Gui Jie Feng

Nitric oxide regulates Th1 cell development through the inhibition of IL-12 synthesis by macrophages

We have previously reported that mice lacking inducible nitric oxide synthase (NOS2) developed enhanced Th1 cell responses. We now investigated the mechanism by which NO modulates Th1 cells differentiation. Peritoneal macrophages from NOS2‐deficient mice infected with Leishmania major in vivo or stimulated with IFN‐γ or lipopolysaccharide (LPS) in vitro produced significantly higher levels of IL‐12 than those from heterozygous or wild‐type mice. A macrophage cell line, J774, produced significant amounts of IL‐12 following activation with LPS, or LPS plus IFN‐γ. This could be markedly enhanced by the NOS inhibitor L‐NG monomethyl arginine (L‐NMMA), but profoundly inhibited by the NO‐generating compound S‐nitroso‐N‐acetyl‐penicillamine (SNAP). The effect of NO in this system is selective, since SNAP enhanced and L‐NMMA decreased TNF‐α synthesis by LPS‐activated J774 cells. The differential effect of NO on IL‐12 and TNF‐α is at the transcriptional level and is activation dependent. Since IL‐12 is a major inducer of Th1 cells which produce IFN‐γ that can activate macrophages to produce IL‐12, our data demonstrate that NO can be an inhibitor of this feedback loop, preventing the excessive amplification of Th1 cells which are implicated in a range of immunopathologies.

REGULATION OF MACROPHAGE IL-12 SYNTHESIS BY LEISHMANIA PHOSPHOGLYCANS

It is now generally accepted that IFN‐γ, secreted by Th1 cells, is the most potent cytokine leading to macrophage activation and host resistance against infection with the intracellular protozoan parasite Leishmania. It is also established that IL‐12 is a critical cytokine involved in the differentiation and expansion of Th1 cells. Therefore, the ability of Leishmania parasites to actively suppress IL‐12 production by host macrophages may be an important strategy for parasite survival. Here we report that a major parasite cell surface molecule, phosphoglycan (PG), of Leishmania could selectively inhibit the synthesis of IL‐12(p40, p70) by activated murine macrophages. Furthermore, synthetic PG (sPG) was able to inhibit IL‐12 release in a dose‐dependent manner. Inhibition was dependent on the galactose(β1‐4)mannose(α1)‐PO4 repeating units and not the glycophosphoinositol lipid anchor of lipophosphoglycan. At the concentration used, sPG had no effect on the release of TNF‐α or IL‐6 in activated macrophages. The inhibition of IL‐12(p40) production was at the transcriptional level, but was not mediated through NFκB inhibition. These data demonstrate that PG may be an important molecule for the establishment and survival of the parasite in permissive hosts.

Lipopolysaccharide-induced M2 to M1 macrophage transformation for IL-12p70 production is blocked by Candida albicans mediated up-regulation of EBI3 expression.

Macrophages are heterogeneous cell populations that are present in all tissues. Macrophages can be divided into classically activated inflammatory macrophages (M1) and alternatively activated anti-inflammatory macrophages (M2). It has been generally accepted that M1 macrophages are polarised in an inflammatory environment to produce pro-inflammatory cytokines, whilst M2 macrophages are involved in anti-inflammation and aid tissue repair in wound healing. Bacterial endotoxin (lipopolysaccharide; LPS) is a potent factor in infection, which induces M1 macrophages resulting in higher levels of iNOS, TNFα and IL-12p70 which dictate inflammatory T cell responses. M2 macrophages can be transformed into M1 macrophages following LPS stimulation to promote inflammation. Candida albicans is a commensal fungal microorganism, which has been suggested to induce immune tolerance; however, the mechanism of C. albicans-induced immune tolerance has not been investigated in detail. IL-35 is a recently identified anti-inflammatory cytokine which is a heterodimeric protein consisting of the Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 shares the protein subunit p35, with IL-12p70. IL-12p70 is the most potent cytokine to induce Th1 responses during inflammation. In this study, we demonstrate that heat-killed C. albicans (HKC) strongly suppressed LPS-induced IL-12p70 production in M2 macrophages. Candida albicans induced a high level of EBI3 expression in M2 macrophages, which served as a mechanism for IL-12p70 suppression by competitive binding of the common protein subunit (p35) of IL-35 and IL-12p70. To demonstrate that EBI3 expression had the ability to block IL-12p70 production intracellularly, a Chinese Hamster Ovary (CHO) cell line with biscistronic expression of IL-12p40 and p35 was constructed, followed by ectopic over-expression of EBI3. The over-expression of EBI3 in the IL-12p70 producing cell line effectively suppressed IL-12p70 production. IL-35 secretion was also detected in the cell line, with suppressed IL-12p70 production by immune-precipitation Western blotting. However, this secretion was not evident in M2 macrophages following stimulation by HKC. This can be explained by the constitutive expression of IL-35 receptors (gp130 and IL-12Rβ2) in M2 macrophages for cytokine consumption. Our results have indicated that C. albicans can suppress host inflammatory responses in mucosal skin by suppressing LPS-induced IL-12p70 production. Lower IL-12p70 production may avoid an unnecessary Th1 response in order to retain immune tolerance, which may be one of the mechanisms by which C. albicans achieves a successful commensal lifestyle without having a detrimental effect on the host’s health.

G protein-coupled receptor fusion proteins in drug discovery

A wide range of peptides and polypeptides can be appended to either the N- or C-terminus of G protein-coupled receptors without disrupting substantially ligand binding and signal transduction. Following fusion of fluorescent proteins, reporter gene constructs or G protein alpha subunits to the C-terminal tail of a receptor high content and G protein activation assays can be employed to identify agonist ligands. Further modification of the receptor fusions to introduce enhanced levels of constitutive activity and to physically destabilise the protein allows antagonist/inverse agonists screens to be developed in parallel. Equivalent C-terminal addition of pairs of complementary, non-functional, polypeptide fragments allows the application of enzyme complementation techniques. Introduction of N-terminal tags to receptors has also allowed the introduction of novel assay techniques based on a pH-sensitive cyanine dye. These have the capacity to overcome certain limitations of GPCR-fluorescent protein fusions.

Conditional disruption of Axin1 leads to development of liver tumors in mice.

BACKGROUND & AIMS Mutations in components of the Wnt signaling pathway, including β-catenin and AXIN1, are found in more than 50% of human hepatocellular carcinomas (HCCs). Disruption of Axin1 causes embryonic lethality in mice. We generated mice with conditional disruption of Axin1 to study its function specifically in adult liver. METHODS Mice with a LoxP-flanked allele of Axin1 were generated by homologous recombination. Mice homozygous for the Axin1fl/fl allele were crossed with AhCre mice; in offspring, Axin1 was disrupted in liver following injection of β-naphthoflavone (Axin1fl/fl/Cre mice). Liver tissues were collected and analyzed by quantitative real-time polymerase chain reaction and immunoprecipitation, histology, and immunoblot assays. RESULTS Deletion of Axin1 from livers of adult mice resulted in an acute and persistent increase in hepatocyte cell volume, proliferation, and transcription of genes that induce the G(2)/M transition in the cell cycle and cytokinesis. A subset of Wnt target genes was activated, including Axin2, c-Myc, and cyclin D1. However, loss of Axin1 did not increase nuclear levels of β-catenin or cause changes in liver zonation that have been associated with loss of the adenomatous polyposis coli (APC) or constitutive activation of β-catenin. After 1 year, 5 of 9 Axin1fl/fl/Cre mice developed liver tumors with histologic features of HCC. CONCLUSIONS Hepatocytes from adult mice with conditional disruption of Axin1 in liver have a transcriptional profile that differs from that associated with loss of APC or constitutive activation of β-catenin. It might be similar to a proliferation profile observed in a subset of human HCCs with mutations in AXIN1. Axin1fl/fl mice could be a useful model of AXIN1-associated tumorigenesis and HCC.

In vivo and in vitro models for the therapeutic targeting of Wnt signaling using a Tet-OΔN89β-catenin system

Although significant progress has been made in understanding the importance of Wnt signaling in the initiation of colorectal cancer, less is known about responses that accompany the reversal of oncogenic Wnt signaling. The aim of this study was to analyze in vivo and in vitro responses to an ‘ideal’ Wnt pathway inhibitor as a model for the therapeutic targeting of the pathway. A tetracycline-inducible transgenic mouse model expressing truncated β-catenin (ΔN89β-catenin) that exhibited a strong intestinal hyperplasia was analyzed during the removal of oncogenic β-catenin expression both in 3D ‘crypt culture’ and in vivo. Oncogenic Wnt signaling was rapidly and completely reversed. The strongest inhibition of Wnt target gene expression occurred within 24 h of doxycycline removal at which time the target genes Ascl2, Axin2 and C-myc were downregulated to levels below that in the control intestine. In vitro, the small molecule Wnt inhibitor CCT036477 induced a response within 4 h of treatment. By 7 days following doxycycline withdrawal, gene expression, cell proliferation and tissue morphology were undistinguishable from control animals.In conclusion, these results demonstrate that the reversal of Wnt signaling by inhibitors should ideally be studied within hours of treatment. The reversible system described, involving medium throughput in vitro approaches and rapid in vivo responses, should allow the rapid advance of early stage compounds into efficacy models that are more usually considered later in the drug discovery pipeline.

WNT-3A regulates an Axin1/NRF2 complex that regulates antioxidant metabolism in hepatocytes.

AIMS Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of oxidant and xenobiotic metabolism, but it is unknown how it is regulated to provide basal expression of this defense system. Here, we studied the putative connection between NRF2 and the canonical WNT pathway, which modulates hepatocyte metabolism. RESULTS WNT-3A increased the levels of NRF2 and its transcriptional signature in mouse hepatocytes and HEK293T cells. The use of short interfering RNAs in hepatocytes and mouse embryonic fibroblasts which are deficient in the redox sensor Kelch-like ECH-associated protein 1 (KEAP1) indicated that WNT-3A activates NRF2 in a β-Catenin- and KEAP1-independent manner. WNT-3A stabilized NRF2 by preventing its GSK-3-dependent phosphorylation and subsequent SCF/β-TrCP-dependent ubiquitination and proteasomal degradation. Axin1 and NRF2 were physically associated in a protein complex that was regulated by WNT-3A, involving the central region of Axin1 and the Neh4/Neh5 domains of NRF2. Axin1 knockdown increased NRF2 protein levels, while Axin1 stabilization with Tankyrase inhibitors blocked WNT/NRF2 signaling. The relevance of this novel pathway was assessed in mice with a conditional deletion of Axin1 in the liver, which showed upregulation of the NRF2 signature in hepatocytes and disruption of liver zonation of antioxidant metabolism. INNOVATION NRF2 takes part in a protein complex with Axin1 that is regulated by the canonical WNT pathway. This new WNT-NRF2 axis controls the antioxidant metabolism of hepatocytes. CONCLUSION These results uncover the participation of NRF2 in a WNT-regulated signalosome that participates in basal maintenance of hepatic antioxidant metabolism.

Role of bacterial lipopolysaccharide in enhancing host immune response to Candida albicans.

Human infections involving yeast of the genus Candida often occur in the presence of bacteria, and, as such, it is important to understand how these bacteria influence innate host immunity towards Candida. Dectin-1 is a cell receptor of macrophages for Candida albicans recognition. The aim of this study was to examine dectin-1 expression by monocytes after stimulation with bacterial lipopolysaccharide (LPS), followed by heat-killed C. albicans (HKC). Freshly isolated human peripheral blood monocytes (PBMCs) and human monocytes cell line (THP-1) cells expressed low levels of dectin-1. Stimulation with LPS and GM-CSF/IL-4 was found to increase dectin-1 expression in both CD14+ human PBMC and THP-1 cells. Enhanced dectin-1 expression resulted in increased phagocytosis of Candida. When THP-1 cells were challenged only with HKC, detectable levels of IL-23 were not evident. However, challenge by LPS followed by varying concentrations of HKC resulted in increased IL-23 expression by THP-1 cells in HKC dose-dependent manner. Increased expression of IL-17 by PBMC also occurred after stimulation with Candida and LPS. In conclusion, bacterial LPS induces an enhanced immune response to Candida by immune cells, and this occurs through increasing dectin-1 expression.

Interactions between G-protein-coupled receptors and periplakin: a selective means to regulate G-protein activation

A substantial number of G-protein-coupled receptor-interacting proteins have been identified initially by the use of yeast two-hybrid screens. Using the C-terminal tail of both opioid receptors and the melanin concentrating hormone receptor-1 as bait, the actin and intermediate filament-binding protein periplakin was isolated. In each case, the site of interaction is within helix VIII of the receptor and periplakin limits agonist-mediated G-protein activation potentially by competing with G-protein for this region of the receptor.

CHAPTER 223 – Using Receptor-G-Protein Chimeras to Screen for Drugs

In the vast majority of cases analysis of GPCR-G protein chimeras takes place following expression in cell systems that also express endogenously the G protein of interest. A range of techniques has been developed to ensure that observed signals represent activation of the G protein within the chimera. Direct measures of the levels of expression of the α subunits of heterotrimeric G proteins indicate that they are generally in considerable excess over the levels of any particular G-protein-coupled receptor (GPCR) that might activate them. Despite this, an emerging strategy that has been used to address both basic questions on the details of GPCR-G protein interactions and to allow screens to be designed for agonist ligands at GPCRs has been to generate chimeric GPCR-G protein constructs that define a 1:1 stoichiometry of the two partner polypeptides. Such constructs provide a convenient means to assess the effects of mutations and polymorphisms in GPCRs on signal transduction effectiveness without altering the ratio of expression of GPCR to G protein. They have also allowed effective means of developing direct assays of GPCR-mediated guanine nucleotide exchange on G proteins of the G s and G q families that historically has been difficult to monitor. Such assays have allowed direct analysis of the extent of constitutive activity of different GPCRs and the detection of inverse agonists.