Interferon-ε is a tumour suppressor and restricts ovarian cancer.

High-grade serous ovarian cancers have low survival rates because of their late presentation with extensive peritoneal metastases and frequent chemoresistance1, and require new treatments guided by novel insights into pathogenesis. Here we describe the intrinsic tumour-suppressive activities of interferon-ε (IFNε). IFNε is constitutively expressed in epithelial cells of the fallopian tube, the cell of origin of high-grade serous ovarian cancers, and is then lost during development of these tumours. We characterize its anti-tumour activity in several preclinical models: ovarian cancer patient-derived xenografts, orthotopic and disseminated syngeneic models, and tumour cell lines with or without mutations in Trp53 and Brca genes. We use manipulation of the IFNε receptor IFNAR1 in different cell compartments, differential exposure status to IFNε and global measures of IFN signalling to show that the mechanism of the anti-tumour activity of IFNε involves direct action on tumour cells and, crucially, activation of anti-tumour immunity. IFNε activated anti-tumour T and natural killer cells and prevented the accumulation and activation of myeloid-derived suppressor cells and regulatory T cells. Thus, we demonstrate that IFNε is an intrinsic tumour suppressor in the female reproductive tract whose activities in models of established and advanced ovarian cancer, distinct from other type I IFNs, are compelling indications of potential new therapeutic approaches for ovarian cancer.

The immune response to RNA suppresses nucleic acid synthesis by limiting ribose 5-phosphate.

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.

Design and Feasibility of an Intervention to Support Cancer Genetic Counselees in Informing their At-Risk Relatives.

Cancer genetic counselees receive individualized information regarding heightened risks and medical recommendations which is also relevant for their at-risk relatives. Unfortunately, counselees often insufficiently inform these relatives. We designed an intervention aimed at improving counselees' knowledge regarding which at-risk relatives to inform and what information to disclose, their motivation to disclose, and their self-efficacy. The intervention, offered by telephone by trained psychosocial workers, is based on the principles of Motivational Interviewing. Phase 1 of the intervention covers agenda setting, exploration, and evaluation, and phase 2 includes information provision, enhancing motivation and self-efficacy, and brainstorming for solutions to disseminate information within the family. Fidelity and acceptability of the intervention were assessed using recordings of intervention sessions and by counselee self-report. A total of 144 counselees participated. Psychosocial workers (n = 5) delivered the intervention largely as intended. Counselees highly appreciated the content of the intervention and the psychosocial workers who delivered the intervention. In the sessions, psychosocial workers provided additional and/or corrective information, and brainstorming for solutions was performed in 70 %. These results indicate that this intervention is feasible and warrants testing in clinical practice. For this, a randomized controlled trial is currently in progress to test the intervention's efficacy.

Distribution patterns of mucosally applied particles and characterization of the antigen presenting cells.

Mucosal application is the most common route of vaccination to prevent outbreaks of infectious diseases like Newcastle disease virus (NDV). To gain more knowledge about distribution and uptake of a vaccine after mucosal vaccination, we studied the distribution pattern of antigens after different mucosal routes of administration. Chickens were intranasally (i.n.), intratracheally (i.t.) or intraocularly (i.o.) inoculated with fluorescent beads and presence of beads in nasal-associated lymphoid tissue (NALT), Harderian gland (HG), conjunctiva-associated lymphoid tissue (CALT), trachea, lungs, air sacs, oesophagus and blood was characterized. The distribution patterns differed significantly between the three inoculation routes. After i.t. inoculation, the beads were mainly retrieved from trachea, NALT and lung. I.n. inoculation resulted in beads found mainly in NALT but detectable in all organs sampled. Finally, after i.o. inoculation, the beads were detected in NALT, CALT, HG and trachea. The highest number of beads was retrieved after i.n. inoculation. Development of novel vaccines requires a comprehensive knowledge of the mucosal immune system in birds in order to target vaccines appropriately and to provide efficient adjuvants. The NALT is likely important for the induction of mucosal immune responses. We therefore studied the phenotype of antigen-presenting cells isolated from NALT after i.n. inoculation with uncoated beads or with NDV-coated beads. Both types of beads were efficiently taken up and low numbers of bead+ cells were detected in all organs sampled. Inoculation with NDV-coated beads resulted in a preferential uptake by NALT antigen-presenting cells as indicated by high percentages of KUL01+-, MHC II+ and CD40+ bead+ cells.

Development of the Informing Relatives Inventory (IRI): Assessing Index Patients' Knowledge, Motivation and Self-Efficacy Regarding the Disclosure of Hereditary Cancer Risk Information to Relatives.

BACKGROUND: Despite the use of genetic services, counselees do not always share hereditary cancer information with at-risk relatives. Reasons for not informing relatives may be categorized as a lack of: knowledge, motivation, and/or self-efficacy. PURPOSE: This study aims to develop and test the psychometric properties of the Informing Relatives Inventory, a battery of instruments that intend to measure counselees' knowledge, motivation, and self-efficacy regarding the disclosure of hereditary cancer risk information to at-risk relatives. METHOD: Guided by the proposed conceptual framework, existing instruments were selected and new instruments were developed. We tested the instruments' acceptability, dimensionality, reliability, and criterion-related validity in consecutive index patients visiting the Clinical Genetics department with questions regarding hereditary breast and/or ovarian cancer or colon cancer. RESULTS: Data of 211 index patients were included (response rate = 62%). The Informing Relatives Inventory (IRI) assesses three barriers in disclosure representing seven domains. Instruments assessing index patients' (positive) motivation and self-efficacy were acceptable and reliable and suggested good criterion-related validity. Psychometric properties of instruments assessing index patients knowledge were disputable. These items were moderately accepted by index patients and the criterion-related validity was weaker. CONCLUSION: This study presents a first conceptual framework and associated inventory (IRI) that improves insight into index patients' barriers regarding the disclosure of genetic cancer information to at-risk relatives. Instruments assessing (positive) motivation and self-efficacy proved to be reliable measurements. Measuring index patients knowledge appeared to be more challenging. Further research is necessary to ensure IRI's dimensionality and sensitivity to change.

Uptake of particulate antigens in a nonmammalian lung: phenotypic and functional characterization of avian respiratory phagocytes using bacterial or viral antigens.

Major distinctive features of avian lungs are the absence of draining lymph nodes and alveoli and alveolar macrophages (MPhs). However, a large network of MPhs and dendritic cells (DCs) is present in the mucosa of the larger airways and in the linings of the parabronchi. For the modulation of respiratory tract immune responses, for example, by vaccination, a better understanding of Ag uptake in the chicken respiratory tract is needed. In this study, we provide detailed characterization of APCs in chicken lungs, including their functional in vivo activities as measured by the uptake of fluorescently labeled 1-µm beads that are coated with either LPS or inactivated avian influenza A virus (IAV) mimicking the uptake of bacterial or viral Ag. We identified different subsets of MPhs and DCs in chicken lungs, based on the expression of CD11, activation markers, and DEC205. In vivo uptake of LPS- and IAV-beads resulted in an increased percentage MHC class II(+) (MHC II(+)) cells and in the upregulation of CD40. The uptake of LPS-beads resulted in the upregulation of CD80 and MHC II on the cell surface, suggesting either uptake of LPS- and IAV-beads by different subsets of phagocytic cells or LPS-mediated differential activation. Differences in phagosomal acidification indicated that in chicken lungs the MHC II(+) and CD80(+) bead(+) cell population includes DCs and that a large proportion of beads was taken up by MPhs. LPS-bead(+) cells were present in BALT, suggesting local induction of immune responses. Collectively, we characterized the uptake of Ags by phagocytes in the respiratory tract of chickens.

Expression of Interferon Epsilon in Mucosal Epithelium is Regulated by Elf3.

Interferon epsilon (IFNε) is a unique type I interferon (IFN) that shows distinct constitutive expression in reproductive tract epithelium. Understanding how IFNε expression is regulated is critical for the mechanism of action in protecting the mucosa from infection. Combined computational and experimental investigation of the promoter of IFNε predicted transcription factor binding sites for the ETS family of transcription factors. We demonstrate here that Ifnε is regulated by Elf3, an epithelial restricted member of the ETS family. It is co-expressed with IFNε at the epithelium of uterus, lung and intestine, and we focused on regulation of IFNε expression in the uterus. Promoter reporter studies demonstrated that Elf3 was a strong driver of Ifnε expression; knockdown of Elf3 reduced expression levels of IFNε; Elf3 regulated Ifnε expression and chromatin immunoprecipitation (ChIP) confirmed the direct binding of Elf3 to the IFNε promoter. These data show that Elf3 is important in regulating protective mucosal immunity by driving constitutive expression of IFNε to protect mucosal tissues from infection in at least three organ systems.

Epithelially Restricted Interferon Epsilon Protects Against Colitis.

BACKGROUND & AIMS: Type I interferon (T1IFN) signalling is crucial for maintaining intestinal homeostasis. We previously found that the novel T1IFN, IFNε, is highly expressed by epithelial cells of the female reproductive tract, where it protects against pathogens. Its function has not been studied in the intestine. We hypothesize that IFNε is important in maintaining intestinal homeostasis. METHODS: We characterized IFNε expression in mouse and human intestine by immunostaining and studied its function in the dextran sulfate sodium (DSS) colitis model using both genetic knockouts and neutralizing antibody. RESULTS: We demonstrate that IFNε is expressed in human and mouse intestinal epithelium, and expression is lost in inflammation. Furthermore, we show that IFNε limits intestinal inflammation in mouse models. Regulatory T cell (Treg) frequencies were paradoxically decreased in DSS-treated IFNε-/- mice, suggesting a role for IFNε in maintaining the intestinal Treg compartment. Colitis was ameliorated by transfer of wild-type Tregs into IFNε-/- mice. This demonstrates that IFNε supports intestinal Treg function. CONCLUSIONS: Overall, we have shown IFNε expression in intestinal epithelium and its critical role in gut homeostasis. Given its known role in the female reproductive tract, we now show IFNε has a protective role across multiple mucosal surfaces.

Interferon-epsilon is a novel regulator of NK cell responses in the uterus.

The uterus is a unique mucosal site where immune responses are balanced to be permissive of a fetus, yet protective against infections. Regulation of natural killer (NK) cell responses in the uterus during infection is critical, yet no studies have identified uterine-specific factors that control NK cell responses in this immune-privileged site. We show that the constitutive expression of IFNε in the uterus plays a crucial role in promoting the accumulation, activation, and IFNγ production of NK cells in uterine tissue during Chlamydia infection. Uterine epithelial IFNε primes NK cell responses indirectly by increasing IL-15 production by local immune cells and directly by promoting the accumulation of a pre-pro-like NK cell progenitor population and activation of NK cells in the uterus. These findings demonstrate the unique features of this uterine-specific type I IFN and the mechanisms that underpin its major role in orchestrating innate immune cell protection against uterine infection.

Systemic distribution of different low pathogenic avian influenza (LPAI) viruses in chicken.

BACKGROUND: Since we were able to isolate viable virus from brain and lung of H7N1 low pathogenic avian influenza virus (LPAIV) infected chickens, we here examined the distribution of different LPAIV strains in chickens by measuring the viral AI RNA load in multiple organs. Subtypes of H5 (H5N1, H5N2), H7 (H7N1, H7N7) and H9 (H9N2), of chicken (H5N2, H7N1, H7N7, H9N2), or mallard (H5N1) origin were tested. The actual presence of viable virus was evaluated with virus isolation in organs of H7N7 inoculated chickens. FINDINGS: Viral RNA was found by PCR in lung, brain, intestine, peripheral blood mononuclear cells, heart, liver, kidney and spleen from chickens infected with chicken isolated LPAIV H5N2, H7N1, H7N7 or H9N2. H7N7 virus could be isolated from lung, ileum, heart, liver, kidney and spleen, but not from brain, which was in agreement with the data from the PCR. Infection with mallard isolated H5N1 LPAIV resulted in viral RNA detection in lung and peripheral blood mononuclear cells only. CONCLUSION: We speculate that chicken isolated LPAI viruses are spreading systemically in chicken, independently of the strain.

Differential expression of adenosine A3 receptors controls adenosine A2A receptor-mediated inhibition of TLR responses in microglia.

Microglia activation is a prominent feature in many neuroinflammatory disorders. Unrestrained activation can generate a chronic inflammatory environment that might lead to neurodegeneration and autoimmunity. Extracellular adenosine modulates cellular activation through adenosine receptor (ADORA)-mediated signaling. There are four ADORA subtypes that can either increase (A(2A) and A(2B) receptors) or decrease (A(1) and A(3) receptors) intracellular cyclic AMP levels. The expression pattern of the subtypes thus orchestrates the cellular response to extracellular adenosine. We have investigated the expression of ADORA subtypes in unstimulated and TLR-activated primary rhesus monkey microglia. Activation induced an up-regulation of A(2A) and a down-regulation of A(3) receptor (A(3)R) levels. The altered ADORA-expression pattern sensitized microglia to A(2A) receptor (A(2A)R)-mediated inhibition of subsequent TLR-induced cytokine responses. By using combinations of subtype-specific agonists and antagonists, we revealed that in unstimulated microglia, A(2A)R-mediated inhibitory signaling was effectively counteracted by A(3)R-mediated signaling. In activated microglia, the decrease in A(3)R-mediated signaling sensitized them to A(2A)R-mediated inhibitory signaling. We report a differential, activation state-specific expression of ADORA in microglia and uncover a role for A(3)R as dynamically regulated suppressors of A(2A)R-mediated inhibition of TLR-induced responses. This would suggest exploration of combinations of A(2A)R agonists and A(3)R antagonists to dampen microglial activation during chronic neuroinflammatory conditions.

Immunization with mannosylated peptide induces poor T cell effector functions despite enhanced antigen presentation.

In this study, we investigated the development of T cell responses in mice after administration of a mannosylated ovalbumin peptide (M-OVA(323-339)). Immunization with M-OVA(323-339) in complete adjuvant resulted in enhanced antigen presentation in draining lymph nodes. Monitoring the fate of CFSE-labeled ovalbumin peptide-specific TCR transgenic CD4(+) T cells revealed that immunization with M-OVA(323-339) induced normal clonal expansion, recirculation and CD62L expression of antigen-specific T cells in vivo. However, these T cells developed only poor effector functions, reflected by minimal IFN-gamma production, low IgG2a levels in serum and poor peptide-specific delayed-type hypersensitivity (DTH) responses. This diminished inflammatory response was associated with decreased infiltration of T cell blasts and macrophages. Importantly, also mice with functional effector T cells did not mount a robust DTH response after a challenge with M-OVA(323-339) in the ear, although their T cells responded normally to M-OVA(323-339) in vitro. In conclusion, mannosylated peptide induces proliferation of T cells with impaired T(h)1 cell effector functions and additionally abrogates the activity of pre-existing effector T cells.

How to support cancer genetics counselees in informing at-risk relatives? Lessons from a randomized controlled trial.

OBJECTIVE: In hereditary and familial cancer, counselees are requested to inform their at-risk relatives. We developed an intervention to support counselees in this task. METHODS: A randomized controlled trial was conducted aimed at improving cancer genetic counselees' i) knowledge, ii) motivation to disclose information, and ii) self-efficacy in this regard. Eligible participants were randomized to telephonic counseling (n = 148), or standard care (n = 157) and assessed at baseline, 1 week post-intervention, and 4 months after study enrolment. RESULTS: No between-group differences were found in participants' knowledge, motivation, and self-efficacy. Knowledge concerning which second-degree relatives to inform was lower compared to first-degree relatives. About 60% of the participants was of the opinion that they needed to inform more relatives than stated in their summary letter and only about 50% were correctly aware of which information to disclose. Of note, at baseline, almost 80% of the participants had already correctly informed their at-risk relatives. CONCLUSIONS: Since, unexpectedly, counselees already informed most of their relatives before the intervention was offered, efficacy of the intervention could not convincingly be determined. Counselees' knowledge about whom to inform about what is suboptimal. PRACTICE IMPLICATIONS: Future interventions should target a more homogeneous sample and address counselees' understanding and recall.

Informing relatives about their hereditary or familial cancer risk: study protocol for a randomized controlled trial.

BACKGROUND: Genetic counseling for hereditary breast or colon cancer has implications for both counselees and their relatives. Although counselees are encouraged by genetic counselors to disclose genetic cancer risk information, they do not always share this information with their at-risk relatives. Reasons for not informing relatives may be generally categorized as a lack of knowledge, motivation and/or self-efficacy. Presented here is the protocol of a randomized controlled trial that aims to establish the effectiveness of an intervention focused on supporting counselees in their disclosure of genetic cancer information to their relatives. METHODS/DESIGN: A multicenter randomized controlled trial with parallel group design will be used to compare the effects of an additional telephone counseling session performed by psychosocial workers to enhance the disclosure of genetic cancer information to at-risk relatives (intervention group) with a control group of standard care. Consecutive index patients with relatives at risk for hereditary or familial breast and/or ovarian cancer or colon cancer, are randomly assigned (block size: 8; 1:1 allocation ratio) to the intervention (n = 132) or control group (n = 132, standard care). Primary outcomes are counselees' knowledge, motivation and self-efficacy regarding informing their relatives. DISCUSSION: This intervention may prove important in supporting counselees to disclose hereditary and/or familial cancer risk information to at-risk relatives and may enable more at-risk relatives to make a well-informed decision regarding genetic services and/or screening. TRIAL REGISTRATION: This trial is registered in the Netherlands National Trial Register (NTR) with trial ID number NTR3745.

Regulation of macrophage and dendritic cell function by pathogens and through immunomodulation in the avian mucosa.

Macrophages (MPh) and dendritic cells (DC) are members of the mononuclear phagocyte system. In chickens, markers to distinguish MPh from DC are lacking, but whether MPh and DC can be distinguished in humans and mice is under debate, despite the availability of numerous markers. Mucosal MPh and DC are strategically located to ingest foreign antigens, suggesting they can rapidly respond to invading pathogens. This review addresses our current understanding of DC and MPh function, the receptors expressed by MPh and DC involved in pathogen recognition, and the responses of DC and MPh against respiratory and intestinal pathogens in the chicken. Furthermore, potential opportunities are described to modulate MPh and DC responses to enhance disease resistance, highlighting modulation through nutraceuticals and vaccination.

Glycans from avian influenza virus are recognized by chicken dendritic cells and are targets for the humoral immune response in chicken.

To increase our understanding of the interaction between avian influenza virus and its chicken host, we identified receptors for putative avian influenza virus (AIV) glycan determinants on chicken dendritic cells. Chicken dendritic cells (DCs) were found to recognize glycan determinants containing terminal αGalNAc, Galα1-3Gal, GlcNAcß1-4GlcNAcß1-4GlcNAcß (chitotriose) and Galα1-2Gal. Infection of chicken dendritic cells with either low pathogenic (LP) or highly pathogenic (HP) AIV results in elevated mRNA expression of homologs of the mouse C-type lectins DEC205 and macrophage mannose receptor (MMR), whereas expression levels of the human dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) homolog remained unchanged. Following uptake and subsequent presentation of avian influenza virus by DCs, adaptive immunity, including humoral immune responses are induced. We have investigated the antibody responses against virus glycan epitopes after avian influenza virus infection. Using glycan micro-array analysis we showed that chicken contained antibodies that predominantly recognize terminal Galα1-3Gal-R, chitotriose and Fucα1-2Galß1-4GlcNAc-R (H-type 2). After influenza-infection, glycan array analysis showed that both levels and repertoire of glycan-recognizing antibodies decreased. However, analysis of the sera by ELISA indicated that the levels of different isotypes of anti-glycan Abs against specific glycan antigens was increased after influenza-infection, suggesting that the presentation of the glycan antigens and iso-type of the Abs are critical parameters to take into account when measuring anti-glycan Abs. This novel approach in avian influenza research may contribute to the development of a broad spectrum vaccine and improves our mechanistic understanding of innate and adaptive responses to glycans.

Differential lung NK cell responses in avian influenza virus infected chickens correlate with pathogenicity.

Infection of chickens with low pathogenicity avian influenza (LPAI) virus results in mild clinical signs while infection with highly pathogenic avian influenza (HPAI) viruses causes death of the birds within 36-48 hours. Since natural killer (NK) cells have been shown to play an important role in influenza-specific immunity, we hypothesise that NK cells are involved in this difference in pathogenicity. To investigate this, the role of chicken NK-cells in LPAI virus infection was studied. Next activation of lung NK cells upon HPAI virus infection was analysed. Infection with a H9N2 LPAI virus resulted in the presence of viral RNA in the lungs which coincided with enhanced activation of lung NK cells. The presence of H5N1 viruses, measured by detection of viral RNA, did not induce activation of lung NK cells. This suggests that decreased NK-cell activation may be one of the mechanisms associated with the enhanced pathogenicity of H5N1 viruses.

Kinetics of the avian influenza-specific humoral responses in lung are indicative of local antibody production.

The role and kinetics of respiratory immunoglobulins in AIV infection has not been investigated. In this study we determined the numbers of both total antibody secreting cells (ASC) and virus-specific ASC in lung, spleen, blood and bone marrow (BM) following low-pathogenic AIV infection. Antiviral humoral immune responses were induced both locally in the lung and systemically in the spleen. Responses in the lung and BM preceded responses in the spleen and in blood, with virus-specific IgY ASC already detected in lung and BM from 1 week post-primary inoculation, indicating that respiratory immune responses are not induced in the spleen, but locally in the lung. ASC present in the blood of the lungs and co-isolated during lymphocyte isolation from the lungs have no major impact on the ASC detected in the lungs based on statistical correlation.

Induction of respiratory immune responses in the chicken; implications for development of mucosal avian influenza virus vaccines.

The risk and the size of an outbreak of avian influenza virus (AIV) could be restricted by vaccination of poultry. A vaccine used for rapid intervention during an AIV outbreak should be safe, highly effective after a single administration and suitable for mass application. In the case of AIV, aerosol vaccination using live virus is not desirable because of its zoonotic potential and because of the risk for virus reassortment. The rational design of novel mucosal-inactivated vaccines against AIV requires a comprehensive knowledge of the structure and function of the lung-associated immune system in birds in order to target vaccines appropriately and to design efficient mucosal adjuvants. This review addresses our current understanding of the induction of respiratory immune responses in the chicken. Furthermore, possible mucosal vaccination strategies for AIV are highlighted.