The effect of induction temperature on aggregation of consensus interferon-alpha expressed by Pichia pastoris was investigated. The cell growth and cIFN level were analyzed and compared when Pichia pastoris was grown at 30,25,20 degrees C during induction phase, using 5.0L fermentor. The result suggested that the cell growth was not affected much under the different induction temperature, but the protein level declined markedly with the decrease of the induction temperature. The total protein ammount induced at 20 degrees C was 67.8 percent of that at 30 degrees C. SDS-PAGE and native-PAGE as well as Western blotting analysis were further conducted. The electrophoresis results revealed that cIFN formed aggregates after secreted into media when protein was induced at 30 degrees C but this problem can be restored by decreasing the induction temperature to 20 degrees C. cIFN monomer in supernatant arrived at 570mg/L and bioactivity of fermentation broth reached 1.05 x 10(9) IU/mL at 20 degrees C of induction temperature. The amount of cIFN monomer and bioactivity in supernatant elevated 7.2 and 38.7 times, respectively, when the induction temperature was controlled at 20 degrees C instead of conventional 30 degrees C.
Fermentation ; Humans ; Interferon Type I ; biosynthesis ; genetics ; Interferon-alpha ; Pichia ; genetics ; growth & development ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Temperature

Type I interferon (IFN) is considered as a bridge between innate and adaptive immunity. Proper activation or inhibition of type I IFN signaling is essential for host defense against pathogen invasion, tumor cell proliferation, and overactive immune responses. Due to intricate and diverse chemical structures, natural products and their derivatives have become an invaluable source inspiring innovative drug discovery. In addition, some natural products have been applied in clinical practice for infection, cancer, and autoimmunity over thousands of years and their promising curative effects and safety have been well-accepted. However, whether these natural products are primarily targeting type I IFN signaling and specific molecular targets involved are not fully elucidated. In the current review, we thoroughly summarize recent advances in the pharmacology researches of natural products for their type I IFN activity, including both agonism/activation and antagonism/inhibition, and their potential application as therapies. Furthermore, the source and chemical nature of natural products with type I IFN activity are highlighted and their specific molecular targets in the type I IFN pathway and mode of action are classified. In conclusion, natural products possessing type I IFN activity represent promising therapeutic strategies and have a bright prospect in the treatment of infection, cancer, and autoimmune diseases.
Biological Products/therapeutic use* ; Immunity, Innate ; Signal Transduction ; Interferon Type I/metabolism*

In order to study the signal pathway secreting type Ⅰ interferon in porcine alveolar macrophages (PAMs) infected with porcine circovirus type 2 (PCV2), the protein and the mRNA expression levels of cGAS/STING pathways were analyzed by ELISA, Western blotting and quantitative reverse transcriptase PCR in PAMs infected with PCV2. In addition, the roles of cGAS, STING, TBK1 and NF-κB/P65 in the generation of type I interferon (IFN-I) from PAMs were analyzed by using the cGAS and STING specific siRNA, inhibitors BX795 and BAY 11-7082. The results showed that the expression levels of IFN-I increased significantly at 48 h after infection with PCV2 (P<0.05), the mRNA expression levels of cGAS increased significantly at 48 h and 72 h after infection (P<0.01), the mRNA expression levels of STING increased significantly at 72 h after infection (P<0.01), and the mRNA expression levels of TBK1 and IRF3 increased at 48 h after infection (P<0.01). The protein expression levels of STING, TBK1 and IRF3 in PAMs infected with PCV2 were increased, the content of NF-κB/p65 was decreased, and the nuclear entry of NF-κB/p65 and IRF3 was promoted. After knocking down cGAS or STING expression by siRNA, the expression level of IFN-I was significantly decreased after PCV2 infection for 48 h (P<0.01). BX795 and BAY 11-7082 inhibitors were used to inhibit the expression of IRF3 and NF-κB, the concentration of IFN-I in BX795-treated group was significantly reduced than that of the PCV2 group (P<0.01), while no significant difference was observed between the BAY 11-7028 group and the PCV2 group. The results showed that PAMs infected with PCV2 induced IFN-I secretion through the cGAS/STING/TBK1/IRF3 signaling pathway.
Animals ; Cells, Cultured ; Circovirus ; Interferon Type I/genetics* ; Macrophages, Alveolar/virology* ; Membrane Proteins/metabolism* ; Nucleotidyltransferases/metabolism* ; Signal Transduction ; Swine

OBJECTIVETo detect the secretions of type I interferon and the expressions of phospho-IRF3 in murine liver dendritic cells intervened by HBV.

METHODSThe murine liver dendritic cells were isolated via anti-CD11c microbeads and were incubated in the presence of GM-CSF and IL-4 to induce the DC generation and proliferation in 24-well cell culture plates. HBV virions were isolated via ultracentrifugation and were detected by quantitative Realtime-PCR. The DCs were divided into two groups: one group was cultured with HBV virions for 24 hours, the other group was cultured without HBV as control group. The cells were harvested at Oh, 1h, 2h, 6h and 24h after being stimulated with poly I:C and the expressions of p-IRF3 and the concentration of IFN beta in supernatants were detected with western blot and ELISA respectively.

RESULTSThe IFN beta concentrations at 0 h, 6 h and 24 h in the supernatants of the HBV group and the control group were (12.38 +/- 3.71) pg/ml, (88.67 +/- 9.01) pg/ml and (69.89 +/- 5.80) pg/ml vs (10.83 +/- 4.11) pg/ml, (137.68 +/- 12.28) pg/ml and (72.25 +/- 8.61) pg/ml, respectively. No statistical differences found at 0 h (t = 0.8398, P > 0.05) and 24 h (t = 0.6820, P > 0.05) between the two groups except that at 6 h (t = 9.653, P < 0.01). The expressions of phospho-IRF3 in HBV group were lower than that in control group.

CONCLUSIONSThe type I interferon secretion and the phospho-IRF3 expression were decreased in murine liver dendritic cells when intervened by HBV.

Animals ; Cells, Cultured ; Coculture Techniques ; Dendritic Cells ; cytology ; metabolism ; Hepatitis B virus ; Hepatocytes ; cytology ; Interferon Regulatory Factor-3 ; metabolism ; Interferon Type I ; secretion ; Male ; Mice ; Mice, Inbred C57BL

Virus infection induces the production of type I interferons (IFNs). IFNs bind to their heterodimeric receptors to initiate downstream cascade of signaling, leading to the up-regulation of interferon-stimulated genes (ISGs). ISGs play very important roles in innate immunity through a variety of mechanisms. Although hundreds of ISGs have been identified, it is commonly recognized that more ISGs await to be discovered. The aim of this study was to identify new ISGs and to probe their roles in regulating virus-induced type I IFN production. We used consensus interferon (Con-IFN), an artificial alpha IFN that was shown to be more potent than naturally existing type I IFN, to treat three human immune cell lines, CEM, U937 and Daudi cells. Microarray analysis was employed to identify those genes whose expressions were up-regulated. Six hundred and seventeen genes were up-regulated more than 3-fold. Out of these 617 genes, 138 were not previously reported as ISGs and thus were further pursued. Validation of these 138 genes using quantitative reverse transcription PCR (qRT-PCR) confirmed 91 genes. We screened 89 genes for those involved in Sendai virus (SeV)-induced IFN-β promoter activation, and PIM1 was identified as one whose expression inhibited SeV-mediated IFN-β activation. We provide evidence indicating that PIM1 specifically inhibits RIG-I- and MDA5-mediated IFN-β signaling. Our results expand the ISG library and identify PIM1 as an ISG that participates in the regulation of virus-induced type I interferon production.
Cells, Cultured ; Gene Library ; Humans ; Interferon Type I ; metabolism ; Interferon-beta ; genetics ; metabolism ; Proto-Oncogene Proteins c-pim-1 ; genetics ; Up-Regulation

To investigate the influence of the fermentation conditions on glycosylation of heterologous recombinant protein in yeast Pichia pastoris, the glycosylation of recombinant human interferon omega (rhIFNomega) under various fermentation conditions, e. g., cell density, initial pH, methanol concentration, duration of the induction, and medium volume were studied. The glycosylation of rhIFNomega in the continuous fermentation process under various pH values and in batch fermentation were also investigated. In 250 mL flask, the optimal cell density, initial pH, medium volume, methanol concentration and frequency of methanol induction were 250 g/L (WCW), pH6.0, less than 30 mL, 15 g/L and 3 (in every 24 h), respectively. In the continuous process, the glycosylation of rhIFNomega could be effectively improved by maintaining the pH value at 7.0-7.5. In the batch fermentation process, the expression level of glycosylated and non-glycosylated rhIFNomega were the same, but the specified value of glycosylation/non-glycosylation was significantly lower than that in the flask culture. The reason of this phenomenon will be further studied. This research lay the foundation for the scale-up of production and the enhancement of rhIFNomega glycosylation in Pichia pastoris.
Bioreactors ; microbiology ; Culture Media ; Fermentation ; Glycosylation ; Humans ; Interferon Type I ; biosynthesis ; genetics ; metabolism ; Methanol ; metabolism ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism

Intrinsic cellular defenses are non-specific antiviral activities by recognizing pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs), one of the pathogen recognize receptor (PRR), sense various microbial ligands. Especially, TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 recognize viral ligands such as glycoprotein, single- or double-stranded RNA and CpG nucleotides. The binding of viral ligands to TLRs transmits its signal to Toll/interleukin-1 receptor (TIR) to activate transcription factors via signal transduction pathway. Through activation of transcription factors, such as interferon regulatory factor-3, 5, and 7 (IRF-3, 5, 7) or nuclear factor-kappaB (NF-kappaB), type I interferons are induced, and antiviral proteins such as myxovirus-resistance protein (Mx) GTPase, RNA-dependent Protein Kinase (PKR), ribonuclease L (RNase L), Oligo-adenylate Synthetase (OAS) and Interferon Stimulated Gene (ISG) are further expressed. These antiviral proteins play an important role of antiviral resistancy against several viral pathogens in infected cells and further activate innate immune responses.
Animals ; GTP-Binding Proteins/metabolism ; Humans ; Interferon Regulatory Factors/metabolism ; Interferon Type I/*metabolism/physiology ; Models, Biological ; NF-kappa B/metabolism ; Toll-Like Receptors/metabolism ; Virus Diseases/*immunology/*metabolism/virology ; eIF-2 Kinase/metabolism

BACKGROUNDBased on the earlier mapping of the epitope recognized by neutralizing antibody, the authors directly replaced binding domain of IFN in AB-loop for enhancement of biological activity.

METHODSTwo unique restriction sites (EcoR? and BsE?) were created into region flanking LoopAB. Casette mutagesis, restriction enzyme digestion, DNA sequencing, antiviral activity assay and antiproliferative activity assay have been used in the project.

RESULTSThe mutated residues M31?D,D32?P of LoopAB in parent IFN were produced. The recombinant phagemid pCANTAB5E/3132IFN 1c/86D and expression plasmid PBV322-132IFN 1c/86D were constructed respectively by replacing the corresponding LoopAB with DNA fragment mutated in the residues M31?D,D32?P, which have been confirmed. The recombinant protein has been expressed in E.coli JM103. The crude 3132IFN 1c/86D has been assayed on human WISH cells challenged with VSV and on HeLa cells by colorimetric MTT. 3132IFN 1c/86D showed 8-old antiviral activity compared to that of parent IFN 1c/86D, while IFN?induced growth inhibition of both types had no difference.

CONCLUSIONSThe authors concluded that a mutant IFN with enhanced antiviral activity can be obtained via a targeted replacement of receptor binding domain in AB-loop.

Amino Acid Substitution ; Antibodies, Viral ; Antiviral Agents ; pharmacology ; Cells, Cultured ; DNA Mutational Analysis ; Humans ; Interferon Type I ; genetics ; pharmacology ; Interferon-alpha ; Mutagenesis, Site-Directed ; Mutation ; Peptide Fragments ; genetics ; pharmacology ; Plasmids ; genetics ; Receptors, Interferon ; metabolism ; Recombinant Proteins

The twenty-first century has already recorded more than ten major epidemics or pandemics of viral disease, including the devastating COVID-19. Novel effective antivirals with broad-spectrum coverage are urgently needed. Herein, we reported a novel broad-spectrum antiviral compound PAC5. Oral administration of PAC5 eliminated HBV cccDNA and reduced the large antigen load in distinct mouse models of HBV infection. Strikingly, oral administration of PAC5 in a hamster model of SARS-CoV-2 omicron (BA.1) infection significantly decreases viral loads and attenuates lung inflammation. Mechanistically, PAC5 binds to a pocket near Asp49 in the RNA recognition motif of hnRNPA2B1. PAC5-bound hnRNPA2B1 is extensively activated and translocated to the cytoplasm where it initiates the TBK1-IRF3 pathway, leading to the production of type I IFNs with antiviral activity. Our results indicate that PAC5 is a novel small-molecule agonist of hnRNPA2B1, which may have a role in dealing with emerging infectious diseases now and in the future.
Animals ; Mice ; Antiviral Agents/pharmacology* ; COVID-19 ; Hepatitis B virus ; Interferon Type I/metabolism* ; SARS-CoV-2/drug effects* ; Heterogeneous-Nuclear Ribonucleoprotein Group A-B/antagonists & inhibitors*

As the first line of immune defense for Mycobacterium tuberculosis (Mtb), macrophages also provide a major habitat for Mtb to reside in the host for years. The battles between Mtb and macrophages have been constant since ancient times. Triggered upon Mtb infection, multiple cellular pathways in macrophages are activated to initiate a tailored immune response toward the invading pathogen and regulate the cellular fates of the host as well. Toll-like receptors (TLRs) expressed on macrophages can recognize pathogen-associated-molecular patterns (PAMPs) on Mtb and mediate the production of immune-regulatory cytokines such as tumor necrosis factor (TNF) and type I Interferons (IFNs). In addition, Vitamin D receptor (VDR) and Vitamin D-1-hydroxylase are up-regulated in Mtb-infected macrophages, by which Vitamin D participates in innate immune responses. The signaling pathways that involve TNF, type I IFNs and Vitamin D are inter-connected, which play critical roles in the regulation of necroptosis, apoptosis, and autophagy of the infected macrophages. This review article summarizes current knowledge about the interactions between Mtb and macrophages, focusing on cellular fates of the Mtb-infected macrophages and the regulatory molecules and cellular pathways involved in those processes.
Animals ; Apoptosis ; Autophagy ; Humans ; Interferon Type I ; metabolism ; Macrophages ; immunology ; metabolism ; Mycobacterium tuberculosis ; physiology ; Receptors, Calcitriol ; metabolism ; Steroid Hydroxylases ; metabolism ; Toll-Like Receptors ; metabolism ; Tuberculosis ; immunology ; metabolism ; pathology ; Tumor Necrosis Factors ; metabolism