Interferon-ß Overexpression in Adipose Tissue-Derived Stem Cells Induces HepG2 and Macrophage Cell Death in Liver Tumor Organoids via Induction of TNF-Related Apoptosis-Inducing Ligand Expression.

Liver tumor organoids derived from liver tumor tissues and pluripotent stem cells are used for liver tumor research but have several challenges in primary cell isolation and stem cell differentiation. Here, we investigated the potential of HepG2-based liver tumor organoids for screening anticancer drugs by evaluating their responsiveness to IFN-ß produced by mesenchymal stem cells (MSCs). Liver tumor organoids were prepared in three days on Matrigel using HepG2, primary liver sinusoidal epithelial cells (LSECs), LX-2 human hepatic stellate cells, and THP-1-derived macrophages at a ratio of 4:4:1:1, with 105 total cells. Hepatocyte-related and M2 macrophage-associated genes increased in liver tumor organoids. IFN-ß treatment decreased the viability of liver tumor organoids and increased M1 macrophage marker expression (i.e., TNF-α and iNOS) and TRAIL. TRAIL expression was increased in all four cell types exposed to IFN-ß, but cell death was only observed in HepG2 cells and macrophages. Further, MSCs overexpressing IFN-ß (ASC-IFN-ß) also expressed TRAIL, contributing to the reduced viability of liver tumor organoids. In summary, IFN-ß or ASC-IFN-ß can induce TRAIL-dependent HepG2 and macrophage cell death in HepG2-based liver tumor organoids, highlighting these liver tumor organoids as suitable for anticancer drug screening and mechanistic studies.

Dexamethasone impairs the expression of antimicrobial mediators in lipopolysaccharide-activated primary macrophages by inhibiting both expression and function of interferon ß.

Glucocorticoids potently inhibit expression of many inflammatory mediators, and have been widely used to treat both acute and chronic inflammatory diseases for more than seventy years. However, they can have several unwanted effects, amongst which immunosuppression is one of the most common. Here we used microarrays and proteomic approaches to characterise the effect of dexamethasone (a synthetic glucocorticoid) on the responses of primary mouse macrophages to a potent pro-inflammatory agonist, lipopolysaccharide (LPS). Gene ontology analysis revealed that dexamethasone strongly impaired the lipopolysaccharide-induced antimicrobial response, which is thought to be driven by an autocrine feedback loop involving the type I interferon IFNß. Indeed, dexamethasone strongly and dose-dependently inhibited the expression of IFNß by LPS-activated macrophages. Unbiased proteomic data also revealed an inhibitory effect of dexamethasone on the IFNß-dependent program of gene expression, with strong down-regulation of several interferon-induced antimicrobial factors. Surprisingly, dexamethasone also inhibited the expression of several antimicrobial genes in response to direct stimulation of macrophages with IFNß. We tested a number of hypotheses based on previous publications, but found that no single mechanism could account for more than a small fraction of the broad suppressive impact of dexamethasone on macrophage type I interferon signaling, underlining the complexity of this pathway. Preliminary experiments indicated that dexamethasone exerted similar inhibitory effects on primary human monocyte-derived or alveolar macrophages.

CD8+ T cells reduce neuroretina inflammation in mouse by regulating autoreactive Th1 and Th17 cells through IFN-γ.

Various regulatory CD8+ T-cell subsets have been proposed for immune tolerance and have been implicated in controlling autoimmune diseases. However, their phenotypic identities and suppression mechanisms are not yet understood. This study found that coculture of T-cell receptor (TCR)- or interferon (IFN)-ß-activated CD8+ T cells significantly suppressed the cytokine production of Th1 and Th17 cells. By experimenting with the experimental autoimmune uveitis (EAU), we found that adoptive transfer of TCR or IFN-ß-activated CD8+ T cells significantly lessened disease development in an IFN-γ-dependent manner with a decreased uveitogenic Th1 and Th17 response. Interestingly, after adoptive transfer into the EAU mice, the IFN-γ+ CD8+ T cells were recruited more efficiently into the secondary lymphoid organs during the disease-priming phase. This recruitment depends on the IFN-γ-inducible chemokine receptor CXCR3; knocking out CXCR3 abolishes the protective effect of CD8+ T cells in EAU. In conclusion, we identified the critical role of IFN-γ for CD8+ T cells to inhibit Th1 and Th17 responses and ameliorate EAU. CXCR3 is necessary to recruit IFN-γ+ CD8+ T cells to the secondary lymphoid organ for the regulation of autoreactive Th1 and Th17 cells.

Interferon beta treatment is a potent and targeted epigenetic modifier in multiple sclerosis.

Introduction: Multiple Sclerosis (MS) has a complex pathophysiology that involves genetic and environmental factors. DNA methylation (DNAm) is one epigenetic mechanism that can reversibly modulate gene expression. Cell specific DNAm changes have been associated with MS, and some MS therapies such as dimethyl fumarate can influence DNAm. Interferon Beta (IFNß), was one of the first disease modifying therapies in multiple sclerosis (MS). However, how IFNß reduces disease burden in MS is not fully understood and little is known about the precise effect of IFNß treatment on methylation. Methods: The objective of this study was to determine the changes in DNAm associated with INFß use, using methylation arrays and statistical deconvolutions on two separate datasets (total ntreated = 64, nuntreated = 285). Results: We show that IFNß treatment in people with MS modifies the methylation profile of interferon response genes in a strong, targeted, and reproducible manner. Using these identified methylation differences, we constructed a methylation treatment score (MTS) that is an accurate discriminator between untreated and treated patients (Area under the curve = 0.83). This MTS is time-sensitive and in consistent with previously identified IFNß treatment therapeutic lag. This suggests that methylation changes are required for treatment efficacy. Overrepresentation analysis found that IFNß treatment recruits the endogenous anti-viral molecular machinery. Finally, statistical deconvolution revealed that dendritic cells and regulatory CD4+ T cells were most affected by IFNß induced methylation changes. Discussion: In conclusion, our study shows that IFNß treatment is a potent and targeted epigenetic modifier in multiple sclerosis.

Polymorphism in interferon alpha/beta receptor contributes to glucocorticoid response and outcome of ARDS and COVID-19.

BACKGROUND: The use of glucocorticoids has given contradictory results for treating acute respiratory distress syndrome (ARDS). The use of intravenous Interferon beta (IFN ß) for the treatment of ARDS was recently tested in a phase III ARDS trial (INTEREST), in which more than half of the patients simultaneously received glucocorticoids. Trial results showed deleterious effects of glucocorticoids when administered together with IFN ß, and therefore, we aimed at finding the reason behind this. METHODS: We first sequenced the genes encoding the IFN α/ß receptor of the patients, who participated in the INTEREST study (ClinicalTrials.gov Identifier:  NCT02622724 , November 24, 2015) in which the patients were randomized to receive an intravenous injection of IFN ß-1a (144 patients) or placebo (152 patients). Genetic background was analyzed against clinical outcome, concomitant medication, and pro-inflammatory cytokine levels. Thereafter, we tested the influence of the genetic background on IFN α/ß receptor expression in lung organ cultures and whether, it has any effect on transcription factors STAT1 and STAT2 involved in IFN signaling. RESULTS: We found a novel disease association of a SNP rs9984273, which is situated in the interferon α/ß receptor subunit 2 (IFNAR2) gene in an area corresponding to a binding motif of the glucocorticoid receptor (GR). The minor allele of SNP rs9984273 associates with higher IFNAR expression, more rapid decrease of IFN γ and interleukin-6 (IL-6) levels and better outcome in IFN ß treated patients with ARDS, while the major allele associates with a poor outcome especially under concomitant IFN ß and glucocorticoid treatment. Moreover, the minor allele of rs9984273 associates with a less severe form of coronavirus diseases (COVID-19) according to the COVID-19 Host Genetics Initiative database. CONCLUSIONS: The distribution of this SNP within clinical study arms may explain the contradictory results of multiple ARDS studies and outcomes in COVID-19 concerning type I IFN signaling and glucocorticoids.

Stability and Activity of Interferon Beta to Treat Idiopathic Pulmonary Fibrosis with Different Nebulizer Technologies.

Background: Idiopathic pulmonary fibrosis (IPF) is a serious lung disease characterized by lung scarring, which results in breathing difficulty. Currently, patients with IPF exhibit a poor survival rate and have access to very limited therapeutic options. Interferon beta (IFN-ß) has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of relapsing forms of multiple sclerosis, and it has also been shown to exhibit therapeutic potential in IPF. However, clinical use of IFN-ß did not lead to improved overall survival in IPF patients in existing studies. One possibility is the limited efficiency of IFN-ß delivery through intravenous or subcutaneous injection. Materials and Methods: The aerosol particle size distribution was determined with a laser diffraction particle size analyzer to characterize the droplet size and fine particle fraction generated by three types of nebulizers: jet, ultrasonic, and mesh. A breathing simulator was used to assess the delivery efficiency of IFN-ß, and the temperature in the medication reservoirs was monitored with a thermocouple during nebulization. To further evaluate the antifibrotic activity of IFN-ß pre- and postnebulization, bleomycin (BLM)- or transforming growth factor-beta (TGF-ß)-treated human lung fibroblast (HLF) cells were used. Cell viability was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Transwell migration assay and Q-PCR analysis were used to evaluate cell migration and the myofibroblast differentiation ability, respectively. IFN-ß protein samples were prepared using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer, and the expression of IFN-ß was assessed by western blotting. Results: Among the current drug delivery systems, aerosolized medication has shown increased efficacy of drug delivery for treating respiratory diseases when compared with parenteral drugs. It was found that neither the structural integrity nor the biological function of nebulized IFN-ß was compromised by the nebulization process of the mesh nebulizer. In addition, in BLM dose-response or TGF-ß-induced lung fibroblast proliferation assays, these effects could be reversed by both parenteral and inhaled IFN-ß nebulized with the mesh nebulizer. Nebulized IFN-ß with the mesh nebulizer also significantly inhibited the migration and myofibroblast differentiation ability of TGF-ß-treated HLF cells. Conclusions: The investigations revealed the potential efficacy of IFN-ß in the treatment of IPF with the mesh nebulizer, demonstrating the higher efficiency of IFN-ß delivered through the mesh nebulizer.

Comparison of the Antiviral Activity of Remdesivir, Chloroquine, and Interferon-ß as Single or Dual Agents Against the Human Beta-Coronavirus OC43.

The human beta-coronavirus strain, OC43, provides a useful model for testing the antiviral activity of various agents. We compared the activity of several antiviral drugs against OC43, including remdesivir, chloroquine, interferon (IFN)-ß, IFN-λ1, and IFN-λ4, in two distinct cell types: human colorectal carcinoma cell line (HCT-8 cells) and normal human bronchial epithelial (NHBE) cells. We also tested whether these agents mediate additive, synergistic, or antagonistic activity against OC43 infection when used in combination. When used as single agents, remdesivir exhibited stronger antiviral activity than chloroquine, and IFN-ß exhibited stronger activity than IFN-λ1 or IFN-λ4 against OC43 in both HCT-8 and NHBE cells. Anakinra (IL-1 inhibitor) and tocilizumab (IL-6 inhibitor) did not mediate any antiviral activity. The combination of IFN-ß plus chloroquine or remdesivir resulted in higher synergy scores and higher expression of IFN-stimulated genes than did IFN-ß alone. In contrast, the combination of remdesivir plus chloroquine resulted in an antagonistic interaction in NHBE cells. Our findings indicate that the combined use of IFN-ß plus remdesivir or chloroquine induces maximal antiviral activity against human coronavirus strain OC43 in primary human respiratory epithelial cells. Furthermore, our experimental OC43 virus infection model provides an excellent method for evaluating the biological activity of antiviral drugs.

IFNß-Induced CXCL10 Chemokine Expression Is Regulated by Pellino3 Ligase in Monocytes and Macrophages.

IFN-I is the key regulatory component activating and modulating the response of innate and adaptive immune system to bacterial as well as viral pathogens. IFN-I promotes the expression of IFN-induced genes (ISG) and, consequently, the production of chemokines, e.g., CXCL10. Those chemokines control migration and localization of immune cells in tissues, and, thus, are critical to the function of the innate immune system during infection. Consequently, the regulation of IFN-I signaling is essential for the proper induction of an immune response. Our previous study has shown that E3 ubiquitin ligase Pellino3 positively regulates IFNß expression and secretion. Herein, we examined the role of Pellino3 ligase in regulating CXCL10 expression in response to IFNß stimulation. Our experiments were carried out on murine macrophage cell line (BMDM) and human monocytes cell line (THP-1) using IFNß as a IFNAR ligand. We demonstrate that Pellino3 is important for IFNß-induced phosphorylation and nuclear translocation of STAT1/STAT2/IRF9 complex which interacts with CXCL10 promoter and enhances its expression. In this study, we characterize a novel molecular mechanism allowing Pellino3-dependent modulation of the IFNß-induced response in BMDM and THP-1 cell lines.

The µ2 and λ1 Proteins of Mammalian Reovirus Modulate Early Events Leading to Induction of the Interferon Signaling Network.

It has been previously shown that amino acid polymorphisms in reovirus proteins µ2 and λ1 are associated with differing levels of interferon induction. In the present study, viruses carrying these polymorphisms in either or both proteins, were further studied. The two viral determinants exert a synergistic effect on the control of ß-interferon induction at the protein and mRNA level, with a concomitant increase in RIG-I. In contrast, levels of phospho-Stat1 and interferon-stimulated genes are increased in singly substituted viruses but with no further increase when both substitutions were present. This suggests that the viral determinants are acting during initial events of viral recognition. Accordingly, difference between viruses was reduced when infection was performed with partially uncoated virions (ISVPs) and transfection of RNA recovered from early-infected cells recapitulates the differences between viruses harboring the different polymorphisms. Altogether, the data are consistent with a redundant or complementary role of µ2 and λ1, affecting either early disassembly or the nature of the viral RNA in the incoming viral particle. Proteins involved in viral RNA synthesis are thus involved in this likely critical aspect of the ability of different reovirus variants to infect various cell types, and to discriminate between parental and transformed/cancer cells.

Combination of oral STING agonist MSA-2 and anti-TGF-ß/PD-L1 bispecific antibody YM101: a novel immune cocktail therapy for non-inflamed tumors.

BACKGROUND: Non-inflamed tumors, including immune-excluded and immune-desert tumors, are commonly resistant to anti-PD-1/PD-L1 (α-PD-1/PD-L1) therapy. Our previous study reported the potent antitumor activity of anti-TGF-ß/PD-L1 bispecific antibody YM101 in immune-excluded tumors. However, YM101 had limited antitumor activity in immune-desert models. MSA-2 is a novel oral stimulator of interferon genes (STING) agonist, which activates the innate immune system and may synergize with YM101 in overcoming immunotherapy resistance. METHODS: The dose-dependent effect of MSA-2 on STING signaling was determined by interferon-ß level. The maturation and function of dendritic cell (DC) were measured by flow cytometry, RNA-seq, one-way mixed lymphocyte reaction (MLR), OVA peptide pulse, and cytokine/chemokine detection. The synergistic effect between MSA-2 and YM101 was assessed by one-way MLR. The macrophage activation was measured by flow cytometry and cytokine/chemokine detection. The in vivo antitumor activity of MSA-2 combined with YM101 was explored in syngeneic murine tumor models. After treatments, the alterations in the tumor microenvironment (TME) were detected by flow cytometry, immunohistochemistry staining, immunofluorescence staining, RNA-seq, and single-cell RNA-seq (scRNA-seq). RESULTS: MSA-2 could promote the maturation and antigen presentation capability of murine DC. In the one-way MLR assay, MSA-2 synergized with YM101 in enhancing naive T cell activation. Moreover, MSA-2 stimulated the classical activation of macrophage, without significant influence on alternative activation. Further in vivo explorations showed that MSA-2 increased multiple proinflammatory cytokines and chemokines in the TME. MSA-2 combined with YM101 remarkedly retarded tumor growth in immune-excluded and immune-desert models, with superior antitumor activity to monotherapies. Flow cytometry, bulk RNA-seq, and scRNA-seq assays indicated that the combination therapy simultaneously boosted the innate and adaptive immunity, promoted antigen presentation, improved T cell migration and chemotaxis, and upregulated the numbers and activities of tumor-infiltrating lymphocytes. CONCLUSION: Our results demonstrate that MSA-2 synergizes with YM101 in boosting antitumor immunity. This immune cocktail therapy effectively overcomes immunotherapy resistance in immune-excluded and immune-desert models.

Interferon Beta-1a versus Combined Interferon Beta-1a and Oligodendrocyte-Specific FGFR1 Deletion in Experimental Autoimmune Encephalomyelitis.

Recombinant beta interferons-1 (IFNß-1) are used as first line therapies in patients with relapsing multiple sclerosis (MS), a chronic inflammatory and neurodegenerative disease of the CNS. IFNß-1a/b has moderate effects on the prevention of relapses and slowing of disease progression. Fibroblast growth factors (FGFs) and FGF receptors (FGFRs) are known to play a key role in the pathology of MS and its model EAE. To investigate the effects of short-term treatment with s.c. IFNß-1a versus the combined application of s.c. IFNß-1a and oligodendrocyte-specific deletion of FGFR1 (Fgfr1ind-/- mice) in MOG35-55-induced EAE. IFNß-1a (30 mg/kg) was applied s.c. from days 0-7 p.i. of EAE in controls and Fgfr1ind-/- mice. FGFR signaling proteins associated with inflammation/degeneration in MS/EAE were analyzed by western blot in the spinal cord. Further, FGFR1 in Oli-neu oligodendrocytes were inhibited by PD166866 and treated with IFNß-1a (400 ng/mL). Application of IFNß-1a over 8 days resulted in less symptoms only at the peak of disease (days 9-11) compared to controls. Application of IFNß-1a in Fgfr1ind-/- mice resulted in less symptoms primarily in the chronic phase of EAE. Fgfr1ind-/- mice treated with IFNß-1a showed increased expression of pERK and BDNF. In Oli-neu oligodendrocytes, treatment with PD166866 and IFNß-1a also showed an increased expression of pERK and BDNF/TrkB. These data suggest that the beneficial effects in the chronic phase of EAE and on signaling molecules associated with ERK and BDNF expression are caused by the modulation of FGFR1 and not by interferon beta-1a. FGFR may be a potential target for therapy in MS.

Interferon-beta induces major histocompatibility complex of class I (MHC-I) expression and a proinflammatory phenotype in cultivated human astrocytes.

Major histocompatibility complex class I (MHC-I) has been implicated in several types of neuroplasticity phenomena. Interferon beta-1b (IFN-ß) increases MHC-I expression by motoneurons after sciatic nerve crush in mice, improving axonal growth and functional recovery. Additionally, IFN-ß induces glial hypertrophy associated with upregulation of glial fibrillary acidic protein (GFAP) and MHC-I in murine astrocytes in vitro. As knowledge about MHC-I and its role in synaptic plasticity in human astrocytes (HAs) is scarce, we investigated these aspects in mature HAs obtained from the neocortex of patients undergoing surgery due to hippocampal sclerosis. Cells were exposed to media in the absence (0 IU/ml) or presence of IFN-ß for 5 days (500 IU/ml). Beta-2 microglobulin (ß2m), a component of the MHC-I, GFAP and vimentin proteins, was quantified by flow cytometry (FC) and increased by 100%, 60% and 46%, respectively, after IFN-ß exposure. We also performed qRT-PCR gene expression analyses for ß2m, GFAP, vimentin, and pro- and anti-inflammatory cytokines. Our data showed that IFN-ß-treated astrocytes displayed ß2m and GFAP gene upregulation. Additionally, they presented a proinflammatory profile with increase in the IL-6 and IL-1ß genes and a tendency to upregulate TNF-α. Moreover, we evaluated the effect of HAs conditioned medium (CM) on the formation/maintenance of neurites/synapses by the PC12 lineage. Synaptophysin protein expression was quantified by FC. The CM of IFN-ß-activated astrocytes was not harmful to PC12 neurites, and there was no change in synaptophysin protein expression. Therefore, IFN-ß activated HAs by increasing GFAP, vimentin and MHC-I protein expression. Like MHC-I modulation and astrocyte activation may be protective after peripheral nerve damage and in some neurodegenerative conditions, this study opens perspectives on the pathophysiological roles of astroglial MHC-I in the human CNS.

PARPi treatment enhances radiotherapy-induced ferroptosis and antitumor immune responses via the cGAS signaling pathway in colorectal cancer.

In cancer cells, poly (ADP-ribose) polymerase (PARP)-1 and PARP2 initiate and regulate DNA repair pathways to protect against DNA damage and cell death caused by radiotherapy or chemotherapy. Radiotherapy and PARP inhibitors (PARPis) have been combined in clinical trials, but their action mechanisms remain unclear. Here, we show that activated by ionizing radiation (IR) generated dsDNA, cyclic GMP-AMP synthase (cGAS) signaling promoted regulated cell death, specifically ferroptosis, via the activating transcription factor 3 (ATF3)-solute carrier family 7 member 11 axis and the antitumor immune response via the interferon-ß-CD8+ T cell pathway. Niraparib, a widely used PARPi, augmented cGAS-mediated ferroptosis and immune activation. In colorectal cancer models, cGAS knockdown (KD) compromised IR-induced ferroptosis via downregulation of ATF3 (key ferroptosis regulator) expression. cGAS depletion reversed IR-induced infiltration of CD8+ T or CD8+GZMB+ T cells in the cGAS KD group. Survival analysis of paired tumor samples before and after standard radiotherapy revealed that high expression levels of cGAS, ATF3, and PTGS2 and high density of CD8+ T cells resulted in a significantly high disease-free survival rate in patients with rectal cancer. Therefore, PARPi treatment increases the cytoplasmic accumulation of dsDNA caused by IR, triggering the cGAS signaling-mediated tumor control in cancer cell lines and mouse xenograft models.

Interferon-ß promotes the survival and function of induced regulatory T cells.

Type I interferons (IFNs) are pleiotropic cytokines and impact various immune cells, including regulatory T cells (Treg cells). The effect of type-I IFNs on the development and function of Treg cells is quite controversial. Here we induced Treg cells (iTreg cells) from naïve CD4+ T cells in vitro in the presence or absence of IFN-ß to elucidate its direct effect on the induction of iTreg cells. We found that IFN-ß suppressed the proliferation of iTreg cells but enhanced their expression of anti-apoptotic genes Bcl-2 and Mcl-1 during the development of iTreg cells. We also found that IFN-ß promoted suppression of conventional T cell proliferation by iTreg cells. These results suggest that IFN-ß promotes the survival and immunomodulatory function of iTreg cells.

Immunomodulatory antitumor effect of interferon­beta combined with gemcitabine in pancreatic cancer.

Resistance to gemcitabine is common and critically limits its therapeutic efficacy in patients with pancreatic cancer. Interferon­beta (IFN­ß) induces numerous antitumor effects and synergizes with gemcitabine treatment. The immunomodulatory effects of this treatment regimen have not yet been described. In the present study, the antitumor effect of IFN­ß combined with gemcitabine was investigated in immune competent mice. Mouse KPC3 cells were used in all experiments. Treatment effects were determined with cell proliferation assay. Reverse transcription­quantitative PCR was used to measure gene expression. For in vivo experiments, cells were subcutaneously injected in immune competent mice. For immune profiling, NanoString analysis was performed on tumor samples of treated and untreated mice. Baseline expression of Ifnar­1 and Ifnar­2c in KPC3 cells was 1.42±0.16 and 1.50±0.17, respectively. IC50 value of IFN­ß on cell growth was high (>1,000 IU/ml). IFN­ß pre­treatment increased the in vitro response to gemcitabine (1.3­fold decrease in EC50; P<0.001). In vivo, tumor size was not statistically significant smaller in mice treated with IFN­ß plus gemcitabine (707±92 mm3 vs. 1,239±338 mm3 in vehicle­treated mice; P=0.16). IFN­ß alone upregulated expression of numerous immune­related genes. This effect was less pronounced when combined with gemcitabine. For the first time, to the best of our knowledge, the immunomodulatory effects of IFN­ß, alone and combined with gemcitabine, in pancreatic cancer were reported. Prognostic markers for predicting effective responses to IFN­ß therapy are urgently needed.

Integrated CRISPR-Cas9 System-Mediated Knockout of IFN-γ and IFN-γ Receptor 1 in the Vero Cell Line Promotes Viral Susceptibility.

The current pandemic and the possible emergence of new viruses urgently require the rapid development of antiviral vaccines and therapeutics. However, some viruses or newly generated variants are difficult to culture in common cell types or exhibit low viral susceptibility in vivo, making it difficult to manufacture viral vector-based vaccines and understand host-virus interactions. To address these issues, we established new cell lines deficient in both type I and type II interferon responses, which are essential for host immunity and interference with virus replication. These cell lines were generated by developing an integrated CRISPR-Cas9 system that simultaneously expresses dual-guide RNA cassettes and Cas9 nuclease in a single plasmid. Using this highly efficient gene-editing system, we successfully established three cell lines starting from IFN-α/ß-deficient Vero cells, deleting the single interferon-gamma (IFNG) gene, the IFNG receptor 1 (IFNGR1) gene, or both genes. All cell lines clearly showed a decrease in IFN-γ-responsive antiviral gene expression and cytokine production. Moreover, production of IFN-γ-induced cytokines remained low, even after HSV-1 or HCoV-OC43 infection, while expression of the receptor responsible for viral entry increased. Ultimately, knockout of IFN-signaling genes in these cell lines promoted cytopathic effects and increased apoptosis after viral infection up to three-fold. These results indicate that our integrated CRISPR-Cas9-mediated IFNG- and IFNGR1-knockout cell lines promote virus replication and will be useful in viral studies used to design novel vaccines and therapies.

Interferon-ß regulates proresolving lipids to promote the resolution of acute airway inflammation.

Aberrant immune responses, including hyperresponsiveness to Toll-like receptor (TLR) ligands, underlie acute respiratory distress syndrome (ARDS). Type I interferons confer antiviral activities and could also regulate the inflammatory response, whereas little is known about their actions to resolve aberrant inflammation. Here we report that interferon-ß (IFN-ß) exerts partially overlapping, but also cooperative actions with aspirin-triggered 15-epi-lipoxin A4 (15-epi-LXA4) and 17-epi-resolvin D1 to counter TLR9-generated cues to regulate neutrophil apoptosis and phagocytosis in human neutrophils. In mice, TLR9 activation impairs bacterial clearance, prolongs Escherichia coli-evoked lung injury, and suppresses production of IFN-ß and the proresolving lipid mediators 15-epi-LXA4 and resolvin D1 (RvD1) in the lung. Neutralization of endogenous IFN-ß delays pulmonary clearance of E. coli and aggravates mucosal injury. Conversely, treatment of mice with IFN-ß accelerates clearance of bacteria, restores neutrophil phagocytosis, promotes neutrophil apoptosis and efferocytosis, and accelerates resolution of airway inflammation with concomitant increases in 15-epi-LXA4 and RvD1 production in the lungs. Pharmacological blockade of the lipoxin receptor ALX/FPR2 partially prevents IFN-ß-mediated resolution. These findings point to a pivotal role of IFN-ß in orchestrating timely resolution of neutrophil and TLR9 activation-driven airway inflammation and uncover an IFN-ß-initiated resolution program, activation of an ALX/FPR2-centered, proresolving lipids-mediated circuit, for ARDS.

Tryptanthrin attenuates TLR3-mediated STAT1 activation in THP-1 cells.

Upon viral infection, dysregulated immune responses are associated with the disease exacerbation and poor prognosis. The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway are essential for the innate immune responses against invading viruses as well as for sustained activation of macrophages. Tryptanthrin, a natural alkaloid, exhibits various bioactivities, including anti-microbial and anti-inflammatory effects. The aim of this study was to elucidate the effects of tryptanthrin on toll-like receptor 3 (TLR3)-mediated STAT1 activation in macrophages in vitro. Using phorbol myristate acetate (PMA)-differentiated THP-1 cells, we analyzed the protein level of phosphorylated-STAT1 (p-STAT1) upon stimulation with polyinosinic-polycytidylic acid (poly IC), a well-known TLR3 ligand, with and without tryptanthrin. We found that tryptanthrin decreased the protein level of p-STAT1 in a concentration-dependent manner after poly IC stimulation. On the other hand, tryptanthrin did not affect the levels of p-STAT1 upon stimulation with lipopolysaccharide from Escherichia coli. Consistently, tryptanthrin suppressed poly IC-induced mRNA expression of interferon (IFN)-stimulated genes which are regulated by STAT1. Moreover, tryptanthrin decreased the protein level of phosphorylated-IFN regulatory factor 3 and the subsequent IFN-ß mRNA induction after poly IC stimulation. Tryptanthrin is a promising therapeutic agent for the aberrant activation of macrophages caused by viral infection.

A synthetic toll-like receptor 7 agonist inhibits porcine reproductive and respiratory syndrome virus replication in piglets.

Toll-like receptor 7 (TLR7) agonists have been shown to exert therapeutic effects against several viruses. However, antiviral potential of TLR7 agonist in inhibiting porcine reproductive and respiratory syndrome virus (PRRSV) infection has not been assessed in vivo. In our previous study, a synthetic TLR7 agonist, SZU101, was confirmed to inhibit PRRSV infection of porcine alveolar macrophages (PAMs). Here, antiviral effects of SZU101 were evaluated in PRRSV-challenged piglets based on assessments of rectal temperature, viremia, gross and microscopic lung lesions, PRRSV-specific antibodies, PRRSV-specific lymphocyte proliferation and serum IFN-ß level. Our results revealed that SZU101 treatment alleviated PRRSV-induced rectal temperature spikes, pulmonary pathologic changes, and serum viral load. Meanwhile, administration of SZU101 led to increased proliferation of PRRSV-specific lymphocytes and serum IFN-ß levels, but did not enhance PRRSV-specific antibody production. These results demonstrate that SZU101 has potential as a therapeutic treatment for PRRS.

Interferon beta attenuates recognition memory impairment and improves brain glucose uptake in a rat model of Alzheimer's disease: Involvement of mitochondrial biogenesis and PI3K pathway.

Interferon beta (IFNß) is a cytokine with anti-apoptotic and anti-inflammatory properties, and its beneficial effects on Alzheimer's disease (AD) have been recently shown. The alterations in cerebral glucose uptake are closely linked to memory deficit and AD progression. The current study was designed to determine if IFNß can improve recognition memory and brain glucose uptake in a rat model of AD. The lentiviruses expressing mutant human amyloid precursor protein were injected bilaterally to the rat hippocampus. From day 23 after virus injection, rats were intranasally treated with recombinant IFNß protein (68,000 IU/rat) every other day until day 50. Recognition memory performance was evaluated by novel object recognition test on days 46-49. The 18F-2- fluoro-deoxy-d-glucose positron emission tomography (18F-FDG-PET) was used to determine changes in brain glucose metabolism on day 50. The expression of the PI3K/Akt pathway components, neurotrophins and mitochondrial biogenesis factors were also measured by qPCR in the hippocampus. Our results showed that IFNß treatment improves recognition memory performance in parallel with increased glucose uptake and neuronal survival in the hippocampus of the AD rats. The neuroprotective effect of IFNß could be attributed, at least partly, to activation of PI3K-Akt-mTOR signaling pathway, increased expression of NGF, and mitochondrial biogenesis. Taken together, our findings suggest the therapeutic potential of IFNß for AD.