Faliang Zhu

A Critical Function of Th17 Proinflammatory Cells in the Development of Atherosclerotic Plaque in Mice

Considerable evidence supports that the CD4+ T cell-mediated immune response contributes to the development of atherosclerotic plaque. However, the effects of Th17 cells on atherosclerosis are not thoroughly understood. In this study, we evaluated the production and function of Th17 and Th1 cells in atherosclerotic-susceptible ApoE−/− mice. We observed that the proportion of Th17 cells, as well as Th1, increased in atherosclerotic ApoE−/− mice compared with nonatherosclerotic wild-type littermates. In ApoE−/− mice with atherosclerosis, the expression of IL-17 and retinoic acid-related orphan receptor γt was substantially higher in the arterial wall with plaque than in the arterial wall without plaque. Increased Th17 cells were associated with the magnitude of atherosclerotic plaque in ApoE−/− mice. Importantly, treatment of ApoE−/− mice with neutralizing anti–IL-17 Ab dramatically inhibited the development of atherosclerotic plaque, whereas rIL-17 application significantly promoted the formation of atherosclerotic plaque. These data demonstrate that Th17 cells play a critical role in atherosclerotic plaque formation in mice, which may have implications in patients with atherosclerosis.

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) mediates natural killer cell suppression in chronic hepatitis B

BACKGROUND & AIMS T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been shown to influence autoimmune diseases; however, its function in viral infection has not been well-defined. We therefore investigated the expression and regulatory function of Tim-3 in natural killer (NK) cells in chronic Hepatitis B (CHB) infection. METHODS Seventy-six CHB patients, 38 healthy controls, and 18 patients with fatty liver disease (FLD) were tested for Tim-3 expression on peripheral blood mononuclear cells (PBMCs) and in the liver tissue by flow cytometry and immunohistochemical stainning. The effects of HBV infection on Tim-3 expression in NK cells and the roles of Tim-3 in regulation of NK-cell function were also studied. RESULTS There was a significant increase of Tim-3 expression in PBMCs, circulating NK cells and liver infiltrating lymphocytes (LILs) from CHB patients compared to that of healthy controls and FLD patients. Increased Tim-3 expression was also detected in NK92 cells that had been transfected with a HBV expression vector and NK cells isolated from the liver of HBV transgenic mice. Importantly, blockage of Tim-3 signaling with anti-Tim-3 antibodies or Tim-3-Fc fusion proteins resulted in an increased cytotoxicity for NK92 cells compared to HepG2 and HepG2.2.15 cells, as well as an elevated interferon-gamma (IFN-gamma) production. Similarly, enhanced cytotoxicity was also observed in PBMCs or NK cells from CHB patients treated with the Tim-3 blockade ex vivo. CONCLUSION HBV infection can up-regulate Tim-3 expression in NK cells, which may in turn suppress NK-cell functions in CHB patients.

Down-regulation of TIPE2 mRNA expression in peripheral blood mononuclear cells from patients with systemic lupus erythematosus

Breakdown of immune homeostasis contributes to the pathogenesis of systemic lupus erythematosus (SLE). Tumor necrosis factor alpha induced protein-8 like-2 (TIPE2) is a very recently identified immune homeostasis maintaining gene. We wondered whether TIPE2 is associated with SLE. We examined the mRNA expression levels of TIPE2 and myxoma resistance protein (MX) 1 (one of type I interferon inducible genes) in peripheral blood mononuclear cells (PBMC) from 39 SLE patients and 35 healthy controls by real-time reverse transcription-polymerase chain reaction analysis (RT-PCR). The TIPE2 mRNA expression was significantly down-regulated in SLE patients compared with healthy controls (P=0.0004), while the MX1 mRNA expression was increased in SLE patients compared with healthy controls (P=0.0031). Furthermore, the TIPE2 mRNA expression levels negatively correlate with the SLE disease activity index (SLEDAI) (r=-0.5016, P=0.0011) and the MX1 mRNA expression levels (r=-0.8083, P<0.0001) in all the SLE patients. These results indicate that decreased expression of TIPE2 gene is closely associated with SLE and suggest an important role for TIPE2 gene in the pathogenesis of SLE.

IL-17 induces apoptosis of vascular endothelial cells — A potential mechanism for human acute coronary syndrome

Th17 cells producing IL-17 are involved in the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. In this study, we investigated the effects of IL-17 on human vascular endothelial cells and showed that IL-17 induced cell death of the vascular endothelial cells, which played a pivotal role in plaque destabilization triggering acute coronary syndrome (ACS). We showed that circulating Th17 cells and IL-17 increased in patients with ACS compared to the patients with stable angina or health individuals; the plasma levels of IL-6 increased but TGF-β decreased in ACS patients, exhibiting a positive and negative correlation with that of IL-17, respectively. Importantly, we uncovered that IL-17 promoted the production of von Willebrand factor by endothelial cells and induced endothelial apoptosis by activating caspase-3, caspase-9 and up-regulating the ratio of Bax/Bcl-2, indicating the function of IL-17 in vascular endothelial damage as a potential mechanism for the pathogenesis of human ACS.

Blockade of Tim-3 Pathway Ameliorates Interferon- γ Production from Hepatic CD8 + T Cells in a Mouse Model of Hepatitis B Virus Infection

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus (HBV) infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8+ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-γ production from hepatic CD8+ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8+ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection.

Increased Tim-3 expression on peripheral lymphocytes from patients with rheumatoid arthritis negatively correlates with disease activity.

Tim-3 has been reported as an important regulatory molecule and plays a pivotal role in several autoimmunity diseases. Here, we demonstrated the increased expression of Tim-3 on peripheral CD4(+) T, CD8(+) T, NKT cells and monocytes from RA patients compared to those from healthy controls. Percentage of Tim-3(+) cells in peripheral blood mononuclear cells (PBMCs) showed an inverse correlation with disease activity score 28 (DAS28) and plasma TNF-α level. Similar negative correlations were found between disease activity and Tim-3 levels on CD4(+) T, CD8(+) T and NKT cells. Consistently, Tim-3 expression on CD3(+) T cells was further increased in patients with disease remission after treatment. Tim-3 expression on CD8(+) T and NKT cells negatively correlates with plasma TNF-α. Our results suggest that Tim-3 might participate in the proceeding of RA by its negative regulation on various T cell subsets. Tim-3 might be a potential new marker for assessing severity of RA.

Upregulation of B7-H1 expression is associated with macrophage infiltration in hepatocellular carcinomas

The overexpression of B7-H1 in hepatocellular carcinoma (HCC) mediates HCC immune escape and obstructs the immunotherapy based on tumor-specific CD8+ T cells. Tumor-associated macrophages (TAM) are a major component of cancer-related inflammation and play a central role in tumor promotion. To classify the mechanism underlying the overexpression of B7-H1 in HCC, we examined B7-H1 expression and TAM infiltration in 63 cases of human HCC samples using immunohistochemistry method and found that B7-H1 overexpression was associated with TAM infiltration in HCC tissues. Furthermore, B7-H1 expression was upregulated at both mRNA level and protein level in HCC cells (BEL-7402 and SMMC-7721) cocultured with macrophages in a transwell system. The upregulation of B7-H1 expression induced by macrophage was inhibited by blocking NF-κB or STAT3 signal pathways. These results suggest that overexpression of B7-H1 in HCC may be induced by inflammatory microenvironment involving macrophages and imply that anti-inflammation therapy might be preventive for immune escape and assistant for immunotherapy of HCC.

PDCD4 gene silencing in gliomas is associated with 5′CpG island methylation and unfavourable prognosis

Programmed cell death 4 (PDCD4) is a newly described tumour suppressor that inhibits oncogenesis by suppressing gene transcription and translation. Loss of PDCD4 expression has been found in several types of human cancers including the most common cancer of the brain, the gliomas. However, the molecular mechanisms responsible for PDCD4 gene silencing in tumour cells remain unclear. Here we report the identification of 5′CpG island methylation as the predominant cause of PDCD4 mRNA silencing in gliomas. The methylation of the PDCD4 5′CpG island was found in 47% (14/30) of glioma tissues, which was significantly associated with the loss of PDCD4 mRNA expression (γ=−1.000, P < 0.0001). Blocking methylation in glioma cells using a DNA methyltransferase inhibitor, 5‐aza‐2′‐deoxycytidine, restored the PDCD4 gene expression, inhibited their proliferation and reduced their colony formation capacity. Longitudinal studies of a cohort of 84 patients with gliomas revealed that poor prognosis of patients with high‐grade tumours were significantly associated with loss of PDCD4 expression. Thus, our current study suggests, for the first time, that PDCD4 5′CpG island methylation blocks PDCD4 expression at mRNA levels in gliomas. These results also indicate that PDCD4 reactivation might be an effective new strategy for the treatment of gliomas.

PDCD4 inhibits the malignant phenotype of ovarian cancer cells.

Programmed cell death 4 (PDCD4) is a newly identified tumor suppressor that can inhibit activator protein (AP)‐1 activation and protein translation. Our previous studies indicate that lost or reduced PDCD4 expression is associated with the progression of ovarian carcinoma. However, direct evidence that PDCD4 inhibits malignant phenotype of human cancer cells is limited. In the present study, we found that PDCD4 expression in ovarian cancer cell lines (SKOV3, 3AO, and CAOV3) inhibited significantly their proliferation and cell cycle progression, and induced apoptosis. More importantly, up‐regulation of PDCD4 expression decreased the colony‐forming capacity of ovarian cancer cells in vitro and tumorigenic capacity in mice. These results demonstrate that PDCD4 can suppress the malignant phenotype of ovarian cancer cells, and may represent a novel therapeutic target for the treatment of ovarian cancer. (Cancer Sci 2009)

The unique expression profile of human TIPE2 suggests new functions beyond its role in immune regulation

TIPE2 (tumor necrosis factor-α-induced protein 8-like 2, or TNFAIP8L2) is a newly identified negative regulator of innate and adaptive immunity. TIPE2 deficiency in mice leads to systemic inflammation and TIPE2 down-regulation in humans is associated with autoimmunity. However, the nature of human TIPE2-expressing cells in various tissues has not been characterized due to the lack of suitable antibodies. In the present study, we generated specific rabbit antibodies against human TIPE2 and examined human TIPE2 expression in various tissues by Western blot, flow cytometry, and immunohistochemistry. We found that unlike murine TIPE2 that is preferentially expressed by hematopoietic cells, human TIPE2 was also expressed in a wide variety of non-hematopoietic cell types, including hepatocytes, neurons in brain and brainstem, squamous epithelial cells in esophagus and cervix, transitional epithelial cells in bladder and ureter, and glandular epithelial cells in stomach, colon and appendix. For squamous epithelium, the level of TIPE2 expression increased dramatically in terminally differentiated cells with the proliferating precursor cells completely devoid of TIPE2. The unique expressional profile of human TIPE2 suggests new functions beyond controlling innate and adaptive immunity.