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We thank Dr. Schwabe and colleagues for interest in our article.1, 2 We have carefully reviewed our published results and also the original data. Our experiments showed that carcinogen-induced apoptosis in mice can be protected by lipopolysaccharide (LPS), an endotoxin (Fig. 1), or other factors induced by diethylnitrosamine (DEN). This figure was originally included in the manuscript, but deleted in revision due to page limitations. The main question concerns why reduction of LPS by antibiotics (figure 7B)1 led to lower liver injury but LPS receptor Toll-like receptor 4 (TLR4) deficiency potentiated liver injury (figure 3A).1 Possible explanations are as follows: (1) The alanine aminotransferase level usually indicates liver damage and does not exclusively represent the degree of cell apoptosis. LPS protection as previously described takes effect by a certain time period (e.g., at 48 hours after injection; figure 6C-F),1 although it even enhances liver injury at 24 hours after DEN injection. We did not claim that no or low LPS levels and antibiotic suppression of LPS will increase liver injury. (2) Because treatment with antibiotics prevented the increase of plasma LPS (figure 7A),1 inhibition of LPS-induced inflammation upon DEN exposure might be responsible for the reduction of liver injury. We found that antibiotic-induced reduction of LPS and lack of TLR4 reduced the number and volume of tumors with the decrease of tumor necrosis factor α and interleukin-6.

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Figure 1. LPS pretreatment prevents death receptor ligands–induced apoptosis in vivo and in vitro. (A,C) Survival curves of LPS-preconditioned or control wild-type mice in response to treatment with (A) tumor necrosis factor/galactosamine (TNF/GaIN) and (C) Jo-2. (B,D) The typical gross and histological appearance of the above treated mice: (B) tumor necrosis factor/galactosamine (TNF/GalN); (D) Jo-2. Scale bars = 100 μm. (E,F) The cleavage of poly(adenosine diphosphate ribose) polymerase (PARP) protein was detected by western blot in LPS-pretreated (1μg/mL for 12 hours) or untreated primary wild-type or TLR4−/− hepatocytes (E) or Hepa1-6 cells (F) upon TNF + cycloheximide (CHX) treatment. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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We had the same concerns about this discrepancy, because it appears to contradict the hypothesis that TLR4−/− mice should have less liver injury. However, in terms of the acute liver damage induced by DEN, the TLR4 knockout mice are different from wild-type mice in response to the short-term antibiotics treatment. In addition to LPS, TLR4 interacts with other endogenous ligands, such as high mobility group box 1 (HMGB1) and heat shock proteins (HSPs). TLR4 deficiency therefore ablated the protective effects of both LPS and endogenous ligands on hepatocytes, thus leading to more severe liver damage. Treatment with antibiotics mainly reduced the acute inflammatory responses to LPS in nonparenchymal cells, which is thought to be the major contributing factor to acute hepatocyte injury. It is conceivable that reduced production of proinflammatory cytokines such as tumor necrosis factor α was responsible for alleviation of DEN-induced acute liver damage. On the other hand, during the multistage process of hepatocarcinogenesis, accumulation of endotoxin promotes chronic tumorigenic inflammation and alleviates apoptosis in precancerous and cancerous cells. So, loss of TLR4 is not equivalent to LPS ablation in terms of the responsive cell types and inflammation status.

As cited by Dr. Schwabe and colleagues, Karin's group at University of California San Diego reported an increase in DEN-induced hepatocellular carcinoma development when inhibitor of nuclear factor kappa B kinase subunit beta (IKKβ) was deleted in hepatocytes. However, the same group also found that hepatocellular carcinoma was suppressed in mice with IKKβ ablated in both hepatocytes and Kupffer cells.3 Furthermore, Pikarsky et al. found that inhibiting nuclear factor κB signaling suppressed hepatocarcinogenesis.4 Resolving these differences will lead to better understanding of hepatocarcinogenesis.

References

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  • 1
    Yu LX, Yan HX, Liu Q, Yang W, Wu HP, Dong W, et al. Endotoxin accumulation prevents carcinogen-induced apoptosis and promotes liver tumorigenesis in rodents. HEPATOLOGY 2010; 52: 1322-1333.
  • 2
    Mencin A, Gwak GY, Schwabe RF. None. HEPATOLOGY 2011.
  • 3
    Maeda S, Kamata H, Luo JL, Leffert H, Karin M. IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis. Cell 2005; 121: 977-990.
  • 4
    Pikarsky E, Porat RM, Stein I, Abramovitch R, Amit S, Kasem S, et al. NF-kappaB functions as a tumour promoter in inflammation-associated cancer. Nature 2004; 431: 461-466.

Lexing Yu Ph.D.*, Hexin Yan Ph.D.*, Wen Yang Ph.D.*, Hongyang Wang M.D.* †, * International Cooperation Laboratory on Signal Transduction, Liver Centre of SMMU, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, China, † National Laboratory for Oncogene and Related Genes, Cancer Institute of Shanghai Jiao Tong University, Shanghai, China.