Expression Of IFN-responsive Genes Research Articles

Epigenomic and transcriptomic analyses reveal differences between low-grade inflammation and severe exhaustion in LPS-challenged murine monocytes

Emerging studies suggest that monocytes can be trained by bacterial endotoxin to adopt distinct memory states ranging from low-grade inflammation to immune exhaustion. While low-grade inflammation may contribute to the pathogenesis of chronic diseases, exhausted monocytes with pathogenic and immune-suppressive characteristics may underlie the pathogenesis of polymicrobial sepsis including COVID-19. However, detailed processes by which the dynamic adaption of monocytes occur remain poorly understood. Here we exposed murine bone-marrow derived monocytes to chronic lipopolysaccharide (LPS) stimulation at low-dose or high-dose, as well as a PBS control. The cells were profiled for genome-wide H3K27ac modification and gene expression. The gene expression of TRAM-deficient and IRAK-M-deficient monocytes with LPS exposure was also analyzed. We discover that low-grade inflammation preferentially utilizes the TRAM-dependent pathway of TLR4 signaling, and induces the expression of interferon response genes. In contrast, high dose LPS uniquely upregulates exhaustion signatures with metabolic and proliferative pathways. The extensive differences in the epigenomic landscape between low-dose and high-dose conditions suggest the importance of epigenetic regulations in driving differential responses. Our data provide potential targets for future mechanistic or therapeutic studies.

Transcriptional differences between JAK2-V617F and wild-type bone marrow cells in patients with myeloproliferative neoplasms

The JAK2-V617F mutation is the most common cause of myeloproliferative neoplasms. Although experiments have revealed that this gain-of-function mutation is associated with myeloid blood cell expansion and increased production of white cells, red cells, and platelets, the transcriptional consequences of the JAK2-V617F mutation in different cellular compartments of the bone marrow have not yet been fully elucidated. To study the direct effects of JAK2-V617F on bone marrow cells in patients with myeloproliferative neoplasms, we performed joint single-cell RNA sequencing and JAK2 genotyping on CD34+-enriched cells from eight patients with newly diagnosed essential thrombocythemia or polycythemia vera. We found that the JAK2-V617F mutation increases the expression of interferon-response genes (e.g., HLAs) and the leptin receptor in hematopoietic progenitor cells. Furthermore, we sequenced a population of CD34- bone marrow monocytes and found that the JAK2 mutation increased expression of intermediate monocyte genes and the fibrocyte-associated surface protein SLAMF7 in these cells.

In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors

The paucity of animal models that simulate the replication of the hepatitis B virus (HBV) is an impediment to advancing new anti-viral treatments. The work reported here employed recombinant adeno-associated viruses (AAVs) to model HBV subgenotype A1 and subgenotype D3 replication in vitro and in vivo. Infection with subgenotype A1 is endemic to parts of sub-Saharan Africa, and it is associated with a high risk of hepatocellular carcinoma. Recombinant AAV serotype 2 (AAV2) and 8 (AAV8) vectors bearing greater-than-genome-length sequences of HBV DNA from subgenotype A1 and D3, were produced. Transduced liver-derived cultured cells produced HBV surface antigen and core antigen. Administration of AAV8 carrying HBV subgenotype A1 genome (AAV8-A1) to mice resulted in the sustained production of HBV replication markers over a six-month period, without elevated inflammatory cytokines, expression of interferon response genes or alanine transaminase activity. Markers of replication were generally higher in animals treated with subgenotype D3 genome-bearing AAVs than in those receiving the subgenotype A1-genome-bearing vectors. To validate the use of the AAV8-A1 murine model for anti-HBV drug development, the efficacy of anti-HBV artificial primary-microRNAs was assessed. Significant silencing of HBV markers was observed over a 6-month period after administering AAVs. These data indicate that AAVs conveniently and safely recapitulate the replication of different HBV subgenotypes, and the vectors may be used to assess antivirals’ potency.

Coevolving JAK2V617F+relapsed AML and donor T cells with PD-1 blockade after stem cell transplantation: an index case

AbstractRelapse of myeloproliferative neoplasms (MPNs) after allogeneic hematopoietic stem cell transplantation (HSCT) is associated with poor outcomes, as therapeutic approaches to reinstate effective graft-versus-leukemia (GVL) responses remain suboptimal. Immune escape through overexpression of PD-L1 in JAK2V617F-mutated MPN provides a rationale for therapeutic PD-1 blockade, and indeed, clinical activity of nivolumab in relapsed MPN post-HSCT has been observed. Elucidation of the features of response following PD-1 blockade in such patients could inform novel therapeutic concepts that enhance GVL. Here, we report an integrated high-dimensional analysis using single-cell RNA sequencing, T-cell receptor sequencing, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq), and assay for transposase-accessible chromatin using sequencing (scATAC-seq), together with mass cytometry, in peripheral blood mononuclear cells collected at 6 timepoints before, during, and after transient response to PD-1 blockade from an index case of relapsed MPN following HSCT. Before nivolumab infusion, acute myeloid leukemia (AML) blasts demonstrated high expression of chemokines, and T cells were characterized by expression of interferon-response genes. This baseline inflammatory signature disappeared after nivolumab infusion. Clinical response was characterized by transient expansion of a polyclonal CD4+ T-cell population and contraction of an AML subpopulation that exhibited megakaryocytic features and elevated PD-L1 expression. At relapse, the proportion of the AML subpopulation with progenitor-like features progressively increased, suggesting coevolution of AML blasts and donor-derived T cells. We thus demonstrate how single-cell technologies can provide complementary insight into cellular mechanisms underlying response to PD-1 blockade, motivating future longitudinal high-dimensional single-cell studies of GVL responses in relapsed myeloid disease.

Abstract 2135: Functional models of chromosome arm aneuploidies in lung squamous cell carcinoma

Abstract Aneuploidy, which we define as whole chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, head and neck, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Although these alterations are frequent, they are not well understood due to difficulty in modeling specific aneuploidy alterations in the matching cell type. However, recent advances in genome engineering allow generation of large chromosomal alterations. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p in human immortalized lung epithelial cells most similar to upper airway basal cells. Deletion of chromosome 3p was validated by whole genome sequencing and karyotyping. Consistent with patient data, expression of 3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p deleted cells. Genome sequencing and karyotype analyses found that evidence of chromosome 3 duplication, transitioning the cell to a state of chromosome 3q gain. We isolated sibling cells with chromosome 3p deletion or chromosome 3q gain and demonstrated that duplication of chromosome 3 could indeed rescue proliferation rates. With our cellular model of chromosome arm-level aneuploidy, we uncovered a selection mechanism that allowed aneuploidy tolerance in vitro, and a possible explanation for joint alteration of both arms of chromosome 3. In conclusion, our genome engineering approach to model chromosome arm-level deletions provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison Marie Taylor, Sejal Jain, Juliann Shih, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Functional models of chromosome arm aneuploidies in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2135.

Abstract NG01: Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in cancers harboring loss of chromosome 18q or 16q

Abstract NG01: Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in cancers harboring loss of chromosome 18q or 16q

Uhrf1 and dnmt1 Loss Induces an Immune Response in Zebrafish Livers Due to Viral Mimicry by Transposable Elements.

Activation of transposable elements (TEs) can cause cellular damage. Cytoplasmic nucleic acid sensing pathways evolved to detect pathogens, but can also serve to cull cells with inappropriate TE activation as TEs can be viral mimetics. Epigenetic silencing of TEs is mediated in part by DNA methylation, but it is not clear if TE activation or the immune system contribute to the cellular damage caused by loss of DNA methylation. Here, we provide mechanistic insight into the observation of an activated interferon response in the liver of zebrafish larvae with deletion in critical components of the DNA methylation machinery, uhrf1 and dnmt1. We focus on dissecting the relationship between DNA methylation, TE activation and induction of an immune response through cytoplasmic DNA and double stranded RNA sensing pathways and identify tnfa as a mediator of cell death in the liver of these mutants. Integrated RNAseq and methylome analysis identified LTR transposons as the most upregulated in these mutants and also the most methylated in control larvae, indicating a direct role of DNA methylation in suppressing this TE subclass. RNAseq analysis from these same samples revealed expression signatures of a type-I interferon response and of tnfa activation, mimicking the pattern of gene expression in virally infected cells. CRISPR/Cas9 mediated depletion of the cellular antiviral sensors sting and mavs reduced expression of interferon response genes and tnfa depletion dramatically reduced cell death in uhrf1 mutant livers. This suggests that the antiviral response induced by DNA hypomethylation and TE activation in the liver is mediated by the signaling pathways activated by both cytoplasmic double stranded RNA and DNA and that tnfa mediates cell death as a potential mechanism to eliminate these damaged cells.

Expression profiles of interferon-related genes in cells infected with influenza A viruses or transiently transfected with plasmids encoding viral RNA polymerase.

Influenza A viruses frequently change their genetic characteristics, which leads to the emergence of new viruses. Consequently, elucidation of the relationship between influenza A virus and host cells has a great importance to cope with viral infections. In this study, it was aimed to determine expression profiles of interferon response genes in human embryonic kidney 293 (HEK293) cells infected with human (A/WSN-H1N1) and avian influenza A viruses (duck/Pennsylvania/10218/84/H5N2) or transfected with plasmids encoding viral RdRP subunits and, to obtain clues about the genes that may be important for the viral pathogenesis. The HEK293 cells cultured in a 12-well plate were infected with influenza A viruses or transfected with plasmids encoding viral polymerase. Total RNA extraction and cDNA preparation were carried out with commercial kits. Qiagen 96-well-RT2 Profiler PCR Array plates designated for interferons response genes were used for quantitation of the transcripts. The relative quantities of transcripts were normalized with STAT3 gen, and the results were evaluated. Quantitative RT-PCR results showed that there are substantial differences of the interferon response gene transcription in cells infected with viruses or transfected with plasmids. A higher number of interferon-related genes were found to be downregulated in the cells infected with DkPen compared to WSN. On the other hand, significant differences in the expression profiles of interferon response genes were observed in the cells expressing viral PA protein. In particular, avian influenza PA protein was found to cause more aggressive changes on the transcript levels. Human and avian influenza A viruses cause a substantial change in interferon response gene expression in HEK293 cells. However, a higher number of genes were downregulated in the cells infected with avian influenza DkPen compared to WSN. It has been also concluded that the viral PA protein is one of the important viral factors affecting the transcript level of host genes.

Temporal and spatial heterogeneity of host response to SARS-CoV-2 pulmonary infection

The relationship of SARS-CoV-2 pulmonary infection and severity of disease is not fully understood. Here we show analysis of autopsy specimens from 24 patients who succumbed to SARS-CoV-2 infection using a combination of different RNA and protein analytical platforms to characterize inter-patient and intra-patient heterogeneity of pulmonary virus infection. There is a spectrum of high and low virus cases associated with duration of disease. High viral cases have high activation of interferon pathway genes and a predominant M1-like macrophage infiltrate. Low viral cases are more heterogeneous likely reflecting inherent patient differences in the evolution of host response, but there is consistent indication of pulmonary epithelial cell recovery based on napsin A immunohistochemistry and RNA expression of surfactant and mucin genes. Using a digital spatial profiling platform, we find the virus corresponds to distinct spatial expression of interferon response genes demonstrating the intra-pulmonary heterogeneity of SARS-CoV-2 infection.

In vivo antiviral host transcriptional response to SARS-CoV-2 by viral load, sex, and age.

Despite limited genomic diversity, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has shown a wide range of clinical manifestations in different patient populations. The mechanisms behind these host differences are still unclear. Here, we examined host response gene expression across infection status, viral load, age, and sex among shotgun RNA sequencing profiles of nasopharyngeal (NP) swabs from 430 individuals with PCR-confirmed SARS-CoV-2 and 54 negative controls. SARS-CoV-2 induced a strong antiviral response with up-regulation of antiviral factors such as OAS1-3 and IFIT1-3 and T helper type 1 (Th1) chemokines CXCL9/10/11, as well as a reduction in transcription of ribosomal proteins. SARS-CoV-2 culture in human airway epithelial (HAE) cultures replicated the in vivo antiviral host response 7 days post infection, with no induction of interferon-stimulated genes after 3 days. Patient-matched longitudinal specimens (mean elapsed time = 6.3 days) demonstrated reduction in interferon-induced transcription, recovery of transcription of ribosomal proteins, and initiation of wound healing and humoral immune responses. Expression of interferon-responsive genes, including ACE2, increased as a function of viral load, while transcripts for B cell–specific proteins and neutrophil chemokines were elevated in patients with lower viral load. Older individuals had reduced expression of the Th1 chemokines CXCL9/10/11 and their cognate receptor CXCR3, as well as CD8A and granzyme B, suggesting deficiencies in trafficking and/or function of cytotoxic T cells and natural killer (NK) cells. Relative to females, males had reduced B cell–specific and NK cell–specific transcripts and an increase in inhibitors of nuclear factor kappa-B (NF-κB) signaling, possibly inappropriately throttling antiviral responses. Collectively, our data demonstrate that host responses to SARS-CoV-2 are dependent on viral load and infection time course, with observed differences due to age and sex that may contribute to disease severity.

The kinase IRAK4 promotes endosomal TLR and immune complex signaling in B cells and plasmacytoid dendritic cells.

The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Bruton's tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex- and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)-responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.

Abstract A44: Functional models of chromosome arm-level aneuploidies in cancer

Abstract Aneuploidy, whole-chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. We previously applied methods that define chromosome arm-level aneuploidy and a global cancer aneuploidy score to 10,522 tumors of 33 types in The Cancer Genome Atlas (TCGA). Aneuploidy level was correlated with TP53 mutation, somatic mutation rate, and expression of proliferation genes. Overall aneuploidy was anti-correlated with expression of immune signaling genes, due to decreased leukocyte infiltrates in high-aneuploidy samples. In contrast, some individual chromosome arm alterations correlated with higher expression of immune signaling genes, including deletion of chromosome 3p, 8p, and 17p. Cancer subtypes are often characterized by tumor specific patterns of arm-level copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Although yeast and mammalian models of whole-chromosome aneuploidies have been extensively investigated, chromosome arm-level aneuploidies have rarely been modeled. However, recent advances in genome engineering and targeting of endonucleases allow generation of large chromosomal alterations. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p in human immortalized lung epithelial cells, validated in 8 clones by whole-genome sequencing. Consistent with patient data, expression of 3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p-deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p-deleted cells; genome sequencing and karyotype analyses suggested that this was partially the result of chromosome 3 duplication. With our cellular model of chromosome arm-level aneuploidy, we uncovered a selection mechanism that allowed aneuploidy tolerance in vitro. We have also grown these cells in spheroid and organoid culture systems to assess the effects of chromosome 3 arm-level aneuploidies on cytokine production and squamous differentiation and are using these systems to identify how chromosome 3p deletion affects drug sensitivity. In conclusion, our genome engineering approach to model chromosome arm-level deletions provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison M. Taylor, Sejal Jain, Juliann Shih, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Functional models of chromosome arm-level aneuploidies in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A44.

Homologous recombination DNA repair deficiency and PARP inhibition activity in primary triple negative breast cancer

Triple negative breast cancer (TNBC) encompasses molecularly different subgroups, with a subgroup harboring evidence of defective homologous recombination (HR) DNA repair. Here, within a phase 2 window clinical trial, RIO trial (EudraCT 2014-003319-12), we investigate the activity of PARP inhibitors in 43 patients with untreated TNBC. The primary end point, decreased Ki67, occured in 12% of TNBC. In secondary end point analyses, HR deficiency was identified in 69% of TNBC with the mutational-signature-based HRDetect assay. Cancers with HRDetect mutational signatures of HR deficiency had a functional defect in HR, assessed by impaired RAD51 foci formation on end of treatment biopsy. Following rucaparib treatment there was no association of Ki67 change with HR deficiency. In contrast, early circulating tumor DNA dynamics identified activity of rucaparib, with end of treatment ctDNA levels suppressed by rucaparib in mutation-signature HR-deficient cancers. In ad hoc analysis, rucaparib induced expression of interferon response genes in HR-deficient cancers. The majority of TNBCs have a defect in DNA repair, identifiable by mutational signature analysis, that may be targetable with PARP inhibitors.

Anti-IHNV activities of recombinant rainbow trout IFNa and its influence on interferon response gene expression in vitro

Our research aimed to evaluate the antiviral activities of prokaryotic-expressed rainbow trout recombinant interferon-a (rtrIFNa) against infectious hematopoietic necrosis virus (IHNV), and the expression change of interferon response genes in vitro following IHNV infection or early stage rtrIFNa stimulation. The antiviral activities of rtrIFNa were evaluated based on cytopathic effect (CPE) reduction assays. Our results showed that the anti-IHNV activity of rtrIFNa was dose related and about 1 × 104 U/mg. RtrIFNa significantly reduced the copy number of the IHNV N gene after infection (P<0.01), indicating that rtrIFNa could suppress replication of the IHNV genome. In addition, rtrIFNa could significantly up-regulate most IFN-stimulated genes early (within 8 h). The strongest significant response was observed in the Mx3 and ISG15 genes. After IHNV infection, these IFN related genes were also significantly induced and then began to decrease within 72 h. The transcript level of these genes in the rtrIFNa-injected group was significantly higher (P<0.05) than in the rpET32a-injected group. Additionally, we observed that the transcription of IFNa and PKR genes was inhibited at early stages after IHNV infection in the absence of rtrIFNa. Finally, we conclude that refolded rtrIFNa has good biological activity, as it could effectively inhibit IHNV replication and activate IFN signaling. The kinetics of interferon related gene expression, elicited by IHNV or rtrIFNa, will facilitate a better understanding of the anti-IHNV activity of rtrIFNa in vitro.

MEK1/2 Inhibitors Unlock the Constrained Interferon Response in Macrophages Through IRF1 Signaling.

Macrophages are immune sentinels essential for pathogen recognition and immune defense. Nucleic acid-sensing toll-like receptors like TLR7 activate tailored proinflammatory and interferon responses in macrophages. Here we found that TLR7 activation constrained itself and other TLRs from inducing interferon response genes in macrophages through MAPK kinase 1/2 (MEK1/2)-dependent IRF1 inhibition. Downstream of the MEK1/2-ERK pathway, TLR7-activated macrophages induced interleukin-10 (IL-10), a signal transducer and activator of transcription 3 (STAT3) signaling axis, which constrained the expression of interferon response genes, immunomodulatory cytokines, and chemokines. Nevertheless, MEK1/2 inhibitors unlocked an IRF1-interferon signature response in an NF-κB-dependent manner. Deficiency in interferon regulatory factor 1 (Irf1) completely abrogated the interferon response and prevented the reprogramming of macrophages into an immunostimulatory phenotype. As a proof of concept, combination treatment with a TLR7 agonist and MEK1/2 inhibitor synergistically extended the survival of wild-type but not Irf1-deficient melanoma-bearing mice. In a retrospective study, higher expression of Irf1 and interferon response genes correlated with more favorable prognosis in patients with cutaneous melanoma. Our findings demonstrated how MEK1/2 inhibitor unlocks IRF1-mediated interferon signature response in macrophages, and the therapeutic potentials of combination therapy with MEK1/2 inhibitor and TLR7 agonist.

Ubiquitination of cGAS by TRAF6 regulates anti-DNA viral innate immune responses

cGAS-STING (stimulator of interferon genes) signaling is crucial for the recognition of cytoplasmic double-stranded DNA by host cells and consequently activating innate immune response by promoting the production cGAMP and type I interferon. However, it remains elusive how the cGAS enzymatic activity is regulated dynamically. In this study, we identified TRAF6 as a regulator of cGAS mediated anti-viral innate immunity. Our data showed that either ectopic expression or knockdown of TRAF6 modulates the double strand DNA induced expression of interferon-responsive genes. Mechanistically, TRAF6 specifically promotes cGAS activation by targeting cGAS for ubiquitination. Knockdown of TRAF6 results in a decrease in cGAS-induced IFNβ production when cells were infected with herpes simplex virus-1 (HSV-1). Together, our data identified TRAF6 as a positive regulator of cGAS-STING pathway by regulating cGAS activity.

Angiotensin-converting enzyme inhibitor suppresses Type I interferon responses, decreases neuroinflammation and improves depression-like behavior in MRL/lpr lupusprone mice

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multi-organ damage. Interferon- α (IFN-α) is a central mediator in disease pathogenesis. Neuropsychiatric lupus (NPSLE) is one of the most common manifestations of human SLE often causing depression. Administration of IFN-α in patients with chronic viral infections or cancers causes depressive symptoms. Angiotensin-converting enzyme (ACE) is part of the kallikrein-kinin/renin-angiotensin (KKS/RAS) system that regulates many physiological processes, including inflammation and brain functions. It is known that ACE degrades bradykinin into inactive peptides. We have previously shown in an in vitro model of mouse bone-marrow-derived dendritic cells that the effects of captopril (an ACE inhibitor) on IFN responsive gene inhibition were reversed when bradykinin receptors were blocked, suggesting that ACE, in part, acts via kinin receptors to bring about the suppressive effects. We used the MRL/lpr lupus-prone mouse model, an established model to study NPSLE, to check the in vivo effects of captopril on IFN-induced immune responses, neuroinflammation, and behavior. Exposing MRL/lpr mice to IFN-α increased depressive-like behavior and enhanced ACE expression in the brain. Administering captopril in MRL/lpr mice decreased expression of IFN responsive genes in brain and kidney, decreased microglial activation (IBA1 expression) and reduced depressive-like behavior as assessed by the forced swim test. Thus, ACE inhibitors may have potential for therapeutic use for systemic and neurolupus.

386 Autocrine IFN-k restricts CRISPR-Cas9 keratinocyte transfection

CRISPR-Cas9 is a unique genome-editing tool but is limited in regards to variable transfection efficiency particularly in keratinocytes (KCs) (1-2%), in contrast to HEK293T cells (62%). Despite this, we have successfully generated >20 knockout lines (KO) in immortalized KCs. Intriguingly, in all KO generated, IFNK mRNA expression (p= 1.7E-06), an endogenous type I IFN secreted by KCs, as well as expression of IFN response genes (IRF7, p<0.01; MX1, FC, p<0.01) were significantly suppressed in all KO lines. The decreased IFNK mRNA expression correlated with hypermethylation of the promoter region in KO lines. Notably, KO KCs (for any gene tested) had almost a 10-fold (p<0.01) increase in transfection efficiency compared to control KCs. In contrast, we observed marked suppression (FC=0.1, p<0.01) of transfection efficiency when recombinant type I IFN was added to cell culture medium prior to transfection. CRISPR plasmid uptake by KCs was highest at 6% at 12 hrs and decreased at later time point (24hrs (3%), 48 hrs (1%)), and accompanied by increased IFNK and MX1 mRNA expression (FC=3.74, p<0.001 and FC=10.72, p<0.001 respectively) at 24 hr time point. This IFN response was dependent upon the function of the intracellular RNA sensor STING. Our data suggest that CRISPR activates IFN-kappa through STING-dependent pathways and that activation of endogenous KC type I IFN signaling is responsible for KC resistance to CRISPR transfection. These findings will have major implications regarding CRISPR research and its use to correct epithelial defects.

CD47 Expression in Natural Killer Cells Regulates Homeostasis and Modulates Immune Response to Lymphocytic Choriomeningitis Virus

CD47 is a ubiquitous cell surface receptor that directly regulates T cell immunity by interacting with its inhibitory ligand thrombospondin-1 and limits clearance of cells by phagocytes that express its counter-receptor signal-regulatory protein-α. Murine natural killer (NK) cells express higher levels of CD47 than other lymphocytes, but the role of CD47 in regulating NK cell homeostasis and immune function remains unclear. Cd47−/− mice exhibited depletion of NK precursors in bone marrow, consistent with the antiphagocytic function of CD47. In contrast, antisense CD47 knockdown or gene disruption resulted in a dose dependent accumulation of immature and mature NK cells in spleen. Mature Cd47−/− NK cells exhibited increased expression of NK effector and interferon gene signatures and an increased proliferative response to interleukin-15 in vitro. Cd47−/− mice showed no defect in their early response to acute Armstrong lymphocytic choriomeningitis virus (LCMV) infection but were moderately impaired in controlling chronic Clone-13 LCMV infection, which was associated with depletion of splenic NK cells and loss of effector cytokine and interferon response gene expression in Cd47−/− NK cells. Broad CD47-dependent differences in NK activation, survival, and exhaustion pathways were observed in NK cell transcriptional signatures in LCMV infected mice. These data identify CD47 as a cell-intrinsic and systemic regulator of NK cell homeostasis and NK cell function in responding to a viral infection.

Abstract 2923: BET inhibitors synergize with HDAC inhibitors and downregulate expression of interferon response genes in glioblastoma

Abstract Glioblastoma (GBM) is the most aggressive brain tumor in adults with a median overall survival of only 15 months. Despite multiple attempts, single agent therapies have failed in clinical trials and new strategies for combination treatments are urgently needed. Here, our aim was to predict rational combination therapies with BET inhibitors (BETi) that target bromodomain and extra-terminal tail (BET) proteins and are currently evaluated as anti-cancer drugs. In order to identify BETi-induced vulnerabilities in cancer relevant pathways that may be targeted with a second drug, we obtained differential expression profiles of glioma sphere lines (GS-lines) treated with the BETi JQ1. Gene set enrichment analysis revealed several significantly disturbed pathways that included IFN-alpha response genes and signatures of response to histone deacetylase inhibitors (HDACi). In order to validate the observed down regulation of the interferon response gene signature, we primed the GS-lines with IFN-alpha and confirmed that interferon-stimulated genes (ISGs), such as MX1, OAS1, and CD274 are down regulated after a 4-hour exposure to JQ1. Importantly, the levels of pSTAT1 in the nucleus remained unchanged upon JQ1 treatment, suggesting that JQ1 was acting directly on the transcriptional level of ISGs and not on the canonical JAK-STAT pathway. Similar results were obtained in adherent GBM cell lines that constitutively express ISGs. Moreover, in U87MG orthotropic xenografts in mice, a single i.p. injection of JQ1 downregulated OAS1 and CD274 expression. Currently we are studying the mechanism of how BETi represses transcription of ISGs by chromatin immuno-precipitation. Finally, we show that HDACi and JQ1 synergize to reduce cell viability of GS-lines in vitro. Further experiments will test HDACi and BETi drug combinations in mouse orthotopic xenografts of GS-lines. Citation Format: Olga Gusyatiner, Minh Diêu Thanh Pham, Yvonne Lei, Jungyeon Park, Pierre Bady, Mauro Delorenzi, Monika Hegi. BET inhibitors synergize with HDAC inhibitors and downregulate expression of interferon response genes in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2923.