Levels Of IFN-β Research Articles

RIG-I and MDA5 are modulated by bone morphogenetic protein (BMP6) and are essential for restricting Zika virus infection in human Sertoli cells.

Sexual transmission of Zika virus (ZIKV) is associated with virus persistence in the testes and shedding in the seminal fluid for months after recovery. We previously demonstrated that ZIKV can establish long-term replication without causing cytotoxicity in human Sertoli cells (SC), responsible for maintaining the immune privileged compartment of seminiferous tubules. Functional gene expression analyses also predicted activation of multiple virus sensing pathways including TLR3, RIG-I, and MDA5. Here, we elucidated which of the RNA virus sensing receptors play a decisive role in restricting ZIKV replication. We show that both poly I:C and IFN-β treatment induced a robust antiviral state and reduced ZIKV replication significantly, suggesting that virus sensing and antiviral signaling are functional in SC. Silencing of TLR3, 7, and 9 did not affect virus replication kinetics; however, both RIG-I and MDA5 played a synergistic role in inducing an anti-ZIKV response. Further, the impact of SC-specific immunosuppressive pathways that collectively regulate SC function, specifically the TGF-β superfamily members, TGF-β, Activin A, and BMP6, on ZIKV replication was investigated. While ZIKV did not modulate the expression of TGF-β and Activin A, BMP6 signaling was suppressed at later stages of infection. Notably, treatment with BMP6 increased IFN-β, p-IRF3, and p-STAT1 levels, and expression of key interferon-stimulated genes including MDA5, suggesting that BMP6 enhances antiviral response in SC. Collectively, this study further delineates the key role of the RIG-I-like receptors in sensing ZIKV in SC, and reveals a novel role of BMP6 in modulating innate immune and antiviral response in the testes.

Lactate-induced mtDNA Accumulation Activates cGAS-STING Signaling and the Inflammatory Response in Sjögren's Syndrome.

Acinar epithelial cell atrophy in secretory glands is a hallmark of primary Sjögren's syndrome (pSS), the cause of which is far from elucidated. We examined the role of acinar atrophy by focusing on the metabolism of glandular epithelial cells and mitochondria in the pSS environment. After confirming the presence of a high-lactate environment in the labial glands of human pSS patients, we used the A253 cell line and NOD/Ltj mice as models to investigate the metabolic changes in salivary gland epithelial cells in a high-lactate environment in vitro and in vivo. We found that epithelial cells produced high levels of IL-6, IL-8, IFN-α, IFN-β and TNF-α and exhibited significant NF-κB and type I IFN-related pathway activation. The results confirmed that lactate damaged mitochondrial DNA (mtDNA) and led to its leakage, which subsequently activated the cGAS-STING pathway. Inflammatory cytokine production and pathway activation were inhibited in vivo and in vitro by the lactate scavenger sodium dichloroacetate (DCA). Our study provides new insights into the etiology and treatment of pSS from the perspective of cell metabolism.

Association between the expression of toll-like receptors, cytokines, and homeostatic chemokines in SARS-CoV-2 infection and COVID-19 severity

The recent identification of the involvement of the immune system response in the severity and mortality of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection highlights the importance of cytokines and chemokines as important factors in the clinical outcomes of COVID-19. However, the impact and roles of the BAFF/APRIL cytokine system, homeostatic chemokines (CXCL12, CXCL13, CCL19, and CCL21), as well as Toll-like receptor (TLR)-3/4 in COVID-19, have not been investigated. We sought to assess the expression levels and roles of TLR3/4, BAFF, APRIL, IFN-β, homeostatic chemokines (CXCL12, CXCL13, CCL19, and CCL21), SARS-CoV-2 IgG and IgM antibodies in patients with critical (ICU) and non-ICU (mild) COVID-19 and their association with mortality and disease severity. Significant high levels of TLR-4 mRNA, IFN-β, APRIL, CXCL13, and IgM and IgG antibodies were observed in ICU patients with severe COVID-19 compared to non-ICU COVID-19 patients and healthy controls. On the other hand, BAFF and CCL21 expression were significantly upregulated in non-ICU patients with COVID-19 compared with that in critical COVID-19 patients. The two groups did not differ in TLR-3, CXCL12, and CCL19 levels. Our findings show high expression levels of some inflammatory chemokines in ICU patients with COVID-19. These findings highlight the potential utility of chemokine antagonists as an immune-based treatment for the severe form of COVID-19. We also believe that selective targeting of TLR/spike protein interactions might lead to the development of a new COVID-19 therapy.

LB1533. Impact of Treatment of Hospitalised COVID-19 Patients With Inhaled Interferon Beta-1a (SNG001) on Long COVID Symptoms: Results From the SPRINTER trial

Abstract Background People with post-COVID conditions can have a wide range of symptoms lasting months and it can affect as many as one in five infected people. Interferon beta (IFN-β) is key in host defence against viruses but can be suppressed by virus or host factors locally at the site of infection. Inhalation of SNG001 (IFN-β-1a nebuliser solution) aims to restore lung IFN-β levels. SPRINTER (NCT04732949) was a RCT of inhaled interferon beta in hospitalised COVID-19. There was no effect of SNG001 on the primary endpoints of time to discharge or recovery most likely due to improvements in the standard of care. However, there was an encouraging signal for the key secondary endpoint of prevention of progression to severe disease or death (ITT 26% relative risk reduction [RRR]; Odds Ratio [95% CI]: 0.71 [0.44, 1.15]; Per Protocol 36% RRR; OR 0.63 [0.35, 1.13]). Post hoc analyses showed enhanced effects favouring SNG001 in subgroups at higher risk of progression. We report on the impact of SNG001 on long COVID symptoms in SPRINTER. Methods Patients requiring low-flow oxygen were randomized to receive SNG001 (314) or placebo (309) once daily for 14 days, plus standard-of-care. Long COVID symptoms were assessed as a secondary endpoint at follow-up visits via telephone/video call on Day 60 and Day 90. The following patient reported outcome (PRO) measures were also assessed: General Anxiety Disorder 7 Questionnaire (GAD-7), Patient Health Questionnaire-9 (PHQ-9), Functional Assessment of Chronic Illness Therapy (FACIT) - Fatigue Scale and Brief Pain Inventory (Short Form). Results When compared to placebo, SNG001 reduced the relative risk of common symptoms of long COVID (fatigue/malaise [RRR=35.4%]; dyspnoea [RRR=28.3%]; loss of smell and/or taste [RRR=61.4%]). Analysis of the PROs is ongoing. Assessment of COVID symptoms at Day 60 and 90 follow up visits Effect of SNG001 treatment on long COVID symptoms Conclusion Long COVID can leave patients with lingering cognitive, respiratory, and functional symptoms months after a SARS-CoV-2 infection. Given the shift from pandemic to endemic status for COVID-19 and the need for new treatments then these findings, suggesting SNG001 may be impacting common long COVID symptoms, provide additional support for the further investigation of SNG001. Disclosures Phillip D. Monk, PhD, Synairgen Plc (Employed by Synairgen Research Ltd which is a subsidiary of Synairgen Plc, Stocks/Bonds): Board Member|Synairgen Plc (Employed by Synairgen Research Ltd which is a subsidiary of Synairgen Plc, Stocks/Bonds): Stocks/Bonds Victoria J. Tear, PhD, Synairgen Plc (Employed by Synairgen Research Ltd which is a subsidiary of Synairgen Plc, Stocks/Bonds): Stocks/Bonds Jody L. Brookes, BSc, Synairgen Plc (Employed by Synairgen Research Ltd which is a subsidiary of Synairgen Plc, Stocks/Bonds): Stocks/Bonds Marcin Mankowski, MD, MFPM (Dis), CytoDyn: Advisor/Consultant|Entasis: Advisor/Consultant|ImmuPharma: Advisor/Consultant|Menarini: Advisor/Consultant|Pfizer: Advisor/Consultant|Synairgen: Advisor/Consultant|Venatorx: Advisor/Consultant Ratko Djukanovic, MD, Synairgen: Advisor/Consultant|Synairgen: Honoraria|Synairgen: Stocks/Bonds Stephen T. Holgate, FMedSci, MD, Synairgen: Board Member|Synairgen: Non executive board director, patent on inhaled interferon beta|Synairgen: Stocks/Bonds chris brightling, FMedSci, Synairgen: Advisor/Consultant|Synairgen: Grant/Research Support Tom Wilkinson, PhD, PhD, AZ: Grant/Research Support|AZ: Honoraria|My mhealth: Board Member|My mhealth: Ownership Interest|My mhealth: Stocks/Bonds|Synairgen: Grant/Research Support|Synairgen: Honoraria.

Kuhuang alleviates liver fibrosis by modulating gut microbiota-mediated hepatic IFN signaling and bile acid synthesis.

Background: Liver fibrosis is a common outcome of the pathological progression of chronic liver disease; however, no specific and effective therapeutic agent has been approved for its treatment. We investigated the effects of Kuhuang on liver fibrosis and the underlying mechanisms of action. Materials and methods: To induce hepatic fibrosis, either 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet was administered, or bile duct ligation (BDL) surgery was performed on C57BL/6 mice. Kuhuang was orally administered to mice for 7days before and after bile duct ligation or 4weeks with a DDC diet. Hematoxylin and eosin, Sirius red staining, and immunohistochemical analyses were performed to evaluate hepatic pathology. Hepatic interferon-β (IFN-β) levels were measured using an enzyme-linked immunosorbent assay. RNA sequencing was performed to examine the gene expression profiles of liver tissues. The mRNA expression of inflammatory, profibrotic, and bile acid (BA)-related genes was further validated by qRT-PCR. A targeted metabolomics assay revealed the alteration of the hepatic bile acid (BA) composition. The composition of the gut microbiota was determined via 16S rRNA sequencing. Results: Treatment with Kuhuang attenuated liver fibrosis and reduced the inflammatory response in bile duct ligation and DDC mouse models. In addition, the hepatic IFN signaling pathway was activated following Kuhuang treatment. Kuhuang treatment also significantly decreased hepatic levels of both primary and secondary BAs. In addition, Kuhuang treatment altered gut microbiota composition, with an increased abundance of interferon-inducing Akkermansia and decreased abundance of bile salt hydrolase-producing Lactobacillus, Clostridium, and Bifidobacterium. Furthermore, the abundance of Akkermansia was positively correlated with the hepatic mRNA expression levels of Ifna4, Ifnb, and Isg15, whereas that of Lactobacillus, Clostridium - sensu - stricto - 1, and Bifidobacterium was positively correlated with levels of bile acid synthesis-related genes. Conclusion: Our results suggest that Kuhuang plays a protective role during the progression of liver fibrosis, potentially by altering the composition of the gut microbiota, which consequently activates interferon signaling and inhibits bile acid synthesis in the liver.

Early plasma interferon-β levels as a predictive marker of COVID-19 severe clinical events in adult patients.

We assessed relationships between early peripheral blood type I interferons (IFN)levels, clinical new early warning scores (NEWS), and clinical outcomes in hospitalized coronavirus disease-19(COVID-19) adult patients. Early IFN-β levels were lower among patients who further required intensive care unit (ICU) admission than those measured in patients who did not require an ICU admission duringsevere acute respiratory syndrome coronavirus type 2infection. IFN-β levels were inversely correlated with NEWS only in the subgroup of patients who further required ICU admission. To assess whether peripheral blood IFN-β levels could be a potential relevant biomarker to predict further need for ICU admission, we performed receiver operating characteristic (ROC) curve analyses that showed for all study patients an area under ROC curve of 0.77 growing to 0.86 (p = 0.003) when the analysis was restricted to a subset of patients with NEWS≥5 at the time of hospital admission. Overall, our findings indicated that early peripheral blood IFN-β levels might be a relevant predictive marker of further need for an ICUadmission in hospitalized COVID-19 adult patients, specifically when clinical score (NEWS) was graded as upper than 5 at the time of hospital admission.

Induction of protective interferon-β responses in murine osteoblasts following Staphylococcus aureus infection.

The refractory and recurrent nature of chronic staphylococcal osteomyelitis may be due, at least in part, to the ability of Staphylococcus aureus to invade and persist within bone-forming osteoblasts. However, osteoblasts are now recognized to respond to S. aureus infection and produce numerous immune mediators and bone regulatory factors that can shape the host response. Type I interferons (IFNs) are best known for their antiviral effects, but it is becoming apparent that they impact host susceptibility to a wide range of pathogens including S. aureus. Here, we have assessed the local expression of IFN-β by specific capture ELISA in an established in vivo mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis, specific capture ELISAs, and/or immunoblot analyses, were then used to assess the expression of type I IFNs and select IFN stimulated genes (ISGs) in S. aureus infected primary murine osteoblasts. The effect of IFN-β on intracellular S. aureus burden was assessed in vitro following recombinant cytokine treatment by serial colony counts of liberated bacteria. We report the presence of markedly elevated IFN-β levels in infected bone tissue in a mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis of S. aureus infected osteoblasts showed enrichment of genes associated with type I IFN signaling and ISGs, and elevated expression of mRNA encoding IFN-β and ISG products. IFN-β production was confirmed with the demonstration that S. aureus induces its rapid and robust release by osteoblasts in a dose-dependent manner. Furthermore, we showed increased protein expression of the ISG products IFIT1 and IFIT3 by infected osteoblasts and demonstrate that this occurs secondary to the release of IFN-β by these cells. Finally, we have determined that exposure of S. aureus-infected osteoblasts to IFN-β markedly reduces the number of viable bacteria harbored by these cells. Together, these findings indicate an ability of osteoblasts to respond to bacteria by producing IFN-β that can act in an autocrine and/or paracrine manner to elicit ISG expression and mitigate S. aureus infection.

Structure-activity relationship study of amidobenzimidazole derivatives as stimulator of interferon genes (STING) agonists

Stimulator of interferon genes (STING) is a crucial adaptor protein that can regulate the innate immune response by inducing the secretion of type Ι interferons and other cytokines after recognizing endogenous or exogenous DNA. Due to the key role of STING in the innate immune system, the activation of STING pathway is expected to be an efficacious immunotherapeutic tactic to treat cancer. In this study, we performed a structure-activity relationship study of amidobenzimidazole monomer, led to a series of ABZI STING agonist derivatives with potent STING-activating effects. Among them, compound 72, as a representative compound, markedly activated the STING-TBK1-IRF3 signaling pathway and significantly increased the mRNA and protein levels of IFN-β, CXCL10 and IL-6 in both WT THP-1 cells and human peripheral blood mononuclear cells (hPBMCs). In addition, it was confirmed that compound 72 was highly selective for human STING, specifically targeting human STING signaling and showing no activation of m-STING.

In Vivo and In Vitro Antiviral Activity of Phlorizin Against Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) is one of the most serious pathogens affecting the cattle industry worldwide. Phlorizin, a kind of flavonoids extracted from apple tree roots, leaves, and fruits, has a variety of biological functions and has been widely used as a herbal supplement and food additive. Here, BALB/c mouse and Madin-Darby bovine kidney (MDBK) cells were used to explore the effect and mechanism of phlorizin against BVDV infection. The results showed that phlorizin significantly inhibited CP BVDV replication and improved the histopathological changes of duodenum and spleen in mice. In vitro studies also confirmed the activity of phlorizin against CP BVDV. Exploration on its potential mechanism suggested that phlorizin inhibited CP BVDV-induced beclin-1 level and the conversion rate of LC3B-I to LC3B-II. Interestingly, although phlorizin also showed a protective effect on MDBK cells, which were treated with 3-methyladenine A (3-MA), the effect was significantly weakened. Furthermore, phlorizin suppressed the stage of BVDV replication but showed no effect on stages of attachment and internalization. Our data further indicated that phlorizin promoted IFN-α and IFN-β levels, decreased IL-1β and IL-6 expression, and regulated RIG-I, MDA5, TLR3, and NLRP3 levels. Similar to CP BVDV results, in vivo and in vitro, phlorizin inhibited NCP BVDV (NY-1 and YNJG2020 strains) infection. These results were the first to be discovered that phlorizin might be used as a new dietary strategy for controlling BVDV infection.

Modulation of the CGAS-Sting Anti-Tumor Immune Response Pathway in an NF-KB-Dependent or -Independent Manner As a Potential Therapeutic Target in Classical Hodgkin Lymphoma (cHL)

Introduction: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through production of the second messenger cGAMP and subsequently activation of the adaptor protein STING. The latter activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The potential role of cGAS-STING pathway in anti-tumor immune responses in classical Hodgkin lymphoma (cHL) remains unknown to date. In this study, we hypothesized that NF-kB pathway, a critical player in cHL oncogenesis, may regulate anti-tumor immune responses through modulation of the cGAS-STING pathway activity and gene expression of the relevant interferons and chemokines. Methods: STING expression was immunohistochemically analyzed in a pilot study group of 32 untreated patients with cHL and available tissue from diagnostic lymph node biopsies using a previously validated monoclonal antibody and standard methods. In addition, control lymphoid tissue (2 reactive lymph nodes, 2 tonsils) were used. At least 500 neoplastic Hodgkin & Reed-Sternberg (HRS) cells were counted and an arbitrary 10% cutoff was used for positivity. The in vitro system included 6 cHL cell lines (MDA-V, L-1236, L-428, L-540, HDLM-2, KM-H2). Gene expression (mRNA level) and protein expression and activation (phosphorylation) of cGAS-STING pathway components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot analysis, respectively. The cHL cell lines were treated with a STING agonist and TBK1/IKK inhibitor (Amlexanox) alone or in combination with other agents. Silencing of STING and NF-kB genes was performed in all cHL cell lines using transient transfection (Nucleofector system, Amaxa, Lonza) with specific STING and NF-kB (RELA) siRNA constructs. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of IFN-β, CXCL10, IFN-γ, and a control gene (GAPDH), with RT-qPCR. Results: In reactive lymphoid tissue, STING expression was restricted to dendritic cells and a subset of T-lymphocytes with a cytoplasmic staining pattern, which served as internal positive controls in all immunostainings. Using an arbitrary 10% cutoff, STING was positive in the neoplastic HRS cells of cHL in 20 of 32 (63%) patients with a predominant cytoplasmic pattern. STING expression at the mRNA and protein level was substantially higher in L-1236, L-428 and HDLM-2 compared to other cHL cell lines. Treatment with STING agonist alone stimulated gene expression of IFN-β and/or CXCL10 at a variable level depending on the cell line indicating functional cGAS-STING anti-tumor immune response pathway in cHL. Knocking down STING gene resulted in dramatic increase in CXCL10 gene expression in cHL cells. Silencing of NF-kB (RELA) resulted in activation of cGAS-STING pathway as shown by IRF3 activation (phosphorylation) and increased IRF3 protein levels in L-428 cells, which was associated with increased CXCL10 mRNA levels. Treatment with Amlexanox resulted in downregulation of IFN-β and/or CXCL10 gene expression in cHL cell lines. Conclusion: The cGAS-STING pathway is activated and, at least in part, regulated by the oncogenic NF-kB pathway. STING agonists and Amlexanox modulate gene expression of type 1 IFNs in cHL with direct therapeutic implications. A large cohort of cHL patients is currently being analyzed for the expression and prognostic significance of STING and the final results will be available at the time of the meeting.

Non-targeting siRNA-mediated responses are associated with apoptosis in chemotherapy-induced senescent skin cancer cells

It is widely accepted that siRNA transfection can promote some off-target effects in the genome; however, little is known about how the cells can respond to the presence of non-viral dsRNA. In the present study, non-targeting control siRNA (NTC-siRNA) was used to evaluate its effects on the activity of pathogen and host-derived nucleic acid-associated signaling pathways such as cGAS-STING, RIG-I, MDA5 and NF-κB in A431 skin cancer cells and BJ fibroblasts. NTC-siRNA treatment promoted cytotoxicity in cancer cells. Furthermore, NTC-siRNA-treated doxorubicin-induced senescent cancer cells were more prone to apoptotic cell death compared to untreated doxorubicin-induced senescent cancer cells. NTC-siRNA stimulated the levels of NF-κB, APOBECs, ALY, LRP8 and phosphorylated STING that suggested the involvement of selected components of nucleic acid sensing pathways in NTC-siRNA-mediated cell death response in skin cancer cells. NTC-siRNA-mediated apoptosis in cancer cells was not associated with IFN-β-based pro-inflammatory response and TRDMT1-based adaptive response. In contrast, in NTC-siRNA-treated fibroblasts, an increase in the levels of RIG-I and IFN-β was not accompanied by affected cell viability. We propose that the use of NTC-siRNA in genetic engineering may provoke a number of unexpected effects that should be carefully monitored. In our experimental settings, NTC-siRNA promoted the elimination of doxorubicin-induced senescent cancer cells that may have implications in skin cancer therapies.

Immune marker expression of irradiated mesothelioma cell lines.

Though immune checkpoint inhibition has recently shown encouraging clinical efficacy in mesothelioma, most patients do not respond. Combining immune checkpoint inhibition with radiotherapy presents an attractive option for improving treatment responses owing to the various immunomodulatory effects of radiation on tumors. However, the ideal dosing and scheduling of combined treatment remains elusive, as it is poorly studied in mesothelioma. The present study characterizes the dose- and time-dependent changes to expression of various immune markers and cytokines important to antitumor responses following irradiation of mesothelioma cell lines. Two murine (AB1, AE17) and two human (BYE, JU77) mesothelioma cell lines were treated with titrated gamma-radiation doses (1-8 Gy) and the expression of MHC class-I, MHC class-II and PD-L1 was measured over a series of post-irradiation timepoints (1-72 hours) by flow cytometry. Levels of cytokines IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17A, IL-23, IL-27, MCP-1, IFN-β, IFN-γ, TNF-α, and GM-CSF were measured by multiplex immunoassay in murine cell lines following 8 Gy radiation. Following irradiation, a dose-dependent upregulation of MHC-I and PD-L1 was observed on three of the four cell lines studied to varying extents. For all cell lines, the increase in marker expression was most pronounced 72 hours after radiation. At this timepoint, increases in levels of cytokines IFN-β, MCP-1 and IL-6 were observed following irradiation with 8 Gy in AB1 but not AE17, reflecting patterns in marker expression. Overall, this study establishes the dose- and time-dependent changes in immune marker expression of commonly studied mesothelioma cell lines following radiation and will inform future study into optimal dosing and scheduling of combined radiotherapy and immune checkpoint inhibition for mesothelioma.

Restoration of dendritic cell homeostasis and Type I/Type III interferon levels in convalescent COVID-19 individuals

BackgroundPlasmacytoid and myeloid dendritic cells play a vital role in the protection against viral infections. In COVID-19, there is an impairment of dendritic cell (DC) function and interferon secretion which has been correlated with disease severity.ResultsIn this study, we described the frequency of DC subsets and the plasma levels of Type I (IFNα, IFNβ) and Type III Interferons (IFNλ1), IFNλ2) and IFNλ3) in seven groups of COVID-19 individuals, classified based on days since RT-PCR confirmation of SARS-CoV2 infection. Our data shows that the frequencies of pDC and mDC increase from Days 15–30 to Days 61–90 and plateau thereafter. Similarly, the levels of IFNα, IFNβ, IFNλ1, IFNλ2 and IFNλ3 increase from Days 15–30 to Days 61–90 and plateau thereafter. COVID-19 patients with severe disease exhibit diminished frequencies of pDC and mDC and decreased levels of IFNα, IFNβ, IFNλ1, IFNλ2 and IFNλ3. Finally, the percentages of DC subsets positively correlated with the levels of Type I and Type III IFNs.ConclusionThus, our study provides evidence of restoration of homeostatic levels in DC subset frequencies and circulating levels of Type I and Type III IFNs in convalescent COVID-19 individuals.

Chicken anemia virus VP1 negatively regulates type I interferon via targeting interferon regulatory factor 7 of the DNA-sensing pathway

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway plays a vital role in sensing viral DNA in the cytosol, stimulating type I interferon (IFN) production and triggering the innate immune response against DNA virus infection. However, viruses have evolved effective inhibitors to impede this sensing pathway. Chicken anemia virus (CAV), a nonenveloped ssDNA virus, is a ubiquitous pathogen causing great economic losses to the poultry industry globally. CAV infection is reported to downregulate type I IFN induction. However, whether the cGAS-STING signal axis is used by CAV to regulate type I IFN remains unclear. Our results demonstrate that CAV infection significantly elevates the expression of cGAS and STING at the mRNA level, whereas IFN-β levels are reduced. Furthermore, IFN-β activation was completely blocked by the structural protein VP1 of CAV in interferon stimulatory DNA (ISD) or STING-stimulated cells. VP1 was further confirmed as an inhibitor by interacting with interferon regulatory factor 7 (IRF7) by binding its C-terminal 143–492 aa region. IRF7 dimerization induced by TANK binding kinase 1 (TBK1) could be inhibited by VP1 in a dose-dependent manner. Together, our study demonstrates that CAV VP1 is an effective inhibitor that interacts with IRF7 and antagonizes cGAS-STING pathway-mediated IFN-β activation. These findings reveal a new mechanism of immune evasion by CAV.

Dengue and Zika virus differential infection of human megakaryoblast MEG-01 reveals unique cellular markers

Platelet count is widely used for the diagnosis and follow-up of patients with dengue. Despite its close viral structural and symptomatic homology, ZIKV infection does not typically induce significant thrombocytopenia. To determine the effect of DENV-2 and ZIKV infection on human platelet precursors we utilized MEG-01 cell line to evaluate the viral infection, viability, innate gene expression and release of platelet-like particles (PLPs). DENV-2 induced a higher proportion of cell death at 48–72 h post-infection than ZIKV. The median range of intracellular NS1+/E+ cells was 11.2% (3.3%–25%) and 5% (3%–8.1%) for DENV-2 and ZIKV, respectively (p = 0.03). MEG-01 cells infected with DENV-2 quickly expressed higher levels of IFN-β, indolamine 2,3-dioxygenase and CXCL10 mRNA compared to ZIKV infected cells and DENV-2 but not ZIKV infection reduced the number PLPs from stimulated MEG-01 cells. The results shed light into mechanisms including thrombocytopenia present in patients with DENV but absent in ZIKV infections.

Manganese Coordination Micelles That Activate Stimulator of Interferon Genes and Capture In Situ Tumor Antigens for Cancer Metalloimmunotherapy.

Cancer immunotherapy holds great promise but is generally limited by insufficient induction of anticancer immune responses. Here, a metal micellar nanovaccine is developed by the self-assembly of manganese (Mn), a stimulator of interferon genes (STING) agonist (ABZI) and naphthalocyanine (ONc) coordinated nanoparticles (ONc-Mn-A) in maleimide-modified Pluronic F127 (malF127) micelles. Owing to synergy between Mn and ABZI, the nanovaccine, termed ONc-Mn-A-malF127, elevates levels of interferon-β (IFNβ) by 324- and 8-fold in vivo, compared to use of Mn or ABZI alone. As such, the activation of the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-STING pathway induces sufficient dendritic cell (DC) maturation, eventually resulting in the death of CD8+ T cell-sensitive tumors and CD8+ T cell-resistant tumors by simultaneously promoting cytotoxic CD8+ T cells and NK cells, respectively. Furthermore, with ONc used as a Mn chelator and an efficient photosensitizer, photoinduced immunogenic cell death (ICD) of tumor cells releases damage-associated molecular patterns (DAMPs) and neoantigens from dying primary tumor cells upon laser irradiation, which are captured in situ by malF127 in tumor cells and then transported to DCs. After laser treatment, in addition to the photothermal therapy, immune responses characterized by the level of IFNβ are further elevated by another 4-fold. In murine cancer models, ICD-based metalloimmunotherapy using the ONc-Mn-A-malF127 nanovaccine in a single dose by intravenous injection achieved eradication of primary and distant tumors. Taken together, ONc-Mn-A-malF127 offers a nanoplatform to enhance anticancer efficacy by metalloimmunotherapy and photoinduced ICD based immunotherapy with strong abscopal effect.

Coriolus versicolor and its bioactive molecule are potential immunomodulators against cancer cell metastasis via inactivation of MAPK pathway

Ethnopharmacological relevanceCoriolus versicolor (CV) has been used in traditional Chinese medicine for over 2000 years as a premium medicine for enhancing good health and longevity. The immunomodulatory and anti-cancer effects of polysaccharopeptides (PSP) from cultured CV have been extensively studied; however, the effect and the mechanism of action of other small molecules from CV remain unknown.Aim of the study: we aim to examine the immunomodulatory and anti-cancer effects of the small molecules from CV (SMCV) and identify the active compounds that are responsible for the biological effects against glioblastoma multiforme cells. Materials and methodsThe effects of SMCV/active compound on cytokine and MMP mRNA expressions and productions were assessed by quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. An active compound from SMCV was identified with a bioassay-guided fractionation scheme. The potential mode of action of the active compound was further investigated by identifying the cell signaling pathway. The protein expressions of phospho-ERK, phospho-JNK and phospho-p38 MAPKs were measured by Western Blotting. The anti-invasive effect of SMCV/bioactive compound against T98G, lung carcinoma (A549), and breast adenocarcinoma (MDA-MB-231) cells were determined using invasion assay. ResultsOur results showed that SMCV had strong immunomodulatory effect by suppressing LPS-induced TNF-α production, whereas increasing poly I:C-induced IFN-β level in PBMac. SMCV not only possessed indirect anti-cancer effect by suppressing TNF-α-induced MMP-3 production in glioblastoma T98G cells, but also directly reduced the invasion ability of malignant cells including T98G, A549 and MDA-MB-231. Using bioassay-guided fractionation scheme, we isolated 9-KODE methyl ester (compound AM) that was responsible for the bioactivity of SMCV. This compound suppressed TNF-α-induced MMP-3 production in T98G cells and the suppression may be correlated with the inactivation of p38 mitogen-activated protein kinase (MAPK) pathway. Moreover, compound AM also directly reduced T98G cell invasion. ConclusionResults of our present study provides scientific evidence that SMCV possesses immunomodulatory and anti-cancer effects. Its bioactive compound, compound AM, is a potential new drug candidate against the invasion and metastasis of glioblastoma cells.

P053: The cGAS-STING anti-tumor immune response pathway as a potential therapeutic target in classical Hodgkin Lymphoma (cHL)

Background: Cytosolic DNA of exogenous or endogenous origin triggers activation of cyclic GMP-AMP (cGAMP) synthase (cGAS), a cytosolic DNA sensor, that activates innate immune responses through production of the second messenger cGAMP and subsequently activation of the adaptor protein STING. The latter activates TBK1 and IKK kinases that, in turn, activate IRF3 and NF-κB transcription factors, which induce expression of interferons (IFNs), chemokines and cytokines involved in anti-tumor immune responses. The potential role of cGAS-STING pathway in anti-tumor immune responses in cHL remains unknown to date. Methods: STING expression was immunohistochemically analysed in a pilot study group of 32 untreated patients with cHL and available tissue. The in vitro system included 6 cHL cell lines (MDA-V, L1236, L428, L540, HDLM2, KMH2). Gene expression (RNA level) and protein expression and activation (phosphorylation) of cGAS-STING pathway components at baseline and experimental conditions were analysed by quantitative RT-PCR (RT-qPCR) and Western blot, respectively. The cHL cell lines were treated with a STING agonist and TBK1/IKK inhibitor (Amlexanox) alone or in combination with other agents. Silencing of STING gene was performed using transient transfection with specific STING siRNA. The cGAS-STING-associated anti-tumor immune responses were evaluated by assessing the RNA levels of IFN-β, CXCL10, IFN-γ, and a control gene (GAPDH) with RT-qPCR. Results: Using an arbitrary 10% cutoff, STING was positive in the neoplastic Hodgkin & Reed-Sternberg (HRS) cells of cHL in 20 of 32 (63%) patients with a membranous and cytoplasmic pattern. STING expression at the mRNA and protein level was substantially higher in L1236, L428 and HDLM2 compared to other cHL cell lines. Treatment with STING agonist alone stimulated gene expression of IFN-β and/or CXCL10 at a variable level depending on the cell line indicating functional cGAS-STING anti-tumor immune response pathway in cHL. Knocking down STING gene resulted in decreased CXCL10 and type 1 IFN gene expression by cHL cells. Amlexanox treatment also resulted in downregulation of IFN-β or CXCL10 gene expression in vitro. Conclusion: STING agonists and Amlexanox modulate gene expression of type 1 IFNs in cHL with direct therapeutic implications. A large cohort of cHL patients is currently being analysed for the expression and prognostic significance of STING, and the final results will be available at the time of ISHL 2022.

Serum level of IFNβ distinguishes early from late relapses after biologics withdrawal in rheumatoid arthritis

Since the advent of biological disease modifying anti-rheumatic drugs (bDMARDs) in the treatment of rheumatoid arthritis (RA), most RA patients receiving such drugs have achieved remission at the expense of cost and infection risk. After bDMARDs are withdrawn, a substantial proportion of patients would have relapses even if they were in complete remission. In our previous report, relapse prediction could be made at the time of bDMARD withdrawal by measuring the serum levels of five cytokines. We report herein that, among 73 cytokines examined, serum levels of only interferon β (IFNβ) at the time of bDMARD withdrawal could predict early relapse (within 5 months) in patients who were categorized to relapse by the five cytokines in our previous report, with a cut-off value of 3.38 in log2 and AUC of 0.833. High serum levels of IFNβ in the early-relapse group remained high until actual relapse occurred. Therefore, patients who relapse early might be biochemically different from those who relapse late or do not relapse at all. We recommend that patients who are predicted to relapse early continue bDMARDs even if they are in complete remission. This finding contributes to shared decision-making regarding how and when bDMARDs should be discontinued.

Interferon Beta (IFN-β)-Modified Bone Marrow Mesenchymal Stem Cells (BMSC) Impede Metastatic Tropism of Prostate Cancer via Modulating Transforming Growth Factor-Beta/Smads (TGF-Beta/Smads) Pathway

The study of Bone marrow mesenchymal stem cells (BMSCs)-based treatment is still unmet needs topic in recent years, especially focusing on the therapeutic effects of genetically modified BMSCs. IFN-β acts as a critical mediator in the occurrence and progress of prostate cancer. Additionally, its related signal transduction pathways affect malignancies. This study aims to discussion the mechanism of IFN-β-modified BMSCs in impeding the metastatic tropism of prostate cancer. A total of 40 male mice (SPF) with a clean grade were randomized into 4 groups (10 mice per group) as follows: control group, BMSCs group, IFN-β modified BMSCs group and TGF-beta/Smads inhibitor group. The following indicators were investigated: the expression level of IFN-β in IFN-β-modified BMSCs, in vitro metastatic tropism of prostate cancer cells, quantification of TGF-beta and Smads protein, along with the targeting of IFN-β and TGF-beta/Smads. The expression of IFN-β level was significantly increased denoted in the modified BMSCs (1.82±0.42) in comparison with those unmodified BMSCs (P < 0.05). After 48- and 72-hour culture, the proportion of migrating cells in the IFN-β-modified BMSCs group was significantly decreased than those in other groups (P < 0.05). Meanwhile, cells in the TGF-beta/Smads inhibitor group exhibited a significantly weaker tendency to migrate in comparison with those in the control group and BMSCs group, but still showed more migrating cells compared to cells in the IFN-β-modified BMSCs group (P < 0.05). Moreover, a significant reduction of migrated cells was denoted in the BMSCs group after 48- and 72-hour culture in comparison with the control group (P < 0.05). The weakest expression of TGF-beta/Smads proteins was denoted in the IFN-β-modified BMSCs group, followed by the TGF-beta/Smads inhibitor group, BMSCs group and control group (P < 0.05). The TGF-beta/Smads inhibitor group exhibited significantly decreased levels of TGF-beta/Smads proteins in comparison with the control group and BMSCs group (P < 0.05). Moreover, a significant decline of TGF-beta/Smads proteins was denoted in the BMSCs group in comparison with the control group (P < 0.05). The IFN-β gene was incubated separately with wild-type and mutant plasmids in the dual-luciferase reporter gene assay. The results indicated that the expression of IFN-β was stronger in the mutant plasmids (P < 0.05) IFN-β-modified BMSCs can boost the entrance of IFN-β into prostate cancer cells, thereby enhancing their expression of IFN-β, which resulted in the expression impediment of TGF-beta/Smads signals, leading to an inhibited metastatic tropism of prostate cancer cells. Its mechanism was mainly related to the TGF-beta/Smads signal transduction pathway.