dissertation, we firstly identified IL-10, a potent anti-inflammatory cytokine, as an important regulator to suppress liver inflammation and regeneration post PHx via regulating production of pro-inflammatory cytokines and subsequently suppressing STAT3 activation in the liver. In addition, another IL-10 family cytokine, interleukin 22 (IL-22), also promotes liver regeneration after PHx when it was overexpressed in the liver. Studies from our lab and other groups showed that IL-6/STAT3 in hepatocytes promote their survival and proliferation during liver regeneration after PHx. In contrast, IFN-γ/STAT1 axis plays an inhibitory role in liver regeneration. Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer (NK) cells. Liver lymphocytes are also enriched in NKT cells, accounting for 20–35% of mouse liver lymphocytes and 10–15% of rat and human liver lymphocytes. NKT cells have been shown to play an important role in regulating innate and adaptive immunity via production of a variety of cytokines, including high amount of IFN-γ. Thus, activation of NKT cells likely contributes to inhibit liver regeneration via production of IFN-γ. However, the exact role of activated NKT cells (typeΙor type II NKT cells) on liver regeneration is still controversial and largely unclear. In the third part of this study, we showed that activated type I natural killer T cells inhibit hepatocyte proliferation and liver regeneration after PHx via IFN-γ/STAT1 -mediated pathway, whereas type II NKT cells may only play a minor role in suppression of liver regeneration.AIMS1. To investigate the effects of IL-10 on the liver regeneration after PHx.2. To investigate the effects of IL-22 on the liver regeneration after PHx.3. To investigate the effects of activated NKT cells on the liver regeneration after PHx.METHODS1. Several strains of transgenic and gene knockout mice were used.2. Hepatocyte proliferation was measured by the Brdu incorporation.3. The levels of proinflammatory cytokines such as TNF-α, IFN-γ, MCP-1, IL-6 and IL-12 following PHx were measured using cytometry bead array.4. The mRNA levels of cytokines were evaluated with real-time PCR.5. The expression of STAT1/3 siganling pathway was detected by Western blot analysis.6. Immunohistochemistry and fluorescence-activated cell sorting (FACS) were used to evaluate the infiltration of liver inflammatory cells.RESULTS1. Enhanced liver regeneration in IL-10-/- mice after PHx via stimulating inflammatory response and activating hepatocyte STAT3After PHx, expression of IL-10 mRNA in the liver and spleen was significantly elevated. Such elevation was diminished in TLR4 mutant mice. Compared with wild-type mice, IL-10-/- mice had higher levels of expression of proinflammatory cytokines (IL-6, TNF-α, and IFN-γ) and inflammatory markers (CCR2 and F4/80) in the liver as well as higher serum levels of proinflammatory cytokines post PHx. The number of neutrophils and macrophages were also higher in the livers of IL-10-/- mice than those in wild-type mice after PHx. Liver regeneration as determined by BrdU incorporation post PHx was higher in IL-10-/- mice than that in wild-type mice, which was associated with higher levels of activation of IL-6 downstream signal STAT3 in the liver. An additional deletion of STAT3 in hepatocytes significantly reduced liver regeneration in IL-10-/- mice after PHx.2. IL-22 transgenic (IL-22TG) mice have accelerated while IL-22 knock out (IL-22-/-) mice have comparable liver regeneration after PHx compared with wild-type miceAfter PHx, liver regeneration was comparable between wild-type mice and IL-22-/-mice. Next, we designed experiments to determine if overexpression of IL-22 expression affects the liver regeneration induced by PHx. The liver/body weight ratio was similar before PHX (0) but was significantly higher in IL-22 TG mice than in WT mice 32h, 48h and 72h post PHx, and returned to the comparable levels 96h after PHx in both groups. BrdU+ incorporation experiments demonstrated that IL-22TG mice had an accelerated peak of hepatocyte proliferation post PHx. In order to define a mechanism responsible for this enhanced liver regeneration capacity in the IL-22TG mice, western blot analyses for STAT3 and STAT1 activation were performed. Our results revealed that the increased levels of cyclin D1 may contribute to the accelerated liver regeneration in IL-22TG mice after PHx.3. Activated type I natural killer T cells inhibit liver regeneration after PHx via IFN-γ/STAT1 -mediated pathway, whereas activated type II NKT cells suppress liver regeneration to a lesser extent.Activation of typeⅠandⅡNKT cells by injection ofα-GalCer and Sulfatide, respectively, significantly inhibit liver regeneration after PHx. The inhibition was most pronounced in the mice withα-GalCer injection right after surgery or 3 days before surgery. Injection of sulfatide also inhibited, to a lesser extent, liver regeneration after PHx. Furthermore,α-GalCer injection resulted in elevated activation of IFN-γ/STAT1 signaling pathway after PHx. Depletion of either the IFN-γgene or STAT1 gene abolishes the negative effects of activated NKT cells on liver regeneration. CONCLUSIONS1. IL-10 plays a negatively regulating role in liver regeneration via limiting inflammatory response and subsequently tempering hepatic STAT3 activation.2. Overexpression of IL-22 in the liver promotes liver regeneration by stimulating hepatic STAT3.3. Activation of natural killer T cells inhibits liver regeneration after PHx via an IFN-γ/STAT1 signaling pathway-dependent manner...