Dimethyl fumarate and 4-octyl itaconate are anticoagulants that suppress Tissue Factor in macrophages via inhibition of Type I Interferon.

Excessive inflammation-associated coagulation is a feature of infectious diseases, occurring in such conditions as bacterial sepsis and COVID-19. It can lead to disseminated intravascular coagulation, one of the leading causes of mortality worldwide. Recently, type I interferon (IFN) signaling has been shown to be required for tissue factor (TF; gene name F3) release from macrophages, a critical initiator of coagulation, providing an important mechanistic link between innate immunity and coagulation. The mechanism of release involves type I IFN-induced caspase-11 which promotes macrophage pyroptosis. Here we find that F3 is a type I IFN-stimulated gene. Furthermore, F3 induction by lipopolysaccharide (LPS) is inhibited by the anti-inflammatory agents dimethyl fumarate (DMF) and 4-octyl itaconate (4-OI). Mechanistically, inhibition of F3 by DMF and 4-OI involves suppression of Ifnb1 expression. Additionally, they block type I IFN- and caspase-11-mediated macrophage pyroptosis, and subsequent TF release. Thereby, DMF and 4-OI inhibit TF-dependent thrombin generation. In vivo, DMF and 4-OI suppress TF-dependent thrombin generation, pulmonary thromboinflammation, and lethality induced by LPS, E. coli, and S. aureus, with 4-OI additionally attenuating inflammation-associated coagulation in a model of SARS-CoV-2 infection. Our results identify the clinically approved drug DMF and the pre-clinical tool compound 4-OI as anticoagulants that inhibit TF-mediated coagulopathy via inhibition of the macrophage type I IFN-TF axis.

SARS-CoV-2 spike and nucleocapsid proteins fail to activate human dendritic cells or γδ T cells.

γδ T cells are thought to contribute to immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but the mechanisms by which they are activated by the virus are unknown. Using flow cytometry, we investigated if the two most abundant viral structural proteins, spike and nucleocapsid, can activate human γδ T cell subsets, directly or in the presence of dendritic cells (DC). Both proteins failed to induce interferon-γ production by Vδ1 or Vδ2 T cells within fresh mononuclear cells or lines of expanded γδ T cells generated from healthy donors, but the same proteins stimulated CD3+ cells from COVID-19 patients. The nucleocapsid protein stimulated interleukin-12 production by DC and downstream interferon-γ production by co-cultured Vδ1 and Vδ2 T cells, but protease digestion and use of an alternative nucleocapsid preparation indicated that this activity was due to contaminating non-protein material. Thus, SARS-CoV-2 spike and nucleocapsid proteins do not have stimulatory activity for DC or γδ T cells. We propose that γδ T cell activation in COVID-19 patients is mediated by immune recognition of viral RNA or other structural proteins by γδ T cells, or by other immune cells, such as DC, that produce γδ T cell-stimulatory ligands or cytokines.

Study protocol for the St James's Hospital, Tallaght University Hospital, Trinity College Dublin Allied Researchers' (STTAR) Bioresource for COVID-19.

Background: The current coronavirus disease 2019 (COVID-19) pandemic began in Ireland with the first confirmed positive case in March 2020. In the early stages of the pandemic clinicians and researchers in two affiliated Dublin hospitals identified the need for a COVID-19 biobanking initiative to support and enhance research into the disease. Through large scale analysis of clinical, regional, and genetic characteristics of COVID-19 patients, biobanks have helped identify, and so protect, at risk patient groups The STTAR Bioresource has been created to collect and store data and linked biological samples from patients with SARS-CoV-2 infection and healthy and disease controls. Aim: The primary objective of this study is to build a biobank, to understand the clinical characteristics and natural history of COVID-19 infection with the long-term goal of research into improved disease understanding, diagnostic tests and treatments. Methods: This is a prospective dual-site cohort study across two tertiary acute university teaching hospitals. Patients are recruited from inpatient wards or outpatient clinics. Patients with confirmed COVID-19 infection as well as healthy and specific disease control groups are recruited.  Biological samples are collected and a case report form detailing demographic and medical background is entered into the bespoke secure online Dendrite database. Impact: The results of this study will be used to inform national and international strategy on health service provision and disease management related to COVID-19. In common with other biobanks, study end points  evolve over time as new research questions emerge. They currently include patient survival, occurrence of severe complications of the disease or its therapy, occurrence of persistent symptoms following recovery from the acute illness and vaccine responses.

Inhibition of the IFN-α JAK/STAT Pathway by MERS-CoV and SARS-CoV-1 Proteins in Human Epithelial Cells.

Coronaviruses (CoVs) have caused several global outbreaks with relatively high mortality rates, including Middle East Respiratory Syndrome coronavirus (MERS)-CoV, which emerged in 2012, and Severe Acute Respiratory Syndrome (SARS)-CoV-1, which appeared in 2002. The recent emergence of SARS-CoV-2 highlights the need for immediate and greater understanding of the immune evasion mechanisms used by CoVs. Interferon (IFN)-α is the body's natural antiviral agent, but its Janus kinase/signal transducer and activators of transcription (JAK/STAT) signalling pathway is often antagonized by viruses, thereby preventing the upregulation of essential IFN stimulated genes (ISGs). Therapeutic IFN-α has disappointingly weak clinical responses in MERS-CoV and SARS-CoV-1 infected patients, indicating that these CoVs inhibit the IFN-α JAK/STAT pathway. Here we show that in lung alveolar A549 epithelial cells expression of MERS-CoV-nsp2 and SARS-CoV-1-nsp14, but not MERS-CoV-nsp5, increased basal levels of total and phosphorylated STAT1 & STAT2 protein, but reduced IFN-α-mediated phosphorylation of STAT1-3 and induction of MxA. While MERS-CoV-nsp2 and SARS-CoV-1-nsp14 similarly increased basal levels of STAT1 and STAT2 in bronchial BEAS-2B epithelial cells, unlike in A549 cells, they did not enhance basal pSTAT1 nor pSTAT2. However, both viral proteins reduced IFN-α-mediated induction of pSTAT1-3 and ISGs (MxA, ISG15 and PKR) in BEAS-2B cells. Furthermore, even though IFN-α-mediated induction of pSTAT1-3 was not affected by MERS-CoV-nsp5 expression in BEAS-2B cells, downstream ISG induction was reduced, revealing that MERS-CoV-nsp5 may use an alternative mechanism to reduce antiviral ISG induction in this cell line. Indeed, we subsequently discovered that all three viral proteins inhibited STAT1 nuclear translocation in BEAS-2B cells, unveiling another layer of inhibition by which these viral proteins suppress responses to Type 1 IFNs. While these observations highlight cell line-specific differences in the immune evasion effects of MERS-CoV and SARS-CoV-1 proteins, they also demonstrate the broad spectrum of immune evasion strategies these deadly coronaviruses use to stunt antiviral responses to Type IFN.

Unravelling the Immunomodulatory Effects of Viral Ion Channels, towards the Treatment of Disease.

The current COVID-19 pandemic has highlighted the need for the research community to develop a better understanding of viruses, in particular their modes of infection and replicative lifecycles, to aid in the development of novel vaccines and much needed anti-viral therapeutics. Several viruses express proteins capable of forming pores in host cellular membranes, termed "Viroporins". They are a family of small hydrophobic proteins, with at least one amphipathic domain, which characteristically form oligomeric structures with central hydrophilic domains. Consequently, they can facilitate the transport of ions through the hydrophilic core. Viroporins localise to host membranes such as the endoplasmic reticulum and regulate ion homeostasis creating a favourable environment for viral infection. Viroporins also contribute to viral immune evasion via several mechanisms. Given that viroporins are often essential for virion assembly and egress, and as their structural features tend to be evolutionarily conserved, they are attractive targets for anti-viral therapeutics. This review discusses the current knowledge of several viroporins, namely Influenza A virus (IAV) M2, Human Immunodeficiency Virus (HIV)-1 Viral protein U (Vpu), Hepatitis C Virus (HCV) p7, Human Papillomavirus (HPV)-16 E5, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) Open Reading Frame (ORF)3a and Polyomavirus agnoprotein. We highlight the intricate but broad immunomodulatory effects of these viroporins and discuss the current antiviral therapies that target them; continually highlighting the need for future investigations to focus on novel therapeutics in the treatment of existing and future emergent viruses.

Deoxycholic acid induces proinflammatory cytokine production by model oesophageal cells via lipid rafts.

The bile acid component of gastric refluxate has been implicated in inflammation of the oesophagus including conditions such as gastro-oesophageal reflux disease (GORD) and Barrett's Oesophagus (BO). Here we demonstrate that the hydrophobic bile acid, deoxycholic acid (DCA), stimulated the production of IL-6 and IL-8 mRNA and protein in Het-1A, a model of normal oesophageal cells. DCA-induced production of IL-6 and IL-8 was attenuated by pharmacologic inhibition of the Protein Kinase C (PKC), MAP kinase, tyrosine kinase pathways, by the cholesterol sequestering agent, methyl-beta-cyclodextrin (MCD) and by the hydrophilic bile acid, ursodeoxycholic acid (UDCA). The cholesterol-interacting agent, nystatin, which binds cholesterol without removing it from the membrane, synergized with DCA to induce IL-6 and IL-8. This was inhibited by the tyrosine kinase inhibitor genistein. DCA stimulated the phosphorylation of lipid raft component Src tyrosine kinase (Src). while knockdown of caveolin-1 expression using siRNA resulted in a decreased level of IL-8 production in response to DCA. Taken together, these results demonstrate that DCA stimulates IL-6 and IL-8 production in oesophageal cells via lipid raft-associated signaling. Inhibition of this process using cyclodextrins represents a novel therapeutic approach to the treatment of inflammatory diseases of the oesophagus including GORD and BO.

Opportunities to Target T Cell Trafficking in Pediatric Inflammatory Bowel Disease.

T cell subsets are considered central orchestrators of inflammation and homeostasis in the intestine and are established targets for the treatment of inflammatory bowel disease. While approaches aimed at the neutralization of T cell effector cytokines have provided significant benefits for pediatric and adult patients, more recent strategies aimed at inhibiting the infiltration of pathogenic T cell subsets have also emerged. In this review, we describe current knowledge surrounding the function of T cell subsets in pediatric inflammatory bowel disease and outline approaches aimed at targeting T cell trafficking to the intestine which may represent a new treatment option for pediatric inflammatory bowel disease.

An Overview of Current Knowledge of Deadly CoVs and Their Interface with Innate Immunity.

Coronaviruses are a large family of zoonotic RNA viruses, whose infection can lead to mild or lethal respiratory tract disease. Severe Acute Respiratory Syndrome-Coronavirus-1 (SARS-CoV-1) first emerged in Guangdong, China in 2002 and spread to 29 countries, infecting 8089 individuals and causing 774 deaths. In 2012, Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) emerged in Saudi Arabia and has spread to 27 countries, with a mortality rate of ~34%. In 2019, SARS-CoV-2 emerged and has spread to 220 countries, infecting over 100,000,000 people and causing more than 2,000,000 deaths to date. These three human coronaviruses cause diseases of varying severity. Most people develop mild, common cold-like symptoms, while some develop acute respiratory distress syndrome (ARDS). The success of all viruses, including coronaviruses, relies on their evolved abilities to evade and modulate the host anti-viral and pro-inflammatory immune responses. However, we still do not fully understand the transmission, phylogeny, epidemiology, and pathogenesis of MERS-CoV and SARS-CoV-1 and -2. Despite the rapid application of a range of therapies for SARS-CoV-2, such as convalescent plasma, remdesivir, hydroxychloroquine and type I interferon, no fully effective treatment has been determined. Remarkably, COVID-19 vaccine research and development have produced several offerings that are now been administered worldwide. Here, we summarise an up-to-date understanding of epidemiology, immunomodulation and ongoing anti-viral and immunosuppressive treatment strategies. Indeed, understanding the interplay between coronaviruses and the anti-viral immune response is crucial to identifying novel targets for therapeutic intervention, which may even prove invaluable for the control of future emerging coronavirus.

Barrett's Registry Collaboration of academic centers in Ireland reveals high progression rate of low-grade dysplasia and low risk from nondysplastic Barrett's esophagus: report of the RIBBON network.

Barrett's esophagus (BE) is the main pathological precursor of esophageal adenocarcinoma (EAC). Progression to high-grade dysplasia (HGD) or EAC from nondysplastic BE (NDBE), low-grade dysplasia (LGD) and indefinite for dysplasia (IND) varies widely between population-based studies and specialized centers for many reasons, principally the rigor of the biopsy protocol and the accuracy of pathologic definition. In the Republic of Ireland, a multicenter prospective registry and bioresource (RIBBON) was established in 2011 involving six academic medical centers, and this paper represents the first report from this network. A detailed clinical, endoscopic and pathologic database registered 3,557 patients. BE was defined strictly by both endoscopic evidence of Barrett's epithelium and the presence of specialized intestinal metaplasia (SIM). A prospective web-based database was used to gather information with initial and follow-up data abstracted by a data manager at each site. A total of 2,244 patients, 1,925 with no dysplasia, were included with complete follow-up. The median age at diagnosis was 60.5 with a 2.1:1 male to female ratio and a median follow-up time of 2.7 years (IQR 1.19-4.04), and 6609.25 person years. In this time period, 125 (5.57%) progressed to HGD/EAC, with 74 (3.3%) after 1 year of follow-up and 38 (1.69%) developed EAC, with 20 (0.89%) beyond 1 year. The overall incidence of HGD/EAC was 1.89% per year; 1.16% if the first year is excluded. The risk of progression to EAC alone overall was 0.57% per year, 0.31% excluding the first year, and 0.21% in the 1,925 patients who had SIM alone at diagnosis. Low-grade dysplasia (LGD) progressed to HGD/EAC in 31% of patients, a progression rate of 12.96% per year, 6.71% with the first year excluded. In a national collaboration of academic centers in Ireland, the progression rate for NDBE was similar to recent population studies. Almost one in two who progressed was evident within 1 year. Crucially, LGD diagnosed and confirmed by specialist gastrointestinal pathologists represents truly high-risk disease, highlighting the importance of expertise in diagnosis and management, and providing indirect support for ablative therapies in this context.

The hepatitis C virus (HCV) protein, p7, suppresses inflammatory responses to tumor necrosis factor (TNF)-α via signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK)-mediated induction of suppressor of cytokine signaling (SOCS)3.

Viruses use a spectrum of immune evasion strategies that enable infection and replication. The acute phase of hepatitis C virus (HCV) infection is characterized by nonspecific and often mild clinical symptoms, suggesting an immunosuppressive mechanism that, unless symptomatic liver disease presents, allows the virus to remain largely undetected. We previously reported that HCV induced the regulatory protein suppressor of cytokine signaling (SOCS)3, which inhibited TNF-α-mediated inflammatory responses. However, the mechanism by which HCV up-regulates SOCS3 remains unknown. Here we show that the HCV protein, p7, enhances both SOCS3 mRNA and protein expression. A p7 inhibitor reduced SOCS3 induction, indicating that p7's ion channel activity was required for optimal up-regulation of SOCS3. Short hairpin RNA and chemical inhibition revealed that both the Janus kinase-signal transducer and activator of transcription (JAK-STAT) and MAPK pathways were required for p7-mediated induction of SOCS3. HCV-p7 expression suppressed TNF-α-mediated IκB-α degradation and subsequent NF-κB promoter activity, revealing a new and functional, anti-inflammatory effect of p7. Together, these findings identify a molecular mechanism by which HCV-p7 induces SOCS3 through STAT3 and ERK activation and demonstrate that p7 suppresses proinflammatory responses to TNF-α, possibly explaining the lack of inflammatory symptoms observed during early HCV infection.-Convery, O., Gargan, S., Kickham, M., Schroder, M., O'Farrelly, C., Stevenson, N. J. The hepatitis C virus (HCV) protein, p7, suppresses inflammatory responses to tumor necrosis factor (TNF)-α via signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK)-mediated induction of suppressor of cytokine signaling (SOCS)3.

HIV-1 Promotes the Degradation of Components of the Type 1 IFN JAK/STAT Pathway and Blocks Anti-viral ISG Induction.

Anti-retroviral therapy successfully suppresses HIV-1 infection, but fails to provide a cure. During infection Type 1 IFNs normally play an essential role in viral clearance, but in vivo IFN-α only has a modest impact on HIV-1 infection, suggesting its possible targeting by HIV. Here, we report that the HIV protein, Vif, inhibits effective IFN-α signalling via degradation of essential JAK/STAT pathway components. We found that STAT1 and STAT3 are specifically reduced in HEK293T cells expressing Vif and that full length, infectious HIV-1 IIIB strain promotes their degradation in a Vif-dependent manner. HIV-1 IIIB infection of myeloid ThP-1 cells also reduced the IFN-α-mediated induction of the anti-viral gene, ISG15, but not MxA, revealing a functional consequence of this HIV-1-mediated immune evasion strategy. Interestingly, while total STAT levels were not reduced upon in vitro IIIB infection of primary human PBMCs, IFN-α-mediated phosphorylation of STAT1 and STAT3 and ISG induction were starkly reduced, with removal of Vif (IIIBΔVif), partially restoring pSTATs, ISG15 and MxB induction. Similarly, pSTAT1 and pSTAT3 expression and IFN-α-induced ISG15 were reduced in PBMCs from HIV-infected patients, compared to healthy controls. Furthermore, IFN-α pre-treatment of a CEM T lymphoblast cells significantly inhibited HIV infection/replication (measured by cellular p24), only in the absence of Vif (IIIBΔVif), but was unable to suppress full length IIIB infection. When analysing the mechanism by which Vif might target the JAK/STAT pathway, we found Vif interacts with both STAT1 and STAT3, (but not STAT2), and its expression promotes ubiquitination and MG132-sensitive, proteosomal degradation of both proteins. Vif's Elongin-Cullin-SOCS-box binding motif enables the formation of an active E3 ligase complex, which we found to be required for Vif's degradation of STAT1 and STAT3. In fact, the E3 ligase scaffold proteins, Cul5 and Rbx2, were also found to be essential for Vif-mediated proteasomal degradation of STAT1 and STAT3. These results reveal a target for HIV-1-Vif and demonstrate how HIV-1 impairs the anti-viral activity of Type 1 IFNs, possibly explaining why both endogenous and therapeutic IFN-α fail to activate more effective control over HIV infection.

A novel anti-viral role for STAT3 in IFN-α signalling responses.

The cytokine, Interferon (IFN)-α, induces a wide spectrum of anti-viral mediators, via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway. STAT1 and STAT2 are well characterised to upregulate IFN-stimulated gene (ISG) expression; but even though STAT3 is also activated by IFN-α, its role in anti-viral ISG induction is unclear. Several viruses, including Hepatitis C and Mumps, reduce cellular STAT3 protein levels, via the promotion of ubiquitin-mediated proteasomal degradation. This viral immune evasion mechanism suggests an undiscovered anti-viral role for STAT3 in IFN-α signalling. To investigate STAT3's functional involvement in this Type I IFN pathway, we first analysed its effect upon the replication of two viruses, Influenza and Vaccinia. Viral plaque assays, using Wild Type (WT) and STAT3-/- Murine Embryonic Fibroblasts (MEFs), revealed that STAT3 is required for the inhibition of Influenza and Vaccinia replication. Furthermore, STAT3 shRNA knockdown also enhanced Influenza replication and hindered induction of several, well characterised, anti-viral ISGs: PKR, OAS2, MxB and ISG15; while STAT3 expression had no effect upon induction of a separate ISG group: Viperin, IFI27, CXCL10 and CCL5. These discoveries reveal, for the first time, an anti-viral role for STAT3 in the IFN-α pathway and characterise a requirement for STAT3 in the expression of specific ISGs. These findings also identify STAT3 as a therapeutic target against viral infection and highlight it as an essential pathway component for endogenous and therapeutic IFN-α responsiveness.

Nuclear factor κB2 p52 protein has a role in antiviral immunity through IκB kinase epsilon-dependent induction of Sp1 protein and interleukin 15.

In this study we describe a previously unreported function for NFκB2, an NFκB family transcription factor, in antiviral immunity. NFκB2 is induced in response to poly(I:C), a mimic of viral dsRNA. Poly(I:C), acting via TLR3, induces p52-dependent transactivation of a reporter gene in a manner that requires the kinase activity of IκB kinase ε (IKKε) and the transactivating potential of RelA/p65. We identify a novel NFκB2 binding site in the promoter of the transcription factor Sp1 that is required for Sp1 gene transcription activated by poly(I:C). We show that Sp1 is required for IL-15 induction by both poly(I:C) and respiratory syncytial virus, a response that also requires NFκB2 and IKKε. Our study identifies NFκB2 as a target for IKKε in antiviral immunity and describes, for the first time, a role for NFκB2 in the regulation of gene expression in response to viral infection.

Cactin targets the MHC class III protein IkappaB-like (IkappaBL) and inhibits NF-kappaB and interferon-regulatory factor signaling pathways.

Toll-like receptors (TLRs) act as primary sensors of the immune system by recognizing specific microbial motifs and inducing proinflammatory genes that facilitate innate and adaptive immunity. TLRs regulate gene expression by activating transcription factors, such as NF-κB and interferon-regulatory factors. Dysregulation of these pathways can lead to inflammatory diseases, and thus they are subject to stringent control by negative regulators of innate immune signaling. Cactin (Cactus interactor) was initially discovered as a novel interactor of Drosophila Cactus, a regulator of Drosophila Toll signaling. We now describe the first functional characterization of the human ortholog of Cactin (hCactin) and show that it acts as a negative regulator of TLRs. Overexpression of hCactin suppresses TLR-induced activation of NF-κB and interferon-regulatory factor transcription factors and induction of TLR-responsive genes, whereas knockdown of endogenous hCactin augments TLR induction of these responses. hCactin also interacts with IκB-like protein and targets other proteins that are encoded by genes in the MHC Class III region of chromosome 6. We demonstrate that hCactin localizes to the nucleus, and this nuclear localization is critical for manifesting its inhibitory effects on TLR signaling. This study thus defines hCactin as a novel negative regulator of TLR signaling and reveals its capacity to target MHC Class III genes at the molecular and functional level.