Strong IFN Research Articles

A novel subpopulation of monocytes with a strong interferon signature indicated by SIGLEC-1 is present in patients with in recent-onset type 1 diabetes.

Type 1 diabetes is a T cell-mediated autoimmune disease characterised by pancreatic beta cell destruction. In this study, we explored the pathogenic immune responses in initiation of type 1 diabetes and new immunological targets for type 1 diabetes prevention and treatment. We obtained peripheral blood samples from four individuals with newly diagnosed latent autoimmune diabetes in adults (LADA) and from four healthy control participants. Single-cell RNA-sequencing (scRNA-seq) was performed on peripheral blood mononuclear cells to uncover transcriptomic profiles of early LADA. Validation was performed through flow cytometry in a cohort comprising 54 LADA, 17 adult-onset type 2 diabetes, and 26 healthy adults, matched using propensity score matching (PSM) based on age and sex. A similar PSM method matched 15 paediatric type 1 diabetes patients with 15 healthy children. Further flow cytometry analysis was performed in both peripheral blood and pancreatic tissues of non-obese diabetic (NOD) mice. Additionally, cell adoptive transfer and clearance assays were performed in NOD mice to explore the role of this monocyte subset in islet inflammation and onset of type 1 diabetes. The scRNA-seq data showed that upregulated genes in peripheral T cells and monocytes from early-onset LADA patients were primarily enriched in the IFN signalling pathway. A new cluster of classical monocytes (cluster4) was identified, and the proportion of this cluster was significantly increased in individuals with LADA compared with healthy control individuals (11.93% vs 5.93%, p=0.017) and that exhibited a strong IFN signature marked by SIGLEC-1 (encoding sialoadhesin). These SIGLEC-1+ monocytes expressed high levels of genes encoding C-C chemokine receptors 1 or 2, as well as genes for chemoattractants for T cells and natural killer cells. They also showed relatively low levels of genes for co-stimulatory and HLA molecules. Flow cytometry analysis verified the elevated levels of SIGLEC-1+ monocytes in the peripheral blood of participants with LADA and paediatric type 1 diabetes compared with healthy control participants and those with type 2 diabetes. Interestingly, the proportion of SIGLEC-1+ monocytes positively correlated with disease activity and negatively with disease duration in the LADA patients. In NOD mice, the proportion of SIGLEC-1+ monocytes in the peripheral blood was highest at the age of 6 weeks (16.88%), while the peak occurred at 12 weeks in pancreatic tissues (23.65%). Adoptive transfer experiments revealed a significant acceleration in diabetes onset in the SIGLEC-1+ group compared with the SIGLEC-1- or saline control group. Our study identified a novel group of SIGLEC-1+ monocytes that may serve as an important indicator for early diagnosis, activity assessment and monitoring of therapeutic efficacy in type 1 diabetes, and may also be a novel target for preventing and treating type 1 diabetes. RNA-seq data have been deposited in the GSA human database ( https://ngdc.cncb.ac.cn/gsa-human/ ) under accession number HRA003649.

TBK1 upregulates the interferon response against virus by the TBK1-IRF3/7 axis in yellow catfish (Pelteobagrus fulvidraco)

Yellow catfish (Pelteobagrus fulvidraco) is an important economic species of freshwater fish, widely distributed in China. Recently, viral diseases of yellow catfish have been identified in Chian (Hubei province), arising more attention to the viral immunity in P. fulvidraco. Tumor necrosis factor (TNF) receptor-associated factor NF-κB activator (TANK)-binding kinase 1 (TBK1) plays an essential role in IFN production and innate antiviral immunity. In the present study, we characterized the P. fulvidraco TBK1 (PfTBK1) and reported its function in interferon response. The full-length open reading frame (ORF) is 2184 bp encoding a protein with 727 amino acids, which is composed of four conserved domains, including KD, ULD, CCD1, and CCD2, similar to TBK1 in other species. Pftbk1 was widely expressed in all detected tissues by qPCR and was not inducible by the spring viremia of carp virus (SVCV), a single-strand RNA virus. In addition, the cellular distribution indicated that PfTBK1 was only located in the cytoplasm. Moreover, PfTBK1 induced strong IFN promoter activities through the Jak-stat pathway, and PfTBK1 interacted with and significantly phosphorylated IFN regulatory factor 3/7 (IRF3/7) in P. fulvidraco, promoting the nuclear translocation of pfIRF3 and PfIRF7, and PfTBK1 upregulated IFN response by PfTBK1-PfIRF3/7 axis. Above all, PfTBK1 triggered IFN response and strongly inhibited the replication of SVCV in EPC cells through induction of IFN downstream IFN-stimulated genes (ISGs). Summarily, this work reveals that PfTBK1 plays a positive regulatory role in IFN induction through the TBK1-IRF3/7 axis, laying a foundation for further exploring the molecular mechanism of the antiviral process in P. fulvidraco.

In vivo CRISPR screens reveal the landscape of immune evasion pathways across cancer.

The immune system can eliminate tumors, but checkpoints enable immune escape. Here, we identify immune evasion mechanisms using genome-scale in vivo CRISPR screens across cancer models treated with immune checkpoint blockade (ICB). We identify immune evasion genes and important immune inhibitory checkpoints conserved across cancers, including the non-classical major histocompatibility complex class I (MHC class I) molecule Qa-1b/HLA-E. Surprisingly, loss of tumor interferon-γ (IFNγ) signaling sensitizes many models to immunity. The immune inhibitory effects of tumor IFN sensing are mediated through two mechanisms. First, tumor upregulation of classical MHC class I inhibits natural killer cells. Second, IFN-induced expression of Qa-1b inhibits CD8+ T cells via the NKG2A/CD94 receptor, which is induced by ICB. Finally, we show that strong IFN signatures are associated with poor response to ICB in individuals with renal cell carcinoma or melanoma. This study reveals that IFN-mediated upregulation of classical and non-classical MHC class I inhibitory checkpoints can facilitate immune escape.

Th2 to Th1 Transition Is Required for Induction of Skin Lesions in an Inducible and Recurrent Murine Model of Cutaneous Lupus-Like Inflammation.

Cutaneous lupus erythematosus (CLE) is an autoimmune skin disease characterized by a strong IFN signature, normally associated with type I IFNs. However, increasing evidence points to an additional role for IFNγ, or at least a pathogenic T effector subset dependent on IFNγ, for disease progression. Nevertheless, Th2 effector subsets have also been implicated in CLE. We have now assessed the role of specific T cell subsets in the initiation and persistence of skin disease using a T cell-inducible murine model of CLE, dependent on KJ1-26 T cell recognition of an ovalbumin fusion protein. We found that only Th2-skewed cells, and not Th1-skewed cells, induced the development of skin lesions. However, we provide strong evidence that the Th2 disease-initiating cells convert to a more Th1-like functional phenotype in vivo by the time the skin lesions are apparent. This phenotype is maintained and potentiates over time, as T cells isolated from the skin, following a second induction of self-antigen, expressed more IFN-γ than T cells isolated at the time of the initial response. Transcriptional analysis identified additional changes in the KJ1-26 T cells at four weeks post injection, with higher expression levels of interferon stimulated genes (ISGs) including CXCL9, IRF5, IFIH1, and MX1. Further, injection of IFN-γ-/- T cells faied to induce skin disease in mice. We concluded that Th2 cells trigger skin lesion formation in CLE, and these cells switch to a Th1-like phenotype in the context of a TLR7-driven immune environment that is stable within the T cell memory compartment.

Genetically programmed alternative splicing of NEMO mediates an autoinflammatory disease phenotype.

Host defense and inflammation are regulated by the NF-κB essential modulator (NEMO), a scaffolding protein with a broad immune cell and tissue expression profile. Hypomorphic mutations in inhibitor of NF-κB kinase regulatory subunit gamma (IKBKG) encoding NEMO typically present with immunodeficiency. Here, we characterized a pediatric autoinflammatory syndrome in 3 unrelated male patients with distinct X-linked IKBKG germline mutations that led to overexpression of a NEMO protein isoform lacking the domain encoded by exon 5 (NEMO-Δex5). This isoform failed to associate with TANK binding kinase 1 (TBK1), and dermal fibroblasts from affected patients activated NF-κB in response to TNF but not TLR3 or RIG-I–like receptor (RLR) stimulation when isoform levels were high. By contrast, T cells, monocytes, and macrophages that expressed NEMO-Δex5 exhibited increased NF-κB activation and IFN production, and blood cells from these patients expressed a strong IFN and NF-κB transcriptional signature. Immune cells and TNF-stimulated dermal fibroblasts upregulated the inducible IKK protein (IKKi) that was stabilized by NEMO-Δex5, promoting type I IFN induction and antiviral responses. These data revealed how IKBKG mutations that lead to alternative splicing of skipping exon 5 cause a clinical phenotype we have named NEMO deleted exon 5 autoinflammatory syndrome (NDAS), distinct from the immune deficiency syndrome resulting from loss-of-function IKBKG mutations.

Reduction of some viral protein's gene expression of recombinant influenza A/H1N1-PR8 virus upon treatment with Punica granatum crude extract

BackgroundIt was we known that Flu infections have a place with the class Orthomyxoviridae furthermore, three distinct sorts (A, B and C sort infections) are known. Among these, influenza viral infections display the broadest host range (birds, people, and different well evolved creatures) also, can develop into profoundly pathogenic strains. So, the scientists tried to find a way to restrict this invasion which can cause certain death to human raise. Subjects & methodsDifferent concentrations of Punica granatum extract (2.5, 5, 10 and 20 μg/ml) prepared from the stock solution were used against human influenza A/Puerto Rico/8/34 H1N1 (IAV/PR8) to test its ability to prevent the virus to grow on MDCKII and A549 cells to check the effect of this plant extract on viral replication titter using plaque assay, gene expression of some viral proteins like non-structural protein NS1, nucleoprotein NP and Protein basic PB1 using reverse transcriptase quantitative real time polymerase chain reaction. Also, localization of NP protein was checked using immune-florescent staining technique. ResultsOur finding showed that there was significant decrease in IAV/PR8 ability to grow and replicate in MDCKII cells at multiplicity of infection (MOI) in both of 0.1 and 0.01 MOI. this finding was confirmed upon checking of expression of NS1, PB1 and NP proteins of IAV/PR8 that were reduced significantly at 10 and 20 μg/ml. Interestingly the localization of viral NP protein affected negatively upon the treatment of cells with elected concentration of plant extract by the weak localization outside the nucleus. ConclusionMethanolic extract of Punica granatum produced a strong anti-viral activity against IAV/PR8 replication ability and that was due to the severe effect on viral proteins expression especially on PB1 and NP which responsible for viral protein synthesis and NS1 protein which is a strong IFN antagonistic protein thus; weakening its replication ability in different cell lines.

The Transient IFN Response and the Delay of Adaptive Immunity Feature the Severity of COVID-19.

COVID-19 patients show heterogeneous and dynamic immune features which determine the clinical outcome. Here, we built a single-cell RNA sequencing (scRNA-seq) dataset for dissecting these complicated immune responses through a longitudinal survey of COVID-19 patients with various categories of outcomes. The data reveals a highly fluctuating peripheral immune landscape in severe COVID-19, whereas the one in asymptomatic/mild COVID-19 is relatively steady. Then, the perturbed immune landscape in peripheral blood returned to normal state in those recovered from severe COVID-19. Importantly, the imbalance of the excessively strong innate immune response and delayed adaptive immunity in the early stage of viral infection accelerates the progression of the disease, indicated by a transient strong IFN response and weak T/B-cell specific response. The proportion of abnormal monocytes appeared early and rose further throughout the severe disease. Our data indicate that a dynamic immune landscape is associated with the progression and recovery of severe COVID-19, and have provided multiple immune biomarkers for early warning of severe COVID-19.

Differential Responsiveness of Monocyte and Macrophage Subsets to Interferon.

Peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients exhibit a gene expression program (interferon [IFN] signature) that is attributed to overproduction of type I IFNs by plasmacytoid dendritic cells. Type I IFNs have been thought to play a role in the pathogenesis of SLE. This study was undertaken to examine an unexpected influence of monocyte/macrophages on the IFN signature. Proinflammatory (classic) and antiinflammatory (nonclassic) monocyte/macrophages were sorted from mice and analyzed by RNA sequencing and quantitative polymerase chain reaction (qPCR). Type I IFN-α/β/ω receptor (IFNAR-1) expression was determined by qPCR and flow cytometry. Macrophages were stimulated in vitro with IFNα, and pSTAT1was measured. Transcriptional profiling of peritoneal macrophages from mice with pristane-induced SLE unexpectedly indicated a strong IFN signature in classic, but not nonclassic, monocyte/macrophages exposed to the same type I IFN concentrations. Ifnar1 messenger RNA and IFNAR surface staining were higher in classic monocyte/macrophages versus nonclassic monocyte/macrophages (P < 0.0001 and P < 0.05, respectively, by Student's t-test). Nonclassic monocyte/macrophages were also relatively insensitive to IFNα-driven STAT1 phosphorylation. Humans exhibited a similar pattern: higher IFNAR expression (P < 0.0001 by Student's t-test) and IFNα-stimulated gene expression (P < 0.01 by paired Wilcoxon's rank sum test) in classic monocyte/macrophages and lower levels in nonclassic monocyte/macrophages. This study revealed that the relative abundance of different monocyte/macrophage subsets helps determine the magnitude of the IFN signature. Responsiveness to IFNα signaling reflects differences in IFNAR expression in classic (high IFNAR) compared to nonclassic (low IFNAR) monocyte/macrophages. Thus, the IFN signature depends on both type I IFN production and the responsiveness of monocyte/macrophages to IFNAR signaling.

Unique Sjögren's syndrome patient subsets defined by molecular features.

To address heterogeneity complicating primary SS (pSS) clinical trials, research and care by characterizing and clustering patients by their molecular phenotypes. pSS patients met American-European Consensus Group classification criteria and had at least one systemic manifestation and stimulated salivary flow of ⩾0.1 ml/min. Correlated transcriptional modules were derived from gene expression microarray data from blood (n = 47 with appropriate samples). Patients were clustered based on this molecular information using an unbiased random forest modelling approach. In addition, multiplex, bead-based assays and ELISAs were used to assess 30 serum cytokines, chemokines and soluble receptors. Eleven autoantibodies, including anti-Ro/SSA and anti-La/SSB, were measured by Bio-Rad Bioplex 2200. Transcriptional modules distinguished three clusters of pSS patients. Cluster 1 showed no significant elevation of IFN or inflammation modules. Cluster 2 showed strong IFN and inflammation modular network signatures, as well as high plasma protein levels of IP-10/CXCL10, MIG/CXCL9, BLyS (BAFF) and LIGHT. Cluster 3 samples exhibited moderately elevated IFN modules, but with suppressed inflammatory modules, increased IP-10/CXCL10 and B cell-attracting chemokine 1/CXCL13 and trends toward increased MIG/CXCL9, IL-1α, and IL-21. Anti-Ro/SSA and anti-La/SSB were present in all three clusters. Molecular profiles encompassing IFN, inflammation and other signatures can be used to separate patients with pSS into distinct clusters. In the future, such profiles may inform patient selection for clinical trials and guide treatment decisions.

Recombinant fusion protein of Heparin-Binding Hemagglutinin Adhesin and Fibronectin Attachment Protein (rHBHA-FAP) of Mycobacterium avium subsp. paratuberculosis elicits a strong gamma interferon response in peripheral blood mononuclear cell culture

BackgroundMycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease in all ruminants worldwide. Economic problems in dairy cattle and sheep industries, public health concern, persistence of MAP in the environment and lack of effective vaccines mentioned necessity of research about various antigens to introduce as vaccine candidates. Based on MAP pathogenesis, it seems that research about the production of new recombinant proteins to stimulate cell-mediated immunity is helpful. This study describes successful expression and purification of a chimeric fusion protein which consists of Heparin-Binding Hemagglutinin Adhesin (HBHA) and high antigenic region of Fibronectin Attachment Protein (FAP-P). Triggered antigen-specific IFN-γ response of isolated PBMCs from immunized goats to rHBHA-FAP and all crude proteins of MAP (PPD), was measured by ELISA.ResultsSignificant increases were observed in the IFN-γ production level of peripheral blood mononuclear cells (PBMCs) stimulated by constructed chimeric protein from rHBHA-FAP and PPD vaccinated goats. Antigen-specific gamma interferon (IFN-γ) secretion in positive group (immunized by PPD) against rHBHA-FAP and test group (immunized by rHBHA-FAP) against PPD, also statistically insignificant rises between stimulation with rHBHA-FAP and PPD, suggested the potential and specificity of our chimeric protein to stimulate cell mediated immunity against MAP.ConclusionsCollectively, these results demonstrate that rHBHA-FAP elicits a strong IFN-γ production in PBMC culture. Therefore, further studies of the present product as a candidate vaccine in naturally infected animals should be conducted, to analyze its potential.

PF438 NKP30‐BASED CHIMERIC ANTIGEN RECEPTOR REDIRECTED HUMAN T LYMPHOCYTES INDUCE EFFECTIVE ANTITUMORAL IMMUNE RESPONSES TO ACUTE MYELOID LEUKEMIA AND MELANOMA

Background:Adoptive cellular therapy (ACT) using chimeric antigen receptor (CAR) redirected human T lymphocytes has achieved remarkable clinical outcome for acute lymphocytic leukemia and also shows great promise for e.g. multiple myeloma. However, suitable antigens for a specific CAR therapy in acute myeloid leukemia (AML) without significant off‐target effects are still warranted as e.g. CD33 and CD123 are also expressed on hematopoietic stem cells (HSC). In contrast, B7H6, a member of the B7 family, is frequently expressed on various cancers including patient‐derived (primary) AML blasts and e.g. melanoma but not found on normal tissues and recognized by the natural killer (NK) cell activating receptor NKp30.Aims:In this study, we therefore evaluated human T cells as well as CRISPR/Cas9 engineered TCR− T cells expressing two different 2nd. generation NKp30 CARs for effective antileukemic immunity to AML in vitro and in vivo using a patient‐derived AML xenograft (PDX) model. Additionally, antitumoral responses of NKp30 CAR T cells to melanoma were examined.Methods:Expression of B7H6 in various AML lines, primary AML samples, solid tumors and primary fibroblasts was examined by RNA‐based qPCR. T cells were polyclonally stimulated and redirected with a CAR containing the extracellular region of the NKp30, a CD8‐ or, alternatively, a IgG1‐spacer, a CD28 costimulatory domain and a CD3ζ signaling domain by retroviral gene transfer. NKp30 CAR+ T cells were antigen‐specifically stimulated with the B7H6 expressing, HLA class I/II negative melanoma cell line MaMel‐86b (courtesy of Dr. C. Wölfel, III. Dept. of Medicine, UMM) and NKp30 expression was determined by flow cytometry. IFN‐γ ELISPOT and cytotoxicity assays were performed to analyze antileukemic responses to different AML samples as well as to Ma‐Mel 86b in vitro. Moreover, NKp30 CAR+ CRISPR/Cas9 engineered TCR− T cells were studied. Biological activity of redirected T cells was tested in an established AML‐PDX NSG mouse model.Results:qPCR assays revealed B7H6 expression at various levels in &gt;95% of primary AML samples tested in addition to e.g. K562 and HL60 myeloid cell lines while no gene bank RNA expression data are reported for healthy tissues. Following transduction and antigen‐specific expansion ≥90% of CD3+ and TCR−CD3− T cells (more CD8+ than CD4+ T cells) expressed both NKp30 CARs. Upon coculture with the B7H6 expressing K562 and HL‐60 cells as well as primary AML samples NKp30‐redirected T cells elicited strong IFN –γ release and exhibited specific cytolytic activity to both leukemia lines and primary AML blasts but not to B7H6 negative U266 cells as shown by 51Cr‐release and colony‐forming cytotoxicity assays. Interestingly, reactivity of NKp30‐CD8‐CD28‐CD3ζ CAR T cells was significantly higher as compared to the other CAR. In addition, NKp30 CAR T cells induced strong responses to MaMel‐86b cells. Moreover, 3rdparty, TCR− NKp30 CAR T cells excluding alloreactivity also elicited potent responses to B7H6. To test for antitumor immunity in vivo, we first examined reactivity of NKp30‐redirected T cells to K562 engrafted into NSG mice and observed reduction of tumor burden. Further studies to evaluate reactivity to primary AML blasts in a PDX model are in progress.Summary/Conclusion:These studies demonstrate that NKp30 CAR T cells and additionally non allogeneic 3rdparty TCR− NKp30 CAR T cells, particularly expressing a CAR with a short spacer, can successfully elicit immunity to AML and also to solid tumors such as melanoma. As the B7H6 is not expressed on CD34+ HSC, this antigen might be a promising target for ACT to AML.

Cationic lipids as one-component vaccine adjuvants: A promising alternative to alum

Effective vaccine formulations consist of several components: an antigen carrier, the antigen, a stimulator of cellular immunity such as a Toll-like Receptors (TLRs) ligand, and a stimulator of humoral response such as an inflammasome activator. Here, we investigated the immunostimulatory and adjuvant properties of lipopolyamines, cationic lipids used as gene carriers. We identified new lipopolyamines able to activate both TLR2 and TLR4 and showed that lipopolyamines interact with TLRs via a mechanism different from the one used by bacterial ligands, activating a strong type-I IFN response, pro-inflammatory cytokines and IL-1β secretion. The TLR and inflammasome stimulations, together with the antigen carrier properties of lipopolyamines, resulted in both humoral and cellular immunity in mice vaccinated against OVA and make lipopolyamines promising one-component vaccine adjuvants.

PO-040 Development of a tunable form of interferon alpha for in vivo cancer gene therapy

IntroductionThe immune system is a double-edge sword in cancer. On the one hand, it exerts immunosurveillance to eradicate transformed cells that occasionally appear in the body; on the other hand, cancer cells can recruit immune cells endowed with pro-tumorigenic activity.Our lab previously developed a strategy for targeted gene-based delivery of interferon alpha (IFNa) to tumours by tumour infiltrating monocytes/macrophages, which induces robust anti-cancer responses in several experimental models without inducing strong IFN responses in normal tissues as compared to systemic administration of recombinant IFNa. Whereas a sustained output could ensure long-term protection from tumour recurrence, it may raise concerns for long-term side effects, especially in case of cancer eradication.To overcome this issue, we are developing inducible strategies to control the amount of IFNa secreted in the tumour microenvironment.Material and methodsBy fusing a destabilising domain (DD) to a protein of interest (POI) the former can confer its instability to the latter. This destabilisation can be rescued in a reversible and dose dependent manner with the addition of a small molecule specifically binding to the DD. To apply this technology to our strategy we have designed and in vitro tested different fusion proteins of IFNa (DD-IFNa). We also developed improved DD-IFNa with the addition of flexible and/or cleavable linkers and selected them for their capacity to be stabilised in a dose dependent manner in presence of their specific ligand in vitro.Results and discussionsThrough this approach, we have identified effective fusion proteins with low basal activity and high fold induction upon ligand treatment. These novel tunable forms of IFNa are functional and their specific activity are comparable to the wild type cytokine in inducing IFN responsive genes.Based on these promising in vitro results we are now translating these new platforms in vivo to test their efficacy in inducing anti-tumour responses in melanoma, colon and glioma models of cancer.ConclusionIn the perspective of clinical translation our approach can be used in the future to switch on/off the levels of IFNa in a tunable and personalised fashion for cancer eradication.

Innate Lymphoid Cells Are Depleted Irreversibly during Acute HIV-1 Infection in the Absence of Viral Suppression

Innate lymphoid cells (ILCs) play a central role in the response to infection by secreting cytokines crucial for immune regulation, tissue homeostasis, and repair. Although dysregulation of these systems is central to pathology, the impact of HIV-1 on ILCs remains unknown. We found that human blood ILCs were severely depleted during acute viremic HIV-1 infection and that ILC numbers did not recover after resolution of peak viremia. ILC numbers were preserved by antiretroviral therapy (ART), but only if initiated during acute infection. Transcriptional profiling during the acute phase revealed upregulation of genes associated with cell death, temporally linked with a strong IFN acute-phase response and evidence of gut barrier breakdown. We found no evidence of tissue redistribution in chronic disease and remaining circulating ILCs were activated but not apoptotic. These data provide a potential mechanistic link between acute HIV-1 infection, lymphoid tissue breakdown, and persistent immune dysfunction.

Additive loss-of-function proteasome subunit mutations in CANDLE/PRAAS patients promote type I IFN production.

Autosomal recessive mutations in proteasome subunit β 8 (PSMB8), which encodes the inducible proteasome subunit β5i, cause the immune-dysregulatory disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE), which is classified as a proteasome-associated autoinflammatory syndrome (PRAAS). Here, we identified 8 mutations in 4 proteasome genes, PSMA3 (encodes α7), PSMB4 (encodes β7), PSMB9 (encodes β1i), and proteasome maturation protein (POMP), that have not been previously associated with disease and 1 mutation in PSMB8 that has not been previously reported. One patient was compound heterozygous for PSMB4 mutations, 6 patients from 4 families were heterozygous for a missense mutation in 1 inducible proteasome subunit and a mutation in a constitutive proteasome subunit, and 1 patient was heterozygous for a POMP mutation, thus establishing a digenic and autosomal dominant inheritance pattern of PRAAS. Function evaluation revealed that these mutations variably affect transcription, protein expression, protein folding, proteasome assembly, and, ultimately, proteasome activity. Moreover, defects in proteasome formation and function were recapitulated by siRNA-mediated knockdown of the respective subunits in primary fibroblasts from healthy individuals. Patient-isolated hematopoietic and nonhematopoietic cells exhibited a strong IFN gene-expression signature, irrespective of genotype. Additionally, chemical proteasome inhibition or progressive depletion of proteasome subunit gene transcription with siRNA induced transcription of type I IFN genes in healthy control cells. Our results provide further insight into CANDLE genetics and link global proteasome dysfunction to increased type I IFN production.

ID: 151: Unique and overlapping roles of type I and type III interferons in influenza pathology and therapy

Type I and type III interferons (IFN α β and IFN λ ) are key antiviral cytokines. In addition, IFN α β is increasingly recognised as potentially disease-promoting in infection. We explored the roles of these IFN families in influenza infection and as anti-influenza treatment. When comparing influenza-infected 129 to C57BL/6 mouse strains, we found increased lung damage, morbidity and mortality, yet higher levels of IFN α β , in 129 mice than in the more resistant C57BL/6 mice. IFN α β receptor deficiency in 129 mice decreased morbidity and lung damage, and ameliorated immune-mediated tissue damage, indicating that IFN α β levels are a host determinant of influenza severity. In contrast, the respiratory epithelial anti-influenza response was unaffected by IFN α β receptor deficiency, as IFN λ replaced IFN α β in these cells. Lack of IFN α β signalling reduced expression of the death-inducing receptor DR5 on lung epithelia and its ligand TRAIL on inflammatory monocytes, and depletion of PDCA-1+ cells or interruption of TRAIL-DR5 interaction protected infected 129 mice. Therefore, excessive IFN α β signalling in response to acute influenza infection may explain morbidity. Our results suggest that humans with propensity to strong IFN α β signalling may be at risk of severe influenza. We assessed the implications of our results for influenza treatment, using C57BL/6 mice with a functional Mx1 allele. Treatment with either IFN α 4 or IFN λ 2 prior to infection blocked influenza replication and protected against host morbidity and mortality. In contrast, outcome of IFN treatment during influenza infection was divergent. Results of IFN therapy will be presented and discussed in the context of treatment options for influenza in humans.

Efficient replication and strong induction of innate immune responses by H9N2 avian influenza virus in human dendritic cells

Avian influenza A (H9N2) viruses have occasionally been identified in humans with upper respiratory tract infections. The novel H7N9/2013 virus identified in China shows that a low pathogenic avian influenza (LPAI) virus can be highly pathogenic in humans. Therefore, it is important to understand virus–host cell interactions and immune responses triggered by LPAI viruses in humans. We found that LPAI A/Hong Kong/1073/99 (H9N2) virus replicated efficiently in human dendritic cells (DCs). The H9N2 virus induced strong IFN gene expression although with different kinetics than seasonal influenza A/Beijing/353/89 (H3N2) virus. IFN inducible antiviral proteins were produced in H9N2 virus-infected cells at the same level as in H3N2 infection. The H9N2 virus was extremely sensitive to the antiviral actions of type I IFNs. These results indicate that the avian influenza H9N2 virus is inducing a strong antiviral IFN response in human DCs.

IFN-α subtypes: distinct biological activities in anti-viral therapy.

During most viral infections, the immediate host response is characterized by an induction of type I IFN. These cytokines have various biological activities, including anti-viral, anti-proliferative and immunomodulatory effects. After induction, they bind to their IFN-α/β receptor, which leads to downstream signalling resulting in the expression of numerous different IFN-stimulated genes. These genes encode anti-viral proteins that directly inhibit viral replication as well as modulate immune function. Thus, the induction of type I IFN is a very powerful tool for the host to fight virus infections. Many viruses evade this response by various strategies like the direct suppression of IFN induction or inhibition of the IFN signalling pathway. Therefore, the therapeutic application of exogenous type I IFN or molecules that induce strong IFN responses should be of great potential for future immunotherapies against viral infections. Type I IFN is currently used as a treatment in chronic hepatitis B and C virus infection, but as yet is not widely utilized for other viral infections. One reason for this restricted clinical use is that type I IFN belongs to a multigene family that includes 13 different IFN-α subtypes and IFN-β, whose individual anti-viral and immunomodulatory properties have so far not been investigated in detail to improve IFN therapy against viral infections in humans. In this review, we summarize the recent achievements in defining the distinct biological functions of type I IFN subtypes in cell culture and in animal models of viral infection as well as their clinical usage in chronic hepatitis virus infections.

ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization

We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non-self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.

Type 1 and 2 Immunity Following Vaccination Is Influenced by Nanoparticle Size: Formulation of a Model Vaccine for Respiratory Syncytial Virus

Previous studies compared uptake by dendritic cells (DC) of 20, 40, 100, 200, 500, 1000, and 2000 nm beads in vivo. When beads were used as antigen carriers, bead size influenced antibody responses and induction of IFN-gamma-producing CD4 and CD8 T cells. Beads of 40-50 nm were taken up preferentially by DC and induced particularly strong immunity. Herein, we examine immunity induced by minute differences in nanobead size, specifically within a narrow viral-sized range (20, 40, 49, 67, 93, 101, and 123 nm), to see if bead carrier size influenced the induction of type 1 or type 2 cells as demonstrated by the production of IFN-gamma or IL-4. In vivo uptake by DC was assessed for selected sizes in this range. Responses to whole ovalbumin (OVA) or the OVA-derived CD8 T cell peptide epitope (SIINFEKL) were tested. After one immunization with beads-OVA, IFN-gamma responses to both OVA and SIINFEKL were significantly better with 40 and 49 nm beads than other sizes, while, in contrast, IL-4 responses to OVA were higher after immunization with OVA conjugated to larger beads (93, 101, and 123 nm). Thus IFN-gamma induction from CD8 T cells was limited to 40-49 nm beads, while CD4 T cell activation and IL-4 were induced by 93-123 nm beads-OVA. After two immunizations, there were comparable high levels of IFN-gamma produced with 40 and 49 beads and IL-4 reactivity was still higher for larger beads (93, 101, 123 nm). Production of IgG1 was seen across the full range of bead sizes, increasing after two immunizations. Since protection against respiratory syncytial virus (RSV) depends on strong IFN responses, while IL-4 responses are reported to cause asthma-like symptoms, immunization with RSV antigens on the 49 nm carrier beads could provide the basis for a suitable vaccine. When the 49 nm beads were conjugated to RSV proteins G88 (surface) or M2.1 (internal capsid), one immunization with G88 induced high levels of IFN-gamma and low levels of IL-4. IL-4 increased with two immunizations. Beads-M2.1 induced only moderate levels of IFN-gamma and low titer antibody after two immunizations. Mice vaccinated once with G88-conjugated 49 nm beads and challenged intranasally with RSV strain A2 subtype showed reduced viral titers and recovered from weight loss more rapidly than mice immunized with M2.1-conjugated 49 nm beads or naive control mice. These results show that precise selection of nanobead size for vaccination can influence the type 1/type 2 cytokine balance after one immunization, and this will be useful in the development of effective vaccines against common human pathogens such as RSV.