IFN Protein Research Articles

Hereditary C1q Deficiency is Associated with Type 1 Interferon-Pathway Activation and a High Risk of Central Nervous System Inflammation

Hereditary C1q deficiency (C1QDef) is a rare monogenic disorder leading to defective complement pathway activation and systemic lupus erythematosus (SLE)-like manifestations. The link between impairment of the complement cascade and autoimmunity remains incompletely understood. Here, we assessed type 1 interferon pathway activation in patients with C1QDef. Twelve patients with genetically confirmed C1QDef were recruited through an international collaboration. Clinical, biological and radiological data were collected retrospectively. The expression of a standardized panel of interferon stimulated genes (ISGs) in peripheral blood was measured, and the level of interferon alpha (IFNα) protein in cerebrospinal fluid (CSF) determined using SIMOA technology. Central nervous system (encompassing basal ganglia calcification, encephalitis, vasculitis, chronic pachymeningitis), mucocutaneous and renal involvement were present, respectively, in 10, 11 and 2 of 12 patients, and severe infections recorded in 2/12 patients. Elevated ISG expression was observed in all patients tested (n = 10/10), and serum and CSF IFNα elevated in 2/2 patients. Three patients were treated with Janus-kinase inhibitors (JAKi), with variable outcome; one displaying an apparently favourable response in respect of cutaneous and neurological features, and two others experiencing persistent disease despite JAKi therapy. To our knowledge, we report the largest original series of genetically confirmed C1QDef yet described. Additionally, we present a review of all previously described genetically confirmed cases of C1QDef. Overall, individuals with C1QDef demonstrate many characteristics of recognized monogenic interferonopathies: particularly, cutaneous involvement (malar rash, acral vasculitic/papular rash, chilblains), SLE-like disease, basal ganglia calcification, increased expression of ISGs in peripheral blood, and elevated levels of CSF IFNα.

Screening for autoimmune diseases in apparently healthy antinuclear antibody positive individuals.

Anti-nuclear antibodies (ANA) assessed by immunofluorescence (IF) microscopy are associated with systemic autoimmune rheumatic diseases (SARD) and can be detected years before onset of clinical symptoms. Recent data indicate dysregulation of the immune system with increased levels of proinflammatory cytokines, including type I interferons (IFN), in ANA-positive versus ANA-negative individuals. Herein, the aims were to investigate IF-ANA, ANA fine specificities, and IFN-α protein levels in relation to self-reported symptoms, as well as clinical signs, of SARD in a large group of healthy blood donors (HBD). Sera from 825 HBD (48.8% females) were included. IF-ANA was assessed, using HEp-2 cells, according to the routine at the accredited laboratory of Clinical Immunology, Linköping University Hospital. All samples were analyzed for IgG-ANA fine specificities using addressable laser bead assay (ALBIA) at the same laboratory. IFN-α was determined using ELISA. Antibody-positive individuals, and their sex- and age-matched antibody-negative controls, were asked to fill a questionnaire regarding symptoms associated with SARD. In total, 130 HBD (15.8%) were positive with IF-ANA and/or ALBIA. Anti-U1RNP was significantly more common among women. Generally, self-reported symptoms correlated poorly with IF-ANA and/or ALBIA results. Two females with high levels of Ro60/SSA, Ro52/SSA and IFN-α reported mild sicca symptoms and were diagnosed with Sjögren's disease after clinical evaluation. A considerable proportion of apparently HBD are autoantibody positive, but without clear association to self-reported symptoms. Nevertheless, the combination of autoantibodies, relevant symptoms and high IFN-α levels identified the small proportion of individuals with SARD in the study population.

Plasma interferon-alpha protein levels during pregnancy are associated with lower birth weight in systemic lupus erythematosus.

Adverse pregnancy outcomes are more common in women with systemic lupus erythematosus (SLE) compared with healthy women, but we lack prognostic biomarkers. Plasma interferon alpha (IFNα) protein levels are elevated in a subgroup of pregnant women with SLE, but whether this is associated with pregnancy outcomes is unknown. We investigated the relationship between IFNα, adverse pregnancy outcomes and the presence of autoantibodies in SLE pregnancy. We followed 76 women with SLE prospectively. Protein levels of IFNα were quantified in plasma collected in the 2nd and 3rd trimester with single-molecule array. Positivity for antinuclear and antiphospholipid antibodies was assessed during late pregnancy with multiplexed bead assay. Clinical outcomes included the adverse pregnancy outcomes small for gestational age (SGA), preterm birth, and preeclampsia. During SLE pregnancy, women with SGA infants compared with those without had higher levels of plasma IFNα protein, and IFNα positivity was associated with lower birth weight of the infant. Preterm birth was associated with autoantibodies against chromatin. IFNα protein levels associated positively with autoantibodies against chromatin, Smith/ribonucleoprotein (SmRNP) and RNP, but negatively with phospholipid antibodies. Elevated IFNα protein in plasma of women with SLE is a potential risk factor for lower birth weight of their infants. The association between IFNα and lower birth weight warrants further investigation regarding the pathophysiological role of IFNα during SLE pregnancy.

Abstract B019: Identification of gene signatures predictive of response to Ad-IFNα/Syn3 in bladder cancer

Abstract Introduction: Interferon α gene therapy (Ad-IFNα/Syn3) is FDA approved for treatment of BCG-unresponsive non-muscle invasive bladder cancer. Ad-IFNα/Syn3 is a non-replicating adenovirus that when transduced into target tissues, produces IFNa2b protein with anti-tumor activity. In the phase 3 multicenter trial, 45.5% of patients with carcinoma-in-situ and 60% of those with Ta/T1 disease who had a complete response to Ad-IFNα/Syn3 at 3 months remained recurrence free at 12 months. Targeting mechanisms of resistance and identifying predictive biomarkers to improve patient selection and overall response rates is a top priority. Towards this, we treated 29 human bladder cancer cell lines with Ad-IFNα/Syn3 and assessed their responses to therapy. We identified sensitive and resistant cell lines and gene signatures predictive of response. Methods: Human bladder cancer cell lines were grown in MEM supplemented with 10% FBS in 96-well plate. After overnight attachment, these were treated with Ad-IFNα/Syn3 (MOI 1000) and cell titre glow assay was performed 72h post-treatment. Cells were also seeded in 6-well plates. and after 24h cell-free supernatants were collected for ELISA and cells were collected for RNA isolation using RNAqueous Total RNA Isolation kit (Thermo Fisher). RNA quality was assessed using Tapestation. RNAseq analysis was carried out using Ion proton Sequencer. Ingenuity pathway analysis (IPA) and gene set enrichment were performed on the sequencing data for the cell lines. ELISA was used to measure IFNα and IL6 protein levels in cell free supernatants. We also tested organoids developed from bladder cancer patients to test drug efficacy and identify gene signatures. Results: Out of the 29 cell lines, we identified that sensitivity to Ad-IFNα/Syn3 was pronounced in luminal cells (8/13 luminal cell lines). Most basal cells were either resistant (>70% viability) or only moderately sensitive to Ad-IFNα/Syn3 (between 70-40% viability) when compared to control. We selected 7 cell lines for sequencing and ELISA measurements: luminal cell lines BV, UC6, Rt4V6 and UC5, and basal cell lines ScaBER, HT1197, and T24. All cell lines mentioned above could be successfully transduced by the adenoviral vector as measured by increased expression of IFNα protein in the cell-free supernatants. Ad-IFNα/Syn3-sensitive luminal cell lines at baseline had lower IFNα and IFNγ scores when compared to resistant basal cell lines. We identified several cell-death pathways enriched in treated cells that were sensitive to Ad-IFNα/Syn3. IL6 measurements in cell free supernatants revealed increased expression of IL6 after treatment in sensitive cells. However, in resistant cells IL6 expression was either high at baseline (ScaBER) or both undetectable at baseline and after treatment (UC5). Conclusion: Our results suggest that the luminal cell lines are more sensitive to Ad-IFNα/Syn3 and that high baseline expression of IFNα and IFNγ in basal cell lines contributes to resistance. IL6 levels are also predictive of response to Ad-IFNα/Syn3. Citation Format: Morgan E. Pierce, Alexis R. Steinmetz, Aruna Nannapaneni, Tanner S. Miest, David J. McConkey, Sharada Mokkapati, Colin P.N. Dinney. Identification of gene signatures predictive of response to Ad-IFNα/Syn3 in bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B019.

Low CD4 + T cell count is related to specific anti-nuclear antibodies, IFNα protein positivity and disease activity in systemic lupus erythematosus pregnancy

BackgroundLymphopenia, autoantibodies and activation of the type I interferon (IFN) system are common features in systemic lupus erythematosus (SLE). We speculate whether lymphocyte subset counts are affected by pregnancy and if they relate to autoantibody profiles and/or IFNα protein in SLE pregnancy.MethodsRepeated blood samples were collected during pregnancy from 80 women with SLE and 51 healthy controls (HC). Late postpartum samples were obtained from 19 of the women with SLE. Counts of CD4 + and CD8 + T cells, B cells and NK cells were measured by flow cytometry. Positivity for anti-nuclear antibodies (ANA) fine specificities (double-stranded DNA [dsDNA], Smith [Sm], ribonucleoprotein [RNP], chromatin, Sjögren’s syndrome antigen A [SSA] and B [SSB]) and anti-phospholipid antibodies (cardiolipin [CL] and β2 glycoprotein I [β2GPI]) was assessed with multiplexed bead assay. IFNα protein concentration was quantified with Single molecule array (Simoa) immune assay. Clinical data were retrieved from medical records.ResultsWomen with SLE had lower counts of all lymphocyte subsets compared to HC throughout pregnancy, but counts did not differ during pregnancy compared to postpartum. Principal component analysis revealed that low lymphocyte subset counts differentially related to autoantibody profiles, cluster one (anti-dsDNA/anti-Sm/anti-RNP/anti-Sm/RNP/anti-chromatin), cluster two (anti-SSA/anti-SSB) and cluster three (anti-CL/anti-β2GPI), IFNα protein levels and disease activity. CD4 + T cell counts were lower in women positive to all ANA fine specificities in cluster one compared to those who were negative, and B cell numbers were lower in women positive for anti-dsDNA and anti-Sm compared to negative women. Moreover, CD4 + T cell and B cell counts were lower in women with moderate/high compared to no/low disease activity, and CD4 + T cell count was lower in IFNα protein positive relative to negative women. Finally, CD4 + T cell count was unrelated to treatment.ConclusionLymphocyte subset counts are lower in SLE compared to healthy pregnancies, which seems to be a feature of the disease per se and not affected by pregnancy. Our results also indicate that low lymphocyte subset counts relate differentially to autoantibody profiles, IFNα protein levels and disease activity, which could be due to divergent disease pathways.

An alternative strategy to increasing influenza virus replication for vaccine production in chicken embryo fibroblast (DF-1) cells by inhibiting interferon alpha and beta using peptide-conjugated phosphorodiamidate morpholino oligomers.

Introduction. Influenza is a global health issue causing substantial health and economic burdens on affected populations. Routine, annual vaccination for influenza virus is recommended for all persons older than 6 months of age. The propagation of the influenza virus for vaccine production is predominantly through embryonated chicken eggs.Hypothesis/Gap Statement. Many challenges face the propagation of the virus, including but not limited to low yields and lengthy production times. The development of a method to increase vaccine production in eggs or cell lines by suppressing cellular gene expression would be helpful to overcome some of the challenges facing influenza vaccine production.Aims. This study aimed to increase influenza virus titres by using a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO), an antisense molecule, to suppress protein expression of the host genes interferon alpha (IFN-α) and interferon beta (IFN-β) in chicken embryo fibroblast (DF-1) cells.Methods. The toxicity of PPMOs was evaluated by cytotoxicity assays, and their specificity to inhibit IFN-α and IFN-β proteins was measured by ELISA. We evaluated the potential of anti-IFN-α and anti-IFN-β PPMOs to reduce the antiviral proteins in influenza virus-infected DF-1 cells and compared the virus titres to untreated controls, nonsense-PPMO and JAK/STAT inhibitors. The effects of complementation and reconstitution of IFN-α and IFN-β proteins in PPMO-treated-infected cells were evaluated, and the virus titres were compared between treatment groups.Results. Suppression of IFN-α by PPMO resulted in significantly reduced levels of IFN-α protein in treated wells, as measured by ELISA and was shown to not have any cytotoxicity to DF-1 cells at the effective concentrations tested. Treatment of the self-directing PPMOs increased the ability of the influenza virus to replicate in DF-1 cells. Over a 2-log10 increase in viral production was observed in anti-IFN-α and IFN-β PPMO-treated wells compared to those of untreated controls at the initial viral input of 0.1 multiplicity of infection. The data from complementation and reconstitution of IFN-α and IFN-β proteins in PPMO-treated-infected cells was about 82 and 97% compared to the combined PPMO-treated but uncomplemented group and untreated group, respectively. There was a 0.5-log10 increase in virus titre when treated with anti-IFN-α and IFN-β PPMO compared to virus titre when treated with JAK/STAT inhibitors.Conclusions. This study emphasizes the utility of PPMO in allowing cell cultures to produce increased levels of influenza for vaccine production or alternatively, as a screening tool to cheaply test targets prior to the development of permanent knockouts of host gene expression.

MicroRNA-122 protects against interferon-α-induced hepatic inflammatory response via the Janus kinase-signal transducer and activator of transcription pathway.

Significant overlap in the epidemiology and coinfection of chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) has been identified, which accelerates the development of severe liver cirrhosis and hepatocellular carcinoma worldwide. Interferon-α (IFN-α), a cytokine with antiviral properties, exerts profound physiological effects on innate immunity by regulating interferon-stimulated genes (ISGs) within cells. However, the underlying mechanism of IFN-α in hepatic inflammation remains to be fully elucidated. Here, we utilized LO2 cells treated with the recombinant IFN-α protein and conducted microRNA (miR) sequencing. MiR-122-3p and miR-122-5p_R+1 were the most enriched miRNAs involved in the pathogenesis of IFN-α-induced inflammatory responses and were significantly downregulated by IFN-α treatment. Furthermore, we identified interferon induced protein with tetratricopeptide repeats 1 (IFIT1) as a potential target gene of miR-122. IFN-α markedly increased the expression of proinflammatory cytokines and fibrogenic genes but decreased the mRNA expression of ISGs. Additionally, IFN-α significantly activated the NF-κB p-p65, MAPK p-p38, and Jak/STAT pathways to trigger inflammation. Importantly, supplementation with a miR-122 mimic significantly alleviated IFN-α-induced inflammation and induced IFIT1 expression in LO2 cells. Conversely, the suppression of miR-122 markedly exacerbated the inflammatory response triggered by IFN-α. Furthermore, silencing IFIT1 via an siRNA elicited an inflammatory response, whereas IFIT1 overexpression ameliorated hepatic inflammation and fibrosis in a manner comparable to that induced by IFN-α treatment. Taken together, our findings suggest that miR-122 and its target, IFIT1, reciprocally regulate the inflammatory response associated with IFN through the Jak/STAT pathway.

Interferon alpha and beta receptor 1 knockout in human embryonic kidney 293 cells enhances the production efficiency of proteins or adenoviral vectors related to type I interferons.

Human embryonic kidney (HEK) 293 cells are widely used in protein and viral vector production owing to their high transfection efficiency, rapid growth, and suspension growth capability. Given their antiviral, anticancer, and immune-enhancing effects, type I interferons (IFNs) have been used to prevent and treat human and animal diseases. However, the binding of type I IFNs to the IFN-α and-β receptor (IFNAR) stimulates the expression of IFN-stimulated genes (ISGs). This phenomenon induces an antiviral state and promotes apoptosis in cells, thereby impeding protein or viral vector production. In this study, we generated an IFNAR subtype 1 knockout (KO) HEK 293 suspension (IFNAR-KO) cell line by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology. Upon treatment with human IFN-α, the IFNAR-KO cells showed a constant expression of ISGs, including 2'-5'-oligoadenylate synthetase 1 (OAS1), myxovirus resistance 1 (Mx1), protein kinase RNA-activated (PKR), and IFN-induced transmembrane protein 1 (IFITM1), when compared with the wild-type HEK 293 (WT) cells, wherein the ISGs were significantly upregulated. As a result, the titer of recombinant adenovirus expressing porcine IFN-α was significantly higher in the IFNAR-KO cells than in the WT cells. Furthermore, the IFNAR-KO cells continuously produced higher amounts of IFN-α protein than the WT cells. Thus, the CRISPR-Cas9-mediated IFNAR1 KO cell line can improve the production efficiency of proteins or viral vectors related to IFNs. The novel cell line may be used for producing vaccines and elucidating the type I IFN signaling pathway in cells.

Low-density granulocytes are related to shorter pregnancy duration but not to interferon alpha protein blood levels in systemic lupus erythematosus

BackgroundAn increased risk of pregnancy complications is seen in women with systemic lupus erythematosus (SLE), but the specific immunopathological drivers are still unclear. Hallmarks of SLE are granulocyte activation, type I interferon (IFN) overproduction, and autoantibodies. Here we examined whether low-density granulocytes (LDG) and granulocyte activation increase during pregnancy, and related the results to IFNα protein levels, autoantibody profile, and gestational age at birth.MethodsRepeated blood samples were collected during pregnancy in trimesters one, two, and three from 69 women with SLE and 27 healthy pregnant women (HC). Nineteen of the SLE women were also sampled late postpartum. LDG proportions and granulocyte activation (CD62L shedding) were measured by flow cytometry. Plasma IFNα protein concentrations were quantified by single molecule array (Simoa) immune assay. Clinical data were obtained from medical records.ResultsWomen with SLE had higher LDG proportions and increased IFNα protein levels compared to HC throughout pregnancy, but neither LDG fractions nor IFNα levels differed during pregnancy compared to postpartum in SLE. Granulocyte activation status was higher in SLE relative to HC pregnancies, and it was increased during pregnancy compared to after pregnancy in SLE. Higher LDG proportions in SLE were associated with antiphospholipid positivity but not to IFNα protein levels. Finally, higher LDG proportions in trimester three correlated independently with lower gestational age at birth in SLE.ConclusionOur results suggest that SLE pregnancy results in increased peripheral granulocyte priming, and that higher LDG proportions late in pregnancy are related to shorter pregnancy duration but not to IFNα blood levels in SLE.

Analysis of the Differential Expression and Antiviral Activity of Porcine Interferon-α In Vitro.

Porcine interferon α (poIFN-α) is a crucial cytokine that can prevent and treat viral infections. Seventeen functional porcine IFN-α subtypes were found in the porcine genome. In this study, multiple sequence alignment was performed to analyze IFN-α protein structure and function. Phylogenetic tree analysis of the poIFN gene family defined the evolutionary relationship of various subtypes. PoIFN-αs, including poIFN-α1-17, were expressed in an Escherichia coli expression system. The antiviral activities of these IFN-α proteins against vesicular stomatitis virus (VSV) and pseudorabies virus (PRV) were examined in PK-15 cells. We found that the antiviral activity of different poIFN-α molecules greatly differed as follows: the poIFN-α14 and 17 subtypes had the greatest antiviral activities against VSV and PRV in PK-15 cells, poIFN-α1, 2, 3, and 8 exhibited lower biological activities, and poIFN-α4, 5, 6, 7, 9, 10, 11, 12, 13, and 16 had minimal or no effect in the tested target cell‒virus systems. Moreover, our studies demonstrated that the antiviral activity of IFN-α was positively correlated with the induction of IFN-stimulated genes, such as 2'-5'oligoadenylate synthetase 1 (OSA1), interferon-stimulated gene 15 (ISG15), myxoma resistance protein 1 (Mx1), and protein kinase R (PKR). Thus, our experimental results provide important information about the antiviral functions and mechanism of poIFN-α.

IFNAR signaling of neuroectodermal cells is essential for the survival of C57BL/6 mice infected with Theiler’s murine encephalomyelitis virus

BackgroundTheiler’s murine encephalomyelitis virus (TMEV) is a single-stranded RNA virus that causes encephalitis followed by chronic demyelination in SJL mice and spontaneous seizures in C57BL/6 mice. Since earlier studies indicated a critical role of type I interferon (IFN-I) signaling in the control of viral replication in the central nervous system (CNS), mouse strain-specific differences in pathways induced by the IFN-I receptor (IFNAR) might determine the outcome of TMEV infection.MethodsData of RNA-seq analysis and immunohistochemistry were used to compare the gene and protein expression of IFN-I signaling pathway members between mock- and TMEV-infected SJL and C57BL/6 mice at 4, 7 and 14 days post-infection (dpi). To address the impact of IFNAR signaling in selected brain-resident cell types, conditional knockout mice with an IFNAR deficiency in cells of the neuroectodermal lineage (NesCre±IFNARfl/fl), neurons (Syn1Cre±IFNARfl/fl), astrocytes (GFAPCre±IFNARfl/fl), and microglia (Sall1CreER±IFNARfl/fl) on a C57BL/6 background were tested. PCR and an immunoassay were used to quantify TMEV RNA and cytokine and chemokine expression in their brain at 4 dpi.ResultsRNA-seq analysis revealed upregulation of most ISGs in SJL and C57BL/6 mice, but Ifi202b mRNA transcripts were only increased in SJL and Trim12a only in C57BL/6 mice. Immunohistochemistry showed minor differences in ISG expression (ISG15, OAS, PKR) between both mouse strains. While all immunocompetent Cre-negative control mice and the majority of mice with IFNAR deficiency in neurons or microglia survived until 14 dpi, lack of IFNAR expression in all cells (IFNAR−/−), neuroectodermal cells, or astrocytes induced lethal disease in most of the analyzed mice, which was associated with unrestricted viral replication. NesCre±IFNARfl/fl mice showed more Ifnb1, Tnfa, Il6, Il10, Il12b and Ifng mRNA transcripts than Cre−/−IFNARfl/fl mice. IFNAR−/− mice also demonstrated increased IFN-α, IFN-β, IL1-β, IL-6, and CXCL-1 protein levels, which highly correlated with viral load.ConclusionsIfi202b and Trim12a expression levels likely contribute to mouse strain-specific susceptibility to TMEV-induced CNS lesions. Restriction of viral replication is strongly dependent on IFNAR signaling of neuroectodermal cells, which also controls the expression of key pro- and anti-inflammatory cytokines during viral brain infection.

Recombinant porcine Interferon-α and Interleukin-2 fusion protein (rPoIFNα+IL-2) shows potent anti-pseudorabies virus activity in vitro and in vivo

Pseudorabies virus (PRV) variants have been widely prevalent since 2011, leading to substantial losses to the swine industry. Although PRV can cause cross-species transmission and induce human infection, no drugs can currently prevent PRV infection. Interferons (IFNs) and interleukin-2 (IL-2) are important cytokines that mediate several biological functions including antiviral activity and immune regulation. In this study, we expressed and purified a recombinant porcine IFN-α and IL-2 fusion protein (rPoIFNα+IL-2), which did not show a cytotoxic effect on PK-15 cells. The antiviral activity was evaluated in PK-15 cells using the cytopathic effect inhibition method, and the results indicated that rPoIFNα+IL-2 can inhibit the replication of PRV, with an antiviral activity of approximately 104 U/mL. Moreover, the proliferation of peripheral blood mononuclear cells was enhanced by rPoIFNα+IL-2. Additionally, rPoIFNα+IL-2 substantially increased the expression of IFN-stimulated genes, including IFIT1, ISG15, MX1, and OAS, which are critical for antiviral activity. Furthermore, rPoIFNα+IL-2 alleviated the clinical symptoms and reduced mortality in mice infected with PRV. Simultaneously, rPoIFNα+IL-2 increased the expression levels of IFN-γ and IL-10 and inhibited the expression of IL-1β and IL-6. Additionally, the viral DNA copies in different tissues in the rPoIFNα+IL-2-treated group were lower than those in the untreated group. These findings indicate that rPoIFNα+IL-2 may serve as an antiviral agent for the prevention and treatment of PRV infection and may expand the potential function of IFN antiviral drugs in the future.

S-39-3: ENDOTHELIAL INFLAMMATION AND DYSFUNCTION INDUCED BY SARS-COV-2 SPIKE PROTEIN 1 INVOLVE ADAM17 AND INTERFERON ACTIVATION PATHWAYS

Objective: COVID19 is associated with vascular inflammation. IFN-alpha (IFNα) and IFN-lambda3 (IFNλ3) are potent cytokines produced in viral infections. Their effects involve interferon-stimulated genes (ISGs) and may influence expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. We hypothesized that S1P-induced immune/inflammatory responses in endothelial cells (EC) are mediated via IFN-activated pathways Design and methods: Human ECs were stimulated with S1P (1 μg/mL), IFNα (100ng/mL) or IFNλ3 (100IU/mL). Because ACE2, ADAM17 and TMPRSS2 are important for SARS-CoV-2 infection, we used inhibitors of ADAM17 (marimastat, 3.8 nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50 μM). Gene and protein expression was investigated by real-time PCR and immunoblotting, respectively. Vascular function was assessed in mesenteric arteries from wild-type (WT) normotensive and hypertensive (LinA3) mice and in ISG15-deficient (ISG15KO) mice. Results: S1P increased expression of IFNα (3-fold), IFNλ3 (4-fold) and ISGs (2-fold) in EC (p < 0.05). EC responses to IFNα (ISG15: 16-fold) were greater than to IFNλ3 (ISG15: 1.7-fold) (p < 0.05). S1P increased gene expression of IL-6 (1.3-fold), TNFα (6.2-fold) and IL-1β (3.3-fold), effects that were amplified by IFNs. Only the ADAM17 inhibitor marimastat inhibited S1P effects. IFNα and IFNλ3 increase protein expression of ADAM17 (27%) and TMPRSS2 (38%). No changes were observed on ACE2 expression. This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). EC production of IL-6 was increased by IFNα (1,230pg/mL) and IFNλ3 (1,124pg/mL) vs control (591pg/mL). Nitric oxide generation and eNOS phosphorylation (Ser1177) were reduced by IFNα (40%) and IFNλ3 (40%). Vascular functional responses demonstrated that endothelium-dependent vasorelaxation (% Emax) in vessels from WT-mice stimulated with IFNα (67%) and IFNλ3 (71%) were reduced vs control (82%) (p < 0.05). Responses were not altered in vessels from ISG15KO mice. Increased contraction was observed only in vessels from hypertensive mice treated with IFNα (9.1 ± 0.5mN vs control: 7.3 ± 0.3mN) (p < 0.05). Conclusions: In ECs, S1P, IFNα and IFNλ3 increased ISG15 and IL-6 by mechanisms dependent on ADAM17. IFNs amplifies endothelial cell inflammatory responses and induced vascular dysfunction through ISG15-dependent mechanisms, with augmented effects in hypertension. Our findings demonstrate that S1P induces immune/inflammatory responses that may be important in endotheliitis associated with COVID-19. This may be especially important in the presence of cardiovascular risk factors, including hypertension.

Inactive disease in patients with lupus is linked to autoantibodies to type I interferons that normalize blood IFNα and B cell subsets

Systemic lupus erythematosus (SLE) is characterized by increased expression of type I interferon (IFN)-regulated genes in 50%-75% of patients. We report that out of 501 patients with SLE analyzed, 73 (14%) present autoantibodies against IFNα (anti-IFN-Abs). The presence of neutralizing-anti-IFN-Abs in 4.2% of patients inversely correlates with low circulating IFNα protein levels, inhibition of IFN-I downstream gene signatures, and inactive global disease score. Hallmarks of SLE pathogenesis, including increased immature, double-negative plasmablast B cell populations and reduction in regulatory B cell (Breg) frequencies, were normalized in patients with neutralizing anti-IFN-Abs compared with other patient groups. Immunoglobulin G (IgG) purified from sera of patients with SLE with neutralizing anti-IFN-Abs impedes CpGC-driven IFNα-dependent differentiation of B cells into immature B cells and plasmablasts, thus recapitulating the neutralizing effect of anti-IFN-Abs on B cell differentiation invitro. Our findings highlight a role for neutralizing anti-IFN-Abs in controlling SLE pathogenesis and support the use of IFN-targeting therapies in patients with SLE lacking neutralizing-anti-IFN-Abs.

Juvenile Neuropsychiatric Systemic Lupus Erythematosus: Identification of Novel Central Neuroinflammation Biomarkers

IntroductionJuvenile systemic lupus erythematosus (j-SLE) is a rare chronic autoimmune disease affecting multiple organs. Ranging from minor features, such as headache or mild cognitive impairment, to serious and life-threatening presentations, j-neuropsychiatric SLE (j-NPSLE) is a therapeutic challenge. Thus, the diagnosis of NPSLE remains difficult, especially in pediatrics, with no specific biomarker of the disease yet validated.ObjectivesTo identify central nervous system (CNS) disease biomarkers of j-NPSLE.MethodsA 5-year retrospective tertiary reference monocentric j-SLE study. A combination of standardized diagnostic criteria and multidisciplinary pediatric clinical expertise was combined to attribute NP involvement in the context of j-SLE. Neopterin and interferon-alpha (IFN-α) protein levels in cerebrospinal fluid (CSF) were assessed, together with routine biological and radiological investigations.ResultsAmong 51 patients with j-SLE included, 39% presented with j-NPSLE. J-NPSLE was diagnosed at onset of j-SLE in 65% of patients. No specific routine biological or radiological marker of j-NPSLE was identified. However, CSF neopterin levels were significantly higher in active j-NPSLE with CNS involvement than in j-SLE alone (p = 0.0008). Neopterin and IFN-α protein levels in CSF were significantly higher at diagnosis of j-NPSLE with CNS involvement than after resolution of NP features (respectively p = 0.0015 and p = 0.0010) upon immunosuppressive treatment in all patients tested (n = 10). Both biomarkers correlated strongly with each other (Rs = 0.832, p < 0.0001, n = 23 paired samples).ConclusionCSF IFN-α and neopterin constitute promising biomarkers useful in the diagnosis and monitoring of activity in j-NPSLE.

Landscape and age dynamics of immune cells in the Egyptian rousette bat.

Bats harbor high-impact zoonotic viruses often in the absence of disease manifestation. This restriction and disease tolerance possibly rely on specific immunological features. In-depth molecular characterization of cellular immunity and imprinting of age on leukocyte compartments remained unexplored in bats. We employ single-cell RNA sequencing (scRNA-seq) and establish immunostaining panels to characterize the immune cell landscape in juvenile, subadult, and adult Egyptian rousette bats (ERBs). Transcriptomic and flow cytometry data reveal conserved subsets and substantial enrichments of CD79a+ B cells and CD11b+ Tcells in juvenile animals, whereas neutrophils, CD206+ myeloid cells, and CD3+ Tcells dominate as bats reach adulthood. Despite differing frequencies, phagocytosis of circulating and tissue-resident myeloid cells and proliferation of peripheral and splenic lymphocytes are analogous in juvenile and adult ERBs. We provide a comprehensive map of the immune landscape in ERBs and show age-imprinted resilience progression and find that variability in cellular immunity only partly recapitulates mammalian archetypes.

Human Platelet Lysate Induces Antiviral Responses against Parechovirus A3.

Human platelet lysate (hPL) contains abundant growth factors for inducing human cell proliferation and may be a suitable alternative to fetal bovine serum (FBS) as a culture medium supplement. However, the application of hPL in virological research remains blank. Parechovirus type-A3 (PeV-A3) belongs to Picornaviridae, which causes meningoencephalitis in infants and young children. To understand the suitability of hPL-cultured cells for PeV-A3 infection, the infection of PeV-A3 in both FBS- and hPL-cultured glioblastoma (GBM) cells were compared. Results showed reduced PeV-A3 infection in hPL-cultured cells compared with FBS-maintained cells. Mechanistic analysis revealed hPL stimulating type I interferon (IFN) antiviral pathway, through which phospho-signal transducer and activator of transcription 1 (STAT1), STAT2, interferon regulatory factor 3 (IRF3) were activated and antiviral genes, such as IFN-α, IFN-β, and Myxovirus resistance protein 1 (MxA), were also detected. In addition, an enhanced PeV-A3 replication was detected in the hPL-cultured GBM cells treated with STAT-1 inhibitor (fludarabine) and STAT1 shRNA. These results in vitro suggested an unexpected effect of hPL-activated type I IFN pathway response to restrict virus replication and that hPL may be a potential antiviral bioreagent.

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

Interferon-α-mediated therapeutic resistance in early rheumatoid arthritis implicates epigenetic reprogramming

ObjectivesAn interferon (IFN) gene signature (IGS) is present in approximately 50% of early, treatment naive rheumatoid arthritis (eRA) patients where it has been shown to negatively impact initial response to...

BacMam Expressing Highly Glycosylated Porcine Interferon Alpha Induces Robust Antiviral and Adjuvant Effects against Foot-and-Mouth Disease Virus in Pigs.

Foot-and-mouth disease (FMD) is an acute contagious disease that affects cloven-hoofed animals and has severe global economic consequences. FMD is most commonly controlled by vaccination. Currently available commercial FMD vaccines contain chemically inactivated whole viruses, which are thought to be slow acting as they are effective only 4 to 7 days following vaccination. Hence, the development of a novel rapid vaccine or alternative measures, such as antiviral agents or the combination of vaccines and antiviral agents for prompt FMD virus (FMDV) outbreak containment, is desirable. Here, we constructed a recombinant baculovirus (BacMam) expressing consensus porcine interferon alpha (IFN-α) that has three additional N-glycosylation sites driven by a cytomegalovirus immediate early (CMV-IE) promoter (Bac-Con3N IFN-α) for protein expression in mammalian cells. Bac-Con3N IFN-α expressing highly glycosylated porcine IFN-α protein increased the duration of antiviral effects. We evaluated the antiviral effects of Bac-Con3N IFN-α in swine cells and mice and observed sustained antiviral effects in pig serum; additionally, Bac-Con3N IFN-α exhibited sustained antiviral effects in vivo as well as adjuvant effects in combination with an inactivated FMD vaccine. Pigs injected with a combination of Bac-Con3N IFN-α and the inactivated FMD vaccine were protected against FMDV at 1, 3, and 7 days postvaccination. Furthermore, we observed that in combination with the inactivated FMD vaccine, Bac-Con3N IFN-α increased neutralizing antibody levels in mice and pigs. Therefore, we suggest that Bac-Con3N IFN-α is a strong potential antiviral and adjuvant candidate for use in combination with inactivated FMD vaccines to protect pigs against FMDV. IMPORTANCE Early inhibition of foot-and-mouth disease (FMD) virus (FMDV) replication in pigs is highly desirable as FMDV transmission and shedding rates are higher in pigs than in cattle. However, commercial FMD vaccines require at least 4 to 7 days postvaccination (dpv) for protection, and animals are vulnerable to heterologous viruses before acquiring high antibody levels after the second vaccination. Therefore, the development of antiviral agents for use in combination with FMD vaccines is essential. We developed a novel antiviral and immunostimulant, Bac-Con3N IFN-α, which is a modified porcine IFN-α-expressing recombinant baculovirus, to improve IFN stability and allow its direct delivery to animals. We present a promising candidate for use in combination with inactivated FMD vaccines as pigs applied to the strategy had early protection against FMDV at 1 to 7 dpv, and their neutralizing antibody levels were higher than those in pigs administered the vaccine only.