Distinct role of interleukin-6 and tumor necrosis factor receptor-1 in oval cell- mediated liver regeneration and inflammation-associated hepatocarcinogenesis

Interleukin 6 (IL6), tumor necrosis factor α (TNFα) and TNF receptor-1(TNFR1) have been shown to involve in oval cell proliferation and hepatocellular carcinoma (HCC) development. However, their role in these processes is still unclear. In the present study, by using hepatocytes-specific DDB1 deletion mouse models, we explored the role and mechanism of IL6, TNFα and TNFR1 in oval cell proliferation and HCC development in the context of inflammation, which is the common features of HCC pathogenesis in humans. Our results showed that IL6 promotes oval cell proliferation and liver regeneration, while TNFα/TNFR1 does not affect this process. Deletion of IL6 accelerates HCC development and increases tumor burden. The number of natural killer(NK) cells is significantly decreased in tumors without IL6, implying that IL6 suppresses HCC by NK cells. In contrast to IL6, TNFR1-mediated signaling pathway promotes HCC development, and deletion of TNFR1 reduced tumor incidence. Increased apoptosis, compensatory proliferation and activation of MAPK/MEK/ERK cascade contribute to the oncogenic function of TNFR1-mediated signaling pathway. Intriguingly, deletion of TNFα accelerates tumor development, which shows divergent roles of TNFα and TNFR1 in hepatocarcinogenesis.


INTRODUCTION
Cytokines modulate various cell types in the liver. Among them, interleukine-6 (IL6) and tumor necrosis factor α (TNFα), which are produced mainly by macrophage, may be the most critical ones [1]. They are implicated in both liver physiological and pathological processes, including regeneration and hepatocellular carcinoma (HCC) [2,3].
Under liver pathological conditions, liver progenitor cells or oval cells, which have bi-potential capacity to differentiate into hepatocytes and cholangiocytes, are activated to restore liver mass [4]. TNFα and IL6 are suggested to promote oval cell proliferation in this process [5,6]. However, due to incapable of distinguishing newborn hepatocytes from pre-exist ones, liver regeneration could not be fully assessed in these chemical injury models [7]. Furthermore, recent studies found that oval cells play minor if any contribution to liver regeneration in conventional chemicals injury models, including thionine-supplemented(CDE) and 3-diethoxycarbonyl-1,4-dihydrocollidine (DDC) [8]. Therefore, the roles of TNFα and IL6 in oval cellsmediated liver regeneration are required to be re-evaluated in an appropriate model, in which oval cells contribute a lot to liver regeneration.
In addition to liver regeneration, IL6 and TNFa/TNFR1 signaling pathways are also involved

Research Paper
Oncotarget 66636 www.impactjournals.com/oncotarget in HCC development [9]. Deletion of IL6 inhibited diethylnitrosamine(DEN)-induced HCC development in mouse [10]. TNFα and TNFR1 do not contribute to HCC development induced by DEN, but promote obesity-and long-term CDE-induced HCC development [11,12]. Thus, IL6 and TNFα/TNFR1 signaling pathways promote HCC development in most mouse models. However, HCC patients with high level of IL6 and TNFα have better prognosis [13][14][15]. 90% of HCC cases in human arised in the context of hepatic inflammation [16], while in chemicals induced HCC models, tumors arise from chemically induced mutation without initial underlying hepatitis [17]. Therefore, re-evaluating the role of IL6, TNFα and TNFR1 in mouse model whose HCC development is accompanied with inflammation is more appropriately.
To re-evaluate the role of cytokines in oval cellsmediated liver regeneration and HCC development in inflammatory condition, we explored IL6 and TNFα/ TNFR1 signaling in genetic induced hepatocyte damage model. Hepatocyte-specific deletion of damaged-binding protein 1(DDB1), an adaptor protein for Cullin4 ubiquitin ligase, blocks the proliferation ability of hepatocytes, resulting in compensatory regeneration mediated by oval cells and progressive tumor development in the context of inflammation [18]. Newborn hepatocytes derived from oval cells could be genetically distinguished in our model, and oval cells were induced independent of chemicals excluding the promiscuous effects of chemicals [7]. In addition, persistent deletion of DDB1 in DDB1 F/F ,Alb-Cre +/− mouse results in HCC arises in aged mice, preceding by intra-hepatic inflammation and immune cells infiltration [18]. Herein, by using hepatocyte-specific DDB1 knockout models, we reported divergent roles of IL6, TNFα and TNFR1 in oval cellsmediated liver regeneration and inflammation-associated hepatocarcinogenesis.

IL6 deficiency delayed liver generation in DDB1 F/ F ,Mx1-Cre +/− Mice after poly(I:C) injection
We previously reported that injection of poly(I:C) into DDB1 F/F ,Mx1-Cre +/− mouse induced hepatocytespecific DDB1 deletion. Oval cells are activated and differentiated into DDB1 positive hepatocytes subsequently [18]. Expression of IL6 was upregulated in the liver of DDB1 F/F ,Mx1-Cre +/− mice after poly(I:C) injection, with activation of downstream STAT3 but not ERK signaling ( Figure 1A, 1B). To investigate the role of IL6 in oval cells mediated liver regeneration, DDB1 F/F ,Mx1-Cre +/− ,IL6 −/− mouse was obtained. DDB1 positive hepatocytes depletion was achieved in both IL6 normal and deficient DDB1 F/F ,Mx1-Cre +/− mice two weeks after poly(I:C) injection ( Figure 1C). Newborn DDB1positive hepatocytes were observed with much fewer in DDB1 F/F ,Mx1-Cre +/− ,IL6 −/− mice at 4 and 6 weeks post injection ( Figure 1C). The level of DDB1 positive hepatocytes in IL6 deficient mice was regenerated to the same level as IL6 normal mice until 8 weeks post injection ( Figure 1C). The delayed regeneration is due to slow proliferation as lower level of proliferation markers PCNA and cyclinD1 in DDB1 F/F ,Mx1-Cre +/− ,IL6 −/− mice at 4 weeks post injection, which was recovered in IL6 deficient mice at 6 weeks post injection ( Figure 1D, 1E). Taken together, these data indicated that IL6 is required for liver regeneration in DDB1 F/F ,Mx1-Cre +/− mouse, loss of IL6 would delay this process.  (Figure 2A). Restricted proliferation of oval cells was further confirmed by significant reduction of CK19 and Thy1 ( Figure 2B). Furthermore, upregulation of HGF and TWEAK, two important factors for oval cell proliferation, was significantly attenuated by IL6 deletion( Figure 2C). Collectively, these results suggested that IL6 is required for oval cell proliferation, partly by promoting the expression of HGF and TWEAK.

TNFR1 is dispensable for oval cell proliferation and liver regeneration in DDB1 F/F ,Mx1-Cre +/− mouse
The role of TNFR1 in oval cell proliferation and liver regeneration was investigated as IL6. Equal DDB1-positive hepatocytes were regenerated at various time points after poly(I:C) injection with similar oval cell distribution in both TNFR1 wildtype or deficient DDB1 F/F ,Mx1-Cre +/− mice ( Figure 3A-3C). The expression of TNFα was also not varied significantly after poly(I:C) injection, as shown in Figure 3D. These results suggested that TNFα/TNFR1-mediated signaling pathway was dispensable for oval cell proliferation and liver regeneration in DDB1 F/F ,Mx1-Cre +/− mouse.

Intra-hepatic inflammation and immune cells infiltration before tumors arise in DDB1 F/F ,Alb-Cre +/− mouse
Various types of cancer arise under context of inflammation, especially for HCC [19]. Inflammation before tumor arised in DDB1 F/F ,Alb-Cre +/− and DEN mouse model was assessed, as shown in Figure 4A, by IHC for biomarkers of leukocytes(CD45) and macrophage (F4/80), more inflammatory cells in 12 months old DDB1 F/F ,Alb-Cre +/− mouse than age-matched control were observed, while no difference of inflammatory cells between DEN-treated and age-match control. We also evaluated the level of inflammation when noticeable HCC developed by IHC and RT-PCR. As shown Figure  4B and C, more inflammatory cells infiltration and T/B cells were detected in DDB1 F/F ,Alb-Cre +/− mouse. These data suggested that compared to DEN model, DDB1 F/F , Alb-Cre +/− mouse model recapitulates key features of human HCC pathogenesis in inflammation.  Figure S1). At age of 21 months, even the tumor incidence was similar, the maximum tumor size increased significantly in DDB1 F/ F , Alb-Cre +/− ,IL6 -/mouse ( Figure 5A, 5B). Although Liver injury was elevated by deletion of IL6 reflected in ALT measurement, the apoptosis and compensatory proliferation was unaltered with similar level of cleaved-caspase3 and PCNA in IL6 wildtype and deficient DDB1 F/F ,Alb-Cre +/− mice (Supplementary Figure S2). Inflammation was also measured by IHC with similar staining for CD45 and F4/80 ( Figure 5C). Interestingly, NK cells infiltration was decreased significantly in  with massive inflammatory cell infiltration, while normal morphology in age-matched DDB1 F/F ,Alb-Cre +/− mice (Figure 9). Taken together, our results suggested that TNFR1 elevated HCC incidence, partially by promoting cells turnover and upregulating P-ERK. Unlike TNFR1, deletion of TNFα accelerated HCC development, which showed divergent role of TNFα and TNFR1 in hepatocarcinogenesis.

DISCUSSION
The involvement of IL6, TNFα and TNFR1 in oval cells mediated liver regeneration and HCC development has been explored by numerous studies with controversial results and discrepancy between experimental and clinical data [13,20,21]. By using hepatocyte-specific DDB1 deletion mouse models, we found IL6 promotes oval cells-mediated liver regeneration, while TNFα/ TNFR1 signaling pathway does not affect this process. Intriguingly, TNFα and TNFR1showed divergent role in inflammation-associated hepatocarcinogenesis, and for the first time, the anti-tumor function of IL6 in HCC was revealed in present study.
Recently, Lu et al. reported that deletion of Mdm2, an E3 ubiquitin ligase responsible for P53 degradation, in hepatocytes resulted in P53 accumulation and elicited cell death and senescence. Subsequently, oval cells were induced and differentiated into P53 negative hepatocytes to restore liver mass [22]. Similarly, hepatocyte-specific deletion of DDB1 Oncotarget 66640 www.impactjournals.com/oncotarget also induced cell senescence(unpublished data) , followed by oval cells-mediated liver regeneration. The regenerative process in DDB1 F/F ,Mx1-Cre +/− mouse is characterized by replenishment of DDB1 negative hepatocytes with positive ones. Distinct from chemicals injury models, oval cells are the major source of newborn hepatocytes, and liver regeneration could be genetically assessed [7,18]. Therefore, DDB1 F/F , Mx1-Cre +/− mouse model suits the investigation for oval cells mediated liver regeneration.
During hepatocytes reconstitution, elevated expression of IL6 was detected, which may secreted by inflammatory cells or senescent hepatocytes [23]. IL6 is shown to promote liver regeneration after partial hepatectomy [24] and oval cell proliferation induced by CDE diet [5]. Therefore, we speculated that IL6 is required for oval cells-mediated liver regeneration in DDB1 F/F, Mx1-Cre +/− mouse. This hypothesis was confirmed by IL6 depletion, which showed delayed liver regeneration and restricted oval cell proliferation. Previous studies have suggested IL6 promotes oval cell proliferation by activating JAK/STAT3 signaling [5]. Consistently, activation of JAK/STAT3 was observed in DDB1 F/F , Mx1-Cre +/− mouse at 1 and 2 weeks post poly(I:C) injection. In addition, expression of HGF and TWEAK, two important mitogens for oval cells, was attenuated by IL6 depletion. HGF and TWEAK are secreted by stellate cells and macrophages respectively, their contribution on oval cell proliferation has been well characterized [25,26]. Therefore, IL6 also modulate oval cell proliferation by promoting HGF and TWEAK expression in vivo. Unlike IL6, TNFR1 depletion did not affect liver regeneration in DDB1 F/F ,Mx1-Cre +/− mouse. The expression of TNFα was also unaltered during regeneration. This results indicated that TNFα/TNFR1 signaling pathway is dispensable for oval cells-mediated liver regeneration in DDB1 F/F , Mx1-Cre +/− mouse.
Although IL6 promotes DEN-induced HCC development, its anti-tumor role in hepatocarcinogenesis was observed in current study. IL6 depletion accelerated tumor development and increased tumor burden in DDB1 F/F ,Alb-Cre +/− mouse. Detailed analysis found Oncotarget 66641 www.impactjournals.com/oncotarget liver injury, compensatory proliferation, and intrahepatic inflammation was not aggravated in DDB1 F/F , Alb-Cre +/− ,IL6 −/− mouse. Interestingly, NK cells were decreased significantly in tumors without IL6. NK cells play an important role in tumor surveillance, it exerts antitumor function by direct cytotoxicity or cytokine secretion such as IFNγ [27]. In HCC patients, high density of intratumoral NK cells correlated with long survival rate [28]. To date, the relationship between NK cells and IL6 is still unclear and controversial [29][30][31]. Based on our results, we postulated that IL6 signaling modulates NK cells to inhibiting inflammation-associated hepatocarcinogenesis, the underlying mechanism will be our next step.
Taken together, by using hepatocyte-specific DDB1 deletion mouse models, the roles of IL6 and TNFα/TNFR1 signaling in liver regeneration and hepatocarcinogenesis were systematically investigated. IL6 promotes liver regeneration by stimulating oval cell proliferation, while TNFα/TNFR1 signaling is dispensable for this process. For the first time, the antitumor role of IL6 in hepatocarcinogenesis was suggested, and NK cells were shown to involve in. Although TNFR1 promotes HCC development in DDB1 F/F ,Alb-Cre +/− mouse, TNFα depletion accelerates rather than inhibits hepatocarcinogenesis. Other ligands may initiate oncogenic function of TNFR1 signaling and special cautions should be paid to anti-IL6 and anti-TNFα therapy in HCC patients.

Real Time-PCR (RT-PCR)
Total RNA was purified from liver tissue samples using Trizol (Invitrogen) according to manufacturer's protocol. Reverse transcript was performed using PrimeScript RT Master Mix kit (Takara). Real time PCR was carried out in SYBR Green PCR Master Mix (Bio-Rad) with ABI PRISM 7500 Sequence Detection System. Primer sequences are listed in Supplementary Table S1. Each measurement was performed in triplicate and results were normalized to the expression of gapdh reference gene.

Serum alanine transaminases assay
Blood collected from mice incubated in room temperature for 2 hours, then centrifuge at 2000 g for 20 minutes. Serum in the upper layer after centrifuge transferred to new tubes. The activity of alanine