IFN-mediated Response Research Articles

African swine fever virus B175L inhibits the type I interferon pathway by targeting STING and 2'3'-cGAMP.

African swine fever virus (ASFV), the only known DNA arbovirus, is the causative agent of African swine fever (ASF), an acutely contagious disease in pigs. ASF has recently become a crisis in the pig industry in recent years, but there are no commercially available vaccines. Studying the immune evasion mechanisms of ASFV proteins is important for the understanding the pathogenesis of ASFV and essential information for the development of an effective live-attenuated ASFV vaccines. Here, we identified ASFV B175L, previously uncharacterized proteins that inhibit type I interferon signaling by targeting STING and 2'3'-cGAMP. The conserved B175L-zf-FCS motif specifically interacted with both cGAMP and the R238 and Y240 amino acids of STING. Consequently, this interaction interferes with the interaction of cGAMP and STING, thereby inhibiting downstream signaling of IFN-mediated antiviral responses. This novel mechanism of B175L opens a new avenue as one of the ASFV virulent genes that can contribute to the advancement of ASFV live-attenuated vaccines.

Abstract 6410: Deciphering the role of MyD88 signaling pathway in regulating type I IFN-mediated responses to radiation therapy in solid tumors

Abstract The balance of innate signaling through adaptor proteins such as MyD88 and TRIF is critical in directing the pattern of inflammatory responses following exposure to endogenous adjuvants. While innate stimuli can cause inflammation that supports adaptive immunity, cancer myeloid cells can be pre-polarized such that the same inflammatory signals cause myeloid cells to suppress adaptive immune responses. We aim to investigate the signals regulating type I IFN secretion by innate immune cells in tumors in order to improve type I IFN secretion, activation of innate immune cells, direct macrophage polarization and improve CD8+ T cell mediated anti-tumor immunity. To better understand the role of MyD88-mediated signaling in driving response to innate adjuvants released following RT in solid tumors, we developed MyD88 conditional knockout mouse models. MyD88 was deleted specifically in CD11c expressing dendritic cells (DC), Lck expressing T cells, or Lyz2 expressing myeloid cells that include macrophages, monocytes and granulocytes. Mice bearing pancreatic tumors (Panc02-SIY or PK5L1940) were randomized to no treatment or 16 Gy CT-guided RT and followed for treatment responses. Lyz2-Cre/MyD88fl/fl mice demonstrated improved responses to RT in pancreatic tumors as compared to control MyD88fl/fl mice, or Lck-Cre mice. The improved responses in Lyz2-Cre/MyD88fl/fl mice were shown to be dependent on CD8+ T cells as well as on type I IFN signaling. To analyze mechanisms of response, tumors were digested into a single cell suspension and CD45+ cells were isolated for single cell sequencing three days post-RT. Lyz2-Cre/MyD88fl/fl mice showed diminished Th1 and Th2-type T cells and had a higher M1/TAM ratio compared to MyD88fl/fl mice. Loss of MyD88 in myeloid cells resulted in increased activation of IFN-dependent transcriptional responses in multiple immune cell populations in the tumor. To model responses ex vivo, bone marrow-derived macrophages (BMDMs) were activated with lipopolysaccharide (LPS) or a synthetic cyclic dinucleotide (CDA) for analysis of cytokine secretion in either monolayer or 3D spheroid culture conditions. BMDM derived from Lyz2-Cre/MyD88fl/fl cultured as a monolayer showed significantly altered secretion of TNF-α, IL-10 and IL-6 on activation with LPS as compared to controls. Preliminary data obtained from 3D spheroid culture systems demonstrated that 3D interactions between cancer cells and macrophages resulted in a significant increase in BMDM co-stimulatory phenotype following activation with innate stimuli compared to 2D cultures. These data suggest that conventional innate signaling through MyD88 in myeloid populations suppresses IFN production and adaptive immune control of irradiated tumors. 3D cultures are an effective tool to study cellular interactions ex vivo and screen novel interventions prior to in vivo translation. Citation Format: Aanchal Preet Kaur, Terry R. Medler, Tiffany C. Blair, Alejandro F. Alice, Megan R. Ball, Alexa K. Dowdell, Brian D. Piening, Marka R. Crittenden, Michael J. Gough. Deciphering the role of MyD88 signaling pathway in regulating type I IFN-mediated responses to radiation therapy in solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6410.

Altered RNA Editing in Atopic Dermatitis Highlights the Role of Double-Stranded RNA for Immune Surveillance

Atopic dermatitis (AD) is associated with dysregulated type 1 IFN‒mediated responses, in parallel with the dominant type 2 inflammation. However, the pathophysiology of this dysregulation is largely unknown. Adenosine-to-inosine RNA editing plays a critical role in immune regulation by preventing double-stranded RNA recognition by MDA5 and IFN activation. We studied global adenosine-to-inosine editing in AD to elucidate the role played by altered editing in the pathophysiology of this disease. Analysis of three RNA-sequencing datasets of AD skin samples revealed reduced levels of adenosine-to-inosine RNA editing in AD. This reduction was seen globally throughout Alu repeats as well as in coding genes and in specific pre-mRNA loci expected to create long double-stranded RNA, the main substrate of MDA5 leading to type I IFN activation. Consistently, IFN signature genes were upregulated. In contrast, global editing was not altered in systemic lupus erythematosus and systemic sclerosis, despite IFN activation. Our results indicate that altered editing leading to impairment of the innate immune response may be involved in the pathogenesis of AD. Possibly, it may be relevant for additional autoimmune and inflammatory diseases.

African Swine Fever Virus EP364R and C129R Target Cyclic GMP-AMP To Inhibit the cGAS-STING Signaling Pathway.

ABSTRACTAfrican swine fever virus (ASFV) is a highly pathogenic swine DNA virus with high mortality that causes African swine fever (ASF) in domestic pigs and wild boars. For efficient viral infection, ASFV has developed complex strategies to evade key components of antiviral innate immune responses. However, the immune escape mechanism of ASFV remains unclear. Upon ASFV infection, cyclic GMP-AMP (2′,3′-cGAMP) synthase (cGAS), a cytosolic DNA sensor, recognizes ASFV DNA and synthesizes the second messenger 2′,3′-cGAMP, which triggers interferon (IFN) production to interfere with viral replication. In this study, we demonstrated a novel immune evasion mechanism of ASFV EP364R and C129R, which blocks cellular cyclic 2′,3′-cGAMP-mediated antiviral responses. ASFV EP364R and C129R with nuclease homology inhibit IFN-mediated responses by specifically interacting with 2′,3′-cGAMP and exerting their phosphodiesterase (PDE) activity to cleave 2′,3′-cGAMP. Particularly notable is that ASFV EP364R had a region of homology with the stimulator of interferon genes (STING) protein containing a 2′,3′-cGAMP-binding motif and point mutations in the Y76S and N78A amino acids of EP364R that impaired interaction with 2′,3′-cGAMP and restored subsequent antiviral responses. These results highlight a critical role for ASFV EP364R and C129R in the inhibition of IFN responses and could be used to develop ASFV live attenuated vaccines.IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic disease in domestic pigs and wild boars caused by African swine fever virus (ASFV). ASF is a deadly epidemic disease in the global pig industry, but no drugs or vaccines are available. Understanding the pathogenesis of ASFV is essential to developing an effective live attenuated ASFV vaccine, and investigating the immune evasion mechanisms of ASFV is crucial to improve the understanding of its pathogenesis. In this study, for the first time, we identified the EP364R and C129R, uncharacterized proteins that inhibit type I interferon signaling. ASFV EP364R and C129R specifically interacted with 2′,3′-cGAMP, the mammalian second messenger, and exerted phosphodiesterase activity to cleave 2′,3′-cGAMP. In this study, we discovered a novel mechanism by which ASFV inhibits IFN-mediated antiviral responses, and our findings can guide the understanding of ASFV pathogenesis and the development of live attenuated ASFV vaccines.

Abstract 1330: Role of ENPP1 mediated extracellular cGAMP hydrolysis in cancer metastasis and immune evasion

Abstract Chromosome instability (CIN) promotes metastasis through sustained tumor-cell autonomous response to cytosolic DNA. Chromosome mis-segregation engenders the formation of micronuclei, which upon rupture chronically activates the cGAS-STING DNA sensing pathway. Tumor cGAS recognizes cytosolic DNA to generate cGAMP, a potent immune-stimulatory molecule that is readily exported into the extracellular space to enforce a type 1 IFN-mediated anti-tumor response in a host STING dependent manner. However, it is unclear how highly aggressive, unstable tumors have evolved to co-opt this chronic inflammatory signaling to drive tumorigenic behaviors and immune evasion. Here, we show that ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) may drive tumor metastasis through degradation of extracellular cGAMP. Using RNA sequencing of isogenic tumors, we uncovered a link between ENPP1 and CIN. We leveraged syngeneic mouse models to demonstrate that genetic perturbation of tumor ENPP1 and overexpression of a catalytically inactive ENPP1 mutant suppress metastasis. Furthermore, loss of ENPP1 increases tumor immune infiltration, reduces extracellular adenosine, and enhances response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. In line with these findings, ENPP1 expression correlates with immune suppression in human cancers. These results suggest that ENPP1-mediated hydrolysis of cGAMP may facilitate metastasis through evasion of immune surveillance, perhaps in part through cGAMP breakdown into immune-suppressive adenosine, in which the contributions of tumor and host adenosine can be further explored. Taken together, our study demonstrates how ENPP1-mediated hydrolysis of cGAMP can transform an otherwise inflammatory pathway into an immune-suppressive mechanism to promote tumor progression and metastasis. Citation Format: John Young Ho Kwon, Jun Li, Mercedes Duran, Ninjit Dhanota, Samuel Bakhoum. Role of ENPP1 mediated extracellular cGAMP hydrolysis in cancer metastasis and immune evasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1330.

Host Cell Restriction Factors of Bunyaviruses and Viral Countermeasures

The Bunyavirales order comprises more than 500 viruses (generally defined as bunyaviruses) classified into 12 families. Some of these are highly pathogenic viruses infecting different hosts, including humans, mammals, reptiles, arthropods, birds, and/or plants. Host cell sensing of infection activates the innate immune system that aims at inhibiting viral replication and propagation. Upon recognition of pathogen-associated molecular patterns (PAMPs) by cellular pattern recognition receptors (PRRs), numerous signaling cascades are activated, leading to the production of interferons (IFNs). IFNs act in an autocrine and paracrine manner to establish an antiviral state by inducing the expression of hundreds of IFN-stimulated genes (ISGs). Some of these ISGs are known to restrict bunyavirus infection. Along with other constitutively expressed host cellular factors with antiviral activity, these proteins (hereafter referred to as “restriction factors”) target different steps of the viral cycle, including viral entry, genome transcription and replication, and virion egress. In reaction to this, bunyaviruses have developed strategies to circumvent this antiviral response, by avoiding cellular recognition of PAMPs, inhibiting IFN production or interfering with the IFN-mediated response. Herein, we review the current knowledge on host cellular factors that were shown to restrict infections by bunyaviruses. Moreover, we focus on the strategies developed by bunyaviruses in order to escape the antiviral state developed by the infected cells.

Heterogeneity of IFN-Mediated Responses and Tumor Immunogenicity in Patients with Cervical Cancer Receiving Concurrent Chemoradiotherapy.

To ask whether the expression of immune markers and IFN signaling in tumor biopsies changes during concurrent chemoradiotherapy (CCRT). Tumor biopsies and peripheral mononuclear blood cells (PMBC) before and immediately after 20 Gy/10 fractions (F) of radiation treatment (RT) from 30 patients with cervical cancer receiving CCRT were evaluated by IHC and qRT-PCR for immune markers and correlated with the short-term response. Tumor immune response to radiation before and after 10F RT as reflected by CD8+ T-cell infiltration had substantial heterogeneity with increases, decreases, and no change all evident. Increases in CD8+ T cells during CCRT correlated with the presence of nuclear IRF1 in tumor cells (r = 0.68, P < 0.0001) and the patient short-term response (P < 0.01). Similarly, in a subset of patients (∼40%) PD-L1 positivity in tumor cells increased, which also correlated with nuclear IRF1 staining (r = 0.48, P < 0.01). Patients with augmented PMBC IFN signature expression after 10F had a significantly higher probability of PD-L1 induction (83% vs. 7%, P < 0.0001). Most patients exhibited abundant expression of SERPINB9 and CD47 in tumor cells, and tumor infiltration by CD68+ cells. SERPINB9 expression correlated with STAT1 signaling in tumor cells. CCRT leads to differential tumor immunogenicity and IFN signaling in patients with cervical cancer, suggesting radiation induction of immunity is limited to a subset of patients and may reflect the heterogeneity of intratumoral induction of IFNs.See related commentary by Mondini and Deutsch, p. 3815.

Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Phlebovirus Inhibits TBK1 to Evade Interferon-Mediated Response.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging phlebovirus of the Phenuiviridae family that has been circulating in the following Asian countries: Vietnam, Myanmar, Taiwan, China, Japan, and South Korea. Despite the increasing infection rates and relatively high mortality rate, there is limited information available regarding SFTSV pathogenesis. In addition, there are currently no vaccines or effective antiviral treatments available. Previous reports have shown that SFTSV suppresses the host immune response and its nonstructural proteins (NSs) function as an antagonist of type I interferon (IFN), whose induction is an essential part of the host defense system against viral infections. Given that SFTSV NSs suppress the innate immune response by inhibiting type I IFN, we investigated the mechanism utilized by SFTSV NSs to evade IFN-mediated response. Our co-immunoprecipitation data suggest the interactions between NSs and retinoic acid inducible gene-I (RIG-I) or TANK binding kinase 1 (TBK1). Furthermore, confocal analysis indicates the ability of NSs to sequester RIG-I and related downstream molecules in the cytoplasmic structures called inclusion bodies (IBs). NSs are also capable of inhibiting TBK1-interferon regulatory factor 3 (IRF3) interaction, and therefore prevent the phosphorylation and nuclear translocation of IRF3 for the induction of type I IFN. The ability of SFTSV NSs to interact with and sequester TBK1 and IRF3 in IBs demonstrate an effective yet unique method utilized by SFTSV to evade and suppress host immunity.

IFNAR2 Deficiency Causing Dysregulation of NK Cell Functions and Presenting With Hemophagocytic Lymphohistiocytosis.

We describe a 2 year old boy with two previously undescribed frameshift mutations in the interferon (IFN)α/β receptor 2 (IFNAR2) gene presenting with hemophagocytic lymphohistiocytosis (HLH) following measles-mumps-rubella vaccination. Functional analyses show the absence of response to type I IFN in the patient’s cells, as revealed by the lack of phosphorylation of STAT1 and the lack of induction of interferon-stimulated genes upon ex vivo stimulation with IFNα. HLH has been reported in patients with inborn errors of type I IFN-mediated immune responses following vaccination with live-attenuated viruses. The relation between HLH and defective type I IFN-mediated responses is unclear. We show that in patient’s natural killer (NK) cells stimulated with IFNα the expected increase in degranulation and inhibition of IFNγ production were affected. These data support a role for NK cell function dysregulation and lack of inhibition of IFNγ production as contributors to the development of HLH in patients with impaired type I IFN signaling.

Abstract CT190: A Phase II basket study of hypomethylating agent oral cc-486 and durvalumab in advanced solid tumors (METADUR)

Abstract Background Aberrant DNA methylation contributes to cancer initiation and progression. In preclinical models of solid tumors, chronic low dose administration of DNA hypomethylating agents can induce T-cell mediated immune activation responses by stimulating expression of endogenous retroviral elements, culminating in an IFN-mediated response. The addition of physiological levels of vitamin C may potentiate viral mimicry and increase anti-tumor immune priming. Immunologically ‘cold’ tumors were selected to evaluate whether these strategies can enhance their otherwise poor responses to immune checkpoint blockade. Methods PD-L1/PD-1 inhibitor naïve patients (pts) with advanced microsatellite stable colorectal cancer (MSS CRC); platinum resistant ovarian cancer (OC); estrogen receptor positive, HER2 negative breast cancer (ER+HER2- BC) were enrolled in this single institution, multi-cohort, investigator-initiated trial. The initial regimen (regimen A) consisted of oral CC-486 300mg QD Days 1-14 (cycles 1-3 only) in combination with durvalumab 1500mg IV Day 15, in 28 day-cycles. A protocol amendment (regimen B) after 19 patients changed CC-486 to 100 mg QD Days 1-21 (cycles 1-3), added oral vitamin C 500mg QD continuously and kept durvalumab 1500mg IV Day 15, in 28-day cycles. Adverse events (AEs) assessed by CTCAEv4.03 grade (G); tumor response by RECIST 1.1 every 2 cycles; paired tumor biopsies at baseline and cycle 2 days 10-14; and serial peripheral blood mononuclear cells (PBMCs) for immune-profiling (IP) and epigenetic analysis. PD-L1 by IHC was assessed using SP263 assay, positivity defined by TC&amp;gt;25%. Results A total of 28 pts with MSS CRC (n=15), OC (n=4), ER+HER2- BC (n=9) were enrolled, 19 pts treated on regimen A, 9 on regimen B. Median age was 56 (range 36-78), ECOG 0:1=7:21, 100% had ≥3 prior lines of therapy, all tumors were PD-L1 negative. Best response was SD (3/28 pts received 3, 3 and 4 cycles respectively) with DCR 7.1%. Median follow-up of 4.7 months, mPFS was 1.9 months (95% CI 1.5-2.3) and mOS was 5 months (95% CI 4.5-10). Three patients (all regimen A) experienced DLTs (2 G4 neutropenia and 1 G3 anemia). Fifteen patients (54%) experienced G1/2 fatigue, 46% experienced G1/2 nausea, vomiting or diarrhea. Toxicity was comparable with addition of vitamin C. Initial analysis of PBMCs by flow cytometry in 3 of 4 OC pts demonstrated an increase in PD-1 and Ki67 expression in CD8 T cells only following administration of durvalumab. EPIC methylation array on 4 pts’ paired tumors and LINE 1 assay on 19 pts’ serial PBMCs (regimen A) demonstrated minimal change in global methylation. Conclusion No meaningful clinical responses to CC-486 plus durvalumab were observed. Tumor tissue and PBMCs both demonstrate minimal global DNA demethylation, with or without physiological dose vitamin C. Incremental immune activation beyond PD-L1 blockade was not demonstrated. Clinical trial information: NCT02811497 Citation Format: Kirsty Taylor, Helen Loo Yau, Ben X. Wang, Philippe L. Bedard, Albiruni R. Razak, Aaron R. Hansen, Anna Spreafico, Dave Cescon, Marcus O. Butler, Amit M. Oza, Stephanie Lheureux, Neda Stjepanovic, Lisa Wang, Brendan Van As, Sarah Boross-Harmer, Trevor Pugh, Lillian L. Siu, Daniel D. De Carvalho. A Phase II basket study of hypomethylating agent oral cc-486 and durvalumab in advanced solid tumors (METADUR) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT190.

A cell type-specific transcriptomic approach to map B cell and monocyte type I interferon-linked pathogenic signatures in Multiple Sclerosis

Alteration in endogenous Interferon (IFN) system may profoundly impact immune cell function in autoimmune diseases.Here, we provide evidence that dysregulation in IFN-regulated genes and pathways are involved in B cell- and monocyte-driven pathogenic contribution to Multiple Sclerosis (MS) development and maintenance.In particular, by using an Interferome-based cell type-specific approach, we characterized an increased susceptibility to an IFN-linked caspase-3 dependent apoptotic cell death in both B cells and monocytes of MS patients that may arise from their chronic activation and persistent stimulation by activated T cells. Ongoing caspase-3 activation functionally impacts on MS monocyte properties influencing the STAT-3/IL-16 axis, thus, driving increased expression and massive release of the bio-active IL-16 triggering and perpetuating CD4+ T cell migration.Importantly, our analysis also identified a previously unknown multi-component defect in type I IFN-mediated signaling and response to virus pathways specific of MS B cells, impacting on induction of anti-viral responses and Epstein-barr virus infection control in patients.Taking advantage of cell type-specific transcriptomics and in-depth functional validation, this study revealed pathogenic contribution of endogenous IFN signaling and IFN-regulated cell processes to MS pathogenesis with implications on fate and functions of B cells and monocytes that may hold therapeutic potential.

Structural Modification in Hepatitis C Virus Envelope Protein; Potential Viral Strategy Against Interferon Therapy

Interferons (IFNs) are the main part of the immune system as a defensive response to viral infection. The phosphorylation domain of Hepatitis C virus (HCV) E2 envelope glycoprotein is homologous to the host key enzymes in IFN-mediated response. So, if host kinases show no preference on E2 protein phosphorylation over IFN triggering factors, the IFN pathway would be competently inhibited. The aim of this study was to see if the resistance of HCV infected patients to IFN-therapy has something to do with HCV E2 envelope glycoprotein. For this purpose, the partially sequenced HCV E2 proteins obtained from 15 patients that were confirmed to be sustainable, semi-resistant (patients with relapsing infection) and resistant to IFN-therapy. The peptide sequences from 15 patients were analysed as a matter of homology, physicochemical properties and predicted structures using MEGA4 software, ProtParam, PSIPRED and I-TASSER tools, respectively. Then, certain point mutations introduced to sequences that acquired from patients resistant to IFN-therapy. Replacement of Asp 653 with Asn 653 in R1 at C-terminal of the E2 protein changes secondary and tertiary structures of downstream regions of E2 protein. Although the sequence of PKR-eIF2alpha phosphorylation homology domain in E2 protein remains intact during IFN therapy, its structure modifies in patients resistant to IFN-therapy, which then puts its phosphorylation potential in jeopardy. Therefore, structural modification in HCV E2 proteins, acquired form patients resistant to IFN-therapy, suggests a mechanism that shows how HCV gets resistant toward IFN-therapy.

Non-redundant ISGF3 Components Promote NK Cell Survival in an Auto-regulatory Manner during Viral Infection

SUMMARYNatural killer (NK) cells are innate lymphocytes that possess adaptive features, including antigen-specific clonal expansion and long-lived memory responses. Although previous work demonstrated that type I interferon (IFN) signaling is crucial for NK cell expansion and memory cell formation following mouse cytomegalovirus (MCMV) infection, the global transcriptional mechanisms underlying type I IFN-mediated responses remained to be determined. Here, we demonstrate that among the suite of transcripts induced in activated NK cells, IFN-α is necessary and sufficient to promote expression of its downstream transcription factors STAT1, STAT2, and IRF9, via an auto-regulatory, feedforward loop. Similar to STAT1 deficiency, we show that STAT2- or IRF9-deficient NK cells are defective in their ability to expand following MCMV infection, in part because of diminished survival rather than an inability to proliferate. Thus, our findings demonstrate that individual ISGF3 components are crucial cell-autonomous and non-redundant regulators of the NK cell response to viral infection.

Dissection of Influenza Infection InVivo by Single-Cell RNA Sequencing.

SummaryThe influenza virus is a major cause of morbidity and mortality worldwide. Yet, both the impact of intracellular viral replication and the variation in host response across different cell types remain uncharacterized. Here we used single-cell RNA sequencing to investigate the heterogeneity in the response of lung tissue cells to in vivo influenza infection. Analysis of viral and host transcriptomes in the same single cell enabled us to resolve the cellular heterogeneity of bystander (exposed but uninfected) as compared with infected cells. We reveal that all major immune and non-immune cell types manifest substantial fractions of infected cells, albeit at low viral transcriptome loads relative to epithelial cells. We show that all cell types respond primarily with a robust generic transcriptional response, and we demonstrate novel markers specific for influenza-infected as opposed to bystander cells. These findings open new avenues for targeted therapy aimed exclusively at infected cells.

The Absent in Melanoma 2-Like Receptor IFN-Inducible Protein 16 as an Inflammasome Regulator in Systemic Lupus Erythematosus: The Dark Side of Sensing Microbes.

Absent in melanoma 2 (AIM2)-like receptors (ALRs) are a newly characterized class of pathogen recognition receptors (PRRs) involved in cytosolic and nuclear pathogen DNA recognition. In recent years, two ALR family members, the interferon (IFN)-inducible protein 16 (IFI16) and AIM2, have been linked to the pathogenesis of various autoimmune diseases, among which systemic lupus erythematosus (SLE) has recently gained increasing attention. SLE patients are indeed often characterized by constitutively high serum IFN levels and increased expression of IFN-stimulated genes due to an abnormal response to pathogens and/or incorrect self-DNA recognition process. Consistently, we and others have shown that IFI16 is overexpressed in a wide range of autoimmune diseases where it triggers production of specific autoantibodies. In addition, evidence from mouse models supports a model whereby ALRs are required for IFN-mediated host response to both exogenous and endogenous DNA. Following interaction with cytoplasmic or nuclear nucleic acids, ALRs can form a functional inflammasome through association with the adaptor ASC [apoptosis-associated speck-like protein containing a caspase recruitment domain (CARD)] and with procaspase-1. Importantly, inflammasome-mediated upregulation of IL-1β and IL-18 production positively correlates with SLE disease severity. Therefore, targeting ALR sensors and their downstream pathways represents a promising alternative therapeutic approach for SLE and other systemic autoimmune diseases.

Age-Associated Differences in Infection of Human Skin in the SCID Mouse Model of Varicella-Zoster Virus Pathogenesis.

Varicella-zoster virus (VZV) is the skin-tropic human alphaherpesvirus responsible for both varicella-zoster and herpes zoster. Varicella-zoster and herpes zoster skin lesions have similar morphologies, but herpes zoster occurs disproportionally in older individuals and is often associated with a more extensive local rash and severe zoster-related neuralgia. We hypothesized that skin aging could also influence the outcome of the anterograde axonal transport of VZV to skin. We utilized human skin xenografts maintained in immunodeficient (SCID) mice to study VZV-induced skin pathology in vivo in fetal and adult skin xenografts. Here we found that VZV replication is enhanced in skin from older compared to younger adults, correlating with clinical observations. In addition to measures of VZV infection, we examined the expression of type I interferon (IFN) pathway components in adult skin and investigated elements of the cutaneous proliferative and inflammatory response to VZV infection in vivo Our results demonstrated that VZV infection of adult skin triggers intrinsic IFN-mediated responses such as we have described in VZV-infected fetal skin xenografts, including MxA as well as promyelocytic leukemia protein (PML), in skin cells surrounding lesions. Further, we observed that VZV elicited altered cell signaling and proliferative and inflammatory responses that are involved in wound healing, driven by follicular stem cells. These cellular changes are consistent with VZV-induced activation of STAT3 and suggest that VZV exploits the wound healing process to ensure efficient delivery of the virus to keratinocytes. Adult skin xenografts offer an approach to further investigate VZV-induced skin pathologies in vivoIMPORTANCE Varicella-zoster virus (VZV) is the agent responsible for both varicella-zoster and herpes zoster. Herpes zoster occurs disproportionally in older individuals and is often associated with a more extensive local rash and severe zoster-related neuralgia. To examine the effect of skin aging on VZV skin lesions, we utilized fetal and adult human skin xenografts maintained in immunodeficient (SCID) mice. We measured VZV-induced skin pathology, examined the expression of type I interferon (IFN) pathway components in adult skin, and investigated elements of the cutaneous proliferative and inflammatory response to VZV infection in vivo Our results demonstrate that characteristics of aging skin are preserved in xenografts; that VZV replication is enhanced in skin from older compared to younger adults, correlating with clinical observations; and that VZV infection elicits altered cell signaling and inflammatory responses. Adult skin xenografts offer an approach to further investigate VZV-induced skin pathologies in vivo.

Immune modulation by combination type I interferon and checkpoint blockade therapy in murine urothelial carcinoma

Abstract Type I interferon (IFN) has potent antitumor effects in bladder cancer and provides an essential alternative treatment option for patients who do not respond to Bacillus Calmette-Guerin (BCG). However, the mechanism of the IFN-stimulated immune response in bladder cancer is not well understood. We hypothesized that type I IFN enhances recruitment and activation of immune cells in murine bladder cancer tumors, and makes them more responsive to anti-PD-1 mAb therapy. To incite an IFN-driven inflammatory response, poly(I:C) was given to murine MB49 bladder cancer tumors peritumorally alone and in combination with anti-PD-1 mAb to determine effects on tumor growth and animal survival. IFN induction in MB49 tumors significantly inhibited tumor growth. This response was reliant on both innate and adaptive immune subsets, shown in the increased influx of intratumoral Ly6G+ neutrophils and the increase in IFNy+CD8 T cells. In addition, depleting specific immune cell subsets indicated that no one type of immune cell was critical for the IFN-mediated anti-tumor response. When used in combination with anti-PD-1 mAb, poly(I:C) prolonged mouse survival and enriched gene pathways involved in metabolism, extracellular matrix formation and organization, and PI3K and AKT signaling. To examine effects of IFN-I in BCG-unresponsive patients, tumor samples were analyzed for RNA expression and immunohistochemical (IHC) staining both before and after treatment with adenoviral interferon-α. Among these samples, 25% showed increased expression of T cell and checkpoint markers, similarly reflected in IHC analysis. Our findings suggest type I IFN grooms the tumor-immune landscape in bladder cancer by activating both innate and adaptive immune cells.

Interleukin-1β Signaling in Dendritic Cells Induces Antiviral Interferon Responses.

ABSTRACTInduction of interferon beta (IFN-β), IFN-stimulated genes (ISGs), and inflammatory responses is critical for control of viral infection. We recently identified an essential linkage of stimulation of the inflammatory cytokine interleukin-1β (IL-1β) and induction of ISGs that function as host restriction pathways against the emerging flavivirus West Nile virus (WNV) in vivo. Here we utilized ex vivo global transcriptome analysis of primary dendritic cells, known targets of WNV replication, to define gene signatures required for this IL-1β-driven antiviral response. Dendritic cells that were deficient in IL-1 receptor signaling showed dysregulation of cell-intrinsic defense genes and loss of viral control during WNV infection. Surprisingly, we found that in wild-type cells, IL-1β treatment, in the absence of infection, drove the transcription of IFN-β and ISGs at late times following treatment. Expression of these antiviral innate immune genes was dependent on the transcription factor IFN regulatory factor 3 (IRF3) and appears to reflect a general shift in IL-1β signaling from an early inflammatory response to a late IFN-mediated response. These data demonstrate that inflammatory and antiviral signals integrate to control viral infection in myeloid cells through a process of IL-1β-to-IRF3 signaling crosstalk. Strategies to exploit these cytokines in the activation of host defense programs should be investigated as novel therapeutic approaches against individual pathogens.

Emerging Alphaviruses Are Sensitive to Cellular States Induced by a Novel Small-Molecule Agonist of the STING Pathway.

The type I interferon (IFN) system represents an essential innate immune response that renders cells resistant to virus growth via the molecular actions of IFN-induced effector proteins. IFN-mediated cellular states inhibit growth of numerous and diverse virus types, including those of known pathogenicity as well as potentially emerging agents. As such, targeted pharmacologic activation of the IFN response may represent a novel therapeutic strategy to prevent infection or spread of clinically impactful viruses. In light of this, we employed a high-throughput screen to identify small molecules capable of permeating the cell and of activating IFN-dependent signaling processes. Here we report the identification and characterization of N-(methylcarbamoyl)-2-{[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}-2-phenylacetamide (referred to as C11), a novel compound capable of inducing IFN secretion from human cells. Using reverse genetics-based loss-of-function assays, we show that C11 activates the type I IFN response in a manner that requires the adaptor protein STING but not the alternative adaptors MAVS and TRIF. Importantly, treatment of cells with C11 generated a cellular state that potently blocked replication of multiple emerging alphavirus types, including chikungunya, Ross River, Venezuelan equine encephalitis, Mayaro, and O'nyong-nyong viruses. The antiviral effects of C11 were subsequently abrogated in cells lacking STING or the type I IFN receptor, indicating that they are mediated, at least predominantly, by way of STING-mediated IFN secretion and subsequent autocrine/paracrine signaling. This work also allowed characterization of differential antiviral roles of innate immune signaling adaptors and IFN-mediated responses and identified MAVS as being crucial to cellular resistance to alphavirus infection.IMPORTANCE Due to the increase in emerging arthropod-borne viruses, such as chikungunya virus, that lack FDA-approved therapeutics and vaccines, it is important to better understand the signaling pathways that lead to clearance of virus. Here we show that C11 treatment makes human cells refractory to replication of a number of these viruses, which supports its value in increasing our understanding of the immune response and viral pathogenesis required to establish host infection. We also show that C11 depends on signaling through STING to produce antiviral type I interferon, which further supports its potential as a therapeutic drug or research tool.

HBV Bypasses the Innate Immune Response and Does Not Protect HCV From Antiviral Activity of Interferon

Hepatitis C virus (HCV) infection is sensitive to interferon (IFN)-based therapy, whereas hepatitis B virus (HBV) infection is not. It is unclear whether HBV escapes detection by the IFN-mediated immune response or actively suppresses it. Moreover, little is known on how HBV and HCV influence each other in coinfected cells. We investigated interactions between HBV and the IFN-mediated immune response using HepaRG cells and primary human hepatocytes (PHHs). We analyzed the effects of HBV on HCV replication, and vice versa, at the single-cell level. PHHs were isolated from liver resection tissues from HBV-, HCV-, and human immunodeficiency virus-negative patients. Differentiated HepaRG cells overexpressing the HBV receptor sodium taurocholate cotransporting polypeptide (dHepaRGNTCP) and PHHs were infected with HBV. Huh7.5 cells were transfected with circular HBV DNA genomes resembling viral covalently closed circular DNA (cccDNA), and subsequently infected with HCV; this served as a model of HBV and HCV coinfection. Cells were incubated with IFN inducers, or IFNs, and antiviral response and viral replication were analyzed by immune fluorescence, reverse-transcription quantitative polymerase chain reaction, enzyme-linked immunosorbent assays, and flow cytometry. HBV infection of dHepaRGNTCP cells and PHHs neither activated nor inhibited signaling via pattern recognition receptors. Incubation of dHepaRGNTCP cells and PHHs with IFN had little effect on HBV replication or levels of cccDNA. HBV infection of these cells did not inhibit JAK-STAT signaling or up-regulation of IFN-stimulated genes. In coinfected cells, HBV did not prevent IFN-induced suppression of HCV replication. In dHepaRGNTCP cells and PHHs, HBV evades the induction of IFN and IFN-induced antiviral effects. HBV infection does not rescue HCV from the IFN-mediated response.