Levels Of IFN-beta Research Articles

Multiple Mechanisms of Action of Sulfodyne®, a Natural Antioxidant, against Pathogenic Effects of SARS-CoV-2 Infection.

Few therapeutic options are available to treat COVID-19. The KEAP1/NRF2 pathway, the major redox-responsive pathway, has emerged as a potential therapeutic target for COVID-19 as it regulates redox homeostasis and inflammation that are altered during SARS-CoV-2 infection. Here, we characterized the effects of NRF2-agonist Sulfodyne®, a stabilized natural Sulforaphane, in cellular and animal models of SARS-CoV-2 infection. In pulmonary or colonic epithelial cell lines, Sulfodyne® elicited a more efficient inhibition of SARS-CoV-2 replication than NRF2-agonists DMF and CDDO. This antiviral activity was not dependent on NRF2 but was associated with the regulation of several metabolic pathways, including the inhibition of ER stress and mTOR signaling, which are activated during SARS-CoV-2 infection. Sulfodyne® also decreased SARS-CoV-2 mediated inflammatory responses by inhibiting the delayed induction of IFNB1 and type I IFN-stimulated genes in infected epithelial cell lines and by reducing the activation of human by-stander monocytes recruited after SARS-CoV-2 infection. In K18-hACE2 mice infected with SARS-CoV-2, Sulfodyne® treatment reduced both early lung viral load and disease severity by fine-tuning IFN-beta levels. Altogether, these results provide evidence for multiple mechanisms that underlie the antiviral and anti-inflammatory activities of Sulfodyne® and pinpoint Sulfodyne® as a potent therapeutic agent against pathogenic effects of SARS-CoV-2 infection.

IFN-beta and EIF2AK2 are potential biomarkers for interstitial lung disease in anti-MDA5 positive dermatomyositis.

Dermatomyositis (DM) with positive anti-melanoma differentiation-related gene 5 (MDA5) antibody is an autoimmune disease with multiple complications. Interstitial lung diseases (ILD) are significantly associated with DM and are particularly related to MDA5+ DM. This article aimed to explore potential molecular mechanisms and develop new diagnostic biomarkers for MDA5+ DM-ILD. The series matrix files of DM and NSIP were downloaded from the Gene Expression Omnibus (GEO) database to identify the differentially expressed genes (DEGs). Gene set enrichment analysis (GSEA) was used to screen the common enriched pathways related to DM and NSIP. Next, the co-expressed differential expressed genes (co-DEGs) between MDA5+, MDA5- and NSIP groups were identified by Venn plots, and then selected for different enrichment analyses and protein-protein interaction (PPI) network construction. The mRNA expression levels of IFN-beta and EIF2AK2 were measured by RT-qPCR. The protein expression levels of IFN-beta were measured by ELISA. Using GSEA, the enriched pathway "herpes simplex virus 1 infection" was both up-regulated in DM and NSIP. Enrichment analysis in MDA5+ DM, MDA5- DM and NSIP reported that the IFN-beta signalling pathway was an important influencing factor in the MDA5+ DM-ILD. We also identified that eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2) was an important gene signature in the MDA5+ DM-ILD by PPI analysis. The expression levels of IFN-beta and EIF2AK2 were significantly increased in MDA5+ DM-ILD patients. IFN-beta and EIF2AK2 contributed to the pathogenesis of MDA5+ DM-ILD, which could be used as potential therapeutic targets.

Study of serum level of kisspeptin and interferon-beta in genital wart patients.

Researchers are interested in genital wart (GW) studies due to their increased incidence. In a single experimental research, virally infected mouse models showed elevated kisspeptin levels and low interferon levels. The objective of the study was to evaluate the serum levels of kisspeptin and interferon (INF)-beta in GW patients. Forty patients with GWs and forty healthy participants of comparable age and sex as a control group were included in this case-control study. Serum levels of kisspeptin and IFN-beta were measured using ELISA during the period from December 2021 to April 2022. Kisspeptin was significantly higher among cases than controls, whereas IFN-beta level was lower among cases than controls (P < 0.001). There were no significant relations between kisspeptin and IFN-beta levels and the clinical data for the studied participants, and there was no significant correlation between both (P > 0.05). The reported increased kisspeptin level which was associated with decreased interferon-beta level in patients with GWs might indicate a new insight into viral infection pathogenesis. Further research including all steps in kisspeptin/G protein-coupled receptor 54 pathway is required. Targeted therapy for this pathway may be of value for those patients.

TMET-10. FATTY ACID METABOLISM REGULATES IMMUNE REACTIVITY OF IRRADIATED GLIOBLASTOMA

Abstract Radiation therapy (RT) is the standard-of-care for glioblastoma (GBM) and is the only treatment for inoperable brain tumors. In multiple cancer, RT stimulates anti-tumor immunity by at least activating cancer cell-intrinsic type I interferon (IFN-I) responses. However, GBM inevitably recur, suggesting that RT promotes immune evasion mechanisms. Altered fatty acid (FA) metabolism is an emerging mechanism that can account for treatment resistance and immune escape of GBM. Therefore, we hypothesize that RT induces a metabolic shift toward the production of FAs to foster immunosuppression of GBM. Supporting this hypothesis, our data show that RT upregulates the expression of the fatty acid synthase (FASN) and the activity of the fatty acid desaturase 2 (FADS2) to generate FAs in murine GBM models, GL261 and CT2A. To determine whether FA metabolism was preventing RT-induced IFN-I, we impaired FA synthesis by inhibiting either FASN or FADS2 using genetic (CRISPR-Cas9) or pharmacological inhibitors (FASNi or FADS2i). As expected, protein quantification (ELISA) as well as gene expression (RT-qPCR) revealed that irradiated GBM cells released greater levels of IFN-beta 24hrs post RT; an effect that was more pronounced when FASN or FADS2 were blocked. Reinforcing the role of FA metabolism in regulating immune activation, in situ immunofluorescence of irradiated intracranial GL261 tumors showed that mice treated with FASNi improved the recruitment of CD8+ T cells into GBM. Along similar lines, flow cytometry of digested GL261 tumors revealed an increase infiltration of CD11c+ CD103+ dendritic cells as well as activated CD86+ CD11b+ cells when FAs synthesis was blocked with FADS2i. Altogether, our data suggest that RT-induced FA synthesis represents a mechanism of resistance by preventing IFN-I and promoting immunological evasion of GBM. Targeting FA metabolism is a promising strategy to promote anti-tumor immunity and sensitize irradiated GBM to immunotherapies.

Profiling transcription factor sub-networks in type I interferon signaling and in response to SARS-CoV-2 infection

Abstract Type I interferons (IFN α/β) play a central role in innate immunity to respiratory viruses, including coronaviruses. In this study, transcription factor profiling in the transcriptome was used to gain novel insights into the role of inducible transcription factors in response to type I interferon signaling in immune cells and in lung epithelial cells after SARS-CoV-2 infection. Modeling the interferon-inducible transcription factor mRNA data in terms of distinct sub-networks based on biological functions such as antiviral response, immune modulation, and cell growth revealed enrichment of specific transcription factors in mouse and human immune cells. Interrogation of multiple microarray datasets revealed that SARS-CoV-2 induced high levels of IFN-beta and interferon-inducible transcription factor mRNA in human lung epithelial cells. Transcription factor mRNA of the three sub-networks were differentially regulated in human lung epithelial cell lines after SARS-CoV-2 infection and in COVID-19 patients. A subset of type I interferon-inducible transcription factors and inflammatory mediators were specifically enriched in the lungs and neutrophils of Covid-19 patients. The emerging complex picture of type I IFN transcriptional regulation consists of a rapid transcriptional switch mediated by the Jak-Stat cascade and a graded output of the inducible transcription factor activation that enables temporal regulation of gene expression.

BVDV-1 induces interferon-beta gene expression through a pathway involving IRF1, IRF7, and NF-κB activation

The bovine viral diarrhea virus (BVDV-1) is a pathogen with the capacity to modulate the interferon type I system. To further investigate the effects of BVDV-1 on the production of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the IFNbeta expression profiles were analyzed.The results showed that cpBVDV-1 was able to induce the production of IFNbeta in a way similar to polyinosinic-polycytidylic acid, but with less intensity. Interestingly, all cpBVDV-1 activities were blocked by pharmacological inhibitors of the IRF-1, IRF-7, and NF-κB signaling pathway, and the level of IFNbeta decreased at the level of transcript and protein. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that cpBVDV-1 regulates IFNbeta expression in bovines through the activation of several key transcription factors. Collectively, the results suggest that during cpBVDV-1 infection, cross talk is evident between various signaling pathways involved in transcriptional activation of IFNbeta in cattle.

The physiologic increase in expression of some type I IFN-inducible genes during pregnancy is not associated with improved disease activity in pregnant patients with rheumatoid arthritis

During pregnancy, most patients with rheumatoid arthritis (RA) experience a spontaneous improvement in their condition. Since type I interferons (IFN) have immunomodulatory properties, we investigated whether type I IFN-inducible genes are upregulated in pregnant patients with RA. Peripheral blood mononuclear cells were evaluated using quantitative real-time polymerase chain reaction for type I IFN-inducible genes (IFI 35, IFI44, IFI44L, IFIT3, OAS1, and Siglec1) in patients with RA and healthy women during and after pregnancy as well as in nonpregnant controls. IFN-alpha and IFN-beta levels in sera of patients and healthy donors were analyzed by enzyme linked immunosorbent assay. It was found that healthy women did not show a change of gene expression levels from the second trimester until postpartum, yet some type I IFN-inducible genes were significantly upregulated in pregnant and postpartum women compared with nonpregnant individuals. In patients with RA, a pronounced upregulation of IFI35 and IFI44 at the second trimester and a peak expression of Siglec1 at the third trimester were observed. Pregnancy levels of IFI35 and IFI44 in patients with RA were higher than those of nonpregnant patients with RA. No significant association of gene expression levels with disease activity was found. In the sera of patients and healthy women, IFN-beta was undetectable and IFN-alpha levels remained stable throughout pregnancy and postpartum. Thus, pregnancy can give rise to an increased expression of type I IFN-inducible genes, reflecting an upregulation of the innate immune system. However, an association of type I IFN-inducible genes with pregnancy induced disease amelioration seems unlikely.

Abstract 412: Role of Interferon Regulatory Factor (IRF)-3 in lung tumor microenvironment

Abstract Cytokines present in the tumor microenvironment influence whether a cancer progresses or is rejected. For example, IFNs promote anti-tumor immunity and thus contribute to tumor rejection whereas IL-10 and TGFb can inhibit anti-tumor immunity and thereby contribute to tumor survival. We find that IFNbeta levels are significantly up-regulated in the microenvironment surrounding lung tumors. In the B16 F10 mouse melanoma lung metastasis model, CD11c+ alveolar cells respond to tumor challenge by rapidly up-regulating their expression of IFNbeta. This induction of IFNbeta was totally absent in IRF3 KO mice but intact in MyD88 and TRIF KO mice. Challenge studies indicate that IRF3 KO mice develop more lung metastasis and have shorter survival than controls. These findings suggest that CD11c+ cells play an important role in tumor recognition, and that they mount an IRF3/IFNbeta pathway specific response that slows tumor progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 412. doi:10.1158/1538-7445.AM2011-412

The PB2 subunit of the influenza virus RNA polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon.

The PB2 subunit of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. However, the molecular mechanisms involved remain unknown. It was previously shown that the PB2 protein, in addition to its nuclear localization, also accumulates in the mitochondria. Here, we demonstrate that the PB2 protein interacts with the mitochondrial antiviral signaling protein, MAVS (also known as IPS-1, VISA, or Cardif), and inhibits MAVS-mediated beta interferon (IFN-beta) expression. In addition, we show that PB2 proteins of influenza viruses differ in their abilities to associate with the mitochondria. In particular, the PB2 proteins of seasonal human influenza viruses localize to the mitochondria while PB2 proteins of avian influenza viruses are nonmitochondrial. This difference in localization is caused by a single amino acid polymorphism in the PB2 mitochondrial targeting signal. In order to address the functional significance of the mitochondrial localization of the PB2 protein in vivo, we have generated two recombinant human influenza viruses encoding either mitochondrial or nonmitochondrial PB2 proteins. We found that the difference in the mitochondrial localization of the PB2 proteins does not affect the growth of these viruses in cell culture. However, the virus encoding the nonmitochondrial PB2 protein induces higher levels of IFN-beta and, in an animal model, is attenuated compared to the isogenic virus encoding a mitochondrial PB2. Overall this study implicates the PB2 protein in the regulation of host antiviral innate immune pathways and suggests an important role for the mitochondrial association of the PB2 protein in determining virulence.

A Phase I Trial of Repeated Intrapleural Adenoviral-mediated Interferon-β Gene Transfer for Mesothelioma and Metastatic Pleural Effusions

We previously showed that a single intrapleural dose of an adenoviral vector expressing interferon-beta (Ad.IFN-beta) in patients with malignant pleural mesothelioma (MPM) or malignant pleural effusions (MPE) resulted in gene transfer, humoral antitumor immune responses, and anecdotal clinical responses manifested by modified Response Evaluation Criteria in Solid Tumors (RECIST) disease stability in 3 of 10 patients at 2 months and an additional patient with significant metabolic response on positron emission tomography (PET) imaging. This phase I trial was conducted to determine whether using two doses of Ad.IFN-beta vector would be superior. Ten patients with MPM and seven with MPE received two doses of Ad.IFN-beta through an indwelling pleural catheter. Repeated doses were generally well tolerated. High levels of IFN-beta were detected in pleural fluid after the first dose; however, only minimal levels were seen after the second dose of vector. Lack of expression correlated with the rapid induction of neutralizing Ad antibodies (Nabs). Antibody responses against tumor antigens were induced in most patients. At 2 months, modified RECIST responses were as follows: one partial response, two stable disease, nine progressive disease, and two nonmeasurable disease. One patient died after 1 month. By PET scanning, 2 patients had mixed responses and 11 had stable disease. There were seven patients with survival times longer than 18 months. This approach was safe, induced immune responses and disease stability. However, rapid development of Nabs prevented effective gene transfer after the second dose, even with a dose interval as short as 7 days.

Expression patterns of Human Interferon-alpha and Interferon-lambda subtypes by monocytes, dendritic cells and B cells. (136.14)

Abstract Human interferon-alpha comprises a family of thirteen highly homologous genes and twelve subtypes (coding sequences of IFN-alpha1 and -13 are identical). We employed two modifications of fluorescent-labeled probe technology--molecular beacons (MB) and locked-nucleic acids (LNA) to develop a highly efficient, sensitive, and specific quantitative RT-PCR assay for each human IFN-alpha subtype as well as the three subtypes of IFN-lambda, and for completion, IFN-beta and -gamma. The assay is formatted to a 384-well plate that measures 16 samples/plate and includes four-point standards for each IFN. Using this assay, we determined expression patterns of these IFN by monocytes, monocyte-derived macrophages and dendritic cells (MDM and MDDC), primary mDC, pDC and B cells in response to ligands for TLR 3, 4, and 9. All the myeloid cells, including mDC expressed high levels of IFN-beta and -lambda1, and moderate levels of IFN-alpha. pDC expressed high levels of almost all IFN, although with quantitative and in one case, a qualitative difference in between the response to CpG (TLR9) vs. imiquod (TLR7). B cells also expressed IFN—more subtypes than myeloid cells, but and fewer than pDC. Type I IFN expression was always accompanied by type III IFN expression. We conclude that IFN expression patterns are both ligand and cell-type specific. Subtypes alone or in combination may have unique roles in the innate immune response.

The activation of liver X receptors inhibits toll‐like receptor‐9‐induced foam cell formation

Toll-like receptors (TLRs) are related to foam cell formation (FCF), key event in the establishment/progression of atherosclerosis. The activation of TLR2 and TLR4 can increase FCF. The aim of this study was to evaluate the role of TLR9 in FCF. Murine macrophages were treated with CpG-ODN, TLR9 agonist, and oxidized particles of LDL (Paz-PC) and FCF was analyzed by means of Oil Red O staining. The administration of CpG-ODN plus Paz-PC onto macrophages increased the amount of lipid droplets, correlated to increased levels of tumor necrosis factor (TNF)-alpha, IFNbeta, and IP-10. The underlying mechanism by which TLR9 ligation influenced Paz-PC in the FCF was NF-kappaB- and IRF7-dependent, as observed by higher levels of phosphorylated IkappaBalpha, increased nuclear translocation of the p65 subunit, lower levels of the total IKKalpha protein and higher release of interferon-dependent cytokines, such as IP-10. Liver X receptors (LXRs) regulate lipid cellular transport and negatively modulate TLR-dependent signaling pathways. Indeed, the addition of GW3965, synthetic LXRs agonist, significantly reduced FCF after CpG-ODN plus Paz-PC stimulation. In this condition, we observed decreased levels of the nuclear translocation of the p65 subunit, related to the higher presence of LXRalpha into the nucleus. TNF-alpha, IP-10, and IFNbeta levels were reduced by the administration of GW3965 following CpG-ODN and Paz-PC treatment. In conclusion, the activation of TLR9 facilitates the formation of foam cells in an NF-kappaB- and IRF7-dependent manner, countered by the activation of LXRs. This study further support LXRs as potential anti-atherosclerotic target.

Focal Distribution of Hepatitis C Virus RNA in Infected Livers

BackgroundHepatitis C virus (HCV) is a plus-strand RNA virus that replicates by amplification of genomic RNA from minus strands leading to accumulation of almost one thousand copies per cell under in vitro cell culture conditions. In contrast, HCV RNA copy numbers in livers of infected patients appear to be much lower, estimated at a few copies per cell.Methodology/Principal FindingsTo gain insights into mechanisms that control HCV replication in vivo, we analyzed HCV RNA levels as well as expression of interferon beta (IFNβ) and several interferon stimulated genes (ISGs) from whole liver sections and micro-dissected subpopulations of hepatocytes in biopsy samples from 21 HCV-infected patients. The results showed that intrahepatic HCV RNA levels range form less than one copy per hepatocyte to a maximum of about eight. A correlation existed between viral RNA levels and IFNβ expression, but not between viral RNA and ISG levels. Also, IFNβ expression did not correlate with ISGs levels. Replication of HCV RNA occurred in focal areas in the liver in the presence of a general induction of ISGs.Conclusion/SignificanceThe low average levels of HCV RNA in biopsy samples can be explained by focal distribution of infected hepatocytes. HCV replication directly induces IFNβ, which then activates ISGs. The apparent lack of a correlation between levels of IFNβ and ISG expression indicates that control of the innate immune response during HCV infections depends on multiple factors.

TLR9‐dependent systemic interferon‐β production by intravenous injection of plasmid DNA/cationic liposome complex in mice

The type I interferon (IFN) response to DNA/cationic liposome complex, or lipoplex, has been reported in cultured cells, but little is known about the response in vivo. Studies of the pro-inflammatory cytokine response to lipoplex have shown the importance of the unmethylated CpG dinucleotide (CpG motif) and its receptor, Toll-like receptor (TLR)-9. CpG- and non-CpG lipoplex consisting of CpG- or non-CpG plasmid DNA, respectively, and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol liposomes were intravenously injected into mice. IFN-beta and interleukin (IL)-6 in the serum and organs were measured by the enzyme-linked immunosorbent assay. The involvement of TLR9, phagocytic cells and the spleen in the responses was evaluated using TLR9(-/-), clodronate liposome-treated-, and splenectomized mice, respectively. Accumulation of blood cells in the lung was evaluated histologically. CpG lipoplex induced a large increase in the levels of IFN-beta and IL-6 in the serum, liver, spleen, lung and kidney, whereas non-CpG lipoplex scarcely had any effect. Neither formulation led to significant cytokine production in TLR9(-/-) mice. Clodronate liposome-treated mice showed a large reduction in both IFN-beta and IL-6 levels. Splenectomized mice receiving CpG lipoplex also showed a significantly low production of IL-6 but a similar level of IFN-beta production to that of unsplenectomized mice. A large number of monocytes were found in the capillary vessels around the pulmonary alveoli of mice receiving lipoplex. These findings indicate that, in contrast to the production of IL-6 from splenic macrophages, IFN-beta is produced from phagocytic cells other than splenic macrophages after the injection of CpG lipoplex through the TLR9-dependent pathway.

Cigarette smoke combined with Toll-like receptor 3 signaling triggers exaggerated epithelial regulated upon activation, normal T-cell expressed and secreted/CCL5 expression in chronic rhinosinusitis

Chronic rhinosinusitis (CRS) is characterized by persistent mucosal inflammation and frequent exacerbations. To determine whether innate epithelial responses to cigarette smoke or bacterial or viral pathogens may be abnormal in CRS leading to an inappropriate inflammatory response. Primary nasal epithelial cells (PNECs) were grown from middle turbinate biopsies of 9 healthy controls and 11 patients with CRS. After reaching 80% to 90% confluence, PNECs were exposed to medium or cigarette smoke extract (CSE) 5% (vol/vol) for 1 hour, washed, then stimulated with staphylococcal lipoteichoic acid, LPS, or double-stranded RNA (dsRNA). After 24 hours, gene expression was quantified by QRT-PCR. At baseline, PNECs revealed elevated TNF-alpha and growth-related oncogene-alpha (a C-X-C chemokine)/CXCL1 (GRO-alpha) (4-fold increase, P = .02; and 16-fold increase, P = .004, respectively) in subjects with CRS compared with controls with normal levels of IL-1beta, IL-6, IL-8/CXCL8, human beta-defensin-2, monocyte chemoattractant protein 2/CCL8, monocyte chemoattractant protein 3/CCL7, and regulated upon activation, normal T-cell expressed and secreted (RANTES)/CCL5. Immunostaining of nasal biopsies, however, revealed comparable epithelial staining for TNF-alpha, GRO-alpha, and RANTES. There were no differences in mRNA induction by CSE, TNF-alpha, lipoteichoic acid, LPS, or dsRNA alone. The combination of CSE+dsRNA induced exaggerated RANTES (12,115-fold vs 1500-fold; P = .03) and human beta-defensin-2 (1120-fold vs 12.5-fold; P = .05) in subjects with CRS. No other genes were differentially induced. Furthermore, CSE+dsRNA induced normal levels of IFN-beta, IFN-lambda1, and IFN-lambda2/3 mRNA in subjects with CRS. Cigarette smoke extract plus dsRNA induces exaggerated epithelial RANTES expression in patients with CRS. We propose that an analogous response to cigarette smoke plus viral infection may contribute to acute exacerbations and eosinophilic mucosal inflammation in CRS.

Cancer gene therapy using mesenchymal stem cells expressing interferon-β in a mouse prostate cancer lung metastasis model

Cell-based therapy for cancer is a promising new field. Among cell types that can be used for this purpose, mesenchymal stem cells (MSCs) appear to hold great advantage for reasons including easier propagation in culture, possible genetic modification to express therapeutic proteins and preferential homing to sites of cancer growth upon in vivo transfer. The present study evaluated the potential of genetically modified MSC, constitutively expressing interferon (IFN)-beta, in an immunocompetent mouse model of prostate cancer lung metastasis. A recombinant adeno-associated virus (rAAV) encoding mouse IFN-beta was constructed and initially tested in vitro for high-level expression and bioactivity of the transgenic protein. MSCs were transduced by the rAAV-IFN-beta or green fluorescent protein ex vivo and used as cellular vehicles to target lung metastasis of TRAMP-C2 prostate cancer cells in a therapy model. Cohorts of mice were killed on days 30 and 75 to determine the effect of therapy by measurement of tumor volume, histology, immunohistochemistry, enzyme-linked immunosorbent assay and flow cytometry. Results indicated a significant reduction in tumor volume in lungs following IFN-beta-expressing MSC therapy. Immunohistochemistry of the lung demonstrated increased tumor cell apoptosis and decreased tumor cell proliferation and blood vessel counts. A significant increase in the natural kill cell activity was observed following IFN-beta therapy correlating the antitumor effect. Systemic level of IFN-beta was not significantly elevated from this targeted cell therapy. These data demonstrate the potential of MSC-based IFN-beta therapy for prostate cancer lung metastasis.

A novel reporter gene assay for interferons based on CHO-K1 cells

Interferons (IFNs) are cytokines playing an important role in the immune response and defence against viruses. They are widely used as biopharmaceuticals. Currently, the anti-viral assay (AVA) is the most commonly used bioassay for determining interferon potency. In the search for rapid and robust but reliable methods, reporter gene assays (RGA) appear to be the most promising approach, therefore we have designed a new reporter cell line, CHO-ISRE-SEAP, suitable for determination of type I interferon potency. Chinese hamster ovary (CHO-K1) cells were stably transfected with secretory alkaline phosphatase (SEAP) gene under the control of interferon stimulated response element (ISRE) promoter. The amount of SEAP in the cell culture medium can be easily measured colorimetrically and has been found to correlate with the amount of IFN added. The new assay is widely applicable for determination of type I IFNs, such as IFN-α, IFN-β and IFN-ω, in research, development of IFN biopharmaceuticals, in batch release, etc. Interestingly, in this assay, IFN-beta shows approximately 6 times higher response than IFN-alpha, which makes it especially appropriate for measuring low levels of IFN-beta. Compared to other known RGAs, the novel CHO-ISRE-SEAP cell line-based RGA appears to have certain advantages with respect to cost and performance.

A possible role of autogenous IFN‐β for cytokine productions in human fibroblasts

It has been already known that human diploid fibroblasts are able to produce not only high levels of IFN-beta but also various kinds of cytokines by poly rI: poly rC, and some inflammatory cytokines are induced by IFN-beta gene activation. We also obtained similar results. However, in our system, cytokine productions were extremely enhanced by treating the cells with a low dose of type 1 IFN and the priming effects on cytokine productions were blocked by cycloheximide similar to those on IFN-beta productions. Most of cytokines were produced later than IFN-beta and synthesis patterns of their mRNA showed the same phenomena. We made clear that cytokine productions by poly rI: poly rC are mediated by secreted IFN-beta at a protein level using a monoclonal antibody against human IFN-beta. Further, it was shown that intra-cellular IFN-beta which is not secreted might also participate in cytokine productions. Meanwhile, IL-1beta induced various kinds of cytokines in human fibroblasts and production time courses of these cytokines were similar to those of poly rI: poly rC induced cytokines. Although secreted IFN-beta was not detected in IL-1beta stimulated culture, expression of IFN-beta mRNA was augmented. These results showed that priming effects of type 1 IFN on cytokine productions by poly rI: poly rC might not be the direct action, but successive IFN-beta production might be essential in the production processes of other cytokines. Further, it was suggested that inducible IFN-beta might also take part in IL-1beta-induced cytokine productions.

Mx1 and IP-10: Biomarkers to Measure IFN-βActivity in Mice Following Gene-Based Delivery

Recombinant interferon-beta (IFN-beta) protein is used successfully for the treatment of multiple sclerosis (MS). Gene therapy might be an alternative approach to overcome drawbacks occurring with IFN-beta protein therapy. A critical issue in developing a new approach is detection of biologically active IFN-beta in preclinical models. The goal of the present study was to determine if Mx1 and IP-10, which are known to be activated after IFN-beta treatment in humans, can be used as biomarkers in mice. In three in vivo experiments, the correlation between different methods of murine IFN-beta (MuIFN-beta) delivery and biomarker induction was studied: (1) bolus protein delivery by intravenous (i.v.) or intramuscular (i.m.) injection, (2) gene-based delivery of IFN- beta by i.m. injection of plasmid DNA, followed by electroporation, and (3) gene-based delivery of IFN-beta by i.m. injection of adenovirus-associated type 1 (AAV1). Short-term induction of Mx1 mRNA and IP-10 was observed after treatment with bolus MuIFN-beta protein. Long-term induction of both biomarkers was observed after IFN-beta plasmid DNA delivery or when AAV1 was used as the vector. The experiments demonstrate that gene-based delivery provides sustained levels of IFN-beta compared with bolus protein injection and that Mx1 RNA and IP-10 can be used to monitor biologically active circulating plasma MuIFN-beta protein in mice.

RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via toll‐like receptor 3

To assess the expression of Toll-like receptor 3 (TLR-3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR-3 ligands. TLR-3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence-activated cell sorting and real-time polymerase chain reaction techniques. TLR-3 signaling was assessed by incubating RASFs with poly(I-C), lipopolysaccharide, palmitoyl-3-cysteine-serine-lysine-4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon-beta (IFNbeta), CXCL10, CCL5, and interleukin-6 (IL-6) protein production in the culture supernatants was performed by enzyme-linked immunosorbent assays. TLR-3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR-3 expression was localized predominantly in the synovial lining, with a majority of the TLR-3-expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR-3 ligand poly(I-C) resulted in the production of high levels of IFNbeta, CXCL10, CCL5, and IL-6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up-regulation of these cytokines and chemokines in a TLR-3-dependent manner. Our findings demonstrate the expression of TLR-3 in RA synovial tissue and the activation of RASFs in vitro by the TLR-3 ligand poly(I-C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR-3 ligand for the stimulation of proinflammatory gene expression in RASFs.