NF-κB Transcription Factor Relish Research Articles

Rel governs loser elimination during stem cell competition in the Drosophila testis

In the Drosophila testis, a group of stromal cells termed hub provides multiple niche signals for the surrounding germline and somatic stem cells. Stem cells of both populations compete for physical retention in the niche, and clones unable to transduce any one niche signal are rapidly eliminated from the stem cell pool by differentiation. We have mapped the transcriptomes of isolated somatic cyst stem cells and differentiated cyst cells, and found that the stem cells but not their differentiated progeny exhibit the signature of an innate immune response including the NF-κB transcription factor Relish (Rel). Related signalling pathways had previously implicated in cell competition in larval epithelia, prompting the question of whether NF-κB signalling was, despite the clear differences between the two competition scenarios, also involved in stem cell competition in the testis. Here we show i) that in the testis Rel is dispensable for stemness, ii) that loss of Rel or the upstream receptor Toll suppresses loser elimination following a variety of different triggers used to induce loser fate, and iii) that clonal Rel activation is sufficient for the displacement of neutral or winner cells from the niche, even if these cells otherwise retain stem cell properties.

Stilbenoid compounds inhibit NF-κB-mediated inflammatory responses in the Drosophila intestine.

Stilbenoid compounds have been described to have anti-inflammatory properties in animal models in vivo, and have been shown to inhibit Ca2+-influx through the transient receptor potential ankyrin 1 (TrpA1). To study how stilbenoid compounds affect inflammatory signaling in vivo, we have utilized the fruit fly, Drosophila melanogaster, as a model system. To induce intestinal inflammation in the fly, we have fed flies with the intestinal irritant dextran sodium sulphate (DSS). We found that DSS induces severe changes in the bacteriome of the Drosophila intestine, and that this dysbiosis causes activation of the NF-κB transcription factor Relish. We have taken advantage of the DSS-model to study the anti-inflammatory properties of the stilbenoid compounds pinosylvin (PS) and pinosylvin monomethyl ether (PSMME). With the help of in vivo approaches, we have identified PS and PSMME to be transient receptor ankyrin 1 (TrpA1)-dependent antagonists of NF-κB-mediated intestinal immune responses in Drosophila. We have also computationally predicted the putative antagonist binding sites of these compounds at Drosophila TrpA1. Taken together, we show that the stilbenoids PS and PSMME have anti-inflammatory properties in vivo in the intestine and can be used to alleviate chemically induced intestinal inflammation in Drosophila.

Partner of neuropeptide bursicon homodimer pburs mediates a novel antimicrobial peptide Ten3LP via Dif/Dorsal2 in Tribolium castaneum

Bursicon is a cystine knot family neuropeptide, composed of two subunits, bursicon (burs) and partner of burs (pburs). The subunits can form heterodimers to regulate cuticle tanning and wing maturation and homodimers to signal different biological functions in innate immunity, midgut stem cell proliferation and energy homeostasis, and reproductive physiology in the model insects Drosophila melanogaster or Tribolium castaneum. Here, we report on the role of the pburs homodimer in signaling innate immunity in T. castaneum larvae. Through transcriptome analysis we identified a set of immune-related genes that respond to pburs RNAi. Treating larvae with recombinant-pburs protein led to up-regulation of antimicrobial peptide (AMP) genes in vivo and in vitro. The upregulation of most AMP genes was dependent on the NF-κB transcription factor Relish. Most importantly, we identified a novel AMP, Tenecin 3-like peptide (Ten3LP), regulated by pburs via NF-κB transcription factor Dorsal-related immunity factor (Dif)/Dorsal2, but not Relish. We conducted Ten3LP RNAi, synthesized recombinant Ten3LP protein for microbial inhibition assays and functionally characterized Ten3LP as an AMP specific for fungi and Gram-positive bacteria. We demonstrate that expression of Ten3LP is activated by pburs via the Toll pathway. These findings identify new molecular targets for development of potential antibiotics for treating microbial infections and perhaps for RNAi based pest management technology.

Cytokine receptor IL27RA is an NF-κB–responsive gene involved in CD38 upregulation in multiple myeloma

AbstractMultiple myeloma (MM) shows constitutive activation of canonical and noncanonical nuclear factor κB (NF-κB) signaling via genetic mutations or tumor microenvironment (TME) stimulations. A subset of MM cell lines showed dependency for cell growth and survival on the canonical NF-κB transcription factor RELA alone, suggesting a critical role for a RELA-mediated biological program in MM pathogenesis. Here, we determined the RELA-dependent transcriptional program in MM cell lines and found the expression of the cell surface molecules interleukin-27 receptor-α (IL-27Rα) and the adhesion molecule JAM2 to be responsive to RELA at the messenger RNA and protein levels. IL-27Rα and JAM2 were expressed on primary MM cells at higher levels than on healthy long-lived plasma cells (PCs) in the bone marrow. IL-27 activated STAT1, and to a lesser extent STAT3, in MM cell lines and in PCs generated from memory B cells in an IL-21–dependent in vitro PC differentiation assay. Concomitant activity of IL-21 and IL-27 enhanced differentiation into PCs and increased the cell-surface expression of the known STAT target gene CD38. In accordance, a subset of MM cell lines and primary MM cells cultured with IL-27 upregulated CD38 cell-surface expression, a finding with potential implications for enhancing the efficacy of CD38-directed monoclonal antibody therapies by increasing CD38 expression on tumor cells. The elevated expression of IL-27Rα and JAM2 on MM cells compared with that on healthy PCs may be exploited for the development of targeted therapeutic strategies that modulate the interaction of MM cells with the TME.

Drosophila Relish-mediated miR-317 expression facilitates immune homeostasis restoration via inhibiting PGRP-LC.

Innate immunity is the first and essential line for resisting pathogens, and the immune intensity and duration need to be strictly regulated to balance excessive or insufficient immune response. MicroRNAs (miRNAs) are crucial regulators of immune response in Drosophila, yet how immune-related miRNAs are regulated remains poorly understood. Herein, we elucidated that the involvement of miR-317 in NF-κB transcription factor Relish mediated Drosophila Imd pathway in response to Gram-negative (G-) bacteria stimulation. Remarkably, the dynamic expression profiling for immune response indicated that Relish simultaneously enhances the expression of the effector antimicrobial peptide Dpt as well as miR-317 post-infection. Upregulation of miR-317 could further down-regulate the expression of PGRP-LC, thereby forming a feedback in Drosophila Imd pathway to prevent over-activation and restore immune homeostasis. Taken together, our study not only uncovers a novel Relish/miR-317/PGRP-LC regulatory axis to attenuate Drosophila Imd immune response and facilitate immune homeostasis restoration, but also provides vital insights into the complex mechanisms of animal innate immune regulation.

Drosophila Relish Activating lncRNA-CR33942 Transcription Facilitates Antimicrobial Peptide Expression in Imd Innate Immune Response.

Long noncoding RNAs (lncRNAs) are an emerging class of regulators that play crucial roles in regulating the strength and duration of innate immunity. However, little is known about the regulation of Drosophila innate immunity-related lncRNAs. In this study, we first revealed that overexpression of lncRNA-CR33942 could strengthen the expression of the Imd pathway antimicrobial peptide (AMP) genes Diptericin (Dpt) and Attacin-A (AttA) after infection, and vice versa. Secondly, RNA-seq analysis of lncRNA-CR33942-overexpressing flies post Gram-negative bacteria infection confirmed that lncRNA-CR33942 positively regulated the Drosophila immune deficiency (Imd) pathway. Mechanistically, we found that lncRNA-CR33942 interacts and enhances the binding of NF-κB transcription factor Relish to Dpt and AttA promoters, thereby facilitating Dpt and AttA expression. Relish could also directly promote lncRNA-CR33942 transcription by binding to its promoter. Finally, rescue experiments and dynamic expression profiling post-infection demonstrated the vital role of the Relish/lncRNA-CR33942/AMP regulatory axis in enhancing Imd pathway and maintaining immune homeostasis. Our study elucidates novel mechanistic insights into the role of lncRNA-CR33942 in activating Drosophila Imd pathway and the complex regulatory interaction during the innate immune response of animals.

Linear ubiquitin as a common regulator of cellular stress.

Linear or M1-ubiquitination (Ub) is required for optimal NF-kB activation and for cell death inhibition. Using Drosophila as a model organism, Aalto et al. found that hypoxia, oxidative and mechanical stress induced M1-Ub by the HOIP homolog, LUBEL. Increased M1-Ub had a protective function driven by activation of the NF-κB transcription factor Relish via the Immune deficiency pathway (Imd). This protective M1-Ub was also induced upon cellular stress in colorectal cancer cells. Collectively, they propose that M1-Ub is a conserved, common response to different forms of stresses. These findings may have important implications for the use of HOIP inhibitors for cancer treatment. Comment on: https://doi.org/10.1111/febs.16425.

M1-linked ubiquitination facilitates NF-κB activation and survival during sterile inflammation.

Methionine 1 (M1)‐linked ubiquitination plays a key role in the regulation of inflammatory nuclear factor‐κB (NF‐κB) signalling and is important for clearance of pathogen infection in Drosophila melanogaster. M1‐linked ubiquitin (M1‐Ub) chains are assembled by the linear ubiquitin E3 ligase (LUBEL) in flies. Here, we have studied the role of LUBEL in sterile inflammation induced by different types of cellular stresses. We have found that the LUBEL catalyses formation of M1‐Ub chains in response to hypoxic, oxidative and mechanical stress conditions. LUBEL is shown to be important for flies to survive low oxygen conditions and paraquat‐induced oxidative stress. This protective action seems to be driven by stress‐induced activation of the NF‐κB transcription factor Relish via the immune deficiency (Imd) pathway. In addition to LUBEL, the intracellular mediators of Relish activation, including the transforming growth factor activating kinase 1 (Tak1), Drosophila inhibitor of apoptosis (IAP) Diap2, the IκB kinase γ (IKKγ) Kenny and the initiator caspase Death‐related ced‐3/Nedd2‐like protein (Dredd), but not the membrane receptor peptidoglycan recognition protein (PGRP)‐LC, are shown to be required for sterile inflammatory response and survival. Finally, we showed that the stress‐induced upregulation of M1‐Ub chains in response to hypoxia, oxidative and mechanical stress is also induced in mammalian cells and protects from stress‐induced cell death. Taken together, our results suggest that M1‐Ub chains are important for NF‐κB signalling in inflammation induced by stress conditions often observed in chronic inflammatory diseases and cancer.

Protein Phosphatase 4 Negatively Regulates the Immune Deficiency-NF-κB Pathway during the Drosophila Immune Response.

The evolutionarily conserved immune deficiency (IMD) signaling pathway shields Drosophila against bacterial infections. It regulates the expression of antimicrobial peptides encoding genes through the activation of the NF-κB transcription factor Relish. Tight regulation of the signaling cascade ensures a balanced immune response, which is otherwise highly harmful. Several phosphorylation events mediate intracellular progression of the IMD pathway. However, signal termination by dephosphorylation remains largely elusive. Here, we identify the highly conserved protein phosphatase 4 (PP4) complex as a bona fide negative regulator of the IMD pathway. RNA interference-mediated gene silencing of PP4-19c, PP4R2, and Falafel, which encode the catalytic and regulatory subunits of the phosphatase complex, respectively, caused a marked upregulation of bacterial-induced antimicrobial peptide gene expression in both Drosophila melanogaster S2 cells and adult flies. Deregulated IMD signaling is associated with reduced lifespan of PP4-deficient flies in the absence of any infection. In contrast, flies overexpressing this phosphatase are highly sensitive to bacterial infections. Altogether, our results highlight an evolutionarily conserved function of PP4c in the regulation of NF-κB signaling from Drosophila to mammals.

B-type allatostatin regulates immune response of hemocytes in mud crab Scylla paramamosain

B-type allatostatins (AST-B) are neuropeptides that have important physiological roles in arthropods, they have also been identified in a number of crustacean species. Recent research on neuroendocrine-immune (NEI) regulatory system in invertebrates has exploded, it reveals that the NEI network plays an indispensable role in optimizing the immune response and maintaining homeostasis. Herein, mud crab Scylla paramamosain provides a primitive and ancient model to study crosstalk between the neuroendocrine and immune systems. In this study, qRT-PCR analysis showed that the nervous system was the main production site for Sp-AST-B mRNA in S. paramamosain, while its receptor gene (Sp-AST-BR) mRNA could be detected in all the analyzed tissues including hemocytes. This reveals that AST-B might act as a pleiotropic neuropeptide. In situ hybridization further confirmed that granular cells of hemocyte subpopulations express Sp-AST-BR. Time-course analysis revealed that bacteria-analog LPS or virus-analog Poly (I:C) challenge significantly induced Sp-AST-B expression in the thoracic ganglion, and the expression of Sp-AST-BR in hemocytes were also positively changed. Furthermore, mud crabs treated with a synthetic AST-B peptide significantly increased the mRNA levels of AST-BR, nuclear factor-κB (NF-κB) transcription factor (Dorsal and Relish), pro-inflammatory cytokine (IL-16) and immune-effector molecules, and also dramatically enhanced the nitric oxide (NO) production and phagocytic activity in hemocytes. Meanwhile dsRNA-mediated knockdown of Sp-AST-B remarkably suppressed the NO concentrations, phagocytic activity and the expression of immune related genes, resulting in markedly impaired ability of crabs to inhibit bacterial proliferation in vivo. Combined, these data demonstrate that AST-B induced innate immune in the mud crab.

2'3'-cGAMP triggers a STING- and NF-κB-dependent broad antiviral response in Drosophila.

We previously reported that an ortholog of STING regulates infection by picorna-like viruses in Drosophila In mammals, STING is activated by the cyclic dinucleotide 2'3'-cGAMP produced by cGAS, which acts as a receptor for cytosolic DNA. Here, we showed that injection of flies with 2'3'-cGAMP induced the expression of dSTING-regulated genes. Coinjection of 2'3'-cGAMP with a panel of RNA or DNA viruses resulted in substantially reduced viral replication. This 2'3'-cGAMP-mediated protection was still observed in flies with mutations in Atg7 and AGO2, genes that encode key components of the autophagy and small interfering RNA pathways, respectively. By contrast, this protection was abrogated in flies with mutations in the gene encoding the NF-κB transcription factor Relish. Transcriptomic analysis of 2'3'-cGAMP-injected flies revealed a complex response pattern in which genes were rapidly induced, induced after a delay, or induced in a sustained manner. Our results reveal that dSTING regulates an NF-κB-dependent antiviral program that predates the emergence of interferons in vertebrates.

The COP9 Signalosome: A Multi-DUB Complex.

The COP9 signalosome (CSN) is a signaling platform controlling the cellular ubiquitylation status. It determines the activity and remodeling of ~700 cullin-RING ubiquitin ligases (CRLs), which control more than 20% of all ubiquitylation events in cells and thereby influence virtually any cellular pathway. In addition, it is associated with deubiquitylating enzymes (DUBs) protecting CRLs from autoubiquitylation and rescuing ubiquitylated proteins from degradation. The coordination of ubiquitylation and deubiquitylation by the CSN is presumably important for fine-tuning the precise formation of defined ubiquitin chains. Considering its intrinsic DUB activity specific for deneddylation of CRLs and belonging to the JAMM family as well as its associated DUBs, the CSN represents a multi-DUB complex. Two CSN-associated DUBs, the ubiquitin-specific protease 15 (USP15) and USP48 are regulators in the NF-κB signaling pathway. USP15 protects CRL1β-TrCP responsible for IκBα ubiquitylation, whereas USP48 stabilizes the nuclear pool of the NF-κB transcription factor RelA upon TNF stimulation by counteracting CRL2SOCS1. Moreover, the CSN controls the neddylation status of cells by its intrinsic DUB activity and by destabilizing the associated deneddylation enzyme 1 (DEN1). Thus, the CSN is a master regulator at the intersection between ubiquitylation and neddylation.

Hyd ubiquitinates the NF-κB co-factor Akirin to operate an effective immune response in Drosophila.

The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor κB (NF-κB) signaling pathway, we undertook an in vitro RNAi screen targeting E3 ubiquitin ligases specifically and identified the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) as a new actor in the IMD pathway. Hyd mediated Lys63 (K63)-linked polyubiquitination of the NF-κB cofactor Akirin was required for efficient binding of Akirin to the NF-κB transcription factor Relish. We showed that this Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-κB transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. We showed further that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-κB pathway for the activation of Interleukin 6 (IL6) transcription by LPS or IL-1β in cultured human cells. Our findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.

Abstract P6-04-06: Protein kinase C beta 1 (PKCβ1), a novel drug target of Z-endoxifen, inhibits growth of estrogen receptor positive (ER+) breast cancer via activation of the NF-kB transcription factor RelA

Abstract Background: The first in-human phase-I clinical trial of the tamoxifen metabolite, Z-endoxifen, demonstrated promising antitumor activity in both aromatase inhibitor (AI) and tamoxifen resistant ER+ breast cancer patients (Goetz M.P., et al; JCO, 2017). By utilizing a letrozole-sensitive MCF7 aromatase expressing (MCF7AC1) cell line, and its corresponding letrozole-resistant (MCF7LR) cell line, we have shown superior antitumor and antiestrogenic activity of Z-endoxifen over tamoxifen in the AI-sensitive and resistant settings in vivo. This study also revealed that Z-endoxifen may target protein kinases in addition to estrogen receptor alpha. Follow-up studies demonstrated that Z-endoxifen, but not tamoxifen, binds to protein kinase C beta 1 (PKCβ1), a member of the serine/threonine-specific protein kinase family that regulates signaling pathways involved in cell proliferation and tumorigenic transformation (Guo C., et al; SABCS, 2017). We have further shown that PKCβ1 silencing, similar to Z-endoxifen, potently inhibits ER+ cell proliferation and additionally causes ERα turnover. Here, we report a potential mechanism by which PKCβ1 may mediate its anti-proliferative effects in ER+ breast cancer cells. Methods: The effects of siRNA-mediated PKCβ1-silencing on gene transcription were evaluated by RNAseq in MCF7AC1 cells. RNAseq results were further validated by quantitative real-time polymerase chain reaction (qRT-PCR) in PKCβ1-silenced MCF7AC1 cells as well as in ER+ T47D cells. The effects of Z-endoxifen on the mRNA expression of genes altered by PKCβ1-silencing in MCF7AC1 cells were evaluated by qRT-PCR analysis. PKCβ1 silencing effects on protein expression in MCF7AC1 cells were evaluated by immunoblot analysis. Results: RNAseq analysis of PKCβ1-silenced MCF7AC1 cells compared to non-silenced cells revealed NF-κB signaling pathway as the top biological pathway significantly altered by PKCβ1 silencing (p=5.93e-08). Enhanced mRNA expression of multiple NF-κB downstream target genes including CXCL10, RELB, MMP1 and IL6 were observed following PKCβ1 silencing. qRT-PCR analyses validated increased mRNA expression of these genes in PKCβ1-silenced MCF7AC1 and T47D cells. Notably, Z-endoxifen treatment in MCF7AC1 cells mirrored PKCβ1 silencing effects, resulting in enhanced mRNA expression of the aforementioned genes. Evaluation of the protein expression of RelA and RelB, the canonical and noncanonical NF-κB transcription factors respectively, in PKCβ1-silenced MCF7AC1 cells revealed significant upregulation of RelA (p=0.0203) but not RelB (p=0.1360) expression. ERα silencing in MCF7AC1 cells did not affect RelA protein expression. Consistent with prior reports, treatment of MCF7 cells with TNFα inhibited cell proliferation. Finally, targeted siRNA-mediated silencing of RelA in PKCβ1-silenced MCF7AC1 cells significantly attenuated PKCβ1-mediated growth inhibition (p=0.0002). Conclusion: Our findings demonstrate that both Z-endoxifen treatment and PKCβ1-silencing commonly activates a NF-κB-mediated gene signature in ER+ breast cancer cells. Notably, PKCβ1-silencing induces the protein expression of the NF-κB transcription factor RelA in an ERα-independent manner, which appears to contribute to PKCβ1-mediated growth inhibition. Current studies are ongoing to evaluate the impact of RelA silencing on Z-endoxifen mediated growth inhibition. This information will foster our understanding of the estrogenic and non-estrogenic mechanisms by which Z-endoxifen mediates its antitumor activity in ER+ breast cancer. Citation Format: Swaathi Jayaraman, Mary J Kuffel, Krishna R Kalari, Kevin J Thompson, Xiaojia Tang, Elizabeth S Bruinsma, John R Hawse, Matthew P Goetz. Protein kinase C beta 1 (PKCβ1), a novel drug target of Z-endoxifen, inhibits growth of estrogen receptor positive (ER+) breast cancer via activation of the NF-kB transcription factor RelA [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-06.

The insect reservoir of diversity for viruses and antiviral mechanisms

The insect reservoir of diversity for viruses and antiviral mechanisms

A tandem death effector domain-containing protein inhibits the IMD signaling pathway via forming amyloid-like aggregates with the caspase-8 homolog DREDD

Negative regulation of the immune signaling pathway involves diverse negative regulators that target different signaling molecules. One of the signaling molecules, DREDD, which activates the NF-κB transcription factor Relish in the IMD pathway, is a homolog of mammalian caspase-8. Some structural related proteins have been identified to regulate the activity of caspase-8 in signaling complex assembly. However, it is unknown in insects whether the IMD pathway undergoes such a down-regulation. In this study, we explored the regulatory role of a newly identified protein BmCaspase-8 like (BmCasp8L) in silkworm, which displays high sequence similarity with the N-terminus of BmDREDD to the IMD pathway, and investigated its mechanism. Domain prediction, phylogenic analysis and gene architecture suggests BmCasp8L acts as a potential inhibitor to BmDREDD. We then found it is highly expressed in the fat body and hemocytes, and suppresses the cleavage of BmRelish and BmIMD mediated by BmDREDD upon PGN stimulation, resulting in deficiency in antimicrobial peptides production. Besides the inhibitory role in the IMD pathway, it also suppresses the BmDREDD-induced apoptosis. By investigating the amyloidal activity of BmCasp8L and its interaction with BmDREDD and BmFADD, we demonstrated that BmCasp8L forms amyloid-like aggregates in vitro as well as in vivo, and it inactivates BmDREDD by blending into the amyloidal speck-like structure formed by BmDREDD and BmFADD that is required for BmDREDD activity. Taken together, our results demonstrate BmCasp8L inhibits the IMD signaling pathway via forming amyloidal aggregates with BmDREDD, suggesting an evolutionarily conserved regulatory mechanism of innate immune signaling pathway.

Association between Single Nucleotide Polymorphism in RelA with Somatic Cell Count and Longevity Supports Importance of NF-κB Signalling in Cattle Health

Mastitis reduces milk production and causes culling. The NF-κB transcription factor RelA plays a central regulatory role in innate immunity. This study used a candidate gene approach to investigate associations between the synonymous C/G SNP rs48035703 in RELA with somatic cell count (SCC) and survival time. Blood samples were collected from 337 Holstein-Friesian heifers on 19 farms and genotyped by PCR-restriction fragment length polymorphism. Animals were monitored from 6 months until 2340 d of age. Pedigree, milk production and disease records were obtained. Genotype frequencies were CC 0.63, CG 0.30 and GG 0.06. The C allele had a favourable additive effect on survival: average longevities from birth were CC, 1872 d; CG, 1745 d and GG 1596 d (P -0.04 ± 0.803, P RELA rs48035703 CC genotype cows were therefore less likely to experience a high SCC and survived longer. These results support a role for RelA in combating mammary gland infection and warrant further studies in additional populations.

Inhibition of NF-\u03baB in astrocytes is sufficient to delay neurodegeneration induced by proteotoxicity in neurons

BackgroundMost neurodegenerative diseases associated with protein aggregation are hallmarked by activation of astrocytes. However, how astrocytes are activated or which signaling pathways in astrocytes contribute to pathogenesis is not clear. One long-standing question is whether the responses in astrocytes are due to stress or damage in astrocytes themselves, or because of astrocytic responses to cellular stress or damage in neurons. Here, we examine responses in astrocytes induced by expression of disease-associated, aggregation-prone proteins in other cells. We also examine the consequences of these responses in astrocytes in a model for neurodegeneration.MethodsWe first examined a role for intracellular astrocytic responses in a Drosophila model for Spinocerebellar ataxia type 3 (SCA3, also known as Machado–Joseph disease), a disease caused by expansion of the polyglutamine (polyQ) stretch in the ATXN3 gene. In this Drosophila SCA3 model, eye-specific expression of a biologically relevant portion of the ATXN3 gene, containing expanded polyQ repeats (SCA3polyQ78) was expressed. In a candidate RNAi screen in the Drosophila SCA3 model, we analyzed whether downregulation of expression of specific genes in astrocytes affected degeneration induced by SCA3polyQ78 expression in Drosophila eyes. We next examined the role of astrocytes in response to proteotoxic stress in neurons induced by SCA3polyQ78 expression or amyloid beta peptides, associated with Alzheimer’s disease.ResultsEye-specific expression of SCA3polyQ78 resulted in the presence of astrocytes in the eye, suggesting putative involvement of astrocytes in SCA3. In a candidate RNAi screen, we identified genes in astrocytes that can enhance or suppress SCA3polyQ78-induced eye degeneration. Relish, a conserved NF-κB transcription factor, was identified as an enhancer of degeneration. Activity of Relish was upregulated in our SCA3 model. Relish can exert its effect via Relish-specific AMPs, since downregulation of these AMPs attenuated degeneration. We next examined Relish signaling in astrocytes on neurodegeneration. Selective inhibition of Relish expression specifically in astrocytes extended lifespan of flies that expressed SCA3polyQ78 exclusively in neurons. Inhibition of Relish signaling in astrocytes also extended lifespan in a Drosophila model for Alzheimer’s disease.ConclusionsOur data demonstrate that astrocytes respond to proteotoxic stress in neurons, and that these astrocytic responses are important contributors to neurodegeneration. Furthermore, our data demonstrate that activation of NF-κB transcription factor Relish in astrocytes, induced by proteotoxic stress in neurons, enhances neurodegeneration, and that specific Relish inhibition in astrocytes extends lifespan. Our data provide direct evidence for cell-non-autonomous contributions of astrocytes to neurodegeneration, with possible implications for therapeutic interventions in multiple neurodegenerative diseases.

Abstract 5065: Cancer derived Escherichia coli induces tumor-promoting inflammatory cytokine IL-6 in cancer associated fibroblasts (CAFs) in a NF-κB/BRD4 dependent manner

Abstract Introduction: Chronic inflammatory responses within the colonic environment are critical to colorectal cancer (CRC) tumor development. While the exact causes of CRC development are unknown, recent data suggest that dysbiosis in the colonic microbiome results in the overgrowth of bacteria from Enterebacteriaceae, Fusobacteriaceae and Bacteroidaceae taxonomic families, contributing to CRC development. Among bacteria of the Enterebacteriaceae family, adherent invasive Esherichia coli has been shown to promote tumor cell growth via production of colibactin. However, the contribution of E.coli to the tumor promoting inflammatory microenvironment (TME) during sporadic CRC development remains unknown. Cancer associated fibroblasts (CAFs) are a major component of TME and contribute to tumor-promoting inflammatory responses via NF-κB dependent production of IL-6. Herein we investigate the effect of E. coli on CAFs. We hypothesize that stimulation of CAFs by cancer derived E.coli is a key processes in supporting tumor-promoting inflammation in CRC. Methods: Bacterial DNA was extracted from tumor and adjacent normal tissue of 28 CRC patients. High throughput sequencing was done using metabarcoding of 16S rDNA for bacteria and analyzed using CLC Genomics Workbench 8.0.5 Microbial Genomics Module, SILVA v119 database for 16S. E.coli PS092717 strain, isolated from CRC tumor site, was used in co-culture experiments. Real time RT-PCR and multiplex cytokine array was used to analyze fibroblasts gene expression and secretion, respectively. Results: We observed a reduction in microbial diversity in tumor vs adjacent normal tissue, with an increased prevalence of Fusobacteria, and Enterobactereacea, but not Bacteroidacea. We then tested the effect of E.coli PS092717 on colonic primary normal fibroblasts and CAFs. Exposure of N-CMFs to E.coli for 24 h only moderately increased IL-6, while CAF responded with a greater increase in IL-6 production. Analysis of the signaling mechanism demonstrated that this process was NF-κB dependent, and was abrogated in the presence of NF-kB specific inhibitor, triptolide (20 ng/mL). Bromodomain containing protein 4 (Brd4) is an epigenetic regulator that can recruit canonical NF-κB transcription factor RelA. Therefore, we analyzed its involvement in the above processes. NF-κB mediated increase in IL-6 expression and secretion induced by E.coli PS092717 was strongly reduced in CAFs than N-CMFs in the presence of BRD4 specific inhibitor (1 µM ZL0590). A similar observation was made when a major components of the E.coli cell wall, lipopolysaccharide (LPS), was used. Conclusion Taken together our data suggests that, E.coli derived from cancer tissue and its cell wall component LPS can contribute to the tumor promoting inflammation via stimulation of IL-6 production by CAFs and this process is BRD4/NF-κB-dependent. Citation Format: Gabriela Uribe, Russel Rourke, Romain Villeger, George Golovko, Kamil Khanipov, Zhiqing Liu, Maria Pimenova, Yuriy Fofanov, Jia Zhou, Allen R. Brasier, Irina V. Pinchuk. Cancer derived Escherichia coli induces tumor-promoting inflammatory cytokine IL-6 in cancer associated fibroblasts (CAFs) in a NF-κB/BRD4 dependent manner [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 5065.

Analysis of Drosophila STING Reveals an Evolutionarily Conserved Antimicrobial Function

The vertebrate protein STING, an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response and the induction of type I interferon during pathogenic infection. Here, we show that a STING ortholog (dmSTING) exists in Drosophila, which, similar to vertebrate STING, associates with cyclic dinucleotides to initiate an innate immune response. Following infection with Listeria monocytogenes, dmSTING activates an innate immune response via activation of the NF-κB transcription factor Relish, part of the immune deficiency (IMD) pathway. DmSTING-mediated activation of the immune response reduces the levels of Listeria-induced lethality and bacterial load in the host. Of significance, dmSTING triggers an innate immune response in the absence of a known functional cyclicguanosine monophosphate (GMP)-AMP synthase (cGAS) ortholog in the fly. Together, our resultsdemonstrate that STING is an evolutionarily conserved antimicrobial effector between flies andmammals, and it comprises a key component of host defense against pathogenic infection in Drosophila.