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

Abstract

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.

Keywords: IL6; NK cell; TNFR1; hepatocellular carcinoma; oval cell.

Figure 1. Deletion of IL6 delayed liver regeneration in DDB1^F/F, Mx1-Cre^+/− mouse after poly(I:C) injection

(A) The mRNA level of IL6 at indicated time points after poly(I:C) injection was detected by RT-PCR. Results are represented as mean ± S.E.M, n = 3–6, *P < .0.05. (B) The activation of IL6 downstream proteins STAT3 and ERK were detected by western blot. (C) IHC staining for DDB1 in liver slides(magnification,100×) and DDB1-positive hepatocytes counting at indicated time points after poly(I:C) injection, cells were counted under 20× objective from eight independently visual fields. Data are represented as mean ± S.E.M, n = 3–6, *P < 0.05, ***P < 0.001. Western blot for detecting DDB1, PCNA and cyclinD1 in liver homogenates of indicated mice at 4 weeks (D) and 6 weeks (E) after poly(I:C) injection.

Figure 2. IL6 promotes oval cell proliferation by elevating the expression of HGF and TWEAK

(A) Liver tissues of indicated mice at 6 weeks after poly(I:C) injection were stained with HE and EpCAM(magnificantion, 400×, arrows indicated positive cells), and (B) the mRNA levels of EpCAM, CK19 and Thy1 were quantified by RT-PCR. Data are represented as mean ± S.E.M, n = 3–6, *P < 0.05. (C) The hepatic mRNA levels of HGF and TWEAK at indicated time points after poly(I:C) injection were measured by RT-PCR. Results are represented as mean ± S.E.M, n = 3–6, *P < 0.05.

Figure 3. TNFR1 was dispensable for oval cell proliferation in DDB1^F/F, Mx1-Cre^+/− mouse

(A) IHC staining for DDB1 in liver tissues at indicated time points after poly(I:C) injection(magnification,100×) and DDB1-positive hepatocytes counting, cells were counted under 20× objective from eight independently visual fields. Data are represented as mean ± S.E.M, n = 4. (B) HE and IHC staining for EpCAM in liver tissues at 6 weeks after poly(I:C) injection (magnificantion, 400×, arrows indicated positive cells). (C) The mRNA levels of EpCAM, CK19 and Thy1 were quantified by RT-PCR. Data are represented as mean ± S.E.M, n = 4. (D) Detecting mRNA level of TNFα at indicated time points after poly(I:C) injection by real time RT-PCR. Results are represented as mean ± S.E.M, n = 4, *P < .0.05.

Figure 4. Established inflammation before HCC arises and more immune cells infiltration in DDB1^F/F, Alb-Cre^+/− mouse

(A and B) Representative pictures of IHC staining for CD45 and F4/80 in liver slides of indicated mice (magnification, 100×). (C) The hepatic mRNA levels of CD3E, B220, F4/80 and Ly6G were measured by RT-PCR. Data are represented as mean ± S.E.M, n = 4, *P < 0.05.

Figure 5. IL6 suppressed inflammation-associated HCC development by modulating NK cells

(A) Representative pictures of liver appearance and HE staining of liver slides(magnification, 400×).(B) Tumor incidence, tumor number(> 1 mm), maximum tumor size and ALT in serum of indicated mice aged 21 months, dotted lines indicated tumor. Data are represented as mean ± S.E.M, n = 4–5, *P < 0.05. (C) Representative pictures of IHC for CD45 and F4/80 (magnification, 100×) and the mRNA levels of CD3E, B220, F4/80 detected by RT-PCR T indicates tumor and PT indicates para-tumor. Data are represented as mean ± S.E.M, n = 4–5. (D) The mRNA level of NK1.1 was detected by RT-PCR. Data are represented as mean ± S.E.M, n = 4–5, *P < 0.05. (E) IF staining for NK1.1 in tumor tissues of indicated mice. Representative pictures are shown (magnification, 200×).

Figure 6. The expression of TNFα and IFNγ was significantly reduced in tumor regions of DDB1^F/F, Alb-Cre^+/−, IL6^−/− mouse

(A) The mRNA levels of TNFα, IFNγ, IL1α and IL1β of liver tissues of indicated mice were detected by RT-PCR. Data are represented as mean ± S.E.M, n = 4–5, *P < 0.05.

Figure 7. Deletion of TNFR1 reduced HCC incidence in DDB1^F/F, Alb-Cre^+/− mouse

All mice were sacrificed at the age of 21 months, tumor incidence, tumor numbers (> 1 mm) and maximum tumor size were calculated. (A) Representative pictures of liver and HE staining of liver slides (magnification, 400×), dotted line indicating tumor. (B) Tumor incidence, tumor number (> 1 mm), maximum tumor size and ALT in serum of indicated mice at the age of 21 months. Data are represented as mean ± S.E.M, n = 3–4. (C) Representative pictures of IHC for CD45 and F4/80 (magnification, 100×) and (D) the mRNA levels of CD3E, B220, F4/80 detected by RT-PCR, T indicates tumor and PT indicates para-tumor. Data are represented as mean ± S.E.M, n = 3–4.

Figure 8. Reduction of cell apoptosis and compensatory proliferation, P-ERK level in DDB1^F/F, Alb-Cre^+/−, TNFR1^−/− mouse

(A) Western blot for detecting cleaved-caspase3, PCNA, β-catenin, P-ERK and ERK. (B) Representative pictures of IHC for cleaved-caspase3, BrdU and P-ERK (magnification, 400×), arrows indicate positive cells

Figure 9. Deletion of TNFα accelerated tumor development in DDB1^F/F, Alb-Cre^+/− mouse

(A) Representative pictures of liver, dotted line indicates tumor. (B) Representative pictures of HE staining and IHC for CD45 and F4/80 (magnification, 200×). T indicates tumor and PT indicates para-tumor.