Transcription Factor IRF3 Research Articles

Abstract PR09: Intratumoral delivery of engineered modified vaccinia virus Ankara expressing Flt3L and OX40L for "in situ" therapeutic cancer vaccination

Abstract Modified vaccinia virus Ankara (MVA) is a highly attenuated, effective, and safe vaccinia strain that is an important vaccine vector for infectious diseases and cancers. We have previously shown that intratumoral (IT) injection of inactivated MVA elicits stronger antitumor immunity compared with live MVA in murine B16-F10 melanoma and MC38 colon cancer models, indicating that viral immune inhibitory factors might be involved in attenuating antitumor effects. Here we provide evidence that vaccinia virulence factor C7 interacts with transcription factors IRF3 and STAT2, which attenuates both type I IFN production and IFN receptor signaling. Infection of conventional dendritic cells (cDCs) with MVAΔC7L in which C7L gene is deleted results in higher levels of IFNB gene induction and IRF3 phosphorylation compared with MVA. IT MVAΔC7L also induced stronger antitumor responses compared with MVA, which correlates with higher levels of tumor infiltrating activated CD4+ and CD8+ T-cells in both injected and noninjected tumors in a bilateral B16-F10 tumor implantation model. We engineered a recombinant MVA in which the C7L gene was replaced with human Flt3L (hFlt3L) and the J2R gene (a.k.a. TK gene) was replaced with murine OX40L (mOX40L) or human OX40L (hOX40L). Using B16-F10 and MC38 tumor models, we show that IT MVAΔC7L-hFlt3L-ΔJ2R-mOX40L is more effective than MVAΔC7L-hFlt3L, or MVAΔC7L, or heat-inactivated MVAΔC7L in eradicating injected tumors, delaying the growth of the noninjected tumors, and prolonging mice survival. ELISPOT analysis demonstrate that IT MVAΔC7L-hFlt3L-ΔJ2R-mOX40L generates the most tumor-specific CD8+ and CD4+ T-cells compared with the other viruses mentioned above. The combination of IT MVAΔC7L-hFlt3L-ΔJ2R-mOX40L with systemic delivery of anti-CTLA-4 or anti-PD-L1 antibodies results in more efficient eradication of injected tumors, higher survival rate compared with IT virus alone in both bilateral tumor implantation and unilateral large established tumor models. MVAΔC7L-hFlt3L-ΔJ2R-hOX40L has been generated and will be investigated in clinical trials for patients with metastatic cancers in the near future. Taken together, we provide proof-of-concept results for the development of immune activating recombinant MVA to alter tumor immunosuppressive microenvironment through the induction of type I IFN via the cytosolic DNA-sensing pathway, activation of CD103+ dendritic cells, as well as direct activation of both CD4 and CD8 T-cells. This approach enhances tumor antigen presentation and T-cell activation through "in situ" therapeutic vaccination effects, either used as monotherapy or in combination with anti-CTLA-4 or anti-PD-L1 antibody therapy. Citation Format: Liang Deng, Ning Yang, Yi Wang, Wei Yan, Jiahu Wang, John Choi, Stewart Shuman, Taha D. Merghoub, Jedd D. Wolchok. Intratumoral delivery of engineered modified vaccinia virus Ankara expressing Flt3L and OX40L for "in situ" therapeutic cancer vaccination [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr PR09.

Abstract A116: Lipid control of DNA-stimulated innate immunity

Abstract Type I interferon (IFN) plays essential roles in both spontaneous and iatrogenic tumor immunogenicity. Tumor-derived DNA is recognized by 2’,3’-cyclic GMP-AMP (cGAMP) synthase (cGAS) that is important in tumor immunogenicity. Upon DNA binding, cGAS produces the endogenous second messenger cGAMP that binds to and activates stimulator of IFN genes (STING), a signaling adaptor. cGAMP binding triggers STING trafficking from the endoplasmic reticulum (ER) to perinuclear compartments, with simultaneous activation of serine/threonine-protein kinase TBK1 that in turn phosphorylates the transcription factor IRF3, leading to upregulation of type I interferons. However, where and how TBK1 is activated by STING upon cGAMP stimulation is unclear. Our study focuses on the regulation of cGAMP-stimulated STING trafficking and activation by lipid messengers with essential roles in subcellular protein/membrane trafficking and signaling. Through in vitro signaling reconstitution, we identified a cellular lipid as an essential factor of STING signaling. We found both STING and TBK1 were lipid effectors. Lipid binding not only promotes STING trafficking but also stimulates TBK1 activation. These results reveal a new component of the STING-TBK1 complex that controls cytosolic DNA-stimulated innate immune signaling. Citation Format: Xiaojun Tan, Conggang Zhang, Zhijian J. Chen. Lipid control of DNA-stimulated innate immunity [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A116.

LSD1 inhibition by tranylcypromine derivatives interferes with GFI1-mediated repression of PU.1 target genes and induces differentiation in AML.

LSD1 has emerged as a promising epigenetic target in the treatment of acute myeloid leukemia (AML). We used two murine AML models based on retroviral overexpression of Hoxa9/Meis1 (H9M) or MN1 to study LSD1 loss of function in AML. The conditional knockout of Lsd1 resulted in differentiation with both granulocytic and monocytic features and increased ATRA sensitivity and extended the survival of mice with H9M-driven AML. The conditional knockout led to an increased expression of multiple genes regulated by the important myeloid transcription factors GFI1 and PU.1. These include the transcription factors GFI1B and IRF8. We also compared the effect of different irreversible and reversible inhibitors of LSD1 in AML and could show that only tranylcypromine derivatives were capable of inducing a differentiation response. We employed a conditional knock-in model of inactive, mutant LSD1 to study the effect of only interfering with LSD1 enzymatic activity. While this was sufficient to initiate differentiation, it did not result in a survival benefit in mice. Hence, we believe that targeting both enzymatic and scaffolding functions of LSD1 is required to efficiently treat AML. This finding as well as the identified biomarkers may be relevant for the treatment of AML patients with LSD1 inhibitors.

Selective Editing of Herpes Simplex Virus 1 Enables Interferon Induction and Viral Replication That Destroy Malignant Cells.

Oncolytic herpes simplex virus 1 (HSV-1), devoid of the γ134.5 gene, exerts antitumor activities. However, the oncolytic effects differ, ranging from pronounced to little responses. Although viral and host factors are involved, much remains to be deciphered. Here we report that engineered HSV-1 ΔN146, bearing amino acids 147 to 263 of γ134.5, replicates competently in and lyses malignant cells refractory to the γ134.5 null mutant. Upon infection, ΔN146 precludes phosphorylation of translation initiation factor eIF2α (α subunit of eukaryotic initiation factor 2), ensuring viral protein synthesis. On the other hand, ΔN146 activates interferon (IFN) regulatory factor 3 (IRF3) and IFN expression, known to prime immunity against virus and tumor. Nevertheless, ΔN146 exhibits sustained replication even exposed to exogenous IFN-α. In a 4T1 tumor model, ΔN146 markedly reduces tumor growth and metastasis formation. This coincides with viral replication or T cell infiltration in primary tumors. ΔN146 is undetectable in normal tissues in vivo Targeted HSV-1 editing results in a unique antineoplastic agent that enables inflammation without major interference of viral growth within tumor cells.IMPORTANCE Oncolytic herpes simplex virus 1 is a promising agent for cancer immunotherapy. Due to a complex virus-host interaction, less is clear about what viral signature(s) constitutes a potent oncolytic backbone. Through molecular or genetic dissection, we showed that selective editing of the γ134.5 gene enables viral replication in malignant cells, activation of transcription factor IRF3, and subsequent induction of type I IFN. This translates into profoundly reduced primary tumor growth and metastasis burden in an aggressive breast carcinoma model in vivo Our work reveals a distinct oncolytic platform that is amendable for further development.

Molecular cloning and preliminary functional analysis of six RING-between-ring (RBR) genes in grass carp (Ctenopharyngodon idellus)

Ubiquitination is a post-translational modification of proteins that is widely present in eukaryotic cells. There is increasing evidence that ubiquitinated proteins play crucial roles in the immune response process. In mammals, RING-between-RING (RBR) proteins play a key role in regulating immune signaling as the important E3 ubiquitin ligases during ubiquitination. However, the function of RBR in fish is still unclear. In the present study, six RBR genes (RNF19A, RNF19B, RNF144AA, RNF144AB, RNF144B and RNF217) of grass carp (Ctenopharyngodon idellus) were cloned and characterized. Similar to mammals, all six members of RBR family contained RING, in-between-ring (IBR) and transmembrane (TM) domains. These genes were constitutively expressed in all studied tissues, but the relative expression level differed. Following grass carp reovirus(GCRV) infection, the expression of six RBR genes in liver, gill, spleen and intestine significantly altered. Additionally, their expression in Ctenopharyngodon idellus kidney (CIK) cells was significantly increased after GCRV infection. And deficiency of RNF144B in CIK with small interference RNA (siRNA) up-regulated polyinosinic:polycytidylic acid poly(I:C))-induced inflammatory cytokines production, including IFN-I, TNF-α, IL-6, and transcription factor IRF3, which demonstrated that RNF144B was a negative regulator of inflammatory cytokines. Our results suggested that the RBR might play a vital role in regulating immune signaling and laid the foundation for the further mechanism research of RBR in fishes.

The EF-Hand Protein CALML6 Suppresses Antiviral Innate Immunity by Impairing IRF3 Dimerization

The transcription factor IRF3 is phosphorylated in response to viral infection, and it subsequently forms a homodimer and translocates into the nucleus toinduce the transcription of genes important for antiviral immunity, such as type I interferons (IFNs). This multistep process is essential for host defense against viral infection, but its regulation remains elusive. Here, we report that the EF-hand protein calmodulin-like 6 (CALML6) directly bound to thephosphorylated serine-rich (SR) region of IRF3 and impaired its dimerization and nuclear translocation. Enforced CALML6 expression suppressed viral infection-induced production of IFN-β and expression of IFN-stimulated genes (ISGs), whereas CALML6 deficiency had the opposite effect. In addition, impaired IFN-β and ISG expression in bone-marrow-derived macrophages and tissues of CALML6 transgenic mice promoted viral replication. These findings identify a phosphorylation-dependent negative feedback loop that maintains the homeostasis of antiviral innate immunity.

IRF8/miR-451a Regulates M-MDSC Differentiation <i>via</i> the AMPK/mTOR Signal Pathway During Lupus Development

Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease. Myeloid-derived suppressor cells (MDSCs) have been found to be involved in the regulation of SLE development. However, little is known about the association between MDSC subsets and the factors that draw MDSCs into abnormal expansion. This study found that M-MDSCs increased in mice with pristane-induced lupus. Toll-like receptor (TLR)7 signal activation and high IFN-α level promoted M-MDSC accumulation in vitro. Moreover, both AMPK agonist metformin and two mTOR inhibitors INK128 and rapamycin inhibited the percentage of M-MDSCs in mice with pristane-induced lupus as well as in the TLR7- and IFN-α-induced bone marrow (BM) differentiation into MDSCs in vitro. In terms of mechanism, whole-genome transcriptome profiling was performed by RNA sequencing, revealing that the expression of the transcription factor IRF-8 was higher in M-MDSCs isolated from mice with pristane-induced lupus compared with control mice. IRF-8 was identified to be crucial for TLR7- and IFN-α-induced BM differentiation into MDSCs in vitro. Furthermore, IRF-8 was targeted by miR-451a in M-MDSC differentiation. Of note, metformin-modified M-MDSCs could relieve lupus symptoms in mice with pristane-induced lupus. The findings revealed a novel mechanism linking IRF8/miR-451a to M-MDSC differentiation via the AMPK/mTOR signal pathway during lupus development. This study might provide an important reference for SLE therapy by targeting M-MDSCs. Funding Statement: This work was supported by the National Natural Science Foundation of China (No. 31872732, 31570909), the Fundamental Research Funds of the Central Universities (No. 021414380342) and the Fund of State Key Laboratory of Trauma, Burns and Combined Injury (No. SKLKF201602). Declaration of Interests: The authors declare no commercial or financial conflicts of interest. Ethics Approval Statement: All procedures involving mice were approved by the institutional license for animal care and use based on the Animal Care Committee at Nanjing University.

TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

Ceramide is one of the important cellular components involved in cancer regulation and exerts its pleiotropic role in the protective immune response without exhibiting any adverse effects during malignant neoplasm. Although, the PKCδ-ceramide axis in cancer cells has been an effective target in reduction of cancer, involvement of PKCδ in inducing nephrotoxicity have become a major questionnaire. In the present study, we have elucidated the mechanism by which cisplatin exploits the ceramide to render cancer cell apoptosis leading to the abrogation of malignancy in a PKCδ independent pathway with lesser toxicity. Our study revealed that cisplatin treatment in PKCδ silenced melanoma cells induces ceramide mediated apoptosis. Moreover, cisplatin induced upregulation of the transcription factor IRF1 leading to the induction of the transcriptional activity of the TNFα promoter was evident from the pharmacological inhibition and RNA interference studies. Increased cellular expression of TNFα resulted in an elevated ceramide generation by stimulating acid-sphingomyelinase and cPLA2. Furthermore, reciprocity in the regulation of sphingosine kinase 1 (Sphk1) and sphingosine kinase 2 (Sphk2) during PKCδ independent ceramide generation was also observed during cisplatin treatment. PKCδ inhibited murine melanoma model showed reduction in nephrotoxicity along with tumor regression by ceramide generation. Altogether, the current study emphasized the unexplored signaling cascade of ceramide generation by cisplatin during PKCδ silenced condition, which is associated with increased TNFα generation. Our findings enlightened the detailed mechanistic insight of ceramide mediated signaling by chemotherapeutic drugs in cancer therapy exploring a new range of targets for cancer treatment strategies.

Differential Roles of the mTOR-STAT3 Signaling in Dermal γδ T Cell Effector Function in Skin Inflammation

Innate dermal γδ T cells play a critical role in skin homeostasis and inflammation. However, the underlying molecular mechanisms by which these cells are activated and differentiated have not been fully understood. In this study, we show that the mTOR signaling and STAT3 pathways are activated in dermal γδ T cells in response to innate stimuli such as IL-1β and IL-23. Although both mTOC1 and mTOC2 are essential for dermal γδ T cell proliferation, mTOC2 deficiency leads to drastically decreased dermal γδT17 cells. Further studies reveal that the IL-1R-MyD88- mTOR pathway is critical in dermal γδ T cell effector function. It appears that mitochondriamediated oxidative phosphorylation is critical in this process. The absence of mTOC2 in dermal γδ T cells ameliorate skin inflammation in an Imiquimod-induced psoriasis-like model. Interestingly, STAT3 pathway while is critical in dermal Vγ4T17 cell effector function but is not required for dermal Vγ6T17 cells. In addition, transcription factor IRF-4 activation promotes dermal γδ T cell IL-17 production by linking IL-1β and IL-23 signaling. Taken together, our results demonstrate that the mTOR-STAT3 signaling differentially regulates dermal γδ T cell effector function in skin inflammation.

Fungal ligands released by innate immune effectors promote inflammasome activation during Aspergillus fumigatus infection.

Invasive pulmonary aspergillosis causes substantial mortality in immunocompromised individuals. Recognition of Aspergillus fumigatus by the host immune system leads to activation of the inflammasome, which provides protection against infection. However, regulation of inflammasome activation at the molecular level is poorly understood. Here, we describe two distinct pathways that coordinately control inflammasome activation during A. fumigatus infection. The C-type lectin receptor pathway activates both MAPK and NF-κB signalling, which leads to induction of downstream mediators, such as the transcription factor IRF1, and also primes the inflammasomes. Toll-like receptor signalling through the adaptor molecules MyD88 and TRIF in turn mediates efficient activation of IRF1, which induces IRGB10 expression. IRGB10 targets the fungal cell wall, and the antifungal activity of IRGB10 causes hyphae damage, modifies the A. fumigatus surface and inhibits fungal growth. We also demonstrate that one of the major fungal pathogen-associated molecular patterns, β-glucan, directly triggers inflammasome assembly. Thus, the concerted activation of both Toll-like receptors and C-type lectin receptors is required for IRF1-mediated IRGB10 regulation, which is a key event governing ligand release and inflammasome activation upon A. fumigatus infection.

JunB regulates homeostasis and suppressive functions of effector regulatory T cells

Foxp3-expressing CD4+ regulatory T (Treg) cells need to differentiate into effector Treg (eTreg) cells to maintain immune homeostasis. T-cell receptor (TCR)-dependent induction of the transcription factor IRF4 is essential for eTreg differentiation, but how IRF4 activity is regulated in Treg cells is still unclear. Here we show that the AP-1 transcription factor, JunB, is expressed in eTreg cells and promotes an IRF4-dependent transcription program. Mice lacking JunB in Treg cells develop multi-organ autoimmunity, concomitant with aberrant activation of T helper cells. JunB promotes expression of Treg effector molecules, such as ICOS and CTLA4, in BATF-dependent and BATF-independent manners, and is also required for homeostasis and suppressive functions of eTreg. Mechanistically, JunB facilitates the accumulation of IRF4 at a subset of IRF4 target sites, including those located near Icos and Ctla4. Thus, JunB is a critical regulator of IRF4-dependent Treg effector programs, highlighting important functions for AP-1 in Treg-mediated immune homeostasis.

IRF4-induced upregulation of lncRNA SOX2-OT promotes cell proliferation and metastasis in cholangiocarcinoma by regulating SOX2 and PI3K/AKT signaling.

The aim of this study was to investigate the role of lncRNA SOX2-OT in the proliferation and metastasis of cholangiocarcinoma (CCA) and its underlying mechanisms. A total of 82 patients with CCA underwent surgery in our hospital were enrolled in this study. Five CCA cell lines (HuH-28, QBC939, HuCCT1, CCLP1, RBE) were used. The ability of proliferation and metastasis of CCA cells were detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay, colony formation assay, and transwell assay, respectively. Additionally, in vivo tumor metastasis assay was done. Furthermore, the Kaplan Meier method was used to validate the prognostic importance of SOX2-OT for patients with cholangiocarcinoma. Besides, the protein and mRNA expression of CCA cells were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. The expression level of lncRNA SOX2-OT was significantly upregulated in cholangiocarcinoma tissues. Functional assays were further conducted to prove the oncogenic role of SOX2-OT on the proliferation and metastasis of cholangiocarcinoma cells. Furthermore, mechanism investigations manifested that transcription factor IRF4 upregulates SOX2-OT by promoting the transcriptional activity of SOX2-OT. SOX2-OT could positively regulate the nearby gene SOX2. SOX2-OT suppressed the nuclear transcription of PTEN, thereby activating PI3K/AKT signaling. lncRNA SOX2-OT upregulated by IRF4 promotes cell proliferation and metastasis in cholangiocarcinoma via upregulating SOX2 and activating PI3K/AKT signaling pathway.

A MAFB Associated Regulatory Network Distinguishes CMML from Other MDS/MPN Overlap Entities

The myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are a unique group of hematologic malignancies characterized by overlapping features of myelodysplastic syndromes and myeloproliferative neoplasms. The category includes atypical chronic myeloid leukemia (aCML), chronic myelomonocytic leukemia (CMML), MDS/MPN, unclassifiable (MDS/MPN, U), and MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T). The recent adaptation of next generation sequencing in genetic testing increased the knowledge of the molecular pattern of MDS/MPN. However, no specific gene signatures have been identified and underlying transcriptional mechanisms are still poorly understood. Therefore, we analyzed the transcriptome of a cohort of 49 aCML, 102 CMML, 50 MDS/MPN-U, and 72 MDS/MPN-RS-T patients, morphologically defined according to the WHO classification.Total RNA was used for 150bp paired-end RNA sequencing with a median read depth of 50 million. The edgeR package was used to perform a combined normalization and differential expression (DE) analysis of the obtained estimated gene counts by integration of trimmed mean of M-values normalization factors into the statistical model used to test for DE. Genes with an FDR < 0.05 and an absolute logFC > 1.5 were considered DE. 273 unique genes were identified differentially expressed between the entities. We found that the transcriptional profile of CMML patients was dominated by an upregulation of MAFB compared to all other entities (p < 0.001). MAFB encodes a transcription factor which is specifically expressed in myeloid cells and a master regulator of human monocytopoiesis.In order to unravel the potential effects of MAFB regulation we performed Spearman's correlation analysis to identify co-expressed genes. 51 genes showed a similar expression (correlation coefficient > 0.7) as MAFB, establishing a CMML gene expression signature (GES) (Figure 1a). The GES included the cell cycle inhibitor CDKN1A, the transcription factors IRF8, EGR2, and MYCL, and the cell surface receptor LRP1. LRP1 represents a therapeutic target and has recently been identified as a key factor in a network for multi-cancer clinical outcome prediction. MAFB was also co-expressed with KYNU, which is synthesized by indoleamine 2,3-dioxygenase, an enzyme expressed by leukemia blasts, and which has been linked with unfavorable outcome in hematologic malignancies. MAFB and KYNU were also co-expressed with multiple CD markers, which are often used as targets of immunotherapy approaches in cancer. High expressions of KYNU can block the anti-tumor effects of chimeric antigen receptor T-cell therapy, indicating complex interactions between the co-expressed genes.To better understand these underlying molecular interactions on a more global scale and to expose potential targets for intervention, a transcriptional network of the 51 MAFB co-expressed genes was reverse engineered. We used STRING, a publicly available database of protein-protein interactions, to build the scaffold of the network and subsequently pruned the network based on our gene expression data. The reconstructed, MAFB associated regulatory network (Figure 1b) identified c-FOS as an additional regulatory link. c-FOS is a known oncogene that heterodimerizes with c-JUN, ATF3, and JDP2 to form the structure of the AP-1 complex. The AP-1 complex is a key regulator of multiple biological processes such as cell proliferation and apoptosis. We observed a considerably higher expression of c-FOS compared to c-JUN, ATF3, and JDP2 in CMMLs. The MafB/cFos heterodimer is known to explicitly repress apoptosis.The co-expression network was significantly enriched for transcriptional activation of p53 responsive genes (p < 0.001). Interestingly, we also found that the p53-regulated long intergenic non-coding RNA lncPRESS1 was down-regulated in our cohort of CMML patients (p < 0.001).Conclusions: 1) MAFB represents a promising gene expression marker to distinguish CMML pts from other MDS/MPN overlap entities. 2) The identified core regulatory signature in CMML pts showed complex interactions to modulate diverse biological processes and might serve as therapeutic targets. [Display omitted] DisclosuresWalter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment.

Transcriptional mechanism of IRF8 and PU.1 governs microglial activation in neurodegenerative condition

ABSTRACTMicroglial activation occurs in divergent neuropathological conditions. Such microglial event has the key involvement in the progression of CNS diseases. However, the transcriptional mechanism governing microglial activation remains poorly understood. Here, we investigate the microglial response to traumatic injury-induced neurodegeneration by the 3D fluorescence imaging technique. We show that transcription factors IRF8 and PU.1 are both indispensible for microglial activation, as their specific post-developmental deletion in microglia abolishes the process. Mechanistically, we reveal that IRF8 and PU.1 directly target the gene transcription of each other in a positive feedback to sustain their highly enhanced expression during microglial activation. Moreover, IRF8 and PU.1 dictate the microglial response by cooperatively acting through the composite IRF-ETS motifs that are specifically enriched on microglial activation-related genes. This action of cooperative transcription can be further verified biochemically by the synergetic binding of IRF8 and PU.1 proteins to the composite-motif DNA. Our study has therefore elucidated the central transcriptional mechanism of microglial activation in response to neurodegenerative condition.

629. Blood Transcriptome Variations Predict Infection and Rejection in the Older Kidney Transplant Recipient

BackgroundCompared with younger patients on similar immunosuppression regimens, older solid-organ transplant recipients experience increased rates of infection and death, but decreased rates of rejection. Our previous findings demonstrated increased T-cell immunosenescence and pro-inflammatory monocytes in older patients. This study sought to define the implications of transcriptome alterations for clinical outcomes. The objective of this abstract is to evaluate older vs. younger solid-organ transplant recipients for differential patterns of gene expression associated with infection and rejection.MethodsPeripheral blood mononuclear cells were isolated from 23 older (≥age 60) and 37 matched younger (ages 30–59) kidney transplant recipients at 3 months after transplantation. RNA extraction was performed on banked PBMCs. Isolated RNA was converted to fluorescent cRNA and hybridized to Illumina Human HT-12 v4 BeadArrays. Gene expression values were quantile-normalized and log2-transformed for mixed effect linear model analyses to identify differential expression as a function of age, adjusted for induction type, donor type, and sex. Statistical analysis was performed using R software.ResultsGenes differentially expressed in older patients revealed an over-representation of pro-inflammatory genes and a down regulation of genes associated with the CD8 immune response. Patients who went on to develop infection demonstrated an increase in IRF transcription factor activation and plasmacytoid dendritic cell activity. Patients who developed rejection demonstrated an increase in myeloid lineage immune cell activity.ConclusionDifferential patterns of gene expression were observed in patients who developed infection in the first year after kidney transplantation. These findings were distinct from the gene expression changes associated with development of rejection. These findings may explain the mechanism behind vulnerability to infection in older transplant patients. In addition, monitoring of changes in gene expression may provide an avenue for patient monitoring after transplantation as well as individualization of immune suppression after solid-organ transplantation.Disclosures All authors: No reported disclosures.

DDX3 directly facilitates IKKα activation and regulates downstream signalling pathways.

DDX3 is a DEAD-box RNA helicase that we and others have previously implicated in antiviral immune signalling pathways leading to type I interferon (IFN) induction. We previously demonstrated that it directly interacts with the kinase IKKε (IκB kinase ε), enhances it activation, and then facilitates phosphorylation of the transcription factor IRF3 by IKKε. However, the TLR7/9 (Toll-like receptor 7/9)-mediated pathway, one of the most physiologically relevant IFN induction pathways, proceeds independently of IKKε or the related kinase TBK1 (TANK-binding kinase 1). This pathway induces type I IFN production via the kinases NIK (NF-κB-inducing kinase) and IKKα and is activated when plasmacytoid dendritic cells sense viral nucleic acids. In the present study, we demonstrate that DDX3 also directly interacts with IKKα and enhances its autophosphorylation and -activation. Modulation of DDX3 expression consequently affected NIK/IKKα-mediated IRF7 phosphorylation and induction of type I interferons. In addition, alternative NF-κB (nuclear factor-κB) activation, another pathway regulated by NIK and IKKα, was also down-regulated in DDX3 knockdown cells. This substantially broadens the effects of DDX3 in innate immune signalling to pathways beyond TBK1/IKKε and IFN induction. Dysregulation of these pathways is involved in disease states, and thus, our research might implicate DDX3 as a potential target for their therapeutic manipulation.

Autocrine–paracrine prostaglandin E2 signaling restricts TLR4 internalization and TRIF signaling

The unique cell biology of Toll-like receptor 4 (TLR4) allows it to initiate two signal transduction cascades: a Mal (TIRAP)–MyD88-dependent signal from the cell surface that regulates proinflammatory cytokines and a TRAM–TRIF-dependent signal from endosomes that drives type I interferon production. Negative feedback circuits to limit TLR4 signals from both locations are necessary to balance the inflammatory response. We describe a negative feedback loop driven by autocrine-paracrine prostaglandin E2 (PGE2), and the PGE2 receptor, EP4, which restricted TRIF-dependent signals and IFN-β induction through regulation of TLR4 trafficking. Inhibition of PGE2 production or EP4 antagonism increased the rate of TLR4 endosomal translocation, and amplified TRIF-dependent IRF3 and caspase 8 activation. This PGE2-driven mechanism restricted TLR4-TRIF signaling in vitro upon infection of macrophages by Gram-negative pathogens Escherichia coli and Citrobacter rodentium and protected mice against Salmonella enteritidis serovar Typhimurium (ST)-induced mortality. Thus, PGE2 restricts TLR4-TRIF signaling specifically in response to lipopolysaccharide.

The Histone Demethylase LSD1 Regulates B Cell Proliferation and Plasmablast Differentiation.

B cells undergo epigenetic remodeling as they differentiate into Ab-secreting cells (ASC). LSD1 is a histone demethylase known to decommission active enhancers and cooperate with the ASC master regulatory transcription factor Blimp-1. The contribution of LSD1 to ASC formation is poorly understood. In this study, we show that LSD1 is necessary for proliferation and differentiation of mouse naive B cells (nB) into plasmablasts (PB). Following LPS inoculation, LSD1-deficient hosts exhibited a 2-fold reduction of splenic PB and serum IgM. LSD1-deficient PB exhibited derepression and superinduction of genes involved in immune system processes; a subset of these being direct Blimp-1 target-repressed genes. Cell cycle genes were globally downregulated without LSD1, which corresponded to a decrease in the proliferative capacity of LSD1-deficient activated B cells. PB lacking LSD1 displayed increased histone H3 lysine 4 monomethylation and chromatin accessibility at nB active enhancers and the binding sites of transcription factors Blimp-1, PU.1, and IRF4 that mapped to LSD1-repressed genes. Together, these data show that LSD1 is required for normal in vivo PB formation, distinguish LSD1 as a transcriptional rheostat and epigenetic modifier of B cell differentiation, and identify LSD1 as a factor responsible for decommissioning nB active enhancers.

RAS P21 Protein Activator 3 (RASA3) Specifically Promotes Pathogenic T Helper 17 Cell Generation by Repressing T-Helper-2-Cell-Biased Programs

Pathogenic Th17 (pTh17) cells drive inflammation and immune-pathology, but whether pTh17 cells are a Th17 cell subset whose generation is under specific molecular control remains unaddressed. We found that Ras p21 protein activator 3 (RASA3) was highly expressed by pTh17 cells relative to non-pTh17 cells and was required specifically for pTh17 generation invitro and invivo. Mice conditionally deficient for Rasa3 in Tcells showed less pathology during experimental autoimmune encephalomyelitis. Rasa3-deficientTcells acquired a Th2 cell-biased program that dominantly trans-suppressed pTh17 cell generation via interleukin 4 production. The Th2 cell bias ofRasa3-deficient Tcells was due to aberrantly elevated transcription factor IRF4 expression. RASA3 promoted proteasome-mediated IRF4 protein degradation by facilitating interaction of IRF4 with E3-ubiquitin ligase Cbl-b. Therefore, a RASA3-IRF4-Cbl-b pathway specifically directs pTh17 cell generation by balancing reciprocal Th17-Th2 cell programs. These findings indicate that a distinct molecular program directs pTh17 cell generation and reveals targets for treating pTh17 cell-related pathology and diseases.

The antineoplastic drug metformin downregulates YAP by interfering with IRF-1 binding to the YAP promoter in NSCLC.

BackgroundActivation of the oncogene YAP has been shown to be related to lung cancer progression and associates with poor prognosis and metastasis. Metformin is a drug commonly used in the treatment of diabetes and with anticancer activity. However, the mechanism through which metformin inhibits tumorigenesis via YAP is poorly understood.MethodsThe mRNA and protein expressions were analyzed by RT-PCR and western blot. The cellular proliferation was detected by CCK8 and MTT. The cell migration and invasion growth were analyzed by wound healing assay and transwell assay. The activities of promoter were analyzed by luciferase reporter assay. Chromatin immunoprecipitation detected the combining ability of IRF-1 and 5′UTR-YAP.FindingsOur immunohistochemistry staining and RT-PCR assays showed that the expression of YAP was higher in lung carcinoma samples. Interestingly, metformin was able to downregulate YAP mRNA and protein expression in lung cancer cells. Mechanistically, we found that metformin depressed YAP promoter by competing with the binding of the transcription factor IRF-1 in lung cancer cells. Moreover, combination of metformin and verteporfin synergistically inhibits cell proliferation, promotes apoptosis and suppresses cell migration/invasion by downregulating YAP, therefore reduces the side effects caused by their single use and improve the quality of life for patients with lung cancer.Interpretationwe concluded that metformin depresses YAP promoter by interfering with the binding of the transcription factor IRF-1. Importantly, verteporfin sensitizes metformin-induced the depression of YAP and inhibition of cell growth and invasion in lung cancer cells.FundThis work was supported by National Natural Science Foundation of China (No.31801085), the Science and Technology Development Foundation of Yantai (2015ZH082), Natural Science Foundation of Shandong Province (ZR2018QH004, ZR2016HB55, ZR2017PH067 and ZR2017MH125), and Research Foundation of Binzhou Medical University (BY2015KYQD29 and BY2015KJ14).