Expression Of IFNβ1 Research Articles

Colorectal cancer-specific IFNβ delivery overcomes dysfunctional dsRNA-mediated type I interferon signaling to increase the abscopal effect of radiotherapy

BackgroundCancer-intrinsic type I interferon (IFN-I) production triggered by radiotherapy (RT) is mainly dependent on cytosolic double-stranded DNA (dsDNA)-mediated cGAS/STING signaling and increases cancer immunogenicity and enhances the antitumor immune response...

Replicative senescence and high glucose induce the accrual of self-derived cytosolic nucleic acids in human endothelial cells.

Recent literature shows that loss of replicative ability and acquisition of a proinflammatory secretory phenotype in senescent cells is coupled with the build-in of nucleic acids in the cytoplasm. Its implication in human age-related diseases is under scrutiny. In human endothelial cells (ECs), we assessed the accumulation of intracellular nucleic acids during in vitro replicative senescence and after exposure to high glucose concentrations, which mimic an in vivo condition of hyperglycemia. We showed that exposure to high glucose induces senescent-like features in ECs, including telomere shortening and proinflammatory cytokine release, coupled with the accrual in the cytoplasm of telomeres, double-stranded DNA and RNA (dsDNA, dsRNA), as well as RNA:DNA hybrid molecules. Senescent ECs showed an activation of the dsRNA sensors RIG-I and MDA5 and of the DNA sensor TLR9, which was not paralleled by the involvement of the canonical (cGAS) and non-canonical (IFI16) activation of the STING pathway. Under high glucose conditions, only a sustained activation of TLR9 was observed. Notably, senescent cells exhibit increased proinflammatory cytokine (IL-1β, IL-6, IL-8) production without a detectable secretion of type I interferon (IFN), a phenomenon that can be explained, at least in part, by the accumulation of methyl-adenosine containing RNAs. At variance, exposure to exogenous nucleic acids enhances both IL-6 and IFN-β1 expression in senescent cells. This study highlights the accrual of cytoplasmic nucleic acids as a marker of senescence-related endothelial dysfunction, that may play a role in dysmetabolic age-related diseases.

Abstract PO-097: Tumor-associated extracellular vesicles from HPV+ oropharyngeal cancer lymphatic fluid trigger an anti-tumor response through activation of the cGAS-STING pathway

Abstract Background: Human papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPC) is increasing rapidly in incidence and is now the most common HPV-associated malignancy in the U.S. Our group previously demonstrated that lymph-enriched fluid from surgical drains in HPV+ OPC patients is rich in tumor-derived cell-free DNA, with prognostic implications. In this study, we characterize extracellular vesicles (EVs) derived from cervical lymph in HPV+ OPC patients and explore the impact on innate immune response. The cyclic GMP-AMP synthase (cGAS) pathway plays a key role in immune response by detecting pathogenic or damaged host DNA. We propose that HPV DNA-containing EVs uptake by HPV-negative cells modulates their immune responses via activation of cGAS-STING signaling. Methods: Surgical lymphatic fluid samples were collected from 31 HPV(+) and 10 HPV(-) OPC patients 24 hours post neck dissection, along with supernatant from HPV(+) and HPV(-) OPC cell lines. EVs were isolated from both biofluids and characterized using immunoblotting, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). EVs were then treated with DNase to eliminate external DNA before extracting EV DNA. Lymph cell-free DNA (cfDNA) was also extracted. We used qPCR to quantify HPV16 levels in supernatant and lymph EVs. Next, we incubated HPV(-) OPC cells with EVs that were isolated from the supernatant from HPV (+) or HPV (-) cells. Finally, we compared cGAS-STING activation induced by HPV (+) EVs to the STING agonist 2,3-GAMP (Sigma) through IFNβ mRNA expression and phosphor-TBK1 immunoblotting. Results: Lymphatic EVs showed typical morphology with diameters ranging 70–150 nm, and were CD63 and TSG101 positive by immunoblot. 27 HPV (+) OPC patients (87%) had detectable HPV16 DNA in their postoperative lymph EVs, while all HPV(-) lymph EVs were negative for HPV16. Comparison of bulk lymph cfDNA and lymph EV DNA showed a significant proportion (39.5±24.4%) of lymph HPV16 DNA is contained by EVs. Further, an EV uptake assay showed HPV16 DNA was trafficked into HPV(-) cells through EVs, and blocking EV internalization with heparin reduced recipient cell HPV16 levels. Crucially, HPV (-) cells cultured with HPV (+) EVs for 8 hours increased IFNβ1 expression, while HPV(-) EVs did not increase IFNβ1 expression. Lastly, we observed TBK1 phosphorylation, a reporter of STING activation, was elevated in these cells treated with HPV(+) EVs. Conclusions: Our study is the first to characterize tumor-associated EVs in lymphatic fluid. We showed many of these tumor-associated lymph EVs in HPV(+) patients harbor HPV16. Additionally, we demonstrate lymph EVs containing HPV16 activate cellular cGAS-STING intermediates. Ongoing studies are investigating the impact of EVs on the tumor microenvironment. Citation Format: Zhongping Xu, Noah Earland, Peter K. Harris, Sophie P. Gerndt, Ricardo Ramirez, Nicholas P. Semenkovich, Aadel A. Chaudhuri, Jose P. Zevallos. Tumor-associated extracellular vesicles from HPV+ oropharyngeal cancer lymphatic fluid trigger an anti-tumor response through activation of the cGAS-STING pathway [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-097.

SP5.1 Paracrine Interferon signalling between cancer associated Fibroblasts and Cancer Cells correlates with poor survival after chemotherapy in Triple Negative Breast Cancer patients

Abstract Aim To determine if expression levels of Interferon-β1 (IFNβ1) in cancer associated fibroblasts, and of Myxovirus Resistance 1 (MX1) – a downstream marker of interferon activity, in cancer cells would be predictive markers of chemotherapy response and subsequent overall survival. Methods Breast cancer tissue was obtained from tissue microarrays of a triple negative cohort. Expression of IFNβ1 and MX1 was determined by immunohistochemistry in 109 patients treated with adjuvant chemotherapy. The cohort was further subdivided into claudin low and high subgroups, based on immunohistochemical assessment of relative expression of claudin-3. Results IFNβ1 in fibroblasts was significantly, positively associated with MX1 expression in tumour cells (Spearman’s correlation r=0.210; p=0.028). High expression of IFNβ1 in fibroblasts, and of MX1 in tumour cells were each significantly associated with poorer disease-free survival (p=0.01). Dividing the cohort into claudin-low (claudin-3 negative; n=49) and claudin-high (claudin-3 positive; n=60), resulted in a stronger correlation between fibroblast IFNβ1 and tumour cell MX1 in the claudin-low group (r=0.375; p=0.008), whereas it was lost in the claudin-high group (r=0.113; p=0.389). Likewise, correlations between survival and expression of IFNβ1 in fibroblasts and MX1 in tumour cells were maintained in claudin-low cases (p<0.05) but lost in claudin-highs. Conclusion IFNβ1 and MX1 may be of value as predictive biomarkers of chemo-response in breast cancer patients. These correlations imitate associations with chemoresistance identified in vitro using cancer associated fibroblasts and claudin-low breast cancer cell lines. Therefore, inhibiting the paracrine cross talk-between the cell types might be a viable strategy for chemo-sensitisation of breast cancers.

Loss of TRIM24 promotes IL-10 expression via CBP/p300-dependent IFNβ1 transcription during macrophage activation.

As an anti-inflammatory cytokine, interleukin 10 (IL-10) plays a vital role in preventing inflammatory and autoimmune pathologies while also maintaining immune homeostasis. IL-10 production in macrophages is tightly regulated by multiple pathways. TRIM24, a member of the Transcriptional Intermediary Factor 1 (TIF1) family, contributes to antiviral immunity and macrophage M2 polarization. However, the role of TRIM24 in regulating IL-10 expression and its involvement in endotoxic shock remains unclear. In vitro, bone marrow derived macrophages cultured with GM-CSF or M-CSF were stimulated with LPS (100ng/ml). Murine models of endotoxic shock were established by challenging the mice with different dose of LPS (i.p). RTPCR, RNA sequencing, ELISA and hematoxylin and eosin staining were performed to elucidate the role and mechanisms of TRIM24 in endotoxic shock. The expression of TRIM24 is downregulated in LPS-stimulated bone marrow-derived macrophages (BMDMs). Loss of TRIM24 boosted IL-10 expression during the late stage of LPS-stimulation in macrophages. RNA-seq analysis revealed the upregulation of IFNβ1, an upstream regulator of IL-10, in TRIM24 knockout macrophages. Treatment with C646, a CBP/p300 inhibitor, diminished the difference in both IFNβ1 and IL-10 expression between TRIM24 knockout and control macrophages. Loss of TRIM24 provided protection against LPS-induced endotoxic shock in mice. Our results demonstrated that inhibiting TRIM24 promoted the expression of IFNβ1 and IL-10 during macrophage activation, therefore protecting mice from endotoxic shock. This study offers novel insights into the regulatory role of TRIM24 in IL-10 expression, making it a potentially attractive therapeutic target for inflammatory diseases.

AB0057 REGULATION OF TYPE I INTERFERON SIGNATURE BY VGLL3 IN THE FIBROBLAST-LIKE SYNOVIOCYTES OF RHEUMATOID ARTHRITIS

BackgroundThe upregulation of interferon (IFN)-stimulated genes (ISGs) induced by type I IFNs (namely type I IFN signature) in RA patients had implications in early diagnosis and prediction of therapy responses. However, factors that modulate the type I IFN signature in RA are largely unknown.ObjectivesTo explore the involvement of VGLL3, a homologue of the vestigial-like gene in Drosophila and a putative regulator of the Hippo pathway, in the modulation of the type I IFN signature in RA.MethodsFLS were isolated from RA and osteoarthritis patients. Expression of VGLL3 in the synovial tissues and FLS was analyzed by immunohistochemistry and PCR. RNA sequencing was performed in RA-FLS upon VGLL3 overexpression. The expression of ISGs was examined by PCR and Western blotting.ResultsVGLL3 was upregulated in the RA synovium and RA-FLS compared to OA. Overexpression of VGLL3 promoted the expression of ISGs in RA-FLS. The expression of STAT1 and MX1 was also upregulated in RA synovium compared to OA. The expression of STAT1 and MX1 was associated with the expression of VGLL3 in RA and OA patients. VGLL3 promoted the IRF3 activation, IFN-β1 expression, and IFN-β1 autocrine signaling in RA-FLS. VGLL3 also modulated the expression of the Hippo pathway molecules WWTR1 and AMOTL2, which mediated the regulation of IRF3 activation and IFN-β1 production by VGLL3 in RA-FLS.ConclusionVGLL3 drives the IRF3-induced IFN-β1 secretion in RA-FLS by inhibiting WWTR1 expression and subsequently promotes the type I IFN signature expression through autocrine IFN-β1 signaling.AcknowledgementsWe would like to thank Prof. Si-feng Chen (Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Fudan University, Shanghai, China) for scientific advice and technical assistance.Disclosure of InterestsNone declared

ID2 inhibits innate antiviral immunity by blocking TBK1- and IKKε-induced activation of IRF3.

The transcription regulator ID2 plays an essential role in the development and differentiation of immune cells. Here, we report that ID2 also negatively regulates antiviral innate immune responses. During viral infection of human epithelial cells, ID2 bound to TANK-binding kinase 1 (TBK1) and to inhibitor of nuclear factor κB kinase ε (IKKε). These interactions inhibited the recruitment and activation of interferon (IFN) regulatory factor 3 (IRF3) by TBK1 or IKKε, leading to a reduction in the expression of IFN-β1 (IFNB1). IFN-β induced the nuclear export of ID2 to form a negative feedback loop. Knocking out ID2 in human cells enhanced innate immune responses and suppressed infection by different viruses, including SARS-CoV-2. Mice with a myeloid-specific deficiency of ID2 produced more IFN-α in response to viral infection and were more resistant to viral infection than wild-type mice. Our findings not only establish ID2 as a modulator of IRF3 activation induced by TBK1 and/or IKKε but also introduce a mechanism for cross-talk between innate immunity and cell development and differentiation.

Regulation of type I interferon signature by VGLL3 in the fibroblast-like synoviocytes of rheumatoid arthritis patients via targeting the Hippo pathway

BackgroundThe upregulation of interferon (IFN)-stimulated genes induced by type I IFNs (namely type I IFN signature) in rheumatoid arthritis (RA) patients had implications in early diagnosis and prediction of therapy responses. However, factors that modulate the type I IFN signature in RA are largely unknown. In this study, we aim to explore the involvement of VGLL3, a homologue of the vestigial-like gene in Drosophila and a putative regulator of the Hippo pathway, in the modulation of type I IFN signature in the fibroblast-like synoviocytes (FLS) of RA patients.MethodsFLS were isolated from RA and osteoarthritis (OA) patients. Expression of VGLL3 in the synovial tissues and FLS was analyzed by immunohistochemistry and PCR. RNA sequencing was performed in RA-FLS upon VGLL3 overexpression. The expression of IFN-stimulated genes was examined by PCR and Western blotting.ResultsVGLL3 was upregulated in the RA synovium and RA-FLS compared to OA. Overexpression of VGLL3 promoted the expression of IFN-stimulated genes in RA-FLS. The expression of STAT1 and MX1 was also upregulated in RA synovium compared to OA and was associated with the expression of VGLL3 in RA and OA patients. VGLL3 promoted the IRF3 activation and IFN-β1 expression in RA-FLS. Increased IFN-β1 induced the expression of IFN-stimulated genes in RA-FLS in an autocrine manner. VGLL3 also modulated the expression of the Hippo pathway molecules WWTR1 and AMOTL2, which mediated the regulation of IRF3 activation and IFN-β1 production by VGLL3 in RA-FLS.ConclusionsVGLL3 drives the IRF3-induced IFN-β1 expression in RA-FLS by inhibiting WWTR1 expression and subsequently promotes the type I IFN signature expression in RA-FLS through autocrine IFN-β1 signaling.

IFNβ1 secreted by breast cancer cells undergoing chemotherapy reprograms stromal fibroblasts to support tumour growth after treatment.

Chemotherapy (CTX) remains the standard of care for most aggressive tumours, including breast cancer (BC). In BC chemotherapeutic regimens, the maximum tolerated dose of cytotoxic drugs is administered at regular intervals, and cancer cells can re‐grow or adapt during the resting periods between cycles. The impact of the tumour microenvironment on the fate of cancer cells after CTX remains poorly understood. Here, we show that paracrine signalling from CTX‐treated cancer cells to stromal fibroblasts can drive cancer cell recovery after cytotoxic drug withdrawal. Interferon β1 (IFNβ1) secreted by cancer cells following treatment with high doses of CTX instigates the acquisition of an anti‐viral state in stromal fibroblasts. This state is associated with an expression pattern here referred to as interferon signature (IFNS), which encompasses several interferon‐stimulated genes (ISGs), including numerous pro‐inflammatory cytokine genes. This crosstalk is an important driver of the expansion of BC cells after CTX, and IFNβ1 blockade in tumour cells abrogated their fibroblast‐dependent recovery potential. Analysis of human breast carcinomas supported a link between CTX‐induced IFNS in tumour stroma and poor response to CTX treatment. First, IFNβ1 expression in human breast carcinomas was found to inversely correlate with recurrence free survival (RFS). Second, using laser capture microdissection data sets, we show a higher expression of IFNS in the stromal tumour compartment compared to the epithelial one and this signature was found to be more prominent in more aggressive subtypes of BC (basal‐like), pointing to a pro‐tumorigenic role of this signature. Moreover, IFNS was associated with higher recurrence rates and a worse outcome in BC patients. Our study unravels a novel form of paracrine communication between cancer cells and fibroblasts that ultimately results in CTX resistance. Targeting this axis has the potential to improve CTX outcomes in patients with BC.

Sanguinarine and Chelidonine Synergistically Induce Endosomal Toll-like Receptor and M1-Associated Mediators Expression

Natural compounds represent the great capability to stimulate several cell types. Macrophage plays an important role for an effective immune response for infection and inflammation. Isoquinoline alkaloid, sanguinarine, and chelidonine are active compounds that exhibit activity on various tumor cells and immune cells. However, the effect of these compounds on the endosomal toll-like receptor (enTLR) and type I interferon (IFN) are still unclear. The monocyte-derived macrophages (MDMs) were cultured and were determined their cell viability and phagocytic activity to Staphylococcus aureus DMST8840. The nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression were also examined. The expression of enTLRs, type I IFN, and cytokines were determined by real-time PCR. Result shows that the compounds did not affect on MDM cell viability. Sanguinarine and chelidonine enhance phagocytic activity of MDM against Staphylococcus aureus DMST8840 by revealing a higher number of bacterial survival than the MDM treated by polyI:C, and the cell control after co-culture for 3 h. The production of NO has no difference amount but iNOS mRNA production was down-regulated in sanguinarine, chelidonine and their mixed treated MDM. The expressions of enTLRs and IFN-β1 mRNA were up-regulated in both compounds and their combination. Additionally, these compounds also enhance M1-liked cytokine by up-regulated IL-6 and down-regulated IL-10 and TGF-β1, respectively. Therefore, sanguinarine and chelidonine enhance enTLR and IFN-β1 expression and trend to stimulate the cell into M1-liked MDM.

Inhibition of respiratory syncytial virus replication and suppression of RSV-induced airway inflammation in neonatal rats by colchicine.

The present study investigated the role of colchicine in the treatment of RSV infection. Treatment of BEAS-2B cells following RSV infection with colchicine caused a significant decrease in the number of viral plaques. In RSV-infected BEAS-2B cells' treatment with colchicine leads to a significant up-regulation of both IFN-β1 and RIG-I genes. The levels of interleukin, NO, and MDA were suppressed in BEAS-2B cells infected with RSV by colchicine. The phosphorylation of Stat3, COX-2, and p38 was also suppressed significantly by colchicine. The phosphorylation of IkBα was promoted in RSV-infected BEAS-2B cells' oncolchicine treatment. In neonatal rats, replication of RSV was inhibited significantly by colchicine treatment which was evident by suppression of RSV-L gene expression. A significant decrease in the level of IL-6 and TNF-α was caused in neonatal rat BALF by colchicine treatment. The production of MDA, NO and MPO in the neonatal rat BALF was suppressed markedly by colchicine treatment. Treatment of the neonatal rats infected by RSV with colchicine suppressed the release of IκBα and COX-2 in the pulmonary epithelial cells. Colchicine treatment of the neonatal rats promoted the expression of IFN-α and IFN-β1. In summary, the current study showed that colchicine inhibited RSV infection in neonatal rats through regulation of anti-oxidative factor production. The expression of IFN-β1 and RIG-I genes was also up-regulated in the RSV-infected alveolar epithelial cells by treatment with colchicine. Therefore, colchicine may be developed as the therapeutic agent for the treatment of RSV infection.

Baicalein triazole prevents respiratory tract infection by RSV through suppression of oxidative damage

Baicalein, an isolate of secutellaria baicalensis is known for its anti-inflammatory activity. In the present study, 12-triazole derivatives of baicalein were synthesized and evaluated against RSV infected BEAS-2B cells in vitro and in mice model in vivo. The preventive effect of most active compound 5f against RSV infection was studied in detail. The compound 5f treatment increased IFN-β1 expression in BEAS-2B cells infected with RSV. In BEAS-2B cells treatment with compound 5f inhibited RSV-induced secretion of interleukin-6 and -8 cytokines. It decreased RSV-induced nitric oxide & malondialdehyde production and inhibited the RSV-mediated activation of NF-κB, COX-2, Stat3 and MAPK. The p38 phosphorylation was enhanced significantly in RSV infected cells by compound 5f pre-treatment. RT-qPCR showed that compound 5f treatment of the RSV-infected mice significantly (P < 0.05) decreased viral load through reduction in the viral replication. In the mice model of RSV-infection compound 5f treatment decreased interleukin-6, -8 and tumor necrosis factor-α expression. The level of MPO, nitric oxide and malondialdehyde was also decreased significantly by compound 5f in the RSV infected mice BALF. It also reduced the infiltration of neutrophils and lymphocytes in the BALF of RVS-infected mice. In summary, compound 5f inhibits RSV-infection and prevents pulmonary airway inflammation through the activation of IFN signalling pathway.

Human cathelicidin peptide LL-37 as a therapeutic antiviral targeting Venezuelan equine encephalitis virus infections

Venezuelan equine encephalitis virus (VEEV), a new world alphavirus belonging to the Togaviridae family, causes periodic disease outbreaks in humans and equines with high associated mortality and morbidity. VEEV is highly infectious via the aerosol route and so has been developed as a biological weapon (Hawley and Eitzen, 2001). Despite its current classification as a category B select agent, there are no FDA approved vaccines or therapeutics to counter VEEV infections. Here we utilize a naturally occurring host defense peptide, LL-37, as a therapeutic strategy to inhibit VEEV multiplication in infected cells. LL-37 has previously demonstrated activity against several viruses by directly interacting with viral particles and indirectly by establishing an antiviral state in the host cell. We show that LL-37 exhibited potent antiviral activity against VEEV by inhibiting viral replication. Genomic RNA copies of the TC-83 strain of VEEV and viral titers were significantly reduced compared to non-treated controls. LL-37 also inhibited the virulent Trinidad Donkey (TrD) strain of VEEV. Entry assays revealed a robust reduction of viral RNA copies at the early stages of TC-83 infection. Pre-incubation of cells with LL-37 and TC-83 resulted in a strong inhibitory response, indicating that LL-37 impacts early stages of the infectious process. Confocal and electron microscopy images confirmed the aggregation of viral particles, which potentially accounts for entry prevention and hence reduced viral infection. LL-37 treatment also modulated type I interferon (IFN) expression in infected cells. LL-37 treatment dramatically increased IFNβ1 expression in treated cells in a time-dependent manner. Our results establish LL-37 as a relevant and novel potential therapeutic strategy for the treatment of VEEV infections.

The in vitro Antiviral Mechanisms of Stronger Neo-Minophafen C, an Established Formulation of Compound Glycyrrhizin

Background: Glycyrrhiza glabra (liquorice) has been extensively used since ancient Egypt, Greek and Roman, and is an important herb in traditional Chinese medicine. Its major compound, Glycyrrhizin (GL) possesses multiple pharmacological activities, such as anti-virus waiting for exploration indepth. Objective: The aim of this research is determining the antiviral mechanisms of Stronger Neo-Minophafen C (SNMC), an established formulation of compound GL based on Interferon (IFN) system, an important cytokine family best known for its antiviral ability. Methods: Four cell lines, A549, Hela, SMMC-7721 and TC-1, were recruited. The relative cell viability (RCV) was measured with 3(4, 5 dimethylthiazol) 2, 5 diphenyltetrazolium bromide (MTT). The gene transcription of key elements on IFN system, such as IFN-β1, IRF3 and ISG15 were evaluated using realtime RT-PCR. The expressions of key enzymes on IFN system were measured by Western blot. The concentrations of IFN-γ and IRF1, representative members of type II interferon, were detected by ELISA. Results: SNMC reduces RCV with concurrent induction of antiviral genes majorly belong to type I IFN pathway, focusing on IRF3-IFN-β1- ISG15 axis. The expression of IFN-β1, IRF3 and ISG15 genes in A549 and Hela cells peak at 12 h post-SNMC incubation, in a time- and dosage- dependent manner. The expression of IFN-β1 protein reaches a peak at 24 h in A549 and SMMC-7721 cells, and peaks at 48 h in Hela and TC-1 cells. The expression of ISG15 reaches a peak at 24 h in A549, Hela and TC-1 cells, and at 48 h in SNMC-7721 cells. The expression of Mx reaches a peak at 24 h in A549 and Hela cells, and at 48 h in SMMC-7721 and TC-1 cells. However, SNMC could not induce the expression of type II IFN signal pathway. Conclusion: We demonstrated that SNMC can induce the expression of important anti-viral genes in type I interferon pathway and indicate the existence of a key pathway response for the anti-viral effects of SNMC.

Abstract LB-131: Activation of innate and adaptive immunity via combinatorial immunotherapy using Synthetic Biotic™ Medicines

Abstract Background: While both T-cell priming and blockade of immune suppression play critical roles in the generation of an efficacious antitumor immune response, most therapies fail to support both processes as a single agent. At Synlogic we are using synthetic biology and engineered bacteria programmed with immune-modulatory circuits to develop treatments or “Synthetic Biotic Medicines” capable of simultaneously manipulating multiple pathways relevant for the treatment of cancer and autoimmunity. Recent studies have demonstrated that activation of the stimulator of interferon genes (STING) pathway can play a critical role in the initiation of an antitumor immune response via activation of antigen presenting cells (APCs) and presentation of tumor antigens. Additionally, metabolites derived from biosynthetic pathways, such as conversion of tryptophan into kynurenine by indoleamine 2,3 dioxygenase (IDO), have also been recently appreciated as major components of the immune-suppressive tumor microenvironment which lead to T cell disfunction and exhaustion. Here we present results showing the development of two circuits, an immune “initiator” STING activating circuit (SYN-STING) and an immune “sustainer” Kynurenine consuming circuit (SYN-Kyn), in engineered strains of E. coli Nissle. Methods and Results: Synthetic biological techniques were employed to generate bacterial strains expressing various protein components capable of (1) generating the STING agonist cyclic-di-AMP (SYN-STING) or (2) actively transporting and metabolizing kynurenine (SYN-Kyn). In in vitro biochemical and functional assays, SYN-STING generated high levels of cyclic-di-AMP and triggered the expression of IFNβ1 and IL-6 upon co-culture with APCs, while SYN-Kyn actively depleted test media containing kynurenine at levels 20-fold higher than those found in tumors of cancer patients. In mice bearing subcutaneous syngeneic tumors the intratumoral administration of SYN-STING resulted in an early rise of innate cytokines which several days later shifted towards molecules indicative of an effector-T-cell response. Additionally the administration of a single dose of SYN-Kyn led to significant decreases in tumor kynurenine levels for up to 72 hours post dose. Finally, administration of a combination of either SYN-STING with anti-PD1 or SYN-Kyn with anti-CTLA4 antibodies in tumor-bearing mice led to significant anti-tumor effects over those observed for the antibodies alone. Conclusions: Taken together, these results demonstrate that the use of synthetic biology to engineer bacteria is a viable approach to deliver profound efficacy in experimental models of cancer. The data supports the further development of Synthetic Biotic Medicines capable of locally targeting multiple immune pathways as single immuno-oncology therapeutic agents. Citation Format: Daniel Leventhal, Kip West, Adam Fisher, Anna Sokolovska, Starsha Kolodziej, Ning Li, Chris Plescia, Carey Gallant, Mary Castillo, Paul Miller, Jose Lora. Activation of innate and adaptive immunity via combinatorial immunotherapy using Synthetic Biotic™ Medicines [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 LB-131.

IFIT1 exerts opposing regulatory effects on the inflammatory and interferon gene programs in LPS-activated human macrophages

Abstract Activation of the TLR4 signaling pathway by LPS leads to activation of both inflammatory and interferon stimulated genes, however the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remains poorly understood. In a genome-wide siRNA screen of the LPS-induced TNF-a response in macrophages, we identified the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identified an unexpected positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of LPS-induced inflammatory and interferon gene expression. We demonstrate that these effects of IFIT1 are mediated through modulation of a Sin3a-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate an unappreciated function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program. IFIT1 depletion led to a significant increase in the replication of B. cenocepacia. Prior treatment of IFIT1 depleted cells with recombinant IFN-β reversed the enhanced replication of B. cenocepacia, indicating a role for autocrine IFN-β in controlling B. cenocepacia infection and a requirement for IFIT1 in interferon induction in response to bacterial infection. We also observed reduced expression of IFNβ1 in response to viral and bacterial infection in IFIT1 depleted cells, suggesting a broad role for IFIT1 in supporting IFNβ1 expression.

Transcriptional repression of IFNβ1 by ATF2 confers melanoma resistance to therapy.

The resistance of melanoma to current treatment modalities represents a major obstacle for durable therapeutic response, and thus, the elucidation of mechanisms of resistance is urgently needed. The crucial functions of Activating Transcription Factor-2 (ATF2) in the development and therapeutic resistance of melanoma have been previously reported, although the precise underlying mechanisms remain unclear. Here, we report a protein kinase C epsilon (PKCε)- and Activating Transcription Factor-2 (ATF2)-mediated mechanism that facilitates resistance by transcriptionally repressing the expression of IFNβ1 and downstream type-I IFN signaling, which is otherwise induced upon exposure to chemotherapy. Treatment of early stage melanomas expressing low levels of PKCε with chemotherapies relieves its transcriptional repression of IFNB1, resulting in impaired S-phase progression, a senescence-like phenotype, and increased cell death. This response is lost in late stage metastatic melanomas expressing high levels of PKCε. Notably, nuclear ATF2 and low expression of IFNβ1 in melanoma tumor samples correlates with poor patient responsiveness to biochemotherapy or neoadjuvant IFN-α2a. Conversely, cytosolic ATF2 and induction of IFNβ1 coincides with therapeutic responsiveness. Collectively, we identify an IFNβ1-dependent, cell autonomous mechanism that contributes to the therapeutic resistance of melanoma via the PKCε-ATF2 regulatory axis.

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

Interaction between allergy and innate immunity: model for eosinophil regulation of epithelial cell interferon expression

BackgroundEosinophils in asthmatic airways are associated with risk of exacerbations. The most common cause of asthma exacerbations is viral respiratory infections, particularly human rhinovirus (HRV). ObjectiveTo determine the mechanism by which eosinophils may influence virus-induced responses. MethodsWe used an in vitro coculture model of primary human eosinophils and the BEAS-2B epithelial cell line either stimulated with HRV1A infection or polyinosinic–polycytidylic acid (poly[I:C]). The messenger RNA (mRNA) expression of interferon (IFN) β1 and IFN-λ1 was assessed by quantitative reverse-transcriptase polymerase chain reaction and the protein level of IFN- λ1 by enzyme-linked immunosorbent assay. ResultsBoth poly(I:C) and HRV1A infection induced BEAS-2B expression of IFN-β1 and IFN-λ1 mRNA. Coculture of eosinophils resulted in suppression of poly(I:C)-stimulated IFN-β1 and IFN-λ1 mRNA expression (2.5-fold and 3.6-fold less, respectively). Separation of cells did not block eosinophil regulatory activity. Coculture of eosinophils with HRV1A-infected BEAS-2B cells also suppressed IFN-β1 and IFN-λ1 mRNA (5.7-fold and 5.0-fold less, respectively) and reduced IFN-λ1 protein secretion (1.6-fold decrease). This corresponded to a 34% increase in the quantity of HRV1A virus RNA on coculture with eosinophils. Recombinant transforming growth factor β suppressed IFN-λ1 from HRV1A-infected BEAS-2B cells. Coculture of eosinophils and BEAS-2B cells induced transforming growth factor β secretion, which may mediate suppression of HRV-induced interferon expression. ConclusionEosinophils suppressed HRV-induced expression of interferons from epithelial cells, resulting in increased quantity of HRV. This represents one mechanism for interaction between allergic inflammation and innate immunity.

In vitro susceptibility to rhinovirus infection is greater for bronchial than for nasal airway epithelial cells in human subjects

Human rhinoviruses (HRVs) characteristically cause upper respiratory tract infection, but they also infect the lower airways, causing acute bronchitis and exacerbating asthma. Our purpose was to study ex vivo the differences in the response to HRV infection of nasal and bronchial epithelial cultures from the same healthy and asthmatic individuals using conditions favoring development of fully differentiated, pseudostratified mucociliary epithelium. Cells from the inferior turbinates and bronchial tree of 5 healthy and 6 asthmatic individuals were cultured at an air-liquid interface. Cultures were infected with HRV-16, and after 48 hours, the degree of infection was measured. Baseline median transepithelial resistance was lower in human bronchial epithelial (HBE) cell cultures than in human nasal epithelial (HNE) cell cultures (195 Omega.cm2 [95% CI, 164-252] vs 366 Omega.cm2 [95% CI, 234-408], respectively; P < .01). Virus replicated more easily in HBE cells than in HNE cells based on virus shedding in apical wash (log tissue culture infective dose of 50%/0.1 mL = 2.0 [95% CI, 1.0-2.5] vs 0.5 [95% CI, 0.5-1.5], P < .01) and on a 20- to 30-fold greater viral load and number of infected cells in HBE cell cultures than in HNE cell cultures. The increases in expression of RANTES and double-stranded RNA-dependent protein kinase were greater in HBE cell cultures than in HNE cell cultures, as were the concentrations of IL-8, IL-1alpha, RANTES, and IP-10 in basolateral medium. However, no significant differences between asthmatic and healthy subjects (including IFN-beta1 expression) were found. Differentiated nasal epithelial cells might have mechanisms of increased resistance to rhinovirus infection compared with bronchial epithelial cells. We could not confirm previous reports of increased susceptibility to HRV infection in epithelial cells from asthmatic subjects.