Xuehao Wang

Interleukin-22 promotes human hepatocellular carcinoma by activation of STAT3†‡§

Interleukin‐22 (IL‐22), one of the cytokines secreted by T helper 17 (Th17) cells, was recently reported to be a novel inflammation driver through STAT3 signaling activation. We aimed to investigate the role of IL‐22 expression in hepatocellular carcinoma (HCC). We demonstrated significant up‐regulation of IL‐22 in human HCC tumor infiltrated leukocytes (TILs) compared to peripheral lymphocytes. Moreover, IL‐22 expression was significantly higher in Edmondson Grade III‐IV HCC patients versus Grade I‐II, confirmed by both real‐time polymerase chain reaction and immunohistochemistry. Both IL‐22 receptor α and IL‐23 were highly expressed in HCC and adjacent cirrhotic tissues compared to normal controls. Enhanced tumor growth and metastasis was found in mice that underwent subrenal transplantation of MHCC‐97H cells cotransplanted with IL‐22+ TILs cells. STAT3 phosphorylation and up‐regulation of downstream genes Bcl‐2, Bcl‐XL, CyclinD1, and vascular endothelial growth factor (VEGF) promoted tumor growth and metastasis. In vitro studies confirmed the tumor‐promoting and antiapoptotic effect of IL‐22, as well as IL‐6. In the mouse chronic hepatitis and HCC model, sustained and increased IL‐22 expression and STAT3 activation were found in liver tissues. A linear correlation was demonstrated between IL‐22 expression and hepatic complementary proliferation. An in vivo diethyl‐nitrosamine‐induced mouse HCC model verified that tumor formation was significantly decreased in IL‐22 knockout mice. Conclusion: Excessive IL‐22 can be found in the HCC microenvironment, leading to tumor growth, inhibition of apoptosis, and promotion of metastasis due to STAT3 activation. (HEPATOLOGY 2011;)

Role of SMAD and Non-SMAD Signals in the Development of Th17 and Regulatory T Cells

Whereas TGF-β is essential for the development of peripherally induced Foxp3+ regulatory T cells (iTreg cells) and Th17 cells, the intracellular signaling mechanism by which TGF-β regulates development of both cell subsets is less understood. In this study, we report that neither Smad2 nor Smad3 gene deficiency abrogates TGF-β–dependent iTreg induction by a deacetylase inhibitor trichostatin A in vivo, although the loss of the Smad2 or Smad3 gene partially reduces iTreg induction in vitro. Similarly, SMAD2 and SMAD3 have a redundant role in development of Th17 in vitro and in experimental autoimmune encephalomyelitis. In addition, ERK and/or JNK pathways were shown to be involved in regulating iTreg cells, whereas the p38 pathway predominately modulated Th17 and experimental autoimmune encephalomyelitis induction. Therefore, selective targeting of these intracellular TGF-β signaling pathways during iTreg and Th17 cell development might lead to the development of therapies in treating autoimmune and other chronic inflammatory diseases.

IL-17A Plays a Critical Role in the Pathogenesis of Liver Fibrosis through Hepatic Stellate Cell Activation

Liver fibrosis is a severe, life-threatening clinical condition resulting from nonresolving hepatitis of different origins. IL-17A is critical in inflammation, but its relation to liver fibrosis remains elusive. We find increased IL-17A expression in fibrotic livers from HBV-infected patients undergoing partial hepatectomy because of cirrhosis-related early-stage hepatocellular carcinoma in comparison with control nonfibrotic livers from uninfected patients with hepatic hemangioma. In fibrotic livers, IL-17A immunoreactivity localizes to the inflammatory infiltrate. In experimental carbon tetrachloride–induced liver fibrosis of IL-17RA–deficient mice, we observe reduced neutrophil influx, proinflammatory cytokines, hepatocellular necrosis, inflammation, and fibrosis as compared with control C57BL/6 mice. IL-17A is produced by neutrophils and T lymphocytes expressing the Th17 lineage–specific transcription factor Retinoic acid receptor–related orphan receptor γt. Furthermore, hepatic stellate cells (HSCs) isolated from naive C57BL/6 mice respond to IL-17A with increased IL-6, α-smooth muscle actin, collagen, and TGF-β mRNA expression, suggesting an IL-17A–driven fibrotic process. Pharmacologic ERK1/2 or p38 inhibition significantly attenuated IL-17A–induced HSC activation and collagen expression. In conclusion, IL-17A+ Retinoic acid receptor–related orphan receptor γt+ neutrophils and T cells are recruited into the injured liver driving a chronic, fibrotic hepatitis. IL-17A–dependent HSC activation may be critical for liver fibrosis. Thus, blockade of IL-17A could potentially benefit patients with chronic hepatitis and liver fibrosis.

IL-22 is related to development of human colon cancer by activation of STAT3

BackgroundIt has been previously reported that IL-22, one of the cytokines secreted by Th17 cells, demonstrates both a protective and inflammatory promotion effect in inflammatory bowel disease (IBD) through STAT3 signaling activation. We sought to investigate the role of IL-22 expression in colon cancer (CC).MethodsThe expression of IL-22 and related molecules were detected in human CC, the detail function and mechanism of IL-22 were investigated by in vivo and in vitro model.ResultsOur results demonstrated significant upregulation of IL-22 in human CC tumor infiltrated leukocytes (TILs) compared to peripheral lymphocytes. Moreover, our findings demonstrated that IL-22 expression was significantly higher in ulcerative colitis (UC) tissues versus normal colon tissues. Both IL-22 receptor α1 (IL-22RA1) and IL-23 were highly expressed in CC and UC tissues compared to normal controls. TILs exhibiting various IL-22 expression levels isolated from CC patients were demonstrated to enhance tumor growth and metastasis co-transplanted with Hct-116 cells underwent subcutaneous transplantation in mice model. Tumor growth and metastasis was promoted by STAT3 phosphorylation and upregulation of its downstream genes such as Bcl-xl, CyclinD1, and VEGF. In vitro studies confirmed the anti-apoptotic and pro-proliferation effect of IL-22 according to the BrdU cooperation assay and peroxide induced apoptosis analysis with or without the presence of IL-22.ConclusionIn this study we demonstrated that excessive IL-22 in the CC and UC microenvironment leads to tumor growth, inhibition of apoptosis, and promotion of metastasis depend on STAT3 activation.

Synergistic effect of TGF-β superfamily members on the induction of Foxp3+ Treg

TGF‐β plays an important role in the induction of Treg and maintenance of immunologic tolerance, but whether other members of TGF‐β superfamily act together or independently to achieve this effect is poorly understood. Although others have reported that the bone morphogenetic proteins (BMP) and TGF‐β have similar effects on the development of thymocytes and T cells, in this study, we report that members of the BMP family, BMP‐2 and ‐4, are unable to induce non‐regulatory T cells to become Foxp3+ Treg. Neutralization studies with Noggin have revealed that BMP‐2/4 and the BMP receptor signaling pathway is not required for TGF‐β to induce naïve CD4+CD25− cells to express Foxp3; however, BMP‐2/4 and TGF‐β have a synergistic effect on the induction of Foxp3+ Treg. BMP‐2/4 affects non‐Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3+ Treg induced by TGF‐β. Data further advocate that TGF‐β is a key signaling factor for Foxp3+ Treg development. In addition, the synergistic effect of BMP‐2/4 and TGF‐β indicates that the simultaneous manipulation of TGF‐β and BMP signaling might have considerable effects in the clinical setting for the enhancement of Treg purity and yield.

Remote Ischemic Preconditioning Protects against Liver Ischemia-Reperfusion Injury via Heme Oxygenase-1-Induced Autophagy

Background Growing evidence has linked autophagy to a protective role of preconditioning in liver ischemia/reperfusion (IR). Heme oxygenase-1 (HO-1) is essential in limiting inflammation and preventing the apoptotic response to IR. We previously demonstrated that HO-1 is up-regulated in liver graft after remote ischemic preconditioning (RIPC). The aim of this study was to confirm that RIPC protects against IR via HO-1-mediated autophagy. Methods RIPC was performed with regional ischemia of limbs before liver ischemia, and HO-1 activity was inhibited pre-operation. Autophagy was assessed by the expression of light chain 3-II (LC3-II). The HO-1/extracellular signal-related kinase (ERK)/p38/mitogen-activated protein kinase (MAPK) pathway was detected in an autophagy model and mineral oil-induced IR in vitro. Results In liver IR, the expression of LC3-II peaked 12–24 h after IR, and the ultrastructure revealed abundant autophagosomes in hepatocytes after IR. Autophagy was inhibited when HO-1 was inactivated, which we believe resulted in the aggravation of liver IR injury (IRI) in vivo. Hemin-induced autophagy also protected rat hepatocytes from IRI in vitro, which was abrogated by HO-1 siRNA. Phosphorylation of p38-MAPK and ERK1/2 was up-regulated in hemin-pretreated liver cells and down-regulated after treatment with HO-1 siRNA. Conclusions RIPC may protect the liver from IRI by induction of HO-1/p38-MAPK-dependent autophagy.

High expression levels of IKKα and IKKβ are necessary for the malignant properties of liver cancer

IKK‐NF‐κB signaling is regarded as an important factor in hepatocarcinogenesis and a potential target for liver cancer therapy. Therefore, in this study, we analyzed the expression of mRNAs encoding components and targets of NF‐κB signaling including IKKα, IKKβ, RANK, RANKL, OPG, CyclinD3, mammary serine protease inhibitor (Maspin), CyclinD1, c‐FLIP, Bcl‐xl, Stat3, Cip1 and Cip2 by real‐time PCR in 40 patients with liver cancer. After statistical analysis, 7 indices including IKKα, IKKβ, RANK, Maspin, c‐FLIP, Cip2 and cyclinD1 were found to show significant differences between tumor tissue and its corresponding adjacent tissue. When IKKα and IKKβ were downregulated in the hepatocellular carcinoma (HCC) cell lines of MHCC‐97L and MHCC‐97H in vitro, the numbers of BrdU positive cells were decreased in both IKKα and IKKβ knockdown cells. Levels of apoptosis were also investigated in IKKα and IKKβ knockdown cells. The growth of HCC was inhibited in the subcutaneous implantation model, and lung metastatogenesis was also significantly inhibited in the kidney capsule transplantation model. Downregulation of IKKα and IKKβ in HCC cultured in vitro revealed that increased Maspin, OPG and RANKL expression was associated with metastasis of HCC. These findings were associated with downregulation of Bcl‐XL and c‐FLIP, which may be the reason for increased apoptosis. The therapeutic effect of IKKα and IKKβ downregulation depends on extent of NF‐κB inhibition and the malignant nature of the HCC. We anticipate that IKK‐targeted gene therapy can be used in the treatment of HCC, a cancer that is notoriously resistant to radiation and chemotherapy.

ATF6 Mediates a Pro-inflammatory Synergy between ER Stress and TLR Activation in the Pathogenesis of Liver Ischemia Reperfusion Injury

Although the roles of the metabolic stress in organ ischemia‐reperfusion injury (IRI) have been well recognized, the question of whether and how these stress responses regulate innate immune activation against IR remains unclear. In a murine liver partial warm ischemia mode, we showed that prolonged ischemia triggered endoplasmic reticulum (ER) stress response, particularly, the activating transcription factor 6 (ATF6) branch, in liver Kupffer cells (KCs) and altered their responsiveness against Toll‐like receptor (TLR) stimulation. Ischemia‐primed cells increased pro‐, but decreased anti‐, inflammatory cytokine productions. Alleviation of ER stress in vivo by small chemical chaperon 4‐phenylbutyrate or ATF6 small interfering RNA (siRNA) diminished the pro‐inflammatory priming effect of ischemia in KCs, leading to the inhibition of liver immune response against IR and protection of livers from IRI. In vitro, ATF6 siRNA abrogated the ER stress‐mediated pro‐inflammatory enhancement of macrophage TLR4 response, by restricting NF‐κB and restoring Akt activations. Thus, ischemia primes liver innate immune cells by ATF6‐mediated ER stress response. The IR‐induced metabolic stress and TLR activation function in synergy to activate tissue inflammatory immune response.

miR-22 Promotes HBV-Related Hepatocellular Carcinoma Development in Males

Purpose: Previous reports have shown that IL-1α–MyD88–IL-6 signaling is essential in promoting hepatocellular carcinoma (HCC) development in a diethylnitrosamine (DEN)-induced mouse model. We aimed to determine whether interleukin (IL)-1α regulates HCC development in humans. Methods: HBV-associated HCC tissue, corresponding adjacent tissue, and normal tissue samples were obtained from 80 male and 36 female patients. IL-1α, ERα, IL-6, and MyD88 were quantified by using real-time PCR and Western blot. Stem-loop PCR was used to quantify miR-22 expression. Luciferase reporter assays were used to study transcriptional regulation. Results: IL-1α was highly expressed in male tumor adjacent tissue compared with normal tissue (P = 0.025); however, this was not the case for female subjects. A linear relationship was observed between increased IL-1α and decreased ERα expression in male tumor adjacent tissue (r = −0.616, P = 0.004). Our results also indicated that estrogen (E2) was suppressed upon IL-1α secretion in ERα-overexpressed HCC cells. We detected high expression of miR-22 in male tumor adjacent tissue compared with controls (P = 0.027); furthermore, we showed that miR-22 downregulates ERα transcription by targeting the 3′-untranslated region. In the DEN-induced model, IL-1α was highly expressed in sprouting tumors and gradually decreased in conjunction with HCC development. Conclusion: Overexpression of miR-22 in male tumor adjacent tissue was associated with downregulated ERα expression, potentially by attenuating the protective effect of estrogen and causing increased IL-1α expression. These results may explain the high incidence of HBV-associated HCC in the male population. Clin Cancer Res; 17(17); 5593–603. ©2011 AACR.

ATF3‐Mediated NRF2/HO‐1 Signaling Regulates TLR4 Innate Immune Responses in Mouse Liver Ischemia/Reperfusion Injury

Activating transcription factor 3 (ATF3) is a stress‐induced transcription factor that has been shown to repress inflammatory gene expression in multiple cell types and diseases. However, little is known about the roles and mechanisms of ATF3 in liver ischemia/reperfusion injury (IRI). In warm and cold liver IRI models, we showed that ATF3 deficiency significantly increased ischemia/reperfusion (IR)‐stressed liver injury, as evidenced by increased serum alanine aminotransferase levels, histological liver damage, and hepatocellular apoptosis. These may correlate with inhibition of the intrahepatic nuclear factor erythroid‐derived 2‐related factor 2/heme oxygenase‐1 (NRF2/HO‐1) signaling pathway leading to enhancing Toll‐like receptor 4/nuclear factor kappa beta (TLR4/NF‐κB) activation, pro‐inflammatory programs and macrophage/neutrophil trafficking, while simultaneously repressing anti‐apoptotic molecules in ischemic liver. Interestingly, activation of NRF2/HO‐1 signaling using an NRF2 activator, oltipraz (M2), during hepatic IRI‐rescued ATF3 anti‐inflammatory functions in ATF3‐deficient mice. For in vitro studies, ATF3 ablation in lipopolysaccharide (LPS)‐stimulated bone marrow‐derived macrophages (BMMs) depressed levels of NRF2/HO‐1 and PI3K/AKT, resulting in enhanced TLR4/NF‐κB activation. Pretreatment of LPS‐stimulated BMMs with M2 increased NRF2/HO‐1 expression, promoted PI3K/AKT, which in turn suppressed TLR4/NF‐κB‐mediated proinflammatory mediators. Thus, our results first demonstrate ATF3‐mediated NRF2/HO‐1 signaling in the regulation of TLR4‐driven inflammatory responses in IR‐stressed livers. Our findings provide a rationale for a novel therapeutic strategy for managing IR‐induced liver injury.