IRF4 Expression Research Articles

The hMeDIP-Seq identifiedINPP4A as a novel biomarker for eosinophilic chronic rhinosinusitis with nasal polyps.

Background: Eosinophilic chronic rhinosinusitis with nasal polyps (ECRSwNP) is an endotype of chronic rhinosinusitis with nasal polyps characterized by more severe symptoms, a stronger association with asthma and a greater recurrence risk. It is unknown whether DNA hydroxymethylation could influence ECRSwNP. Methods: Hydroxymethylated DNA immunoprecipitation sequencing was carried out in three distinct groups (control, ECRSwNP and NECRSwNP). Additional qRT-PCR, immunohistochemistry and analysis of the receiver operating characteristic curve were performed. Results: Between ECRSwNP and NECRSwNP, 26 genes exhibited differential DNA hydroxymethylation. Consistent with their hydroxymethylation level, GNAL, INPP4A and IRF4 expression levels were significantly different between ECRSwNP and the other two groups. The receiver operating characteristic curve revealed that INPP4A mRNA has a high predictive accuracy for ECRSwNP. Conclusion: DNA hydroxymethylation regulates the expression of multiple genes in ECRSwNP. INPP4A mRNA was markedly decreased in ECRSwNP polyps and can predict ECRSwNP.

Anti-Inflammatory Effects of Red Rice Bran Extract Ameliorate Type I Interferon Production via STING Pathway.

Type I interferons (IFNs-I) are inflammatory cytokines that play an essential role in the pathogenesis of inflammation and autoimmune diseases. Signaling through nucleic acid sensors causes the production of IFNs-I. A stimulator of interferon genes (STING) is a DNA sensor that signals transduction, leading to the production of IFNs-I after their activation. This study aims to determine the anti-inflammatory effects of red rice bran extract (RRBE) on macrophages through the activation of STING signaling. RAW264.7 macrophage cells were stimulated with STING agonist (DMXAA) with and without RRBE. Cells and supernatant were collected. The level of mRNA expression was determined by qPCR, and inflammatory cytokine production was investigated by ELISA. The results indicate that RRBE significantly lowers the transcription of STING and interferon-stimulated genes (ISGs). Moreover, RRBE suppresses the phosphorylation of STING, leading to a decrease in the expression of Irf3, a transcription factor that initiates IFN-I signaling. Our results provide evidence that red rice bran extract may be a protective compound for inflammatory diseases by targeting STING signaling.

Correlation of the transcription factors IRF4 and BACH2 with the abnormal NFATC1 expression in T cells from chronic myeloid leukemia patients

ABSTRACT Objective T cell dysfunction is a common characteristic of patients with myeloid leukemia and is closely related to clinical efficacy and prognosis. In order to clarify the mechanisms leading to the T cell dysfunction, we characterized the gene expression profile of T cells from chronic myelogenous leukemia (CML) patients by microarray analysis and investigated the related regulating pathway. Methods We employed gene expression profiling, bioinformatics and real-time quantitative reverse transcription PCR (RT-qPCR) to detect genes differentially expressed in CML patients versus healthy donors. Results There were 1704 genes differentially expressed between CD3+ T cells from CML patients and healthy donors, including 868 up-regulated genes and 836 down-regulated genes, which mostly related to T cell functional pathways. In particular, lower expression of NFATC1, a member of the TCR signaling pathway, was detected in CD3+ T cells from CML patients. We further found that the expression of IRF4 and BACH2, transcription factors that potentially regulate NFATC1, in CD3+ T cells from CML patients was significantly lower than that in healthy donors. Conclusion We for the first time observed the altered gene expression profiles of CD3+ T cells from CML patients, and the results suggested that IRF4, BACH2 and NFATC1 may be involved in regulating T cell dysfunction in CML patients in the form of a transcriptional regulatory network. These findings may provide potential targets for tyrosine kinase inhibitors in combination with other targeted immunotherapies .

SLE risk variant regulates IRF8 expression.

SLE risk variant regulates IRF8 expression.

TBK1 Mediates Innate Antiviral Immune Response against Duck Enteritis Virus.

Duck enteritis virus (DEV) can infect several types of waterfowl can cause high mortality and huge economic losses to the global waterfowl industry. Type I interferons (IFN) are important for host defense against virus infection through induction of antiviral effector molecules. TANK-binding kinase 1 (TBK1) is a key kinase required for the induction of type I IFNs; however, the role of TBK1 on DEV infection remains unclear. Here, we observed that the expression levels of TBK1 and IFN-β were upregulated during DEV infection in vivo and in vitro. Thus, the function of TBK1 on DEV infection was determined. The results showed that overexpression of TBK1 reduced DEV infection and knockdown of TBK1 resulted in the increased of DEV infection. Additionally, TBK1 overexpression upregulated the expression of IFN-β and a few interferon-stimulated genes (ISGs), which thus inhibited the synthesis of DEV glycoprotein B. On the other hand, the TBK1 inhibitor Amlexanox down-regulated the expression levels of IFN-β and IRF3. Interestingly, the expression levels of MAVS and GSK-3β were decreased in the cells treated with Amlexanox. Furthermore, overexpression of TBK1 activated the expression of upstream molecules MAVS and GSK-3β. Whereas, the expression of TBK1, IRF3 and IFN-β was inhibited by the GSK-3β inhibitor SB216763. Our findings suggest that DEV–stimulated TBK1 may be involved in defense against DEV infection.

Single-Cell Transcriptome Analysis Reveals the Importance of IRF1/FSTL1 in Synovial Fibroblast Subsets for the Development of Rheumatoid Arthritis

Objectives This study aimed to investigate the potential role of synovial fibroblasts (SFs) in the development of rheumatoid arthritis (RA) to identify potential molecular targets and provide a theoretical basis for the treatment of RA. Methods GSE109449, a fibroblast transcriptome dataset of synovial tissue from RA and osteoarthritis (OA), were obtained from the GEO database. After standard cell quality control, this single-cell transcriptome data was used to perform routine single-cell analysis processes. After completing dimensionality reduction, clustering, and cell subset identification of fibroblasts, the SCENIC analysis helped calculate the significant gene regulatory networks in fibroblasts and their subsets. From these computed gene regulatory networks, the regulon in which follistatin-like protein 1 (FSTL1) resides was extracted and used to analyze the transcriptional regulatory status of fibroblasts. Finally, the gene set enrichment analysis (GSEA) was used to calculate the respective enriched gene sets of IRF1 and FSTL1. Results Three SF subgroups were identified from the single-cell transcriptome analysis; SF subset 3 was more abundant in RA than in OA (p < 0.001). From the SCENIC analysis, we obtained 269 regulons and the corresponding gene regulatory networks in SF from the RA datasets. Next, we screened and obtained a regulon-containing FSTL1, where IRF1 was the major transcription factor. The top five regulons in SF subset 3 were TWIST1, MECOM, KLF6, MAFB, and RUNX1. Among the 3 SF subsets, IRF1 regulon was ranked the highest in SF subset 3. Differential analysis of pseudobulk RNA-seq showed that IRF1 was up-regulated in RA compared to OA. Between the three SF subgroups, IRF1 and FSTL1 expression was more up-regulated in SF subset 3 compared to the other two subgroups. Conclusions IRF1 was found to regulate the invasiveness of SFs by regulating FSTL1, which may influence the disease progression of RA.

Scorpion and centipede alleviates severe asthma through M2 macrophage-derived exosomal miR-30b-5p.

Asthma is one of the most common chronic inflammatory diseases. Although the scorpion and centipede (SC) significantly ameliorates asthma and changes exosomal miRNAs, the molecular mechanism is still obscure. Here, we show that SC improves inflammation in asthmatic mice and increases M2 macrophage-derived exosomes (M2Φ-Exos) by promoting M2 macrophage polarization. The M2Φ-Exos remarkably inhibits airway epithelial cell pyroptosis by reducing the expression of NLRP3, caspase-1, and LI-1β and mitochondrial swelling. Furthermore, miR-30b-5p is up-regulated in M2Φ-Exos compared with M1Φ-Exos. Overexpression of miR-30b-5p in M2Φ-Exos prevents airway epithelial cell pyroptosis, while down-regulation of miR-30b-5p promotes pyroptosis. We also uncover that pyroptosis is increased in asthmatic mice, while SC blocks pyroptosis. Moreover, miR-30b-5p overexpressed M2Φ-Exos further enhances the ameliorative effect of SC, which significantly down-regulates IRF7 expression. Our results collectively reveal that M2Φ-Exos induced by SC could carry miR-30b-5p to mitigate severe asthma by inhibiting airway epithelial cell pyroptosis. Most importantly, our findings may provide a potential clinical application of M2Φ-Exos for treating severe asthma.

Cell-specific regulation of antigen-specific germinal center formation and antibody responses by thymic stromal lymphopoietin

Abstract Germinal centers (GCs) are dynamic immune structures that expand upon T-dependent immune challenge to generate high-affinity antibody-producing plasma cells and long-lived memory. GCs require stringent regulation to optimize GC-derived antibody responses. Here, we identified thymic stromal lymphopoietin (TSLP), as a novel regulator of GCs. After immunization with NP-OVA in Alum, TSLP receptor-deficient (TSLPR−/−) mice fail to form antigen-specific GCs and antibodies. TSLPR expression is elevated on T follicular helper cells (Tfh) and GC B cells during inflammation, so we asked how cell-specific loss of TSLPR signaling could disrupt overall GC activity. When TSLPR was deleted in CD4 T cells with a tamoxifen-inducible Cre (CD4CreERT2/+TSLPRFLOX/FLOX), we observed a similar reduction in T follicular helper cells (Tfh), GC formation, and antibody responses after immunization when compared to CD4CreERT2/+ and TSLPRFLOX/FLOX controls. Conversely, when TSLPR signaling was lost on mature B cells (CD21Cre/+TSLPRFLOX/FLOX) and GC B cells (AIDCre/+TSLPRFLOX/FLOX), we observed an increase in antigen-specific GC B cells and antigen-specific memory B cells. TSLPR signaling regulates IRF4 expression in dendritic cells, and we observed a similar reduction in IRF4 expression in Tfh and GC B cells from TSLPR−/− mice compared to TSLPR+/+ controls. Collectively, these findings suggest that TSLPR signaling promotes IRF4 expression in GC B cells and Tfh cells, resulting in distinct roles in the GC. TSLPR and IRF4 signaling in T cells is required for the establishment and maintenance of antigen-specific Tfh cells, whereas TSLPR/IRF4 signaling in B cells inhibits antigen-specific GC B cell retention and the establishment of B cell memory. Supported by a grant from the NIH (F32 AI154787)

An unexpected role for IRF8 during human T cell development.

Abstract Lineage tracing studies or other genetic approaches have allowed investigators to obtain detailed insights into the molecular mechanisms that control T cell development in the mouse. However, our understanding of how this process is regulated in human has remained unclear. Using various genome-wide approaches, such as bulk RNAseq, ATACseq, ChIPseq and scRNAseq, we have recently obtained important novel insights into the molecular mechanisms through which human hematopoietic progenitor cells are converted into T-lineage committed precursors, and further into the TCRαβ and −γδ lineages. Specifically, we identified two distinct populations of thymus seeding precursor cells (TSPs) of which one population, TSP2, expressed IRF8, a transcription factor that is mainly associated with dendritic cell development. Consistently, IRF8-expressing TSPs have both T− and DC-linage potential and are characterised by the coexpression of CD127 and CD123. We discovered that Notch activation, the main initiator of T-lineage specification, induces IRF8 expression in human ETPs, in contrast to in mouse, and that IRF8 is transiently expressed during early human T cell development. As T cell precursors commit to the T-cell lineage, IRF8 is repressed by GATA3, while IRF8 levels rise for ETPs that differentiate into plasmacytoid dendritic cells. Importantly, IRF8 is required for the generation of CD34+ CD127+ CD123+ T/DC precursors and low level IRF8 activity enhances their generation from hematopoietic progenitors. Thus, using these approaches, we have identified an IRF8-dependent T cell precursor subset and revealed unique molecular events that control early T cell development in human. Supported by grants from the Fund for Scientific Research Flanders (FWO), the Foundation Against Cancer (STK), the Chan Zuckerberg Initiative (CZI) and Ghent University (BOF Ugent).

Chilblains observed during the COVID-19 pandemic cannot be distinguished from classic, cold-related chilblains

Type 1 interferon (IFN-I) response induced by SARS-CoV-2 has been hypothesized to explain the association between chilblain lesions (CL) and SARS-CoV-2 infection. To explore direct cytopathogenicity of SARS-CoV-2 in CL and to focus on IFN-I expression in patients with chilblains. A monocentric cohort of 43 patients presenting with CL from April 2020 to May 2021 were included. During this period, all CL were, a priori, considered to be SARS-CoV-2-related. RT-qPCR on nasopharyngeal swabs and measurements of anti-SARS-CoV-2 antibodies were performed. Anti-SARS-CoV-2 immunostainings as well as SARS-CoV-2 RT-qPCR were performed on biopsy specimens of CL and controls. Expression of MX1 and IRF7 was analysed on patients’ biopsy specimens and/or PBMC and compared with controls and/or chilblains observed before the pandemic. Serum IFN-α was also measured. RT-qPCR was negative in all patients and serological tests were positive in 11 patients. Immunostaining targeting viral proteins confirmed the lack of specificity. SARS-CoV-2 RNA remained undetected in all CL specimens. MX1 immunostaining was positive in CL and in pre-pandemic chilblains compared to controls. MX1 and IRF7 expression was significantly increased in CL specimens but not in PBMC. Serum IFN-α was undetected in CL patients. CL observed during the pandemic do not appear to be directly related to SARS-CoV-2 infection, either based on viral cytopathogenicity or high IFN-I response induced by the virus.

Abstract 363: Irf8 And Securinine: Seeking Treatments For Abdominal Aortic Aneurysm

Objective: Abdominal aortic aneurysm (AAA) is defined as local weakening and dilatation of the abdominal aorta ≥50% and has high incidence in elderly. Current therapy is limited to surgical repair, only, no pharmacological therapy has shown effectiveness against AAA development and growth. Untreated AAA rupture is highly associated with death, and aged patients are at augmented risk for surgical complications. We therefore sought to identify novel agents that could be employed for medical AAA therapy. Methods: Using whole genome DNA methylation analysis (RRBS-Seq) in a transgenerational model of murine AAA, combined with transcriptional analysis of murine AAA tissue, we identified interferon regulatory factors (IRFs: a group of immune-system-related transcription factors (TF)), as major drivers of AAA risk and pathophysiology. We performed TF enrichment analysis that specifically suggested IRF8 as being crucial for AAA development. Drug Signatures Database (DSigDB) and Connectivity Map (CMap) analysis identified securinine, an alkaloid and traditional Chinese herbal medicine derived from Securinega suffruticosa , as a probable IRF8 inhibitor. Its vascular effects are relatively unknown. Results: We studied the effects of securinine using the porcine pancreatic elastase (PPE) model of AAA. Sonographic imaging data of 28-day securinine-treated mice (15 mg/kg) showed smaller aneurysm diameters and significantly downregulated gene expression of IRF8 and IFN-γ compared to control PPE groups. Conclusion: These data suggest that securinine may have therapeutic benefits on AAA development, potentially through regulation of IRF8.

Reduced IRF4 expression promotes lytic phenotype in Type 2 EBV-infected B cells.

Humans are infected with two types of EBV (Type 1 (T1) and Type 2 (T2)) that differ substantially in their EBNA2 and EBNA 3A/B/C latency proteins and have different phenotypes in B cells. T1 EBV transforms B cells more efficiently than T2 EBV in vitro, and T2 EBV-infected B cells are more lytic. We previously showed that both increased NFATc1/c2 activity, and an NFAT-binding motif within the BZLF1 immediate-early promoter variant (Zp-V3) contained in all T2 strains, contribute to lytic infection in T2 EBV-infected B cells. Here we compare cellular and viral gene expression in early-passage lymphoblastoid cell lines (LCLs) infected with either T1 or T2 EBV strains. Using bulk RNA-seq, we show that T2 LCLs are readily distinguishable from T1 LCLs, with approximately 600 differentially expressed cellular genes. Gene Set Enrichment Analysis (GSEA) suggests that T2 LCLs have increased B-cell receptor (BCR) signaling, NFAT activation, and enhanced expression of epithelial-mesenchymal-transition-associated genes. T2 LCLs also have decreased RNA and protein expression of a cellular gene required for survival of T1 LCLs, IRF4. In addition to its essential role in plasma cell differentiation, IRF4 decreases BCR signaling. Knock-down of IRF4 in a T1 LCL (infected with the Zp-V3-containing Akata strain) induced lytic reactivation whereas over-expression of IRF4 in Burkitt lymphoma cells inhibited both NFATc1 and NFATc2 expression and lytic EBV reactivation. Single-cell RNA-seq confirmed that T2 LCLs have many more lytic cells compared to T1 LCLs and showed that lytically infected cells have both increased NFATc1, and decreased IRF4, compared to latently infected cells. These studies reveal numerous differences in cellular gene expression in B cells infected with T1 versus T2 EBV and suggest that decreased IRF4 contributes to both the latent and lytic phenotypes in cells with T2 EBV.

P234 The effect of calcitriol on the expression of interferon signature genes in dendritic cells and macrophages in systemic lupus erythematosus

Abstract Background/Aims A number of studies have demonstrated the inverse association between vitamin D and (i) disease activity and (ii) interferon (IFN) signature gene expression in systemic lupus erythematosus (SLE). The aim of the study was to establish the effect of calcitriol supplementation to cultured macrophages and dendritic cells (DC) from a SLE patient and control, on the expression of 12 IFN signature genes, interferon regulatory factor (IRF)7 and IRF8. Methods Blood samples were collected from a 41-year-old female SLE patient who fulfilled the SLICC classification criteria for SLE and from a 36-year-old healthy female control after obtaining informed consent. Both patients had a serum 25-hydroxyvitamin D level of 25ng/ml. Peripheral blood mononuclear cells (PBMC) were isolated by using histopaque®-1077. CD83 and CD14 expression was measured by flow cytometry and cell count and viability were assessed. The PBMC suspension was cultured in 6-well plates using: (i) ImmunoCultTM DC culture kit (following kit protocol) for DC differentiation, (ii) culture medium for macrophage differentiation containing RPMI 1640 Medium, DMEM, fetal bovine serum and LPS. Following 7 days of incubation, differentiation of cells was confirmed using microscopy and flow cytometry to measure CD83 and CD14 expression. Calcitriol at a concentration of 20nM was added to half of the wells of each sample category. Following 24 and 48 hours, RNA extraction was carried out on treated and untreated samples. QuantiGene Plex technology was used to measure the expression of 12 IFN signature genes, IRF7, IRF8 and four housekeeping genes in the extracted RNA. The normalised median florescence intensity for the 12 IFN signature genes was used to calculate the IFN score. Results A significantly lower IFN score was noted at 24 hours in SLE patient dendritic cell and macrophage cell cultures and in control macrophage cell cultures treated with calcitriol when compared with respective untreated samples (p = 0.014, p = 0.034, p = 0.031 respectively). On comparing IRF8 expression in treated and untreated cell cultures, a significantly lower IRF8 expression was noted in treated macrophage cell cultures obtained from both SLE patient and control (p = 0.034, p = 0.041). No difference in IRF7 expression was noted in treated and untreated cultures for all samples. Conclusion The suppression of the interferon signature genes in dendritic cell and macrophage cell cultures with vitamin D was confirmed in the in vitro experiment carried out. In this experiment a decrease in the expression of IRF8 (known to have a VDR binding site) was also observed in macrophage cultures from both SLE patient and control. Disclosure R. Magro: None. E. Seria: None. G. Grech: None. A.A. Borg: None. C. Scerri: None.

High-Fat Diet-Induced Dysregulation of Immune Cells Correlates with Macrophage Phenotypes and Chronic Inflammation in Adipose Tissue.

Obesity is a complex disease associated with various metabolic abnormalities, cardiovascular diseases, and low-grade chronic inflammation. Inflammation associated with T helper 1 (Th1) immune cells is dominant in adipose tissue (AT) and exerts metabolically deleterious impacts. The precise mechanism of alteration in AT immune system and its effect on metabolic homeostasis remains unclear. In this study, we investigated how a high-fat diet (HFD) alters the AT immune response and influences inflammation during obesity. HFD consumption amends the metabolic parameters, including body weight, glucose, and insulin levels. We observed increased infiltration of Th17 cells, a subset of dendritic cells (CD103+), and M1 macrophages in AT of mice fed HFD compared to those fed a normal diet (ND). In mice that were fed HFD, we also observed a reduction in regulatory T cells (Tregs) relative to the numbers of these cells in mice fed ND. Corresponding with this, mice in the HFD group exhibited higher levels of proinflammatory cytokines and chemokines than those in the ND group. We also observed alterations in signaling pathways, including increased protein expression of IRF3, TGFβ1, and mRNA expression of IL-6, KLF4, and STAT3 in the AT of the mice fed HFD as compared to those fed ND. Further, HFD-fed mice exhibited decreased protein expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) compared to mice fed ND, suggesting that PPAR-γ functions as a negative regulator of Th17 cell differentiation. These results suggest that HFD induces increased levels of inflammatory cytokines and key immune cells, including Th17, M1 macrophages, and CD103+ dendritic cells, and reduces levels of PPAR-γ and Tregs to sustain AT inflammation. This study supports the notion that dysregulation of Th17/Tregs, which polarizes macrophages towards M1 phenotypes in part through TGFβ1-IRF3-STAT3 and negatively regulates PPAR-γ mediated pathways, results in AT inflammation during obesity.

Candidate genes for polycystic ovary syndrome are regulated by TGFβ in the bovine foetal ovary.

STUDY QUESTIONCould changes in transforming growth factor β (TGFβ) signalling during foetal ovary development alter the expression of polycystic ovary syndrome (PCOS) candidate genes leading to a predisposition to PCOS?SUMMARY ANSWERTGFβ signalling molecules are dynamically expressed during foetal ovary development and TGFβ1 inhibits expression of the androgen receptor (AR) and 7 (INSR, C8H9orf3, RAD50, ERBB3, NEIL2, IRF1 and ZBTB16) of the 25 PCOS candidate genes in foetal ovarian fibroblasts in vitro, whilst increasing expression of the AR cofactor TGFβ-induced transcript 1 (TGFB1I1 or Hic5).WHAT IS KNOWN ALREADYThe ovarian stroma arises from the mesonephros during foetal ovary development. Changes in the morphology of the ovarian stroma are cardinal features of PCOS. The ovary is more fibrous and has more tunica and cortical and subcortical stroma. It is not known why this is and when this arises. PCOS has a foetal origin and perhaps ovarian stroma development is altered during foetal life to determine the formation of a polycystic ovary later in life. PCOS also has a genetic origin with 19 loci containing 25 PCOS candidate genes. In many adult tissues, TGFβ is known to stimulate fibroblast replication and collagen deposition in stroma, though it has the opposite effect in the non-scaring foetal tissues. Our previous studies showed that TGFβ signalling molecules [TGFβs and their receptors, latent TGFβ binding proteins (LTBPs) and fibrillins, which are extracellular matrix proteins that bind LTBPs] are expressed in foetal ovaries. Also, we previously showed that TGFβ1 inhibited expression of AR and 3 PCOS candidate genes (INSR, C8H9orf3 and RAD50) and stimulated expression of TGFB1I1 in cultured foetal ovarian fibroblasts.STUDY DESIGN, SIZE, DURATIONWe used Bos taurus for this study as we can ethically collect foetal ovaries from across the full 9-month gestational period. Foetal ovaries (62–276 days, n = 19) from across gestation were collected from pregnant B. taurus cows for RNA-sequencing (RNA-seq) analyses. Foetal ovaries from B. taurus cows were collected (160–198 days, n = 6) for culture of ovarian fibroblasts.PARTICIPANTS/MATERIALS, SETTING, METHODSRNA-seq transcriptome profiling was performed on foetal ovaries and the data on genes involved in TGFβ signalling were extracted. Cells were dispersed from foetal ovaries and fibroblasts cultured and treated with TGFβ1. The effects of TGFβ regulation on the remaining eight PCOS candidate genes not previously studied (ERBB3, MAPRE1, FDFT1, NEIL2, ARL14EP, PLGRKT, IRF1 and ZBTB16) were examined.MAIN RESULTS AND THE ROLE OF CHANCEMany TGFβ signalling molecules are expressed in the foetal ovary, and for most, their expression levels increased accross gestation (LTBP1/2/3/4, FBN1, TGFB2/3, TGFBR2/3 and TGFB1I1), while a few decreased (FBN3, TGFBR3L, TGFBI and TGFB1) and others remained relatively constant (TGFBRAP1, TGFBR1 and FBN2). TGFβ1 significantly decreased expression of PCOS candidate genes ERBB3, NEIL2, IRF1 and ZBTB16 in cultured foetal ovarian fibroblasts.LARGE SCALE DATAThe FASTQ files, normalized data and experimental information have been deposited in the Gene Expression Omnibus (GEO) accessible by accession number GSE178450.LIMITATIONS, REASONS FOR CAUTIONRegulation of PCOS candidate genes by TGFβ was carried out in vitro and further studies in vivo are required. This study was carried out in bovine where foetal ovaries from across all of the 9-month gestational period were available, unlike in the human where it is not ethically possible to obtain ovaries from the second half of gestation.WIDER IMPLICATIONS OF THE FINDINGSFrom our current and previous results we speculate that inhibition of TGFβ signalling in the foetal ovary is likely to (i) increase androgen sensitivity by enhancing expression of AR, (ii) increase stromal activity by stimulating expression of COL1A1 and COL3A1 and (iii) increase the expression of 7 of the 25 PCOS candidate genes. Thus inhibition of TGFβ signalling could be part of the aetiology of PCOS or at least the aetiology of polycystic ovaries.STUDY FUNDING/COMPETING INTEREST(S)Funding was received from Adelaide University China Fee Scholarship (M.L.), Australian Research Training Program (R.A.) and the Faculty of Health and Medical Science Divisional Scholarship (R.A.), Adelaide Graduate Research Scholarships (R.A. and N.A.B.), Australia Awards Scholarship (M.D.H.), Robinson Research Institute Career Development Fellowship (K.H.) and Building On Ideas Grant (K.H.), National Health and Medical Research Council of Australia Centre for Research Excellence in the Evaluation, Management and Health Care Needs of Polycystic Ovary Syndrome (N.A.B., M.D.H. and R.J.R.; GTN1078444) and the Centre for Research Excellence on Women’s Health in Reproductive life (R.A., R.J.R. and K.H.; GTN1171592) and the UK Medical Research Council (R.A.A.; grant no. G1100357). The funders did not play any role in the study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors of this manuscript have nothing to declare and no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Transcriptomic Analysis of Fumarate Compounds Identifies Unique Effects of Isosorbide Di-(Methyl Fumarate) on NRF2, NF-kappaB and IRF1 Pathway Genes.

Dimethyl fumarate (DMF) has emerged as a first-line therapy for relapsing-remitting multiple sclerosis (RRMS). This treatment, however, has been limited by adverse effects, which has prompted development of novel derivatives with improved tolerability. We compared the effects of fumarates on gene expression in astrocytes. Our analysis included diroximel fumarate (DRF) and its metabolite monomethyl fumarate (MMF), along with a novel compound isosorbide di-(methyl fumarate) (IDMF). Treatment with IDMF resulted in the largest number of differentially expressed genes. The effects of DRF and MMF were consistent with NRF2 activation and NF-κB inhibition, respectively. IDMF responses, however, were concordant with both NRF2 activation and NF-κB inhibition, and we confirmed IDMF-mediated NF-κB inhibition using a reporter assay. IDMF also down-regulated IRF1 expression and IDMF-decreased gene promoters were enriched with IRF1 recognition sequences. Genes altered by each fumarate overlapped significantly with those near loci from MS genetic association studies, but IDMF had the strongest overall effect on MS-associated genes. These results show that next-generation fumarates, such as DRF and IDMF, have effects differing from those of the MMF metabolite. Our findings support a model in which IDMF attenuates oxidative stress via NRF2 activation, with suppression of NF-κB and IRF1 contributing to mitigation of inflammation and pyroptosis.

The Long Non-Coding RNA Nostrill Regulates Transcription of Irf7 Through Interaction With NF-κB p65 to Enhance Intestinal Epithelial Defense Against Cryptosporidium parvum.

The cells of the intestinal epithelium establish the frontline for host defense against pathogens in the gastrointestinal tract and play a vital role in the initiation of the immune response. Increasing evidence supports the role of long non-coding RNAs (lncRNAs) as critical regulators of diverse cellular processes, however, their role in antimicrobial host defense is incompletely understood. In this study, we provide evidence that the lncRNA Nostrill is upregulated in the intestinal epithelium following infection by Cryptosporidium parvum, a globally prevalent apicomplexan parasite that causes significant diarrheal disease and an important opportunistic pathogen in the immunocompromised and AIDS patients. Induction of Nostrill in infected intestinal epithelial cells was triggered by NF-κB signaling and was observed to enhance epithelial defense by decreasing parasitic infection burden. Nostrill participates in the transcriptional regulation of C. parvum-induced Irf7 expression through interactions with NF-κB p65, and induction of Nostrill promotes epigenetic histone modifications and occupancy of RNA polymerase II at the Irf7 promoter. Our data suggest that the induction of Nostrill promotes antiparasitic defense against C. parvum and enhances intestinal epithelial antimicrobial defense through contributions to transcriptional regulation of immune-related genes, such as Irf7.

Lupus enhancer risk variant causes dysregulation of IRF8 through cooperative lncRNA and DNA methylation machinery

Despite strong evidence that human genetic variants affect the expression of many key transcription factors involved in autoimmune diseases, establishing biological links between non-coding risk variants and the gene targets they regulate remains a considerable challenge. Here, we combine genetic, epigenomic, and CRISPR activation approaches to screen for functional variants that regulate IRF8 expression. We demonstrate that the locus containing rs2280381 is a cell-type-specific enhancer for IRF8 that spatially interacts with the IRF8 promoter. Further, rs2280381 mediates IRF8 expression through enhancer RNA AC092723.1, which recruits TET1 to the IRF8 promoter regulating IRF8 expression by affecting methylation levels. The alleles of rs2280381 modulate PU.1 binding and chromatin state to regulate AC092723.1 and IRF8 expression differentially. Our work illustrates an integrative strategy to define functional genetic variants that regulate the expression of critical genes in autoimmune diseases and decipher the mechanisms underlying the dysregulation of IRF8 expression mediated by lupus risk variants.

SUMOylation inhibition enhances multiple myeloma sensitivity to lenalidomide

Despite the potent effect of lenalidomide (Len) in multiple myeloma (MM) treatment, patients develop Len resistance leading to progressive disease, demanding an urgent need to investigate the mechanisms mediating Len resistance. Our study identified SUMOylation as a potential mechanism regulating Len resistance in MM. Len-resistant MM cell line MMR10R presented much higher SUMO E1 (SAE2) expression and more global SUMOylation than Len-sensitive MM1S cell line. SUMOylation inhibition by using TAK-981, a novel and specific SUMO E1 inhibitor, significantly enhances myeloma sensitivity to Len in MM cell lines. Moreover, the enhanced anti-MM activity by TAK-981 and Len combination has been validated using primary relapsing MM patient samples. Overexpression of IRF4 and c-Myc is a major mechanism of Len resistance. Len showed limited effect on IRF4 and c-Myc level in Len-resistance cell line, but TAK-981 treatment reduced IRF4 and c-Myc expression in Len-resistant line and caused further decrease when combined with Len. We found SUMOylation inhibition decreases IRF4 at transcriptional and post-translational level. SUMOylation inhibition reduced DOT1L with decreased methylation of histone H3 lysine 79, to suppress IRF4 gene transcription. SUMOylation inhibition also reduced IRF4 protein level by enhancing degradation. Overall, our data revealed SUMOylation inhibition enhances Len sensitivity through downregulating IRF4.

RUNX1 inhibits the antiviral immune response against influenza A virus through attenuating type I interferon signaling

BackgroundInfluenza A viruses (IAVs) are zoonotic, segmented negative-stranded RNA viruses. The rapid mutation of IAVs results in host immune response escape and antiviral drug and vaccine resistance. RUNX1 is a transcription factor that not only plays essential roles in hematopoiesis, but also functions as a regulator in inflammation. However, its role in the innate immunity to IAV infection has not been well studied.MethodsTo investigate the effects of RUNX1 on IAV infection and explore the mechanisms that RUNX1 uses during IAV infection. We infected the human alveolar epithelial cell line (A549) with influenza virus A/Puerto Rico/8/34 (H1N1) (PR8) and examined RUNX1 expression by Western blot and qRT-PCR. We also knocked down or overexpressed RUNX1 in A549 cells, then evaluated viral replication by Western blot, qRT-PCR, and viral titration.ResultsWe found RUNX1 expression is induced by IAV H1N1 PR8 infection, but not by poly(I:C) treatment, in the human alveolar epithelial cell line A549. Knockdown of RUNX1 significantly inhibited IAV infection. Conversely, overexpression of RUNX1 efficiently promoted production of progeny viruses. Additionally, RUNX1 knockdown increased IFN-β and ISGs production while RUNX1 overexpression compromised IFN-β and ISGs production upon PR8 infection in A549 cells. We further showed that RUNX1 may attenuate the interferon signaling transduction by hampering the expression of IRF3 and STAT1 during IAV infection.ConclusionsTaken together, we found RUNX1 attenuates type I interferon signaling to facilitate IAV infection in A549 cells.