Type-1 Interferon Signaling Research Articles

Disruption of polyunsaturated fatty acid biosynthesis drives STING-dependent acute myeloid leukemia cell maturation and death

The role of polyunsaturated fatty acid (PUFA) biosynthesis in acute myeloid leukemia (AML) remains largely undefined. A comparative expression analysis of 35 genes encoding fatty acid biosynthesis enzymes showed that fatty acid desaturase 1 (FADS1) was highly expressed across multiple AML subtypes relative to healthy controls and that elevated FADS1 expression correlates with worse overall AML patient survival. Functionally, shRNA-mediated inhibition of FADS1 reduced AML cell growth invitro and significantly delayed leukemia onset in an AML mouse model. AML cell lines depleted of FADS1 arrested in the G1/S-phase of the cell cycle, acquired characteristics of myeloid maturation and subsequently died. To understand the molecular consequences of FADS1 inhibition, a combination of mass spectrometry-based analysis of complex lipids and gene expression analysis (RNA-seq) was performed. FADS1 inhibition caused AML cells to exhibit significant lipidomic remodeling, including depletion of PUFAs from the phospholipids, phosphatidylserine, and phosphatidylethanolamine. These lipidomic alterations were accompanied by an increase induction of inflammatory and stimulator of interferon genes (STING)-mediated type-1 interferon signaling. Remarkably, genetic deletion of STING largely prevented the AML cell maturation and death phenotypes mediated by FADS1 inhibition. Highlighting the therapeutic implications of these findings, pharmacological blockade of PUFA biosynthesis reduced patient-derived AML cell numbers exvivo but not that of healthy donor cells. Similarly, STING agonism attenuated patient-derived-AML survival; however, STING activation also reduced healthy granulocyte numbers. Collectively, these data unveil a previously unrecognized importance of PUFA biosynthesis in leukemogenesis and that imbalances in PUFA metabolism can drive STING-mediated AML maturation and death.

Rare IFNAR1 variant in patient with autoinflammatory disorder and elevated type-1 interferon signaling

Case ReportWe present the case of a 30-year-old female with a history of chronic urticaria, fevers, mild hearing loss, and seronegative arthritis, who was found to have a rare variant in IFNAR1 and elevated type-1 interferon gene expression. She initially presented for evaluation of an autoinflammatory phenotype, and was found to have significantly elevated IL-18 (3473 pg/mL), IgG hypergammaglobulinemia (1680 mg/dL), and a variant of unknown significance in IFNAR1 (p.Ala424Thr). This variant is seen in 0.01% of East Asians in gnomAD. Further investigation revealed elevated expression of type-1 interferon stimulated genes (score 0.83, normal <–0.5).IFNAR1 codes for one subunit of the interferon alpha/beta receptor. Our patient’s rare variant in IFNAR1, in combination with her elevated type 1 interferon gene expression, is concerning for a gain-of-function variant in IFNAR1 leading to increased interferon pathway signaling. Gain-of-function variants in IFNAR1 have not been described previously. Further experiments are needed to validate whether this variant is gain of function. If so, she could benefit from inhibition of the type-1 interferon receptor signal, for example, with a JAK inhibitor such as baricitinib or with the monoclonal anifrolumab. This case demonstrates the utility of genetic testing in combination of functional assays to develop novel targeted therapies for patients in the field of clinical immunology.

Heterogeneous RNA editing and influence of ADAR2 on mesothelioma chemoresistance and the tumor microenvironment.

We previously observed increased levels of adenosine-deaminase-acting-on-dsRNA (Adar)-dependent RNA editing during mesothelioma development in mice exposed to asbestos. The aim of this study was to characterize and assess the role of ADAR-dependent RNA editing in mesothelioma. We found that tumors and mesothelioma primary cultures have higher ADAR-mediated RNA editing compared to mesothelial cells. Unsupervised clustering of editing in different genomic regions revealed heterogeneity between tumor samples as well as mesothelioma primary cultures. ADAR2 expression levels are higher in BRCA1-associated protein 1 wild-type tumors, with corresponding changes in RNA editing in transcripts and 3'UTR. ADAR2 knockdown and rescue models indicated a role in cell proliferation, altered cell cycle, increased sensitivity to antifolate treatment, and type-1 interferon signaling upregulation, leading to changes in the microenvironment invivo. Our data indicate that RNA editing contributes to mesothelioma heterogeneity and highlights an important role of ADAR2 not only in growth regulation in mesothelioma but also in chemotherapy response, in addition to regulating inflammatory response downstream of sensing nucleic acid structures.

POS0390 GENES OF “DEFENSE RESPONSE TO VIRUS” IN PERIPHERAL BLOOD OF ANTI-MDA5 POSITIVE DERMATOMYOSITIS WERE UPREGULATED AS COMPARE WITH OTHER FORMS OF DERMATOMYOSITIS. ~SUPPRESSING RIG-I LIKE RECEPTOR SIGNALING OR TYPE 1/2 INTERFERON SIGNALING WERE THE KEYS FOR SURVIVAL.

BackgroundAnti-melanoma differentiation-associated gene 5–positive dermatomyositis (MDA5 DM) is a rare but distinct subtype of idiopathic inflammatory myopathy (IIM) that is characterized by high mortality due to rapid progressive interstitial lung disease (ILD). MDA5 is a cytosolic protein and a family of retinoic-acid inducible gene-I (RIG-I) like receptor, which functions as a virus RNA sensor and induces the production of type I interferons (IFN-I) and proinflammatory cytokines. This leads to the death of infected cells and the activation of the antigen-specific antiviral immune response. Although the pathogenesis of MDA5 DM is largely unknown, a hypothesis that pathogenic involvement of anti-MDA5 antibodies has been proposed. Recently, similarities have been noted between multifaceted features of COVID-19 and MDA5 DM, which suggests shared underlying autoinflammatory mechanisms.ObjectivesTo detect the critical actors in the pathogenesis of MDA5 DM by gene expression analysis of peripheral blood.MethodsTotal of 31 DM cases were investigated, including anti-aminoacyl-tRNA synthetase positive (ARS) DM (n=12), MDA5 DM (n=7, survivor=3) and others (n=12). Peripheral blood was drawn at baseline and 2 to 3 months after treatments. Total RNAs were then extracted with using PAXgene miRNA kit. After quantifying the expressions of transcripts by multiplex sequencing. And then, hierarchical clustering analysis, enrichment analysis using gene ontology (GO) terms, single sample gene set enrichment analysis (ssGSEA) and weighted gene co-expression network analysis (WGCNA) were performed.ResultsThe hierarchical clustering with expression profiles of peripheral blood at baseline showed major 3 clusters. Interestingly, ARS DM cases were segregated into right side of the 3rd cluster while MDA5 DM cases fell into 1st and 2nd clusters. ARS and MDA5 DM were clearly discriminated if differentially expressed genes (DEGs) between these subtypes of DM were analyzed. By GO enrichment analysis, the terms, such as related to “defense response to virus” including “type1 interferon signaling pathway” were found in the DEGs. In the MDA5 DM cases, ssGSEA revealed that genes of “Fcγ receptor mediated phagocytosis pathway” or “complement and coagulation cascade” were significantly enriched and WGCNA showed that pathways of “T-cell antigen receptor signaling” or “lung fibrosis” were significantly upregulated. Next, we also investigated the DEGs of peripheral blood at 2-3 months after treatment between survival and fatal cases in MDA5 DM. We found that suppressing RIG-I like receptor and type 1 and type 2 interferon (IFN) signaling were the keys for survival.ConclusionMDA5 is a key sensor of several RNA viruses including coronavirus families and then activate antiviral gene transcription such as type 1 IFN genes, leading to establish an antiviral host response. As the pulmonary damage of COVID-19 is known to be difficult to distinguish from the ILD associated with anti-MDA5 DM, the life-threating ILD of MDA5 DM may be caused by the over-activation of RIG-I like receptor signaling via MDA5. The hypothesis is supported by our findings that the defining features of MDA5 DM are activation of “type 1 IFN pathways” and antigen-specific antiviral immune responses including “Fcγ receptor mediated phagocytosis pathway” or “T-cell antigen receptor signaling”. As the levels of anti-MDA5 antibodies reported to be important prognostic parameter, it may be involved in pathogenesis of MDA5 DM. As we found that suppression of type 1 and type 2 IFN signaling were the keys for survival, it seems to be reasonable to use inhibitors of Janus Kinases (JAK) for treatment of MDA5 DM.Disclosure of InterestsYoshinobu Koyama Speakers bureau: Abbvie, Asahikasei, Ayumi, BMS, Eli-Lilly, Mitsubishi Tanabe, Grant/research support from: Abbvie, GSK, Yoshiharu Sato: None declared, Moe Sakamoto: None declared

Type I Interferon signaling controls the accumulation and transcriptomes of monocytes in the aged lung.

Aging is paradoxically associated with a deteriorated immune defense (immunosenescence) and increased basal levels of tissue inflammation (inflammaging). The lung is particularly sensitive to the effects of aging. The immune cell mechanisms underlying physiological lung aging remain poorly understood. Here we reveal that aging leads to increased interferon signaling and elevated concentrations of chemokines in the lung, which is associated with infiltration of monocytes into the lung parenchyma. scRNA‐seq identified a novel Type‐1 interferon signaling dependent monocyte subset (MO‐ifn) that upregulated IFNAR1 expression and exhibited greater transcriptomal changes with aging than the other monocytes. Blockade of type‐1 interferon signaling by treatment with anti‐IFNAR1 neutralizing antibodies rapidly ablated MO‐ifn cells. Treatment with anti‐IFNAR1 antibodies also reduced airway chemokine concentrations and repressed the accumulation of the overall monocyte population in the parenchyma of the aged lung. Together, our work suggests that physiological aging is associated with increased basal level of airway monocyte infiltration and inflammation in part due to elevated type‐1 interferon signaling.

Abstract LB118: Mechanisms underlying synergistic activity in MYD88MTDLBCL of KT-413, a targeted degrader of IRAK4 and IMiD substrate

Abstract Relapsed/refractory DLBCL remains an incurable disease, and single-agent therapies typically show low response rates and/or transient clinical responses. Oncogenic MYD88 mutations occur in ~25% of DLBCL and drive constitutive NFkB activation, promoting proliferation and survival. Despite its role in tumor biology, targeting MYD88MT by inhibiting or degrading IRAK4 alone, a key component of the MYD88 complex, does not drive significant antitumor activity in preclinical models. One potential reason is the frequent redundant NFkB pathway activation by co-mutations, highlighting the need for combination therapies to effectively target the NFkB pathway in DLBCL. To address this challenge, we have developed IRAKIMiDs, heterobifunctional degraders that simultaneously degrade both IRAK4 and IMiD substrates, as a rational therapeutic combination in a single molecule. IRAKIMiDs show increased antitumor activity in vitro and in vivo in MYD88MT cells as compared to IMiD or IRAK4-targeting alone, highlighting suggesting their potential as single agents in R/R DLBCL.Our lead IRAKIMiD, KT-413, is a potent degrader of IRAK4 (DC50 6nM) and IMiD substrates (Ikaros/Aiolos DC50 2nM), inducing rapid and potent cell killing in vitro and complete and sustained tumor regressions in vivo in MYD88MT models of DLBCL. This activity is superior to the IMiD CC-220, which has similar activity against IMiD substrates (Ikaros/Aiolos DC50 1 nM), supporting the synergistic role of IRAK4 degradation in the context of IMiD biology. We show here that the combined activity of these 2 mechanisms drives a synergistic effect on NFkB and IRF4 signaling with greater downstream effect on NFkB and type 1 interferon (IFN) signaling and cell cycle gene expression than either mechanism alone. In THP1 cells engineered with NFkB and IRF4 reporters, KT-413 but not CC-220 inhibits TLR-stimulated NFkB and IRF4 transcription, supporting a role for IRAK4 but not IMiDs in MYD88-driven survival and proliferation signals. IMiDs have previously been shown to modulate type1 IFN signaling through downregulation of IRF4. We propose that simultaneous targeting of both NFkB and type 1 IFN signaling with KT-413 drives synergistic cell killing in MYD88MT cells. In MYD88MT OCI-Ly10 cells, KT-413 leads to greater IRF4 downregulation, increased type 1 IFN signaling, and preferential downregulation of NFkB pathway and cell cycle transcripts when compared to CC-220. These data support the hypothesis that the synergistic activity of targeting IRAK4 and IMiD substrates by KT-413 in MYD88MT DLBCL is a result of dual targeting of NFkB and IRF4/Type1 IFN through degradation of both IRAK4 and IMiD substrates,driving significantly greater cell killing as compared to either mechanism alone, supporting the potential for the first single agent targeted therapy in MYD88MT DLBCL. KT-413 is on track for initiation of a Phase 1 trial in B cell lymphoma in 2H 2021. Citation Format: Christine R. Klaus, Scott F. Rusin, Kirti Sharma, Samyabrata Bhaduri, Matthew M. Weiss, Alice A. McDonald, Michele F. Mayo, Duncan Walker, Rahul Karnik. Mechanisms underlying synergistic activity in MYD88MTDLBCL of KT-413, a targeted degrader of IRAK4 and IMiD substrate [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 LB118.

Abstract LB169: Systemic Administration of an antibody/RNA complex results in tumor specific delivery of immunostimulatory RNAs and tumor growth suppression in a mouse model of melanoma

Abstract There is intense interest in the development of nucleic acid ligands for immune stimulation of the tumor microenvironment via pattern recognition receptors, especially for the treatment of “cold” tumors. However, delivery of these ligands in a tumor-specific manner to avoid systemic toxicity has been a challenge. Many current efforts rely on direct intra-tumoral injection of RNAs or other stimulatory immune ligands, which is therapeutically sub-optimal, especially for metastatic disease. Here we report studies evaluating the utility of a lupus-derived, cell-penetrating antibody, to deliver nucleic acids including mRNA to tumors in vivo. This antibody, a modified version of 3E10-D31N, forms non-covalent complexes with RNAs and can mediate highly specific delivery broadly into tumors via intravenous injection by targeting the nucleoside transporter ENT2. Studies with labeled RNAs and with mRNAs expressing a GFP reporter gene show tumor specific delivery and functional expression by imaging, with minimal delivery to healthy tissues. Cell culture studies show antibody-mediated delivery of a series of RIG-I ligands, including hairpin RNAs and poly(I:C), resulting in robust RIG-I stimulation and induction of type-1 interferon signaling. Using mouse B16 melanoma tumors as a model, we observe substantial tumor growth suppression upon i.v. injection of a combination of 3E10 complexed with poly(I:C) (synthetic double-stranded RNA). These results highlight a novel approach for the systemic delivery of immunostimulatory RNAs in a targeted manner that may offer treatment advantages over current approaches. Citation Format: Elias Quijano, Yanfeng Liu, Stephen Squinto, Bruce C. Turner, Peter M. Glazer. Systemic Administration of an antibody/RNA complex results in tumor specific delivery of immunostimulatory RNAs and tumor growth suppression in a mouse model of melanoma [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 LB169.

LAMP-5 is an essential inflammatory-signaling regulator and novel immunotherapy target for mixed lineage leukemia-rearranged acute leukemia.

Although great advances have been made in understanding the pathobiology of mixed lineage leukemia-rearranged (MLL-r) leukemias, therapies for this leukemia have remained limited, and clinical outcomes remain bleak. In order to identify novel targets for immunotherapy treatments, we compiled a lineage-independent MLL-r leukemia gene signature using publicly available data sets. Data from large leukemia repositories were filtered through the in silico human surfaceome, providing a list of highly predicted cell surface proteins overexpressed in MLL-r leukemias. LAMP5, a lysosomal associated membrane protein, is expressed highly and specifically in MLL-r leukemia. We found that LAMP5 is a direct target of the oncogenic MLL-fusion protein. LAMP5 depletion significantly inhibited leukemia cell growth in vitro and in vivo. Functional studies showed that LAMP-5 is a novel modulator of innate-immune pathways in MLL-r leukemias. Downregulation of LAMP5 led to inhibition of NF-kB signaling and increased activation of type-1 interferon signaling downstream of Toll-like receptor/interleukin 1 receptor activation. These effects were attributable to the critical role of LAMP-5 in transferring the signal flux from interferon signaling endosomes to pro-inflammatory signaling endosomes. Depletion of IRF7 was able to partially rescue the cell growth inhibition upon LAMP5 downregulation. Lastly, LAMP-5 was readily detected on the surface of MLL-r leukemia cells. Targeting surface LAMP-5 using an antibody-drug conjugate leads to significant cell viability decrease specifically in MLL-r leukemias. Overall, based on the limited expression throughout human tissues, we postulate that LAMP-5 could potentially serve as an immunotherapeutic target with a wide therapeutic window to treat MLL-r leukemias.

Analysis of Multiplexed Single Cell RNA Sequencing Clinical Correlative Data in AML Reveals Biomarkers of Resistance

Single cell RNA sequencing (scRNA-seq) is a powerful method to understand gene expression changes in specific subsets of heterogeneous cell populations and to provide novel insight into mechanisms of drug resistance. However, substantial cost and interpatient variability have limited its application as a correlate for prospective clinical trials. To overcome these challenges, we employed a genetic polymorphism-based multiplexing approach to assay peripheral blood samples prospectively collected from patients with relapsed/refractory AML treated on a Phase 1b study using the anti-hepatocyte growth factor monoclonal antibody, Ficlatuzumab (NCT02109627). Ficlatuzumab was administered on study days 1, 15, 29 and 42-44 in combination with high-dose cytarabine. Peripheral blood was collected from 11 subjects on study days 0, 1, 2, 3 and 42-44. N = 7 subjects achieved a complete response to therapy and N = 4 did not respond to therapy. A peripheral blood sample from a healthy volunteer was collected and processed in parallel. 57 samples were multiplexed into 5 pools of 11 to 12 samples using a Latin square design to maximize genetic diversity within each pool and minimize batch effects. Each pool was assayed in duplicate on a 10X Chromium single cell isolation system using 3'-capture technology, targeting 40,000 cells per replicate pool. After initial bioinformatic processing, 175,667 unique cell barcodes were recovered. Genotype-free demultiplexing was performed using Freemuxlet (https://github.com/yelabucsf/popscle). Single nucleotide variants were annotated within the aligned reads from each pool using the 1000 Genomes variant database. A Bayesian clustering algorithm was applied to assign each cell to a group of cells with like genotype. Reference sample genotypes were determined using the Infinium OmniExpressExome-8 BeadChip (Illumina) and these were used to map each cluster to known subject identity. This approach unambiguously assigned 105,363 cells (60.0%) to single subjects and 60,787 (34.6%) to inter-sample doublets. 103,415 cells were retained after removal of intra-sample doublets and low-quality cells. Dimensionality reduction and clustering identified 3 groups of cells that expressed AML blast markers including CD33, CD34, KIT and HLA-DR. We hypothesized that induction of HGF expression by AML blasts might be a mechanism of resistance to anti-HGF therapy. AML blasts were stratified by treatment day and multivariable regression was performed using cluster assignment as a potential confounder. This showed that non-response to study therapy was independently correlated with higher HGF levels on study D0 (q = 0.008), D1 (q = 0.02), and D42-44 (q = 5x10-7). To identify additional biomarkers of resistance to anti-HGF therapy, we performed unsupervised gene module analysis and found 20 groups of genes that were co-regulated within the dataset. One module was strongly induced in non-responders compared to responders at D0 (q = 0), D1 (q = 1x10-278), and D42-44 (q = 0). This module was highly enriched for genes related to type-1 interferon signaling and antiviral response including IL6, IRF7, IRF2, STAT1, STAT2 and APOBEC3A. Unsupervised hierarchical clustering based on gene module scores segregated non-responders from complete responders and showed that the normal control cells were most similar to complete responders at D42-44. These data suggest that multiplexing using genetic polymorphisms increases the number of scRNA-seq samples it is feasible to analyze in the context of a prospective clinical trial. Gene module analysis is able to identify pathways as potential biomarkers of drug resistance in an unsupervised way. These approaches will aid studies in which interpatient variability is a challenge to interpretation of scRNA-seq data. Disclosures Andreadis: Merck: Research Funding; Gilead/Kite: Consultancy; Karyopharm: Honoraria; Jazz Pharmaceuticals: Honoraria; Novartis: Research Funding; Genentech: Consultancy, Current equity holder in publicly-traded company; BMS/Celgene/Juno: Honoraria, Research Funding; Incyte: Consultancy.

Aberrant ER Stress Induced Neuronal-IFNβ Elicits White Matter Injury Due to Microglial Activation and T-Cell Infiltration after TBI.

Persistent endoplasmic reticulum (ER) stress in neurons is associated with activation of inflammatory cells and subsequent neuroinflammation following traumatic brain injury (TBI); however, the underlying mechanism remains elusive. We found that induction of neuronal-ER stress, which was mostly characterized by an increase in phosphorylation of a protein kinase R-like ER kinase (PERK) leads to release of excess interferon (IFN)β due to atypical activation of the neuronal-STING signaling pathway. IFNβ enforced activation and polarization of the primary microglial cells to inflammatory M1 phenotype with the secretion of a proinflammatory chemokine CXCL10 due to activation of STAT1 signaling. The secreted CXCL10, in turn, stimulated the T-cell infiltration by serving as the ligand and chemoattractant for CXCR3+ T-helper 1 (Th1) cells. The activation of microglial cells and infiltration of Th1 cells resulted in white matter injury, characterized by impaired myelin basic protein and neurofilament NF200, the reduced thickness of corpus callosum and external capsule, and decline of mature oligodendrocytes and oligodendrocyte precursor cells. Intranasal delivery of CXCL10 siRNA blocked Th1 infiltration but did not fully rescue microglial activation and white matter injury after TBI. However, impeding PERK-phosphorylation through the administration of GSK2656157 abrogated neuronal induction of IFNβ, switched microglial polarization to M2 phenotype, prevented Th1 infiltration, and increased Th2 and Treg levels. These events ultimately attenuated the white matter injury and improved anxiety and depressive-like behavior following TBI.SIGNIFICANCE STATEMENT A recent clinical study showed that human brain trauma patients had enhanced expression of type-1 IFN; suggests that type-1 IFN signaling may potentially influence clinical outcome in TBI patients. However, it was not understood how TBI leads to an increase in IFNβ and whether induction of IFNβ has any influence on neuroinflammation, which is the primary reason for morbidity and mortality in TBI. Our study suggests that induction of PERK phosphorylation, a characteristic feature of ER stress is responsible for an increase in neuronal IFNβ, which, in turn, activates microglial cells and subsequently manifests the infiltration of T cells to induce neuroinflammation and subsequently white matter injury. Blocking PERK phosphorylation using GSK2656157 (or PERK knockdown) the whole cascade of neuroinflammation was attenuated and improved cognitive function after TBI.

Murine host response to Neisseria gonorrhoeae upper genital tract infection reveals a common transcriptional signature, plus distinct inflammatory responses that vary between reproductive cycle phases

BackgroundThe emergence of fully antimicrobial resistant Neisseria gonorrhoeae has led global public health agencies to identify a critical need for next generation anti-gonococcal pharmaceuticals. The development and success of these compounds will rely upon valid pre-clinical models of gonorrhoeae infection. We recently developed and reported the first model of upper genital tract gonococcal infection. During initial characterization, we observed significant reproductive cycle-based variation in infection outcome. When uterine infection occurred in the diestrus phase, there was significantly greater pathology than during estrus phase. The aim of this study was to evaluate transcriptional profiles of infected uterine tissue from mice in either estrus or diestrus phase in order to elucidate possible mechanisms for these differences.ResultsGenes and biological pathways with phase-independent induction during infection showed a chemokine dominant cytokine response to Neisseria gonorrhoeae. Despite general induction being phase-independent, this common anti-gonococcal response demonstrated greater induction during diestrus phase infection. Greater activity of granulocyte adhesion and diapedesis regulators during diestrus infection, particularly in chemokines and diapedesis regulators, was also shown. In addition to a greater induction of the common anti-gonococcal response, Gene Set Enrichment Analysis identified a diestrus-specific induction of type-1 interferon signaling pathways.ConclusionsThis transcriptional analysis of murine uterine gonococcal infection during distinct points in the natural reproductive cycle provided evidence for a common anti-gonococcal response characterized by significant induction of granulocyte chemokine expression and high proinflammatory mediators. The basic biology of this host response to N. gonorrhoeae in estrus and diestrus is similar at the pathway level but varies drastically in magnitude. Overlaying this, we observed type-1 interferon induction specifically in diestrus infection where greater pathology is observed. This supports recent work suggesting this pathway has a significant, possibly host-detrimental, function in gonococcal infection. Together these findings lay the groundwork for further examination of the role of interferons in gonococcal infection. Additionally, this work enables the implementation of the diestrus uterine infection model using the newly characterized host response as a marker of pathology and its prevention as a correlate of candidate vaccine efficacy and ability to protect against the devastating consequences of N. gonorrhoeae-associated sequelae.

Modulation of HIV replication in monocyte derived macrophages (MDM) by steroid hormones.

Significant sex specific differences in the progression of HIV/AIDS have been reported. Several studies have implicated steroid hormones in regulating host factor expression and modulating HIV transmission and replication. However, the exact mechanism exerted by steroid hormones estrogen and progesterone in the regulation of HIV-1 replication is still unclear. Results from the current study indicated a dose dependent down regulation of HIV-1 replication in monocyte derived macrophages pre-treated with high concentrations of estrogen or progesterone. To elucidate the molecular mechanisms associated with the down regulation of HIV-1 replication by estrogen and progesterone we used PCR arrays to analyze the expression profile of host genes involved in antiviral responses. Several chemokines, cytokines, transcription factors, interferon stimulated genes and genes involved in type-1 interferon signaling were down regulated in cells infected with HIV-1 pre-treated with high concentrations of estrogen or progesterone compared to untreated HIV-1 infected cells or HIV-1 infected cells treated with low concentrations of estrogen or progesterone. The down regulation of CXCL9, CXCL10 and CXCL11 chemokines and IL-1β, IL-6 cytokines in response to high concentrations of estrogen and progesterone pre-treatment in HIV-1 infected cells was confirmed at the protein level by quantitating chemokine and cytokine concentrations in the culture supernatant. These results demonstrate that a potent anti-inflammatory response is mediated by pre-treatment with high concentrations of estrogen and progesterone. Thus, our study suggests a strong correlation between the down-modulation of anti-viral and pro-inflammatory responses mediated by estrogen and progesterone pre-treatment and the down regulation of HIV-1 replication. These findings may be relevant to clinical observations of sex specific differences in patient populations and point to the need for further investigation.

Pharmacokinetics, Pharmacodynamics, and Proposed Dosing of the Oral JAK1 and JAK2 Inhibitor Baricitinib in Pediatric and Young Adult CANDLE and SAVI Patients.

Population pharmacokinetic (popPK) modeling was used to characterize the PK profile of the oral Janus kinase (JAK)1/JAK2 inhibitor, baricitinib, in 18 patients with Mendelian interferonopathies who are enrolled in a compassionate use program. Patients received doses between 0.1 to 17 mg per day. Covariates of weight and renal function significantly influenced volume-of-distribution and clearance, respectively. The half-life of baricitinib in patients less than 40 kg was substantially shorter than in adult populations, requiring the need for dosing up to 4 times daily. On therapeutic doses, the mean area-under-the-concentration-vs.-time curve was 2,388 nM*hr, which is 1.83-fold higher than mean baricitinib exposures in adult patients with rheumatoid arthritis receiving doses of 4 mg once-daily. Dose-dependent decreases in interferon (IFN) biomarkers confirmed an in vivo effect of baricitinib on type-1 IFN signaling. PopPK and pharmacodynamic data support a proposal for a weight- and estimated glomerular filtration rate-based dosing regimen in guiding baricitinib dosing in patients with rare interferonopathies.

Type 1 Interferon in the Human Intestine-A Co-ordinator of the Immune Response to the Microbiota.

The human gut is in constant complex interaction with the external environment. Although much is understood about the composition and function of the microbiota, much remains to be learnt about the mechanisms by which these organisms interact with the immune system in health and disease. Type 1 interferon (T1IFN), a ubiquitous and pleiotropic family of cytokines, is a critical mediator of the response to viral, bacterial, and other antigens sampled in the intestine. Although inflammation is enhanced in mouse model of colitis when T1IFN signaling is lost, the action of T1IFN is context specific and can be pro- or anti-inflammatory. In humans, T1IFN has been used to treat inflammatory diseases, including multiple sclerosis and inflammatory bowel disease but intestinal inflammation can also develop after the administration of T1IFN. Recent findings indicate that "tonic" or "endogenous" T1IFN, induced by signals from the commensal microbiota, modulates the local signaling environment to prime the intestinal mucosal immune system to determine later responses to pathogens and commensal organisms. This review will summarize the complex immunological effects of T1IFN and recent the role of T1IFN as a mediator between the microbiota and the mucosal immune system, highlighting human data wherever possible. It will discuss what we can learn from clinical experiences with T1IFN and how the T1IFN pathway may be manipulated in the future to maintain mucosal homeostasis.

Characterization of the Role of Regulatory T Cells (Tregs) in Inducing Progression of Multiple Myeloma

Introduction. Growing evidence suggests that immune cells that reside within the tumor microenvironment are dysregulated and functionally impaired, leading to defective anti-tumor immunity of the host. One of the major immunosuppressive mechanisms during tumor progression is expansion of regulatory immune cells. Here, we analyzed the immune cells within the bone marrow (BM) and the peripheral blood (PB) of 2 immunocompetent multiple myeloma (MM) mouse models. We next studied the role of regulatory T cells (Tregs) in MM pathogenesis.Materials and methods. To study the immune cell populations of the BM and PB, we used two immuncompetent mouse models and transplanted VK*MYC cells or 5TGM1 cells into C57BL/6 and C57BL/Kalwrij mice respectively. The immune cell populations and checkpoint receptor expressions were analyzed by CyTOF mass cytometer or flow-cytometry. Treg induction assay was performed in vitro to study the mechanism of Treg increase in the BM of myeloma injected mice. CD4+ CD25- cells were obtained from C57BL/Kalwrij mice and were co-cultured with 5TGM1 cells or B cells from C57BL/Kalwrij mice in vitro. Treg induction was compared by flow-cytometry.Transplantable VK*MYC cells were injected into "depletion of regulatory T cell" (DEREG) mice, which expresses a simian diphtheria toxin (DT) receptor-enhanced GFP fusion protein under the control of the FOXP3 gene locus, or their wild type littermates. DT injection into these mice leads to depletion of Tregs as previously described (J Exp Med. 2007; 204: 57-63). DT was given once every week for a total 3 times i.p to the DEREG mice or the littermate controls to specifically deplete Tregs and to study the role of Tregs during MM progression. Tregs (CD4+ FOXP3-GFP+ cells) were sorted from VK*MYC injected mice or non-injected DEREG mice BM using FACSAria cell sorter. Cells isolated were subjected to RNA sequencing. Gene Set Enrichment Analysis (GSEA) was performed to define differences in molecular signatures between MM-associated and normal Tregs.Results. The Treg proportion was significantly increased within the CD4+ T cells in the BM of myeloma cell injected mice from the early stage of disease compared to control mice, while in the PB, the increase was observed only at the late stages of disease progression. The effector T cell (Teff)/Treg ratio was significantly decreased in the BM at the end-stage myeloma bearing mice (P<0.01). Checkpoint related molecules (PD-1, LAG-3 and Tim-3) on Tregs and Teffs were up-regulated at the protein level in the BM of myeloma injected mice compared to control (P<0.01). These data indicate the activation of Tregs and decrease of Teff activity, leading to suppression of anti-myeloma T cell activity in the MM BM. Under in vitro co-culture conditions, 5TGM1 cells induced a significant increase in the number of Tregs from non-Treg CD4+ T cells compared to controls (P<0.01). 5TGM1 cell-induced Tregs presented with enhanced ki-67 expression, thus suggesting the ability of MM cells to induce Treg proliferation. Additionally, the trans-well co-culture experiment showed that the major mechanism for Treg induction by 5TGM1 was by direct contact with T cells rather than secreting factors. Significant increase in survival was observed in the VK*MYC injected DEREG mice under Treg depletion compared to the DEREG mice without Treg depletion and wild type littermates under DT treatment (P<0.001). The Treg depleted DEREG mice were accompanied with an increase of Teffs (P<0.01), indicating recovery of anti-myeloma T cell activity. RNA sequencing of BM Tregs from VK*MYC injected mice showed increased expression of immune checkpoint related molecules and increased Treg effector molecules (IL-10, Granzyme B) compared to BM Tregs from control mice, indicating a more functionally active phenotype of VK*MYC associated Tregs. GSEA showed an enrichment of genes involved in type-1 interferon signaling in VK*MYC associated Tregs.Conclusions. We used 2 immunocompetent MM mouse models to study the characterization and significance of Tregs in MM progression. CyTOF analysis and RNA sequencing data indicated Treg activation in the MM BM microenvironment. The Treg in vivo depletion experiment showed that Tregs have a significant role in MM progression. These data indicate that immunotherapy targeting Tregs may represent a novel therapeutic strategy for MM. Studies are ongoing to understand the roles of Tregs in human MM patients. DisclosuresNo relevant conflicts of interest to declare.

Abstract 4091: Interfering with interferon: An axis of ATF2-mediated chemoresistance

Abstract Malignant melanoma is currently the most lethal skin cancer and comprises approximately 8% of total U.S. cancers cases. Despite the advent of even the most effective targeted monotherapies to date (e.g., mutant B-RAF inhibitors), clinical responses have largely been transient, as melanomas rapidly develop therapeutic resistance, highlighting the need to target other critical signaling pathways. In melanoma, Activating Transcription Factor 2 (ATF2) elicits an oncogenic function when nuclear but a tumor suppressor function when it is cytosolic/mitochondrial. In progressively malignant melanoma, increased PKCϵ-mediated phosphorylation of ATF2 on threonine 52 (T52) promotes its nuclear localization and transcriptional activity, and renders the cells more resistant to genotoxic stress. To determine how PKCϵ-phosphorylated ATF2 contributes to melanoma biology, we performed gene expression profiling of melanoma cells that were reconstituted with wild-type, T52 phosphomutant or phosphomimic ATF2, in the presence or absence of genotoxic stress (which attenuates the T52 phosphorylation of ATF2). We report that in melanoma cells, genotoxic stress induces the expression of a cluster of Interferon β1 (IFNβ1) and related genes, which is suppressed by the expression of ATF2T52E. ATF2 binds to the 5′-promoter region of IFNβ1, and we identified the ATF2/AP-1 binding site required for transcriptional repression. The stress-induced expression of IFNβ1 mediates S-phase accumulation during genotoxic stress with a concomitant induction of a senescence-like phenotype. In contrast, blockade of type-1 interferon signaling by shRNA-mediated depletion of the IFNAR receptor abrogates the S-phase accumulation, blocking cells in G1 phase and reducing the extent of stress-induced cell death. The expression of or treatment with exogenous IFNβ1 blunts the proliferation of melanoma cells in both 2- and 3-dimensional spheroid cultures. Notably, the treatment of melanoma cells with mutant BRAF inhibitor (Vemurafenib/PLX4720) also induces the expression of IFNβ1 and senescence markers, and the co-treatment of melanoma cells with IFNβ1 and etoposide or PLX4720 significantly enhances the cell death induced by either etoposide or PLX4720 alone. Lastly, we found that the stress-induced IFNβ1 expression by melanoma cells elicits paracrine effects, enhancing the melanoma cell clearance by lymphocytes in an in vitro co-culture system. Our data demonstrate a novel transcriptional regulatory role exhibited by ATF2 on IFNβ1, which influences cell cycle dynamics and stress/treatment resistance of melanoma cells. The therapeutic and prognostic implications of these findings will be discussed. Citation Format: Eric Lau, Giuseppina Claps, David S. Hoon, Ze'ev A. Ronai. Interfering with interferon: An axis of ATF2-mediated chemoresistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4091. doi:10.1158/1538-7445.AM2014-4091

Type-1 interferon signaling mediates neuro-inflammatory events in models of Alzheimer's disease

A neuro-inflammatory response has been implicated in human patients and animal models of Alzheimer's disease (AD). Type-1 interferons are pleiotropic cytokines involved in the initiation and regulation of the pro-inflammatory response; however, their role in AD is unknown. This study investigated the contribution of type-1 IFN signaling in the neuro-inflammatory response to amyloid-beta (Aβ) in vitro and in the APP/PS1 transgenic mouse model of AD. Enzyme-linked immunosorbent assay confirmed a 2-fold increase in IFNα in APP/PS1 brains compared with control brains. Quantitative polymerase chain reaction also identified increased IFNα and IFNβ expression in human pre-frontal cortex from AD patients. In vitro studies in primary neurons demonstrated Aβ-induced type-1 IFN expression preceded that of other classical pro-inflammatory cytokines, IL1-β, and IL-6. Significantly, ablation of type-1 interferon-α receptor 1 expression in BE(2)M17 neuroblastoma cells and primary neurons afforded protection against Aβ-induced toxicity. This study supports a role for type-1 interferons in the pro-inflammatory response and neuronal cell death in AD and suggests that blocking type-1 interferon-α receptor 1 maybe a therapeutic target to limit the disease progression.

Heme Oxygenase-1 Mediated Cytoprotection Against Liver Ischemia and Reperfusion Injury: Inhibition of Type-1 Interferon Signaling

Toll-like receptor (TLR)-4 signaling plays a key role in initiating exogenous antigen-independent innate immunity-dominated liver ischemia/reperfusion injury (IRI). Heme oxygenase (HO)-1, a heat-shock protein 32, exerts potent adaptive anti-oxidant and anti-inflammatory functions. Signal transducers and activator of transcription (STAT)-1 activation triggers interferon (IFN)-inducible protein 10 (CXCL-10), one of major products of type-1 IFN pathway downstream of TLR4. This study focuses on the role of type-1 IFN pathway in the mechanism of HO-1 cytoprotection during liver IRI. Cobalt protoporphyrin (CoPP)-induced HO-1 overexpression ameliorated liver damage in a well-defined mouse model of liver warm IRI, as evidenced by improved hepatic function (serum alanine aminotransferase levels) and liver histology (Suzuki's scores). HO-1 downregulated phospho-STAT-1 and its key product, CXCL-10. In contrast, TLR4 expression remained elevated regardless of the IRI status. To dissect the mechanism of HO-1 upon CXCL-10, we cultured RW 264.7 (macrophage) cells with exogenous rIFN-beta to stimulate CXCL-10 production via TLR4 pathway in vitro. Indeed, CoPP-induced HO-1 suppressed otherwise highly upregulated rIFN-beta-triggered CXCL-10. Moreover, consistent with our in vitro data, CoPP pretreatment diminished rIFN-beta-induced CXCL-10 production in normal mouse livers. Hepatic IRI activates TLR4 signaling in vivo to elaborate CXCL-10. HO-1 overexpression downregulates activation of STAT1 via type-1 IFN pathway downstream of TLR4, which in turn decreases CXCL-10 production. This study provides evidence for a novel mechanism by which HO-1 exerts adaptive cytoprotective and anti-inflammatory functions in the context of innate TLR4 activation.