Immune Interferon Production Research Articles

A Degradation Motif in STAU1 Defines a Novel Family of Proteins Involved in Inflammation.

Cancer development is regulated by inflammation. Staufen1 (STAU1) is an RNA-binding protein whose expression level is critical in cancer cells as it is related to cell proliferation or cell death. STAU1 protein levels are downregulated during mitosis due to its degradation by the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C). In this paper, we map the molecular determinant involved in STAU1 degradation to amino acids 38–50, and by alanine scanning, we shorten the motif to F39PxPxxLxxxxL50 (FPL-motif). Mutation of the FPL-motif prevents STAU1 degradation by APC/C. Interestingly, a search in databases reveals that the FPL-motif is shared by 15 additional proteins, most of them being involved in inflammation. We show that one of these proteins, MAP4K1, is indeed degraded via the FPL-motif; however, it is not a target of APC/C. Using proximity labeling with STAU1, we identify TRIM25, an E3 ubiquitin ligase involved in the innate immune response and interferon production, as responsible for STAU1 and MAP4K1 degradation, dependent on the FPL-motif. These results are consistent with previous studies that linked STAU1 to cancer-induced inflammation and identified a novel degradation motif that likely coordinates a novel family of proteins involved in inflammation. Data are available via ProteomeXchange with the identifier PXD036675.

Host Innate Immunity Against Hepatitis Viruses and Viral Immune Evasion.

Hepatitis viruses are primary causative agents of hepatitis and represent a major source of public health problems in the world. The host innate immune system forms the first line of defense against hepatitis viruses. Hepatitis viruses are sensed by specific pathogen recognition receptors (PRRs) that subsequently trigger the innate immune response and interferon (IFN) production. However, hepatitis viruses evade host immune surveillance via multiple strategies, which help compromise the innate immune response and create a favorable environment for viral replication. Therefore, this article reviews published findings regarding host innate immune sensing and response against hepatitis viruses. Furthermore, we also focus on how hepatitis viruses abrogate the antiviral effects of the host innate immune system.

Targeted Mitochondrial Therapy With Over-Expressed MAVS Protein From Mesenchymal Stem Cells: A New Therapeutic Approach for COVID-19.

The SARS-CoV-2, the virus that causes COVID-19, has infected millions of people worldwide. The symptoms of this disease are primarily due to pulmonary involvement, uncontrolled tissue inflammation, and inadequate immune response against the invader virus. Impaired interferon (IFN) production is one of the leading causes of the immune system’s inability to control the replication of the SARS-CoV-2. Mitochondria play an essential role in developing and maintaining innate cellular immunity and IFN production. Mitochondrial function is impaired during cellular stress, affecting cell bioenergy and innate immune responses. The mitochondrial antiviral-signaling protein (MAVS), located in the outer membrane of mitochondria, is one of the key elements in engaging the innate immune system and interferon production. Transferring healthy mitochondria to the damaged cells by mesenchymal stem cells (MSCs) is a proposed option for regenerative medicine and a viable treatment approach to many diseases. In addition to mitochondrial transport, these cells can regulate inflammation, repair the damaged tissue, and control the pathogenesis of COVID-19. The immune regulatory nature of MSCs dramatically reduces the probability of an immune rejection. In order to induce an appropriate immune response against the SARS-CoV-2, we hypothesize to donate mitochondria to the host cells of the virus. We consider MSCs as an appropriate biological carrier for mitochondria. Besides, enhancing the expression of MAVS protein in MSCs and promoting the expression of SARS-CoV-2 viral spike protein as a specific ligand for ACE2+ cells will improve IFN production and innate immune responses in a targeted manner.

RIG-I-like receptor activation drives type I IFN and antiviral signaling to limitHantaan orthohantavirus replication.

Pathogenic hantaviruses, genus Orthohantaviridae, are maintained in rodent reservoirs with zoonotic transmission to humans occurring through inhalation of rodent excreta. Hantavirus disease in humans is characterized by localized vascular leakage and elevated levels of circulating proinflammatory cytokines. Despite the constant potential for deadly zoonotic transmission to humans, specific virus-host interactions of hantaviruses that lead to innate immune activation, and how these processes impart disease, remain unclear. In this study, we examined the mechanisms of viral recognition and innate immune activation of Hantaan orthohantavirus (HTNV) infection. We identified the RIG-I-like receptor (RLR) pathway as essential for innate immune activation, interferon (IFN) production, and interferon stimulated gene (ISG) expression in response to HTNV infection in human endothelial cells, and in murine cells representative of a non-reservoir host. Our results demonstrate that innate immune activation and signaling through the RLR pathway depends on viral replication wherein the host response can significantly restrict replication in target cells in a manner dependent on the type 1 interferon receptor (IFNAR). Importantly, following HTNV infection of a non-reservoir host murine model, IFNAR-deficient mice had higher viral loads, increased persistence, and greater viral dissemination to lung, spleen, and kidney compared to wild-type animals. Surprisingly, this response was MAVS independent in vivo. Innate immune profiling in these tissues demonstrates that HTNV infection triggers expression of IFN-regulated cytokines early during infection. We conclude that the RLR pathway is essential for recognition of HTNV infection to direct innate immune activation and control of viral replication in vitro, and that additional virus sensing and innate immune response pathways of IFN and cytokine regulation contribute to control of HTNV in vivo. These results reveal a critical role for innate immune regulation in driving divergent outcomes of HTNV infection, and serve to inform studies to identify therapeutic targets to alleviate human hantavirus disease.

Quantitative theory of entropic forces acting on constrained nucleotide sequences applied to viruses

We outline a theory to quantify the interplay of entropic and selective forces on nucleotide organization and apply it to the genomes of single-stranded RNA viruses. We quantify these forces as intensive variables that can easily be compared between sequences, outline a computationally efficient transfer-matrix method for their calculation, and apply this method to influenza and HIV viruses. We find viruses altering their dinucleotide motif use under selective forces, with these forces on CpG dinucleotides growing stronger in influenza the longer it replicates in humans. For a subset of genes in the human genome, many involved in antiviral innate immunity, the forces acting on CpG dinucleotides are even greater than the forces observed in viruses, suggesting that both effects are in response to similar selective forces involving the innate immune system. We further find that the dynamics of entropic forces balancing selective forces can be used to predict how long it will take a virus to adapt to a new host, and that it would take H1N1 several centuries to adapt to humans from birds, typically contributing many of its synonymous substitutions to the forcible removal of CpG dinucleotides. By examining the probability landscape of dinucleotide motifs, we predict where motifs are likely to appear using only a single-force parameter and uncover the localization of UpU motifs in HIV. Essentially, we extend the natural language and concepts of statistical physics, such as entropy and conjugated forces, to understanding viral sequences and, more generally, constrained genome evolution.

Chicken interferon-mediated induction of major histocompatibility complex Class II antigens on peripheral blood monocytes

Conditioned medium containing immune interferon (IFN) activity was prepared by stimulating spleen lymphocytes obtained from inbred SC chickens with 10 μg concanavalin A (Con A) for 48 h. Pretreatment of spleen cells with monoclonal antibody against CD4, but not CD8, abrogated IFN production suggesting that CD4+ lymphocytes are responsible for immune IFN production. Immune IFN was purified 25-fold from Con A conditioned medium using controlled-pore glass column chromatography resulting in an increase in specific antiviral activity from 7 to 3290 units mg −1. Partially purified immune IFN retained antiviral and macrophage-activating factor (MAF)-like activities. Normal peripheral blood macrophages, when cultured in the presence of partially purified immune IFN, showed a dose-dependent increase in cell surface major histocompatibility complex Class II antigen expression by flow cytometry. Northern blot analysis of mRNA obtained from IFN-treated macrophages showed a concomitant increase in Class II gene expression. This effect was more obvious in cells induced for 48 h than in those induced for 24 h. These results strongly suggest the existence of an avian homologue of the MAF-like activity.

Production of human immune interferon by recombinant mammalian cells cultivated on microcarriers

Recombinant Chinese hamster ovary (rCHO) cells were cultivated on microcarriers for the production of human immune (Gamma) interferon. The effect of basal medium, serum, and microcarrier concentration on interferon production was investigated. The specific interferon productivity in the post-confluent stage was similar to that in the growth stage. Control of the pH results in a significant improvement in the volumetric interferon production. The volumetric production rate of interferon by these rCHO cells did not decrease after one month of cultivation on microcarriers.

Cerebral Toxoplasmosis in the Acquired Immunodeficiency Syndrome: Case Report and Review of the Literature

Cerebral Toxoplasmosis in the Acquired Immunodeficiency Syndrome: Case Report and Review of the Literature

Antigen-specific inhibition of immune interferon production by suppressor cells of autoimmune encephalomyelitis.

Previous work from this laboratory has revealed that spleen and/or lymph node cells from Lewis rats, that have recovered from an acute episode of experimental autoimmune encephalomyelitis (EAE), suppress the development of EAE when injected into syngeneic recipients subsequently challenged with myelin basic protein (MBP) in CFA. In an effort to understand the mechanism of this suppression, we measured the production of immune IFN-gamma, which may be required for the induction of an immune response, by EAE effector T cells (which transfer disease) and EAE suppressor cells when cultured in vitro with MBP. We now report that EAE effector T cells produce IFN-gamma when cultured in vitro with MBP. In contrast, spleen cells from recovered rats (which manifest suppressor activity in vivo) do not produce IFN-gamma. Moreover, in cell mixing experiments, these suppressor spleen cells inhibited the production of IFN-gamma by EAE effector cells. This inhibition was not eliminated by the removal of macrophages nor by the inhibition of PG synthesis by indomethacin. Furthermore, the inhibition was shown to be Ag-specific and mediated by nylon-adherent, radiation-sensitive splenic T cells. The findings suggest that suppressor cells regulate EAE by inhibiting IFN-gamma production by effector cells. This inhibition may result in the down-regulation of IFN-gamma-induced expression of class II major histocompatibility Ag on cells of the central nervous system, thus reducing the presentation of tissue-specific Ag (i.e., MBP) to autoreactive lymphocytes.

Microcarrier culture of recombinant Chinese hamster ovary cells for production of human immune interferon and human tissue-type plasminogen activator

Recombinant Chinese hamster ovary cells were successfully cultured semi-continuously on microcarriers of gelatin or modified dextran under non-selective conditions for up to three weeks. High and constant production rates for human immune interferon and tissue-type plasminogen activator were obtained. For cells that produced interferon, the highest cell concentration and interferon production was obtained with gelatin microcarriers though the specific production when grown in the presence of 0.2% fetal calf serum was slightly higher for cells cultured on dextran microcarriers (0.12 U/cell day versus 0.11 U/cell day). For cells that produced plasminogen activator, a slightly higher cell concentration was obtained for cells grown on dextran microcarriers (9x105 cells/ml versus 7x105 cells/ml). However, the specific and total production rates were significantly higher for cells cultured on gelatin microcarriers (6.7 pg/cell day versus 2.1 pg/cell day). The maximum cell concentration and specific production rate could be increased to 2.3x106 cells/ml and 3.4 pg/cell day for dextran microcarriers by adding 6-aminohexanoic acid to the medium. For gelatin microcarriers, the addition of 6-aminohexanoic acid increased the specific production rate to 14.4 pg/cell day. Cell growth, however, was inhibited.

Effect of erythromycin on the immune response and interferon production

The influence of erythromycin on some aspects of humoral and cell-mediated immunity has been examined employing human as well as murine models, both in vivo and in vitro. No significant differences in antibody synthesis, α- and γ-interferon yield, cutaneous delayed hypersensitivity or lymphocyte blastogenic response to mitogens have been detected between erythromycin-treated subjects and controls. Similarly, in vitro tests on interferon production and blastogenic response to mitogens showed no significant differences when performed with and without erythromycin. Therefore, in contrast with many other antibacterial drugs, erythromycin seems to be devoid of any adverse effects on the immune system.

Interleukin 1 and interferon-gamma: cytokines that provide reciprocal regulation of macrophage and T cell function.

Interactions between macrophages and T cells are symbiotic, since optimal functions of both cell types require interchange of soluble mediators. Upon activation, macrophages release interleukin-1 (alias lymphocyte activating factor, leukocytic endogenous mediator, and endogenous pyrogen), a family of molecules with multivarious biological effects, ranging from induction of fever and the acute phase response to lymphocyte activation and concomitant release of interleukin-2. Interleukin-2 induces activation and replication of several subsets of precursor lymphocytes, including cytotoxic T cells, lymphokine-activated killer (LAK) cells, and natural killer (NK) cells, and enhances their cytotoxic activity for tumor cells. Both interleukin-2 and leukotrienes enhance production of immune interferon (interferon-gamma) by activated T cells. Aside from antiviral activity, interferon-gamma produces a number of immunomodulatory effects, including macrophage activation (Ia induction, antimicrobial effector function, and activation of oxidative metabolism) and augmentation of NK function. Expression of Ia on accessory cell membranes is required for the initiation of many antigen-specific, T-dependent immune responses. Interferon-gamma also synergizes with a variety of microbial agents to augment macrophage tumoricidal function and enhance interleukin-1 secretion. The production of interferon-gamma appears to have a critical role in feeding back the cascade of interleukins in a loop of amplification. Both interleukin-1 and interferon-gamma modulate release of arachidonate metabolites in various cells. This cascade of cytokines, in collaboration with arachidonate oxygenation products, regulates immunity and sets the stage for many of the events underlying inflammation. Various anti-inflammatory drugs and immunopotentiators appear to act by modulating cytokine pathways.

Production of Immune (Gamma) Interferon by Bronchoalveolar Cells and Peripheral Blood Mononuclear Cells of Subjects with Sarcoidosis

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10] Production of human immune interferon with normal human leukocytes by the inducers A-23187 and mezerein

Human leukocytes are an abundant and available source for the production of natural immune interferon. This chapter describes the induction of these leukocytes with the combination of mezerein and the calcium ionophore, A-23187. This novel procedure offers the use of small organic compounds which, by virtue of their physicochemical properties, can be readily removed from the final product. Over the past two years, both spinner culture systems and stationary culture systems have been utilized. The advantage in employing the spinner system is its yield per volume, whereas the stationary system requires considerably less A-23187. The method described in this chapter for the production of human immune interferon has several advantages. First, the reagents employed (e.g., A-23187, mezerein, calf serum (CS), and Dulbecco's minimal essential medium (DMEM)) are readily available and inexpensive. Second, unlike proteinaceous inducers, such as phytohemagglutinin (PHA), con-canavalin A, or staphylococcal enterotoxin A, these low-molecular-weight organic compounds can be readily removed from the final product. Last, because this procedure does not require lysis of erythrocytes and is suitable in spinner culture, this method is quite adaptable to large-scale practice.

12] Large-scale production, concentration, and partial purification of human immune interferon

This chapter discusses the large-scale production, concentration, and partial purification of human immune interferon. Induction of the synthesis of gamma interferon by lymphocytes is connected with the activation of a variety of peripheral white blood cells (PWBC) with nonspecific mitogens. Normally PWBC from a large number of donors are employed for large-scale production. In such situations, a mixed lymphocyte reaction occurs in addition to mitogen activation. As a result of the combined activation, different types of PWBC release into the media gamma interferon plus a variety of enzymes (proteases, phosphatases, oxygenases, phosphorylases, and lipases) and other lymphokines (interleukins 1, 2, and 3, inhibitors, other molecules, and growth factors). Isolation of gamma interferon from the products of activated PWBC and the proteins and mitogens introduced in the incubation medium creates formidable purification problems. To achieve success in purification, it is necessary to achieve adequate induction of gamma interferon by the PWBC. Gamma interferon batches gave an initial titer below 300 units/m. The above purification method is simple, effective, and relatively fast (purification time does not exceed 48 hr). This method can be applied to large quantities and can be totally automated without need for intermediate steps that would otherwise require adjustment of the material between steps of purification.

22] Production and purification of mouse immune interferon

This chapter discusses the production and purification of mouse immune interferon. Crude murine immune interferon preparations contain a variety of lymphokines and biologically active molecules other than murine gamma-interferon (Mu-IFN-γ). The execution of in vivo experiments demands large quantities of Mu-IFN-γ, which should be largely devoid of these substances. The investigations on the biochemical properties of the Mu-IFN-γ molecules, and the generation of both polyclonal and monoclonal antibodies (e.g., in rat), require highly purified antigen preparations. Methods for obtaining such preparations are described in this chapter. The highest levels of Mu-IFN-γ production are obtained with the lysate of S. aureus 13N2909. On average, a level of 10 3.1 -10 4.3 units/ml is reached after 72 hr of incubation. This crude Mu-IFN-y is purified by a sequence of silicic acid adsorption/elution, affinity chromatography on Concanavalin A (Con A)-Sepharose and anion-exchange chromatography on fast protein liquid chromatography (FPLC) mono Q. The average purification factor, recovery, and specific activity obtained after these different steps are summarized in the chapter.

7] Production and partial purification of human immune interferon

This chapter mimics the routine methodology for the production of alpha interferons so that both natural leukocyte interferon and natural immune interferon could be conveniently prepared in the same laboratory and avoids the use of reagents, which would make the product unsuitable for clinical use. Human leukocytes from normal donors are used in many laboratories around the world for the production of alpha interferons for clinical use. Either of two paramyxoviruses––Sendai or newcastle disease virus (NDV)––is an effective inducer of many subtypes of alpha interferons, and monocytes are the main producer cells. Human leukocytes are also used for the production of immune or gamma interferon for clinical studies. Mitogens are commonly used as inducers, and the immune interferon is produced by T lymphocytes. Human leukocytes can be collected and purified by the same procedure for the production of both the leukocyte and the immune interferons. However, the subsequent production steps for the interferons differ substantially as far as the inducer, cell concentration, medium, conditions, and time of the incubation are concerned.

8] Production of human immune interferon from leukocytes cocultured with exogenous cells

Publisher Summary This chapter discusses the production of human immune interferon from leukocytes cocultured with exogenous cells. Human gamma interferon (IFN-γ) is produced in vitro from peripheral blood leukocytes stimulated with mitogens or with the calcium ionophore A-23187. A detailed procedure for the isolation of human leukocytes, the initiation of the leukocytes into 10-liter suspension cell cultures, and induction of the cells with a lectin, phytohemagglutinin-A (PHA), to produce IFN-γ is described. Yields of interferon are augmented by the addition of a phorbol ester to the culture medium and by the cocultivation of the leukocytes with an established human promyloblastic cell line. An average titer of 10,000 reference units/ml of IFN-γ is obtained from human leukocytes cocultivated with HL-60 cells in spinner culture, however, due to the variation of the leukocyte pools, titers ranged from 2500 to 40,000 units/ml. HL-60 cells proliferate in suspension culture; therefore, the time of addition of the cells to the leukocyte induction medium and the final concentration of the HL-60 cells is critical for optimum IFN-γ yields. Human leukocytes, obtained by leukapheresis of normal donors, may be used for the production of human IFN-γ in place of the normal buffy coat leukocytes.

11] Large-scale production and recovery of human immune interferon

Publisher Summary This chapter discusses the development of a method for large-scale production of human immune interferon based on induction scheme with the addition of a cell fractionation process and a novel method for product recovery. A 10- to 15-fold enhancement in interferon yield and a 50-fold increase in purity of crude product were accomplished by nylon wool column fractionation of lymphocytes combined with simultaneous reduction of autologous plasma protein levels prior to induction. The production method described is suitable for large-scale preparation of natural human immune interferon. Its principal advantages include greater interferon yield and purity without substantial increase in process costs. The process provides a method for the production of both higher and more consistent yields of interferon. A major factor in the higher yield from nylon wool fractionated buffy coat cells is the prolongation of the active production period compared to unfractionated cell preparations. Higher purity without concomitant yield loss results from removal of about 70% of buffy coat plasma, a step which results in reduced yields when using unfractionated cells. This sequential application of nylon wool fractionation and protein removal steps combines to provide 50-fold improvement in initial purity, which leads to higher purification yields and a reduction in purification costs.

55] Expression of human interferon genes in E. coli with the lambda PL promoter

This chapter describes the utilization of the expression vector pRC23 containing the phage lambda P L promoter for the high-level production of human immune interferon (IFN-γ) in E. coli . The capability of producing mammalian proteins in bacteria has become a valuable tool for studying biologically interesting polypeptides that otherwise cannot be isolated in sufficient amounts. Expression of cloned human interferon genes in E. coli is now a classical case in point. Leukocyte interferon is currently being produced and purified in gram quantities, allowing the application of standard biochemical approaches for studying the protein at the molecular level and expanding the number and kinds of experiments designed to understand the mechanisms of its biological activities. The first requirement for obtaining high-level expression of a heterologous gene in E. coli is that the coding sequences be placed under the transcriptional control of a strong promoter, preferably one that can be efficiently and conveniently regulated. Tight transcriptional regulation is important for at least three reasons: (1) the synthesis of some heterologous proteins may be toxic to the bacterial cell, (2) transcription from a strong promoter can interfere with plasmid replication, leading to plasmid instability, and (3) a high rate of synthesis during a short time period can be crucial for avoiding proteolytic degradation of the foreign gene product.