Innate Immune Signaling Pathways Research Articles

MicroRNA (miR-KT-635) encoded by Cyprinid herpesvirus 2 regulates the viral replication with targeting to the ORF23.

Herpesviruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. In our previous studies, we found a new miRNA miR-KT-635 encoded by Cyprinid herpesvirus 2, which is predicted to target viral genes and cellular genes involved in innate immune signalling pathway and apoptosis. However, the function and target gene of miR-KT-635 are not proved. In this study, the regulating target gene of miR-KT-635 was proved as the viral gene ORF23 directly, the target point sequence on gene was verified and miR-KT-635 was identified to regulate the expression of ORF23 protein. According to the bioinformatics analysis, the tRNA domain and ribosome domain in the protein sequence of ORF23 were found to share high homology with R2i and P53R2i, which are related to the ribonucleotide reductase small subunit in the host (transform NTP to dNTP). Within expectations, silencing of viral ORF23 or transfecting miR-KT-635 mimics in Carassius auratus gibelio caudal fin cell line (GiCF) could suppress viral propagation significantly.

SARS-CoV-2-mediated evasion strategies for antiviral interferon pathways.

With global expansion of the COVID-19 pandemic and the emergence of new variants, extensive efforts have been made to develop highly effective antiviral drugs and vaccines against SARS-CoV-2. The interactions of coronaviruses with host antiviral interferon pathways ultimately determine successful viral replication and SARS-CoV-2-induced pathogenesis. Innate immune receptors play an essential role in host defense against SARS-CoV-2 via the induction of IFN production and signaling. Here, we summarize the recent advances in innate immune sensing mechanisms of SARS-CoV-2 and various strategies by which SARS-CoV-2 antagonizes antiviral innate immune signaling pathways, with a particular focus on mechanisms utilized by multiple SARS-CoV-2 proteins to evade interferon induction and signaling in host cell. Understanding the underlying immune evasion mechanisms of SARS-CoV-2 is essential for the improvement of vaccines and therapeutic strategies.

The Emerging Role of RNA Modifications in the Regulation of Antiviral Innate Immunity.

Posttranscriptional modifications have been implicated in regulation of nearly all biological aspects of cellular RNAs, from stability, translation, splicing, nuclear export to localization. Chemical modifications also have been revealed for virus derived RNAs several decades before, along with the potential of their regulatory roles in virus infection. Due to the dynamic changes of RNA modifications during virus infection, illustrating the mechanisms of RNA epigenetic regulations remains a challenge. Nevertheless, many studies have indicated that these RNA epigenetic marks may directly regulate virus infection through antiviral innate immune responses. The present review summarizes the impacts of important epigenetic marks on viral RNAs, including N6-methyladenosine (m6A), 5-methylcytidine (m5C), 2ʹ-O-methylation (2ʹ-O-Methyl), and a few uncanonical nucleotides (A-to-I editing, pseudouridine), on antiviral innate immunity and relevant signaling pathways, while highlighting the significance of antiviral innate immune responses during virus infection.

DDX3X functionally and physically interacts with Estrogen Receptor-alpha

DEAD-box protein 3X (DDX3X) is a human DEAD-box protein with conventional roles in RNA metabolism and unconventional functions in signalling pathways that do not require its enzymatic activity. For example, DDX3X acts as a multifunctional adaptor molecule in anti-viral innate immune signalling pathways, where it interacts with and regulates the kinase IKB-kinase-epsilon (IIKKε). Interestingly, both DDX3X and IKKɛ have also independently been shown to act as breast cancer oncogenes. IKKɛ's oncogenic functions are likely multifactorial, but it was suggested to phosphorylate the transcription factor Estrogen receptor alpha (ERα) at Serine 167, which drives expression of Erα target genes in an estrogen-independent manner. In this study, we identified a novel physical interaction between DDX3X and ERα that positively regulates ERα activation. DDX3X knockdown in ER+ breast cancer cell lines resulted in reduced ERα phosphorylation, reduced Estrogen Response Element (ERE)-controlled reporter gene expression, decreased expression of ERα target genes, and decreased cell proliferation. Vice versa, overexpression of DDX3X resulted in enhanced ERα phosphorylation and activity. Furthermore, we provide evidence that DDX3X physically binds to ERα from co-immunoprecipitation and pulldown experiments. Based on our data, we propose that DDX3X acts as an adaptor to facilitate IKKε-mediated ERα activation, akin to the mechanism we previously elucidated for IKKε-mediated Interferon Regulatory factor 3 (IRF3) activation in innate immune signalling. In conclusion, our research provides a novel molecular mechanism that might contribute to the oncogenic effect of DDX3X in breast cancer, potentially linking it to the development of resistance against endocrine therapy.

Evasion of Host Antiviral Innate Immunity by Paramyxovirus Accessory Proteins.

For efficient replication, viruses have developed multiple strategies to evade host antiviral innate immunity. Paramyxoviruses are a large family of enveloped RNA viruses that comprises diverse human and animal pathogens which jeopardize global public health and the economy. The accessory proteins expressed from the P gene by RNA editing or overlapping open reading frames (ORFs) are major viral immune evasion factors antagonizing type I interferon (IFN-I) production and other antiviral innate immune responses. However, the antagonistic mechanisms against antiviral innate immunity by accessory proteins differ among viruses. Here, we summarize the current understandings of immune evasion mechanisms by paramyxovirus accessory proteins, specifically how accessory proteins directly or indirectly target the adaptors in the antiviral innate immune signaling pathway to facilitate virus replication. Additionally, some cellular responses, which are also involved in viral replication, will be briefly summarized.

Viral and cellular oncogenes promote immune evasion.

Thirteen percent of cancers worldwide are associated with viral infections. While many human oncogenic viruses are widely endemic, very few infected individuals develop cancer. This raises the question why oncogenic viruses encode viral oncogenes if they can replicate and spread between human hosts without causing cancer. Interestingly, viral infection triggers innate immune signaling pathways that in turn activate tumor suppressors such as p53, suggesting that tumor suppressors may have evolved not primarily to prevent cancer, but to thwart viral infection. Here, we summarize and compare several major immune evasion strategies used by viral and non-viral cancers, with a focus on oncogenes that play dual roles in promoting tumorigenicity and immune evasion. By highlighting important and illustrative examples of how oncogenic viruses evade the immune system, we aim to shed light on how non-viral cancers avoid immune detection. Further study and understanding of how viral and non-viral oncogenes impact immune function could lead to improved strategies to combine molecular therapies targeting oncoproteins in combination with immunomodulators.

The Significance of Toll-Like Receptors in the Neuroimmunologic Background of Alcohol Dependence.

Toll-like receptors (TLR) are a group of protein belonging to the family of Pattern Recognition Receptors (PRR) which have the ability to distinguish between an organism's own antigens and foreign ones and to induce immunological response. TLR play a significant part in non-specific immunity but at the same time they are also a vital element linking non-specific response to the specific one. A growing number of data seems to indicate that the non-specific immunity mechanisms affect the development and sustenance of alcohol addiction. Alcohol damages the organism's cells not only directly but also through an increase inintestinal permeability which induces innate immune response of peripheral blood cells. The signaling pathway of Toll-like receptors located on the surface of brain immune cells intensifies the inflammatory reaction and, through modifying gene expression of proinflammatory factors, unnaturally supports it. This overly protracted “sterile inflammatory reaction” positively correlates with alcohol craving affecting also the functioning of the reward system structures and increasing the risk of relapse of alcoholism. Recurrent alcoholic binges sensitize the microglia and cause an escalation in inflammatory reaction which also leads to neurodegeneration. The induction of innate immunity signaling pathways exposes clinical symptoms of alcohol addiction such as increased impulsivity, loss of behavioral control, depressive-anxiety symptoms and cognitive dysfunctions. Traditional methods of treating alcohol addiction have tended to focus predominantly on reducing symptoms which—given the frequency of relapses—seems insufficient. The aim of the present paper is to discuss the role of toll-like receptors as elements of the immunity system which, together with the nervous system, plays a crucial part in the pathogenesis of alcohol addiction. We also wish to present pharmacotherapeutic perspectives targeted at the neuroimmunological mechanisms of alcohol addiction.

Structural basis of K63-ubiquitin chain formation by the Gordon-Holmes syndrome RBR E3 ubiquitin ligase RNF216

An increasing number of genetic diseases are linked to deregulation of E3 ubiquitin ligases. Loss-of-function mutations in the RING-between-RING (RBR) family E3 ligase RNF216 (TRIAD3) cause Gordon-Holmes syndrome (GHS) and related neurodegenerative diseases. Functionally, RNF216 assembles K63-linked ubiquitin chains and has been implicated in regulation of innate immunity signaling pathways and synaptic plasticity. Here, we report crystal structures of key RNF216 reaction states including RNF216 in complex with ubiquitin and its reaction product, K63 di-ubiquitin. Our data provide a molecular explanation for chain-type specificity and reveal the molecular basis for disruption of RNF216 function by pathogenic GHS mutations. Furthermore, we demonstrate how RNF216 activity and chain-type specificity are regulated by phosphorylation and that RNF216 is allosterically activated by K63-linked di-ubiquitin. These molecular insights expand our understanding of RNF216 function and its role in disease and further define the mechanistic diversity of the RBR E3 ligase family.

Molecular Insights into Acute Pancreatitis Systemic Inflammation: 3-Hydroxykynurenine Enhances the Response of THP-1 Cells to Toll-Like Receptor Activation

Introduction: The kynurenine pathway is a critical immuno-metabolic regulator of inflammation and is implicated in the pathogenesis of tissue injury and systemic inflammation. Increased flux through the kynurenine pathway contributes to organ damage in acute pancreatitis, and blockade of kynurenine monooxygenase (KMO) protects against this. High levels of 3-hydroxykynurenine (3HK), the product of KMO, produces a pro-inflammatory state with upregulation of innate immune signalling pathways including Toll-like receptor (TLR) cascades. Here we demonstrate that 3HK does not activate TLRs, but instead enhances the response of immune cells to TLR ligands. Methods: Ramos-Blue and THP-1 reporter cells were treated with 3HK and other kynurenine metabolites for 24 hours. The effect of 3HK on TLR 1 through 9, NOD 1, NOD 2, the STING pathway and NLRP3 inflammasome activation was measured. In addition, the effect of 3HK on the response of reporter cells to specific TLR ligands was assessed. Results: 3HK did not directly (ANOVA p=0.3140) or indirectly (p=0.0599) activate TLRs 1/2, 2/6, 2, 3, 4, 5, 7, 8, 9, NOD 1 or NOD2 receptors. Nor did it activate the STING pathway (ANOVA p=0.2676) or NLRP3 inflammasome. 3HK enhanced the response of THP-1 cells to TLR 2/1 (Welch’s t-test p=0.0177), TLR 2/6 (p=0.0136), TLR 2 (p=P<0.0001), TLR 4 (p=0.0002), TLR 8 (p=0.0436) and NOD 2 (p=0.0076) specific ligands. Conclusions: 3HK plays an important role in propagating organ damage in acute pancreatitis by enhancing the response of immune cells to circulating DAMPs.

DExD/H-box helicases: multifunctional regulators in antiviral innate immunity.

DExD/H-box helicases play critical roles in multiple cellular processes, including transcription, cellular RNA metabolism, translation, and infections. Several seminal studies over the past decades have delineated the distinct functions of DExD/H-box helicases in regulating antiviral innate immune signaling pathways, including Toll-like receptors, retinoic acid-inducible gene I-like receptors, cyclic GMP-AMP synthase-the stimulator of interferon gene, and NOD-like receptors signaling pathways. Besides the prominent regulatory roles, there is increasing attention on their functions as nucleic acid sensors involved in antiviral innate immunity. Here we summarize the complex regulatory roles of DExD/H-box helicases in antiviral innate immunity. A better understanding of the underlying molecular mechanisms of DExD/H-box helicases’ regulatory roles is vital for developing new therapeutics targeting DExD/H-box helicases and their mediated signaling transduction in viral infectious diseases.

Innate immunity and inflammophagy: balancing the defence and immune homeostasis.

Extensive crosstalk exists between autophagy and innate immune signalling pathways. The stimuli that induce pattern recognition receptor (PRR)-mediated innate immune signalling pathways, also upregulate autophagy. The purpose of this increased autophagy is to eliminate the stimuli and/or suppress the inflammatory pathways by targeted degradation of PRRs or intermediary proteins (termed 'inflammophagy'). By executing these functions, autophagy dampens excess inflammation triggered by the innate immune signalling pathways. Thus, autophagy helps in the maintenance of the body's innate immune homeostasis to protect from inflammatory and autoimmune diseases. Many autophagy-dependent mechanisms that could control innate immune signalling have been studied over the last few years. However, still, the understanding is incomplete, and studies that are more systematic should be undertaken to delineate the mechanisms of inflammophagy. Here, we discuss the available knowledge of crosstalk between autophagy and PRR signalling pathways.

Advances in Multi-Omics Approaches for Molecular Breeding of Black Rot Resistance in Brassica oleracea L.

Brassica oleracea is one of the most important species of the Brassicaceae family encompassing several economically important vegetables produced and consumed worldwide. But its sustainability is challenged by a range of pathogens, among which black rot, caused by Xanthomonas campestris pv. campestris (Xcc), is the most serious and destructive seed borne bacterial disease, causing huge yield losses. Host-plant resistance could act as the most effective and efficient solution to curb black rot disease for sustainable production of B. oleracea. Recently, ‘omics’ technologies have emerged as promising tools to understand the host-pathogen interactions, thereby gaining a deeper insight into the resistance mechanisms. In this review, we have summarized the recent achievements made in the emerging omics technologies to tackle the black rot challenge in B. oleracea. With an integrated approach of the omics technologies such as genomics, proteomics, transcriptomics, and metabolomics, it would allow better understanding of the complex molecular mechanisms underlying black rot resistance. Due to the availability of sequencing data, genomics and transcriptomics have progressed as expected for black rot resistance, however, other omics approaches like proteomics and metabolomics are lagging behind, necessitating a holistic and targeted approach to address the complex questions of Xcc-Brassica interactions. Genomic studies revealed that the black rot resistance is a complex trait and is mostly controlled by quantitative trait locus (QTL) with minor effects. Transcriptomic analysis divulged the genes related to photosynthesis, glucosinolate biosynthesis and catabolism, phenylpropanoid biosynthesis pathway, ROS scavenging, calcium signalling, hormonal synthesis and signalling pathway are being differentially expressed upon Xcc infection. Comparative proteomic analysis in relation to susceptible and/or resistance interactions with Xcc identified the involvement of proteins related to photosynthesis, protein biosynthesis, processing and degradation, energy metabolism, innate immunity, redox homeostasis, and defence response and signalling pathways in Xcc–Brassica interaction. Specifically, most of the studies focused on the regulation of the photosynthesis-related proteins as a resistance response in both early and later stages of infection. Metabolomic studies suggested that glucosinolates (GSLs), especially aliphatic and indolic GSLs, its subsequent hydrolysis products, and defensive metabolites synthesized by jasmonic acid (JA)-mediated phenylpropanoid biosynthesis pathway are involved in disease resistance mechanisms against Xcc in Brassica species. Multi-omics analysis showed that JA signalling pathway is regulating resistance against hemibiotrophic pathogen like Xcc. So, the bonhomie between omics technologies and plant breeding is going to trigger major breakthroughs in the field of crop improvement by developing superior cultivars with broad-spectrum resistance. If multi-omics tools are implemented at the right scale, we may be able to achieve the maximum benefits from the minimum. In this review, we have also discussed the challenges, future prospects, and the way forward in the application of omics technologies to accelerate the breeding of B. oleracea for disease resistance. A deeper insight about the current knowledge on omics can offer promising results in the breeding of high-quality disease-resistant crops.

Isolates of Salmonella typhimurium circumvent NLRP3 inflammasome recognition in macrophages during the chronic phase of infection

Inflammasome signaling results in cell death and release of cytokines from the IL-1 family, which facilitates control over an infection. However, some pathogens such as Salmonella typhimurium (ST) activate various innate immune signaling pathways, including inflammasomes, yet evade these cell death mechanisms, resulting in a chronic infection. Here we investigated inflammasome signaling induced by acute and chronic isolates of ST obtained from different organs. We show that ST isolated from infected mice during the acute phase displays an increased potential to activate inflammasome signaling, which then undergoes a protracted decline during the chronic phase of infection. This decline in inflammasome signaling was associated with reduced expression of virulence factors, including flagella and the Salmonella pathogenicity island I genes. This reduction in cell death of macrophages induced by chronic isolates had the greatest impact on the NLRP3 inflammasome, which correlated with a reduction in caspase-1 activation. Furthermore, rapid cell death induced by Casp-1/11 by ST in macrophages limited the subsequent activation of cell death cascade proteins Casp-8, RipK1, RipK3, and MLKL to prevent the activation of alternative forms of cell death. We observed that the lack of the ability to induce cell death conferred a competitive fitness advantage to ST only during the acute phase of infection. Finally, we show that the chronic isolates displayed a significant attenuation in their ability to infect mice through the oral route. These results reveal that ST adapts during chronic infection by circumventing inflammasome recognition to promote the survival of both the host and the pathogen.

C. elegans: out on an evolutionary limb.

The natural environment of the free-living nematode Caenorhabditis elegans is rich in pathogenic microbes. There is now ample evidence to indicate that these pathogens exert a strong selection pressure on C.elegans, and have shaped its genome, physiology, and behaviour. In this short review, we concentrate on how C.elegans stands out from other animals in terms of its immune repertoire and innate immune signalling pathways. We discuss how C.elegans often detects pathogens because of their effects on essential cellular processes, or organelle integrity, in addition to direct microbial recognition. We illustrate the extensive molecular plasticity that is characteristic of immune defences in C.elegans and highlight some remarkable instances of lineage-specific innovation in innate immune mechanisms.

Orelabrutinib, a Selective Bruton's Tyrosine Kinase (BTK) Inhibitor in the Treatment of Primary Immune Thrombocytopenia (ITP)

Background:Primary immune thrombocytopenia (ITP) is the most common autoimmune hemorrhagic disorder characterized by decreased platelet count. increased risk of bleeding, and poor quality of life. Only about 70% patients response to first-line treatments, some patients are still refractory or relapsed after combined therapies, therefore it is necessary to explore new therapeutic targets. Bruton's tytosine kinase (BTK) is a non-receptor tyrosine kinase of Tec family, which is widely expressed in hematopoietic cells including B cells, monocytes/macrophages, and others. BTK participates in a variety of signaling pathways of innate and adaptive immunity, and plays an important role in cell survival and maturation. Platelet destruction mediated by anti-platelet glycoprotein antibodies is considered to be the main cause of ITP. B cells differentiate into plasma cells and produce autoantibodies due to the intolerance to autoantigens, which are important effectors in the pathogenesis of ITP. We speculated that inhibition of BTK may reduce platelet destruction by inhibiting B cell activation and autoantibody production. Orelabrutinib is a new generation of BTK inhibitor which has been used in hematological malignancies, this is the first study to explore the mechanisms of BTK inhibitor in the treatment of ITP.Methods:The concentrations of orelabrutinib were set at 1 nM, 10 nM, 100 nM and 1 μM in the in vitro study. Peripheral blood mononuclear cells (PBMCs) were isolated from active ITP patients and healthy controls and cultured for 72 hours, the apoptosis rate of PBMCs in each group was measured by Annexin V/PI double staining. CD19 + B cells of ITP patients were sorted by magnetic beads and stimulated with anti-human IgM to evaluate the activation of B-cell receptor (BCR) pathway and differentiation of plasma cells, respectively. Further, we transfused the splenocytes of immunized CD61-KO mice (C57BL/6) into the severe combined immunodeficient (SCID) mice to establish the active ITP murine models. Orelabrutinib was administered intragastrically at 10mg/kg, once a day. The control group was treated with 0.5% CMC at the same volume and frequency. Platelet count was measured weekly, the peripheral blood was collected and the B cell subsets in spleen were detected by flow cytometry at days 28 after splenocyte transfusion.Results:The proportion of early apoptotic cells (Annexin V +PI -) in PBMCs from both ITP patients and healthy controls was increased by orelabrutinib at 1μM,but there was no statistical difference. Orelabrutinib significantly inhibited the expression of CD69 in a dose-dependent manner at the concentrations of 10nM, 100nM and 1μM. Another early activation marker of BCR signaling pathway, CD86, was also found to be inhibited by orelabrutinib at 100nM and 1μM. The number of CD138 + plasma cells was decreased after treated with orelabrutinib at 10nM, 100nM and 1μM without dose-dependent manner. In the murine models, mice administered with orelabrutinib had significantly higher platelet count than the control mice at days 7, 14, 28 after splenocyte transfusion. The frequency of total B cells in peripheral blood leukocytes, the proportion of GL-7 + germinal center B cells and plasma cells in splenocytes were all determined to be lower in mice treated with orelabrutinib than the control group, though did not reach the statistical significance.Conculsion:Orelabrutinib could effectively suppress the activation and differentiation of B cells in vitro and in vivo, thus alleviate the thrombocytopenia in active ITP murine models. It could be the new treatment strategy for refractory/relapsed ITP patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Transcriptomic Signitures of Azacitidine (AZA) and Decitabine (DAC) Resistance in MDS and CMML

INTRODUCTION: Myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML) are heterogeneous myeloid neoplastic disorders characterized by ineffective hematopoiesis leading to cytopenias and increased risk of transformation to acute myeloid leukemia (AML) . The hypomethylating agents (HMA) azacitidine (AZA) and decitabine (DAC) improve the natural history of MDS and CMML patients (Fenaux et al 2009). However, over half of the cases experience primary failure defined by a lack of response to HMA treatment which is associated with poor prognosis and a median survival of 4-6 months (Garcia-Manero et al. 2016, Jabbour et al. 2010). The highly heterogeneous pathophysiology of myeloid neoplasms and poorly understood mechanisms underlying therapeutic action of HMAs pose substantial challenges in understanding the biology of HMA failure in MDS and CMML.METHODS: We established a cohort of baseline bone marrow (BM) cells that were collected from 17 CMML and 34 MDS patients with excessive blasts (MDS-EB) prior to their HMA based therapies (Figure 1). RNA-Seq based transcriptomic analysis was performed in CD34+ BM hematopoietic stem and progenitor cells (HSPCs) of patients, known to be the cellular origin of these diseases, to identify biological signatures of primary HMA resistance.RESULTS: Similar to the recent reports regarding the lack of common gene expression signatures in HMA resistant AML cell lines (Leung et al. 2019) , RNA-Seq in patient cohort detected fewer than 0.5% of the total number of differentially expressed genes in non-responders in common for both AZA and DAC. Of note, all the AZA and a large portion of DAC associated genes with down-regulations in non-responders encode immunoglobulins, which is consistent with several recent findings indicating impaired differentiation B cells in association with unfavorable outcomes in MDS and CMML (Ribeiro et al. 2006 and Kahn et al. 2015). We performed flow cytometry analysis in BM cells available for 13 patients prior to start of HMA treatments (5 non-responders and 8 responders), and detected that HMA non-responders had a strong tendency (P=0.07) of decreased baseline frequencies of B cells in their BM than HMA responders.GSEA analysis based on leading edge genes identified over 200 and 300 biological signaling pathways to be associated with AZA and DAC failure respectively, with 78 pathways commonly correlated with treatment failure of both drugs (28 up-regulated and 60 down-regulated, Table 1). Clustering of these commonly altered pathways based on biological functions revealed that most of them are known to have a role in MDS and CMML pathogenesis and/ or drug resistance. For instance, neurotransmitter, olfactory pathways, and associated G-protein coupled receptor signals was recently reported to play a role in regulating the maintenance and differentiation of BM HSPCs (Shao et al. 2021 and Shim et al. 2013), whereas cell junction signaling that involves integrins and increased MAP2K-MAPK signaling were also reported to be associated with AZA resistance in MDS and CMML (Unnikrishnan et al. 2017).Among commonly down-regulated biological pathways in non-responders of AZA and DAC, there were clusters of immunoglobulin-associated signals, protein translation regulatory pathways that involve ribosomal proteins, cell cycle signaling, respiratory chain, and Golgi transport signals. Decreased expression of ribosomal proteins and related impairment of ribosomal functions were known mechanism in MDS and CMML development (Ebert et al. 2008 and Schneider et al. 2016). Furthermore, decreased respiratory chain and Golgi transport signals were also identified by transcriptomic investigation in the DAC resistant TF1-RES cell lines.In addition to the commonly altered signals for both AZA and DAC resistance, innate immune signaling pathways including interferon and toll-like receptor signals were significantly up-regulated in DAC non-responders but down-regulated in AZA non-responders.CONCLUSIONS: In this study, the transcriptomic data between responders and non-responders to AZA and DAC were investigated separately, thereby drug-specific as well as the common biomarkers associated with treatment failures of both drugs could be identified. The relatively low proportion of genes and pathways shared by AZA and DAC non-responders suggest the difference underlying biological mechanisms of AZA and DAC failure. [Display omitted] DisclosuresSasaki: Novartis: Consultancy, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees. Wei: Daiichi Sanko: Research Funding.

Combination of Ras Modulator and Azacitidine Impacts Innate Immune Signaling Pathway in MDS-L Cell Line

Background: Myelodysplastic syndrome (MDS) is a clinically heterogenous disease of hematopoietic stem cells (HSC) characterized by ineffective hematopoiesis, uni/multi-lineage dysplasia and a high tendency to transform into acute myeloid leukemia. Aberrant chromosomal and genetic lesions contribute to MDS pathogenesis which has been associated with chronic activation of the innate immune response and a hyperinflammatory microenvironment (Barryero L, et al. Blood, 2018). Dysfunction of Toll like receptors (TLR) and downstream effectors has been associated with the loss of progenitor function and differentiation of bone marrow (BM) cells in MDS patients. Azacitidine (AZA), a hypomethylating agent (HMA), is the mainstay of therapy for patients with higher-risk MDS (Silverman LR, The Myelodysplastic Syndrome in Cancer Medicine, Editors: R.J. Bast, et al. 2017) and carries an overall response rate (ORR) of 50% in patients with significant effects on hematopoiesis, ranging from improvement in a single lineage to complete restoration of blood counts and transfusion independence with survival benefits (Silverman LR, et al., Leukemia, 1993). The response to AZA is not durable and all patients relapse with worsening bone marrow failure.The paradigm of MDS therapy is now shifting to combinatorial drug treatment to overcome single-agent HMA resistance in higher-risk MDS patients. Rigosertib (RIGO), a Ras mimetic which had been shown to interfere with the Ras-Raf binding domain, has limited single-agent activity (ORR 15%) and failed to provide a survival benefit compared to standard of care in MDS patients failing an HMA. RIGO combined with AZA produced an ORR of 90% in HMA naïve patients and 54% in patients who failed HMA (Navada SC, et al. EHA Library 2019). This represents a critical observation in overcoming the epigenetic clinical resistance phenotype. The mechanism is still unclear.Method: We therefore investigated the pathways that are perturbed by AZA and RIGO monotherapy and in combination (RIGO-AZA). We used the MDS-L cell line as a model to limit the heterogeneity observed in MDS patients. The cells were treated with RIGO, AZA, and RIGO-AZA for 48 hrs and further analyzed by qPCR and western blot.Result: We found an increase in H3K9ac protein expression with RIGO and RIGO-AZA; AZA and RIGO alone each had similar effects on H3K4me3, however, its expression was markedly upregulated with RIGO-AZA (Figure A). Effects on H3K36me3 were comparable in all treated cells. We observed marked effects on the repression marks H3K9me3 and H3K27me3 by RIGO and RIGO-AZA combination (Figure A). Furthermore, we studied the expression of bacterial sensing TLRs (1, 2 and 6), viral sensing endosomal TLRs (3 and 9), and cytosolic viral particle sensing receptors like Retinoic acid inducible gene (RIG)-I, Melanoma differentiation-associated protein 5 (MDA5) and Stimulator of interferon genes (STING); their intermediate adaptor molecules Myeloid differentiation factor 88 (MYD88) (for all TLRs except TLR3), mitochondrial antiviral signaling (MAVS) gene (for RIG-I and MDA5); and interferon regulatory factor (IRF)-3 and -7 by qPCR. We observed that AZA, RIGO, and RIGO-AZA significantly inhibit the expression of TLR1, 2 and 6 (Figure B). However, TLR-3, 9, RIG-I, MDA5, STING, MAVS, MYD88, and IRF-3, 7 were significantly inhibited by RIGO and RIGO-AZA (Figure C-E).Conclusion: RIGO has effects on innate immune signaling and histone modification of both activator and repressor marks. Further studies are underway to determine the correlation of the histone modification and innate immune signaling changes, and if these mechanisms contribute to the improvement in hematopoiesis in MDS patients. [Display omitted] DisclosuresNavada: Janssen Pharmaceuticals, Inc.: Current Employment. Reddy: Onconova Therapeutics, Inc.: Current equity holder in publicly-traded company.

Slc39a2-Mediated Zinc Homeostasis Modulates Innate Immune Signaling in Phenylephrine-Induced Cardiomyocyte Hypertrophy

Zinc dyshomeostasis has been involved in the pathogenesis of cardiac hypertrophy; however, the dynamic regulation of intracellular zinc and its downstream signaling in cardiac hypertrophy remain largely unknown. Using Zincpyr1 staining, we found a significant decrease of intracellular Zinc concentration in phenylephrine (PE)-induced hypertrophy of neonatal rat ventricular myocytes (NRVMs). We then screened SLC39 family members responsible for zinc uptake and identified Slc39a2 as the only one altered by PE treatment. Slc39a2 knockdown in NRVMs reduced the intracellular Zinc level, and exacerbated the hypertrophic responses to PE treatment. In contrast, adenovirus-mediated Slc39a2 overexpression enhanced zinc uptake and suppressed PE-induced Nppb expression. RNA sequencing analysis showed a pro-hypertrophic transcriptome reprogramming after Slc39a2 knockdown. Interestingly, the innate immune signaling pathways, including NOD signaling, TOLL-like receptor, NFκB, and IRFs, were remarkably enriched in the Slc39a2-regulated genes. Slc39a2 deficiency enhanced the phosphorylation of P65 NFκB and STAT3, and reduced the expression of IκBα. Finally, the expression of IRF7 was significantly increased by Slc39a2 knockdown, which was in turn suppressed by IRF7 knockdown. Our data demonstrate that zinc homeostasis mediated by a Slc39a2/IRF7 regulatory circuit contributes to the alteration of innate immune signaling in cardiomyocyte hypertrophy.

PKACα negatively regulates TAK1/IRF7 signaling in black carp Mylopharyngodon piceus

Protein Kinase A catalytic subunit α (PKACα), plays an important role in the PKA and NF-κB signaling pathway in mammals. However, the function of PKACα in teleost fish remains largely unknown. In this study, PKACα from black carp (bcPKACα) has been cloned and its role in the innate immune antiviral signaling pathway was investigated. The open reading frame of bcPKACα gene contains 1056 nucleotides and the immunofluorescence assay verified that PKACα was mainly distributed in the cytoplasm. The reporter assay showed that bcPKACα expression and co-expression of bcPKACα and black carp TAK1 (bcTAK1) could activate the transcription of NF-κB. However, bcTAK1/bcIRF7-mediated IFN transcription was inhibited by bcPKACα. Knockdown of bcPKACα showed slightly enhanced antiviral activity against spring viremia of carp virus (SVCV) compared with control group. Accordingly, the antiviral activity against SVCV and grass carp reovirus (GCRV) of EPC cells co-expressing bcPKACα, bcTAK1 and bcIRF7 was obviously lower than that of EPC cells co-expressing bcTAK1 and bcIRF7. The similar subcellular distribution and interaction between bcPKACα and bcTAK1 were detected by immunofluorescent staining and co-immunoprecipitation assay separately. The data generated in this study demonstrates that bcPKACα associates with bcTAK1 and positively regulates NF-κB signaling, however, negatively regulates TAK1/IRF7 signaling pathway.

The crosstalk between the caspase family and the cGAS‒STING signaling pathway.

Edited by Jiarui Wu Cytosolic nucleic acid sensors are critical for sensing nucleic acids and initiating innate immunity during microbial infections and/or cell death. Over the last decade, several key studies have characterized the conserved mechanism of cyclic guanosine monophosphate‒adenosine monophosphate synthase (cGAS) and the downstream signaling adaptor stimulator of interferon genes (STING) initiating the innate immune signaling pathways. Aside from its primary involvement in microbial infections and inflammatory diseases, there is growing interest in the alternate roles of cGAS‒STING-mediated signaling. Caspase family members are powerful functional proteins that respond to cellular stress, including cell death signals, inflammation, and innate immunity. Recent studies have uncovered how the caspase family cooperates with the cGAS‒STING signaling pathway. Most caspase family members negatively regulate the cGAS‒STING signaling pathway. In turn, some caspase family members can also be modulated by cGAS‒STING. This review gives a detailed account of the interplay between the caspase family and the cGAS‒STING signaling pathway, which will shed light on developing novel therapeutics targeting the caspase family and cGAS‒STING signaling in antiviral innate immunity, cancer, inflammatory, and autoimmunity.