Critical Role for STAT4 Activation by Type 1 Interferons in the Interferon-Y Response to Viral Infection

Interferons (IFNs) are essential for host defense. Although the antiviral effects of the type 1 IFNs IFN- and IFN- (IFN-/) have been established, their immunoregulatory functions, especially their ability to regulate IFN- production, are poorly understood. Here we show that IFN-/ activate STAT4 directly (STAT, signal transducers and activators of transcription) and that this is required for IFN- production during viral infections of mice, in concert with T cell receptor-derived signals. In contrast, STAT1 appears to negatively regulate IFN-/ induction of IFN-. Thus, type 1 IFNs, in addition to interleukin-12, provide pathways for innate regulation of adaptive immunity, and their immunoregulatory functions are controlled by modulating the activity of individual STATs.

BRCA1 regulates the interferon gamma-mediated apoptotic response

BRCA1 is a tumor suppressor gene implicated in transcriptional regulation. We have generated cell lines with inducible expression of BRCA1 as a tool to identify downstream targets that may be important mediators of BRCA1 function. Oligonucleotide array-based expression profiling identified 11 previously described interferon regulated genes that were up-regulated following inducible expression of BRCA1. Northern blot analysis revealed that a subset of the identified targets including IRF-7, MxA, and ISG-54 were synergistically up-regulated by BRCA1 in the presence of interferon gamma (IFN-gamma) but not interferons alpha or beta. Importantly, IFN-gamma-mediated induction of IRF-7 and MxA was attenuated in the BRCA1 mutant cell line HCC1937, an effect that was rescued following reconstitution of exogenous wild type BRCA1 in these cells. Furthermore, reconstituted BRCA1 sensitized HCC1937 cells to IFN-gamma-induced apoptotic cell death. This study identifies BRCA1 as a component of the IFN-gamma-regulated signaling pathway and suggests that BRCA1 may play a role in the regulation of IFN-gamma-mediated apoptosis.

Polymorphims in the Interferon-y/Interleukin 26 Gene Region Contribute to Sex Bias in Susceptibility to Rheumatoid Arthritis

Objective:

To determine whether polymorphisms in the interferon-? (IFN?)/interleukin-26 (IL-26; formerly, AK155) gene cluster contribute to sex-based differential susceptibility to rheumatoid arthritis (RA).

Methods:

Four microsatellite markers, located in a 118-kb interval that contains both the IFN? and IL-26 genes on chromosome 12q15, were typed in 251 patients with RA and 198 unrelated healthy controls (all of whom lived in Northern Ireland) by means of polymerase chain reaction–based fragment analysis.

Results:

Marker D12S2510, which is located 3 kb 3' from the IL-26 gene, was significantly associated with RA in women (corrected P [Pcorr] = 0.008, 2 degrees of freedom [2 df]) but not in men (P = 0.99, 2 df). A 3-marker haplotype, IFNGCA*13;D12S2510*8;D12S2511*9, was inferred that showed significant underrepresentation in women with RA (odds ratio 0.50, 95% confidence interval 0.32–0.78; P = 0.002, Pcorr = 0.03) but not in men with RA.

Conclusion:

Our results demonstrate that common polymorphisms in the IFN?/IL-26 gene region may contribute to sex bias in susceptibility to RA, by distorting the propensity of female carriers versus male carriers to contract this disease. These results conform to our recent observations of a role for this gene cluster in sex-based differential susceptibility to another Th1-type inflammatory disease, multiple sclerosis.

Clinical Features of Dominant and Recessive Interferon γ Receptor 1 Deficiencies

Background Interferon ? receptor 1 (IFN? R1) deficiency is a primary immunodeficiency with allelic dominant and recessive mutations characterised clinically by severe infections with mycobacteria. We aimed to compare the clinical features of recessive and dominant IFN?R1 deficiencies. Methods We obtained data from a large cohort of patients worldwide. We assessed these people by medical histories, records, and genetic and immunological studies. Data were abstracted onto a standard form. Findings We identified 22 patients with recessive complete IFN?R1 deficiency and 38 with dominant partial deficiency. BCG and environmental mycobacteria were the most frequent pathogens. In recessive patients, 17 (77%) had environmental mycobacterial disease and all nine BCG-vaccinated patients had BCG disease. In dominant patients, 30 (79%) had environmental mycobacterial disease and 11 (73%) of 15 BCG-vaccinated patients had BCG disease. Compared with dominant patients, those with recessive deficiency were younger at onset of first environmental mycobacterial disease (mean 3·1 years [SD 2·5] vs 13·4 years [14·3], p=0·001), had more mycobacterial disease episodes (19 vs 8 per 100 person-years of observation, p=0·0001), had more severe mycobacterial disease (mean number of organs infected by Mycobacterium avium complex 4·1 [SD 0·8] vs 2·0 [1·1], p=0·004), had shorter mean disease-free intervals (1·6 years [SD 1·4] vs 7·2 years [7·6], p

Cellular FLICE-inhibitory protein regulates chemotherapy-induced apoptosis in breast cancer cells

Combination treatment regimens that include topoisomerase-II-targeted drugs, such as doxorubicin, are widely used in the treatment of breast cancer. Previously, we demonstrated that IFN-� and doxorubicin co-treatment synergistically induced apoptosis in MDA435 breast cancer cells in a STAT1-dependent manner. In this study, we found that this synergy was caspase 8-dependent. In addition, we found that IFN-γ down-regulated the expression of the caspase 8 inhibitor c-FLIP. Furthermore, IFN-� down-regulated c-FLIP in a manner that was dependent on the transcription factors STAT1 and IRF1. However, IFN-� had no effect on c-FLIP mRNA levels, indicating that c-FLIP was down-regulated at a post-transcriptional level following IFN-� treatment. Characterisation of the functional significance of c-FLIP modulation by siRNA gene silencing and stable over-expression studies, revealed it to be a key regulator of IFN-γ- and doxorubicin-induced apoptosis in MDA435 cells. Analysis of a panel of breast cancer cell lines indicated that c-FLIP was an important general determinant of doxorubicin- and IFN-�-induced apoptosis in breast cancer cells. Furthermore, c-FLIP gene silencing sensitised MDA435 cells to other chemotherapies, including etoposide, mitoxantrone and SN-38. These results suggest that c-FLIP plays a pivotal role in modulating drug-induced apoptosis in breast cancer cells.

Epidermal Langerhans Cells are Dispensable for Humoral and Cell-Mediated Immunity Elicited by Gene Gun Immunization.

Gene gun immunization, i.e., bombardment of skin with DNA-coated particles, is an efficient method for the administration of DNA vaccines. Direct transfection of APC or cross-presentation of exogenous Ag acquired from transfected nonimmune cells enables MHC-I-restricted activation of CD8(+) T cells. Additionally, MHC-II-restricted presentation of exogenous Ag activates CD4(+) Th cells. Being the principal APC in the epidermis, Langerhans cells (LC) seem ideal candidates to accomplish these functions. However, the dependence on LC of gene gun-induced immune reactions has not yet been demonstrated directly. This was primarily hampered by difficulties to discriminate the contributions of LC from those of other dermal dendritic cells. To address this problem, we have used Langerin-diphtheria toxin receptor knockin mice that allow for selective inducible ablation of LC. LC deficiency, even over the entire duration of experiments, did not affect any of the gene gun-induced immune functions examined, including proliferation of CD4(+) and CD8(+) T cells, IFN-gamma secretion by spleen cells, Ab production, CTL activity, and development of protective antitumor immunity.

THE IMPACT OF VIRUS INFECTION IN DEREGULATION OF CYTOKINE PRODUCTION UPON SECONDARY BACTERIAL INFECTION

Dendritic cells (DCs) secrete cytokines such as interleukin-23 (IL-23) when stimulated with certain Toll-like receptor (TLR) agonists and infected with pathogens such as P. aeruginosa. IL- 23 is a proinflammatory cytokine that plays a critical role in the proliferation and differentiation of the IL-17 producing Th17- CD4 T helper cells. The lack of efficient cytokine production from antigen-presenting cells, such as DCs, can impact CD4 differentiation and thus impair the immune responses against pathogens. Clearance of some bacterial infections, such as Klebsiella pneumonia and Listeria monocytogenes has been shown to be dependent on the induction of IL-23 and therefore, deregulation of these cytokines as a direct result of virus infection may impede immune responses to secondary infections. Here, an inhibition of TLR ligand or P. aeruginosa-induced IL- 23 expression in Lymphocytic Choriomeningitis Virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs) has been demonstrated, indicating that an important function of these cells is disrupted during virus/bacterial coinfection. While production of TNF-α was unaffected in LPS stimulated cells, TNF-α was significantly inhibited in bacterium infected cells by LCMV. Type I IFN in LPS or LCMV infected cell was not detected and therefore, ruling out the possibility of cytokine suppression by Type I IFN. The production of IL-10 was high in BMDCs infected with LCMV and stimulated with LPS or bacteria. Analysis of multiple cytokines produced in this coinfection model demonstrated that LCMV infection impacts specific cytokine production upon LPS or bacterium infection, which may be important for bacterial clearance. This data is important for future immunotherapy use in viral/bacterial coinfection scenarios.

Tumor immunotherapy by epicutaneous immunization requires langerhans cells.

A role for Langerhans cells (LC) in the induction of immune responses in the skin has yet to be conclusively demonstrated. We used skin immunization with OVA protein to induce immune responses against OVA-expressing melanoma cells. Mice injected with OVA-specific CD8(+) T cells and immunized with OVA onto barrier-disrupted skin had increased numbers of CD8(+) T cells in the blood that produced IFN-gamma and killed target cells. These mice generated accelerated cytotoxic responses after secondary immunization with OVA. Prophylactic or therapeutic immunization with OVA onto barrier-disrupted skin inhibited the growth of B16.OVA tumors. LC played a critical role in the immunization process because depletion of LC at the time of skin immunization dramatically reduced the tumor-protective effect. The topically applied Ag was presented by skin-derived LC in draining lymph nodes to CD8(+) T cells. Thus, targeting of tumor Ags to LC in vivo is an effective strategy for tumor immunotherapy.

Langerhans Cells Prime IL-17-Producing T Cells and Dampen Genital Cytotoxic Responses following Mucosal Immunization

Langerhans cells (LCs) are dendritic cells (DCs) localized in stratified epithelia, such as those overlaying skin, buccal mucosa, and vagina. The contribution of LCs to the promotion or control of immunity initiated at epithelial sites remains debated. We report in this paper that an immunogen comprising OVA linked to the B subunit of cholera toxin, used as delivery vector, was efficient to generate CTLs after vaginal immunization. Using Lang-EGFP mice, we evaluated the contribution of distinct DC subsets to the generation of CD4 and CD8 T cell responses. We demonstrate that the vaginal epithelium, unlike the skin epidermis, includes a minor population of LCs and a major subset of langerin(-) DCs. Intravaginally administered Ag is taken up by LCs and langerin(-) DCs and carried up to draining lymph nodes, where both subsets prime CD8 T cells, unlike blood-derived DCs, although with distinct capabilities. LCs prime CD8 T cells with a cytokine profile dominated by IL-17, whereas Lang(-) DCs induce IFN-gamma-producing T cells. Using Lang-DTR-EGFP mice to ensure a transient ablation of LCs, we found that these cells not only are dispensable for the generation of genital CTL responses but also downregulate these responses, by a mechanism that may involve IL-10 and IL-17 cytokines. This finding has implications for the development of mucosal vaccines and immunotherapeutic strategies designed for the targeting of DCs.

Langerin(+) Dermal Dendritic Cells Are Critical for CD8(+) T Cell Activation and IgH gamma-1 Class Switching in Response to Gene Gun Vaccines

The C-type lectin langerin/CD207 was originally discovered as a specific marker for epidermal Langerhans cells (LC). Recently, additional and distinct subsets of langerin(+) dendritic cells (DC) have been identified in lymph nodes and peripheral tissues of mice. Although the role of LC for immune activation or modulation is now being discussed controversially, other langerin(+) DC appear crucial for protective immunity in a growing set of infection and vaccination models. In knock-in mice that express the human diphtheria toxin receptor under control of the langerin promoter, injection of diphtheria toxin ablates LC for several weeks whereas other langerin(+) DC subsets are replenished within just a few days. Thus, by careful timing of diphtheria toxin injections selective states of deficiency in either LC only or all langerin(+) cells can be established. Taking advantage of this system, we found that, unlike selective LC deficiency, ablation of all langerin(+) DC abrogated the activation of IFN-gamma producing and cytolytic CD8(+) T cells after gene gun vaccination. Moreover, we identified migratory langerin(+) dermal DC as the subset that directly activated CD8(+) T cells in lymph nodes. Langerin(+) DC were also critical for IgG1 but not IgG2a Ab induction, suggesting differential polarization of CD4(+) T helper cells by langerin(+) or langerin-negative DC, respectively. In contrast, protein vaccines administered with various adjuvants induced IgG1 independently of langerin(+) DC. Taken together, these findings reflect a highly specialized division of labor between different DC subsets both with respect to Ag encounter as well as downstream processes of immune activation. The Journal of Immunology, 2011, 186: 1377-1383.

Immunological Interactions between 2 Common Pathogens, Th1-Inducing Protozoan Toxoplasma gondii and the Th2-Inducing Helminth Fasciola hepatica

Background: The nature of the immune response to infection is dependent on the type of infecting organism. Intracellular organisms such as Toxoplasma gondii stimulate a Th1-driven response associated with production of IL-12, IFN-gamma, nitric oxide and IgG2a antibodies and classical activation of macrophages. In contrast, extracellular helminths such as Fasciola hepatica induce Th2 responses characterised by the production of IL-4, IL-5, IL-10 and IgG1 antibodies and alternative activation of macrophages. As co-infections with these types of parasites commonly exist in the field it is relevant to examine how the various facets of the immune responses induced by each may influence or counter-regulate that of the other.

Intravenous tolerance effectively overcomes enhanced pro-inflammatory responses and EAE severity in the absence of IL-12 receptor signaling

Intravenous (i.v.) administration of autoantigen effectively induces Ag-specific tolerance against experimental autoimmune encephalomyelitis (EAE). We and others have shown enhanced EAE severity in mice lacking IL-12 or its receptor, strongly suggesting an immunoregulatory effect of IL-12 signaling. To examine the role of IL-12 responsiveness in autoantigen-induced tolerance in EAE, we administered autoantigen i.v. in two distinct treatment regimes to wildtype and IL-12Rβ2(-/-) mice, immunized to develop EAE. Administration at the induction phase suppressed EAE in wildtype and IL-12Rβ2(-/-) mice however the effect was somewhat less potent in the absence of IL-12Rβ2. Expression of pro-inflammatory cytokines such as IFN-γ, IL-17 and IL-2, was inhibited in wild-type tolerized mice but less so in IL-12Rβ2(-/-) mice. I.v. antigen was also effective in suppressing disease in both genotypes when given during the clinical phase of disease with similar CNS inflammation, demyelination and peripheral inflammatory cytokine profiles observed in both genotypes. There was however a mild impact of a lack of IL-12 signaling on Treg induction during tolerance induction compared to WT mice in this treatment regime. These findings show that the enhanced severity of EAE that occurs in the absence of IL-12 signaling can be effectively overcome by i.v. autoantigen, indicating that this therapeutic effect is not primarily mediated by IL-12 and that i.v. tolerance could be a powerful approach in suppressing severe and aggressive MS.

Regulation of Foxp3+ Inducible Regulatory T Cell Stability by SOCS2

Suppressor of cytokine signaling (SOCS) proteins are key regulators of CD4+ T cell differentiation, and in particular, we have recently shown that SOCS2 inhibits the development of Th2 cells and allergic immune responses. Interestingly, transcriptome analyses have identified SOCS2 as being preferentially expressed in both natural regulatory T cells (Tregs) and inducible Tregs (iTregs); however, the role of SOCS2 in Foxp3+ Treg function or development has not been fully elucidated. In this study, we show that despite having no effect on natural Treg development or function, SOCS2 is highly expressed in iTregs and required for the stable expression of Foxp3 in iTregs in vitro and in vivo. Indeed, SOCS2-deficient CD4+ T cells upregulated Foxp3 following in vitro TGF-ß stimulation, but failed to maintain stable expression of Foxp3. Moreover, in vivo generation of iTregs following OVA feeding was impaired in the absence of SOCS2 and could be rescued in the presence of IL-4 neutralizing Ab. Following IL-4 stimulation, SOCS2-deficient Foxp3+ iTregs secreted elevated IFN-? and IL-13 levels and displayed enhanced STAT6 phosphorylation. Therefore, we propose that SOCS2 regulates iTreg stability by downregulating IL-4 signaling. Moreover, SOCS2 is essential to maintain the anti-inflammatory phenotype of iTregs by preventing the secretion of proinflammatory cytokines. Collectively, these results suggest that SOCS2 may prevent IL-4-induced Foxp3+ iTreg instability. Foxp3+ iTregs are key regulators of immune responses at mucosal surfaces; therefore, this dual role of SOCS2 in both Th2 and Foxp3+ iTregs reinforces SOCS2 as a potential therapeutic target for Th2-biased diseases.

Immunotoxicity of aflatoxin B1: Impairment of the cell-mediated response to vaccine antigen and modulation of cytokine expression

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus or A. parasiticus, is a frequent contaminant of food and feed. This toxin is hepatotoxic and immunotoxic. The present study analyzed in pigs the influence of AFB1 on humoral and cellular responses, and investigated whether the immunomodulation observed is produced through interference with cytokine expression. For 28 days, pigs were fed a control diet or a diet contaminated with 385, 867 or 1807 mu g pure AFB1/kg feed. At days 4 and 15, pigs were vaccinated with ovalbumin. AFB1 exposure, confirmed by an observed dose-response in blood aflatoxin-albumin adduct, had no major effect on humoral immunity as measured by plasma concentrations of total IgA, IgG and IgM and of anti-ovalbumin IgG. Toxin exposure did not impair the mitogenic response of lymphocytes but delayed and decreased their specific proliferation in response to the vaccine antigen, suggesting impaired lymphocyte activation in pigs exposed to AFB1. The expression level of pro-inflammatory (TNF-alpha, IL-1 beta, IL-6, IFN-gamma) and regulatory (IL-10) cytokines was assessed by real-time PCR in spleen. A significant up-regulation of all 5 cytokines was observed in spleen from pigs exposed to the highest dose of AFB1. In pigs exposed to the medium dose, IL-6 expression was increased and a trend towards increased IFN-gamma and IL-10 was observed. In addition we demonstrate that IL-6 impaired in vitro the antigenic- but not the mitogenic-induced proliferation of lymphocytes from control pigs vaccinated with ovalbumin. These results indicate that AFB1 dietary exposure decreases cell-mediated immunity while inducing an inflammatory response. These impairments in the immune response could participate in failure of vaccination protocols and increased susceptibility to infections described in pigs exposed to AFB1. (C) 2008 Elsevier Inc. All rights reserved.

Galectin-9 protein expression in endothelial cells is positively regulated by histone deacetylase 3

Galectin-9 expression in endothelial cells can be induced in response to inflammation. However, the mechanism of its expression remains unclear. In this study, we found that interferon-? (IFN-?) induced galectin-9 expression in human endothelial cells in a time-dependent manner, which coincided with the activation of histone deacetylase (HDAC). When endothelial cells were treated with the HDAC3 inhibitor, apicidin, or shRNA-HDAC3 knockdown, IFN-?-induced galectin-9 expression was abolished. Overexpression of HDAC3 induced the interaction between phosphoinositol 3-kinase (PI3K) and IFN response factor 3 (IRF3), leading to IRF3 phosphorylation, nuclear translocation, and galectin-9 expression. HDAC3 functioned as a scaffold protein for PI3K/IRF3 interaction. In addition to galectin-9 expression, IFN-? also induced galectin-9 location onto plasma membrane, which was HDAC3-independent. Importantly, HDAC3 was essential for the constitutive transcription of PI3K and IRF3, which might be responsible for the basal level of galectin-9 expression. The phosphorylation of IRF3 was essential for galectin-9 expression. This study provides new evidence that HDAC3 regulates galectin-9 expression in endothelial cells via interaction with PI3K-IRF3 signal pathway.