Interferon-induced transmembrane 3 binds osteopontin in vitro: expressed in vivo IFITM3 reduced OPN expression

Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein, which has an important role in tumour progression. We have shown that Wnt, Ets, AP-1, c-jun and beta-catenin/Lef-1/Tcf-1 stimulates OPN transcription in rat mammary carcinoma cells by binding to a specific promoter sequence. However, co-repressors of OPN have not been identified. In this study, we have used the bacterial two-hybrid system to isolate cDNA-encoding proteins that bind to OPN and modulate its role in malignant transformation. Using this approach we isolated interferon-induced transmembrane protein 3 gene (IFITM3) as a potential protein partner. We show that IFITM3 and OPN interact in vitro and in vivo and that IFITM3 reduces osteopontin (OPN) mRNA expression, possibly by affecting OPN mRNA stability. Stable transfection of IFITM3 inhibits OPN, which mediates anchorage-independent growth, cell adhesion and cell invasion. Northern blot analysis revealed an inverse mRNA expression pattern of IFITM3 and OPN in human mammary cell lines. Inhibition of IFITM3 by antisense RNA promoted OPN protein expression, enhanced cell invasion by parental benign non-invasive Rama 37 cells, indicating that the two proteins interact functionally as well. We also identified an IFITM3 DNA-binding domain, which interacts with OPN, deletion of which abolished its inhibitive effect on OPN. This work has shown for the first time that IFITM3 physically interacts with OPN and reduces OPN mRNA expression, which mediates cell adhesion, cell invasion, colony formation in soft agar and metastasis in a rat model system. Oncogene (2010) 29, 752-762; doi: 10.1038/onc.2009.379; published online 9 November 2009

Enhanced lymphocyte interferon (IFN)-γ responses in a PTEN mutation-negative Cowden disease kindred.

Identification of immune modifiers of inherited cancer syndromes may provide a rationale for preventive therapy. Cowden disease (CD) is a genetically heterogeneous inherited cancer syndrome that arises predominantly from germline phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation and increased phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signalling. However, many patients with classic CD diagnostic features are mutation-negative for PTEN (PTEN M-Neg). Interferon (IFN)-gamma can modulate the PI3K/mTOR pathway, but its association with PTEN M-Neg CD remains unclear. This study assessed IFN-gamma secretion by multi-colour flow cytometry in a CD kindred that was mutation-negative for PTEN and other known susceptibility genes. Because IFN-gamma responses may be regulated by killer cell immunoglobulin-like receptors (KIR) and respective human leucocyte antigen (HLA) ligands, KIR/HLA genotypes were also assessed. Activating treatments induced greater IFN-gamma secretion in PTEN M-Neg CD peripheral blood lymphocytes versus healthy controls. Increased frequency of activating KIR genes, potentially activating KIR/HLA compound genotypes and reduced frequency of inhibitory genotypes, were found in the PTEN M-Neg CD kindred. Differences of IFN-gamma secretion were observed among PTEN M-Neg CD patients with distinct KIR/HLA compound genotypes. Taken together, these findings show enhanced lymphocyte secretion of IFN-gamma that may influence the PI3K/mTOR CD causal molecular pathway in a PTEN mutation-negative CD kindred.

Altered Toll-like Receptor 2-mediated Endotoxin Tolerance Is Related to Diminished Interferon β Production

Induction of endotoxin tolerance leads to a reduced inflammatory response after repeated challenge by LPS and is important for resolution of inflammation and prevention of tissue damage. Enterobacterial LPS is recognized by the TLR4 signaling complex, whereas LPS of some non-enterobacterial organisms is capable of signaling independently of TLR4 utilizing TLR2-mediated signal transduction instead. In this study we report that Porphyromonas gingivalis LPS, a TLR2 agonist, fails to induce a fully endotoxin tolerant state in a human monocytic cell line (THP-1) and mouse bone marrow-derived macrophages. In contrast to significantly decreased production of human IL-8 and TNF-alpha and, in mice, keratinocyte-derived cytokine (KC), macrophage inflammatory protein-2 (MIP-2), and TNF-alpha after repeated challenge with Escherichia coli LPS, cells repeatedly exposed to P. gingivalis LPS responded by producing less TNF-alpha but sustained elevated secretion of IL-8, KC, and MIP-2. Furthermore, in endotoxin-tolerant cells, production of IL-8 is controlled at the signaling level and correlates well with NF-kappa B activation, whereas TNF-alpha expression is blocked at the gene transcription level. Interferon beta plays an important role in attenuation of chemokine expression in endotoxin-tolerized cells as shown in interferon regulatory factor-3 knock-out mice. In addition, human gingival fibroblasts, commonly known not to display LPS tolerance, were found to be tolerant to repeated challenge by LPS if pretreated with interferon beta. The data suggest that the inability of the LPS-TLR2 complex to induce full endotoxin tolerance in monocytes/macrophages is related to diminished production of interferon beta and may partly explain the involvement of these LPS isoforms in the pathogenesis of chronic inflammatory diseases.

Interferon regulatory factor (IRF) 3 is critical for the development of experimental autoimmune encephalomyelitis.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) is an animal model of autoimmune inflammatory demyelination that is mediated by Th1 and Th17 cells. The transcription factor interferon regulatory factor 3 (IRF3) is activated by pathogen recognition receptors and induces interferon-beta production.

METHODS: To determine the role of IRF3 in autoimmune inflammation, we immunised wild-type (WT) and irf3-/- mice to induce EAE. Splenocytes from WT and irf3-/- mice were also activated in vitro in Th17-polarising conditions.

RESULTS: Clinical signs of disease were significantly lower in mice lacking IRF3, with reduced Th1 and Th17 cells in the central nervous system. Peripheral T-cell responses were also diminished, including impaired proliferation and Th17 development in irf3-/- mice. Myelin-reactive CD4+ cells lacking IRF3 completely failed to transfer EAE in Th17-polarised models as did WT cells transferred into irf3-/- recipients. Furthermore, IRF3 deficiency in non-CD4+ cells conferred impairment of Th17 development in antigen-activated cultures.

CONCLUSION: These data show that IRF3 plays a crucial role in development of Th17 responses and EAE and warrants investigation in human multiple sclerosis.

Anti-TNF antibody-induced psoriasiform skin lesions in patients with inflammatory bowel disease are characterised by interferon-γ-expressing Th1 cells and IL-17A/IL-22-expressing Th17 cells and respond to anti-IL-12/IL-23 antibody treatment

Background We analysed incidence, predictors, histological features and specific treatment options of anti-tumour necrosis factor alpha (TNF-alpha) antibody-induced psoriasiform skin lesions in patients with inflammatory bowel diseases (IBD).

Design Patients with IBD were prospectively screened for anti-TNF-induced psoriasiform skin lesions. Patients were genotyped for IL23R and IL12B variants. Skin lesions were examined for infiltrating Th1 and Th17 cells. Patients with severe lesions were treated with the anti-interleukin (IL)-12/IL-23 p40 antibody ustekinumab.

Results Among 434 anti-TNF-treated patients with IBD, 21 (4.8%) developed psoriasiform skin lesions. Multiple logistic regression revealed smoking (p=0.007; OR 4.24, 95% CI 1.55 to 13.60) and an increased body mass index (p=0.029; OR 1.12, 95% CI 1.01 to 1.24) as main predictors for these lesions. Nine patients with Crohn's disease and with severe psoriasiform lesions and/or anti-TNF antibody-induced alopecia were successfully treated with the anti-p40-IL-12/IL-23 antibody ustekinumab (response rate 100%). Skin lesions were histologically characterised by infiltrates of IL-17A/IL-22-secreting T helper 17 (Th17) cells and interferon (IFN)-gamma-secreting Th1 cells and IFN-alpha-expressing cells. IL-17A expression was significantly stronger in patients requiring ustekinumab than in patients responding to topical therapy (p=0.001). IL23R genotyping suggests disease-modifying effects of rs11209026 (p.Arg381Gln) and rs7530511 (p.Leu310Pro) in patients requiring ustekinumab.

Conclusions New onset psoriasiform skin lesions develop in nearly 5% of anti-TNF-treated patients with IBD. We identified smoking as a main risk factor for developing these lesions. Anti-TNF-induced psoriasiform skin lesions are characterised by Th17 and Th1 cell infiltrates. The number of IL-17A-expressing T cells correlates with the severity of skin lesions. Anti-IL-12/IL23 antibody therapy is a highly effective therapy for these lesions.

Generation of replication-proficient influenza virus NS1 point mutants with interferon-hyperinducer phenotype

The NS1 protein of influenza A viruses is the dedicated viral interferon (IFN)-antagonist. Viruses lacking NS1 protein expression cannot multiply in normal cells but are viable in cells deficient in their ability to produce or respond to IFN. Here we report an unbiased mutagenesis approach to identify positions in the influenza A NS1 protein that modulate the IFN response upon infection. A random library of virus ribonucleoproteins containing circa 40 000 point mutants in NS1 were transferred to infectious virus and amplified in MDCK cells unable to respond to interferon. Viruses that activated the interferon (IFN) response were subsequently selected by their ability to induce expression of green-fluorescent protein (GFP) following infection of A549 cells bearing an IFN promoter-dependent GFP gene. Using this approach we isolated individual mutant viruses that replicate to high titers in IFN-compromised cells but, compared to wild type viruses, induced higher levels of IFN in IFN-competent cells and had a reduced capacity to counteract exogenous IFN. Most of these viruses contained not previously reported NS1 mutations within either the RNA-binding domain, the effector domain or the linker region between them. These results indicate that subtle alterations in NS1 can reduce its effectiveness as an IFN antagonist without affecting the intrinsic capacity of the virus to multiply. The general approach reported here may facilitate the generation of replication-proficient, IFN-inducing virus mutants, that potentially could be developed as attenuated vaccines against a variety of viruses.