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Interferon regulatory factor (IRF) 3 is a transcription factor that binds the interferon-sensitive response element (ISRE) and is activated by Toll-like receptor 3 (TLR3) and TLR4. We have found that a dominant negative form of I kappa B kinase 2 and a mutant form of I kappa B, which acts as a super-repressor of NF-kappa B, blocked activation of the ISRE by the TLR4 ligand lipopolysaccharide but not the TLR3 ligand poly(I-C). TLR4 failed to activate the ISRE in mouse embryonic fibroblasts bearing a targeted deletion of p65, whereas the response to TLR3 in these cells was normal. The p65 subunit of NF-kappa B was detected in the lipopolysaccharide-activated but not poly(I-C)-activated ISRE-binding complex. Finally, p65 promoted transactivation of gene expression by IRF-3. These results therefore indicate that IRF-3-mediated activation of the ISRE by TLR4 but not TLR3 requires the p65 subunit of NF-kappa B.

Background: Fetal sex and placental development impact pregnancy outcomes and fetal–maternal health, but the critical timepoint of placenta establishment in first trimester is understudied in human pregnancies. Methods: Pregnant subjects were recruited in late first trimester (weeks 10–14) at time of chorionic villus sampling, a prenatal diagnostic test. Leftover placenta tissue was collected and stored until birth outcomes were known, then DNA and RNA were isolated from singleton, normal karyotype pregnancies resulting in live births. DNA methylation was measured with the Illumina Infinium MethylationEPIC BeadChip array (n = 56). Differential methylation analysis compared 25 females versus 31 males using a generalized linear model on 743,461 autosomal probes. Gene expression sex differences were analyzed with RNA-sequencing (n = 74). An integrated analysis was performed using linear regression to correlate gene expression and DNA methylation in 51 overlapping placentas. Results: Methylation analysis identified 151 differentially methylated probes (DMPs) significant at false discovery rate < 0.05, including 89 (59%) hypermethylated in females. Probe cg17612569 (GABPA, ATP5J) was the most significant CpG site, hypermethylated in males. There were 11 differentially methylated regions affected by fetal sex, with transcription factors ZNF300 and ZNF311 most significantly hypermethylated in males and females, respectively. RNA-sequencing identified 152 genes significantly sexually dimorphic at false discovery rate < 0.05. The 151 DMPs were associated with 18 genes with gene downregulation (P < 0.05) in the direction of hypermethylation, including 2 genes significant at false discovery rate < 0.05 (ZNF300 and CUB and Sushi multiple domains 1, CSMD1). Both genes, as well as Family With Sequence Similarity 228 Member A (FAM228A), showed significant correlation between DNA methylation and sexually dimorphic gene expression, though FAM228A DNA methylation was less sexually dimorphic. Comparison with other sex differences studies found that cg17612569 is male-hypermethylated across gestation in placenta and in human blood up to adulthood. Conclusions: Overall, sex dimorphic differential methylation with associated differential gene expression in the first trimester placenta is small, but there remain significant genes that may be regulated through methylation leading to differences in the first trimester placenta. Plain language summary: Fetal sex and placenta development affect pregnancy outcomes for both the fetus and mother throughout pregnancy, including risk of miscarriages, preterm birth, preeclampsia, and other outcomes. Epigenetics, the "overlay" of regulatory signals on DNA which affects how DNA is read, is not well understood in early pregnancy when critical placenta developments are happening that affect the rest of pregnancy. Here, we use leftover placenta biopsy samples (n = 56) donated by Cedars-Sinai patients with informed consent to learn about first trimester human placenta DNA methylation differences due to fetal sex. Out of the total 743,461 sites analyzed, we identified 151 sites significantly affected by fetal sex after correcting p-values to reduce false positives (false discovery rate < 0.05). We also performed an analysis to look at multiple sites and identified 11 regions across the genome with significant DNA methylation changes due to fetal sex. Furthermore, because DNA methylation is a regulatory mark on DNA which typically dampens gene expression, we also compared the DNA methylation sex differences to placental RNA-sequencing gene expression analysis using the same tissue from a mostly overlapping patient group (n = 74 total sequenced, n = 51 overlap). We identify 18 genes which show both significant DNA methylation differences and gene expression changes. The most significant gene was transcription factor ZNF300 with higher DNA methylation in males and reduced gene expression in males (and thus higher gene expression in females). This study identifies some sex differences that continue until later pregnancy and others that are unique to first trimester. Highlights: We identify sex differences in first trimester human placenta DNA methylation (n = 56) and total RNA-sequencing (n = 74), with a sample overlap of n = 51. At late first trimester, there are 151 differentially methylated probes (DMPs), significantly different between female and male placenta at false discovery rate < 0.05. These encompass 11 differentially methylated regions. The gene with placental DNA methylation most affected by fetal sex is transcription factor ZNF300, hypermethylated in males, and with significantly female upregulated gene expression in first trimester placenta, consistent with previous third trimester studies. In addition to ZNF300, other genes in significantly differentially methylated regions at first trimester include GABPA/ATP5J (hypermethylated in males); ZNF311, CCDC178, ATP5G2, FOXG1 (females); ZNF175 (males); SGCE (females); C6orf47-AS1/C6orf47, REC8, and FOXA1 (females). We identify 18 genes with significant fetal sex-associated changes in both placenta DNA methylation and placenta gene expression. The most significant are ZNF300, tumor suppressor CSMD1, and transcription factor ZNF311. [ABSTRACT FROM AUTHOR]

Regulation of IFN signaling is critical in host recognition and response to pathogens while its dysregulation underlies the pathogenesis of several chronic diseases. STimulator of IFN Genes (STING) has been identified as a critical mediator of IFN inducing innate immune pathways, but little is known about direct coregulators of this protein. We report here that TMEM203, a conserved putative transmembrane protein, is an intracellular regulator of STING-mediated signaling. We show that TMEM203 interacts, functionally cooperates, and comigrates with STING following cell stimulation, which in turn leads to the activation of the kinase TBK1, and the IRF3 transcription factor. This induces target genes in macrophages, including IFN-β. Using Tmem203 knockout bone marrow-derived macrophages and transient knockdown of TMEM203 in human monocyte-derived macrophages, we show that TMEM203 protein is required for cGAMPinduced STING activation. Unlike STING, TMEM203 mRNA levels are elevated in T cells from patients with systemic lupus erythematosus, a disease characterized by the overexpression of type I interferons. Moreover, TMEM203 mRNA levels are associated with disease activity, as assessed by serum levels of the complement protein C3. Identification of TMEM203 sheds light into the control of STING-mediated innate immune responses, providing a potential novelmechanism for therapeutic interventions in STING-associated inflammatory diseases. [ABSTRACT FROM AUTHOR]

Transcription of the type I IFN genes is regulated by members of the Interferon Regulatory Factor (IRF) family of transcription factors, composed in humans of 9 distinct proteins. In addition to IRF3 and IRF7, the transcription factor IRF5 has been shown to be involved in type I IFN production and interestingly, polymorphisms of the IRF5 gene in humans can result in risk or protective haplotypes with regard to SLE susceptibility. In addition to regulation of type I IFN expression, IRF5 is involved in other signaling pathways, including IgG switching in B cells, macrophage polarization and apoptosis, and its role in SLE pathogenesis may therefore not be limited to dysregulated control of IFN expression. In this review we will comprehensively discuss the role of IRF5 in immune-mediated responses and its potential multifaceted role in conferring SLE susceptibility. [ABSTRACT FROM AUTHOR]

IRF5 is a member of the Interferon Regulatory Factor (IRF) family of transcription factors activated downstream of the Toll-Like receptors (TLRs). Polymorphisms in IRF5 have been shown to be associated with the autoimmune disease Systemic Lupus Erythematosus (SLE) and other autoimmune conditions, suggesting a central role for IRF5 in the regulation of the immune response. Four different IRF5 isoforms originate due to alternative splicing and to the presence or absence of a 30 nucleotide insertion in IRF5 exon 6. Since the polymorphic region disturbs a PEST domain, a region associated with protein degradation, we hypothesized that the isoforms bearing the insertion might have increased stability, thus explaining the association of individual IRF5 isoforms with SLE. As the E3 ubiquitin ligase TRIpartite Motif 21 (TRIM21) has been shown to regulate the stability and hence activity of members of the IRF family, we investigated whether IRF5 is subjected to regulation by TRIM21 and whether dysregulation of this mechanism could explain the association of IRF5 with SLE. Our results show that IRF5 is degraded following TLR7 activation and that TRIM21 is involved in this process. Comparison of the individual IRF5 variants demonstrates that isoforms generated by alternative splicing are resistant to TRIM21-mediated degradation following TLR7 stimulation, thus providing a functional link between isoforms expression and stability/activity which contributes to explain the association of IRF5 with SLE. [ABSTRACT FROM AUTHOR]

Neuronal survival and plasticity critically depend on constitutive activity of the transcription factor nuclear factor-kB (NF-kB). We here describe a role for a small intracellular fibroblast growth factor homologue, the fibroblast growth factor homologous factor 1 (FHF1/ FGF12), in the regulation of NF-kB activity in mature neurons. FHFs have previously been described to control neuronal excitability, and mutations in FHF isoforms give rise to a form of progressive spinocerebellar ataxia. Using a protein-array approach, we identified FHF1b as a novel interactor of the canonical NF-kB modulator IKKγ/NEMO. Co-immunoprecipitation, pull-down and GAL4-reporter experiments, as well as proximity ligation assays, confirmed the interaction of FHF1 and NEMO and demonstrated that a major site of interaction occurred within the axon initial segment. Fhf1 gene silencing strongly activated neuronal NF-kB activity and increased neurite lengths, branching patterns and spine counts in mature cortical neurons. The effects of FHF1 on neuronal NF-kB activity and morphology required the presence of NEMO. Our results imply that FHF1 negatively regulates the constitutive NF-kB activity in neurons. [ABSTRACT FROM AUTHOR]

Patients suffering from Systemic Lupus Erythematous (SLE) have elevated type I interferon (IFN) levels which correlate with disease activity and severity. TRIM21, an autoantigen associated with SLE, has been identified as an ubiquitin E3 ligase that targets the transcription factor IRF3 in order to turn off and limit type I IFN production following detection of viral and bacterial infection by Toll Like Receptors (TLRs). However, how the activity of TRIM21 is regulated downstream of TLRs is unknown. In this study we demonstrate that TRIM21 is tyrosine phosphorylated following TLR3 and TLR4 stimulation, suggesting that its activity is potentially regulated by tyrosine phosphorylation. Using Netphos, we have identified three key tyrosines that are strongly predicted to be phosphorylated, two of which are conserved between the human and murine forms of TRIM21, at residues 343, 388, and 393, all of which have been mutated from tyrosine to phenylalanine (Y343F, Y388F, and Y393F). We have observed that tyrosine phosphorylation of TRIM21 only occurs in the substrate binding PRY/ SPRY domain, and that Y393, and to a lesser extent, Y388 are required for TRIM21 to function as a negative regulator of IFNb promoter activity. Further studies revealed that mutating Y393 to phenylalanine inhibits the ability of TRIM21 to interact with its substrate, IRF3, thus providing a molecular explanation for the lack of activity of Y393 on the IFN-b promoter. Our data demonstrates a novel role for tyrosine phosphorylation in regulating the activity of TRIM21 downstream of TLR3 and TLR4. Given the pathogenic role of TRIM21 in systemic autoimmunity, these findings have important implications for the development of novel therapeutics. [ABSTRACT FROM AUTHOR]

The concept that viral sensing systems, via their ability to drive pro-inflammatory cytokine and interferon production, contribute to the development of autoimmune and autoinflammatory disease is supported by a wide range of clinical and experimental observations. Recently, the tripartite motif-containing proteins (TRIMs) have emerged as having key roles in antiviral immunity - either as viral restriction factors or as regulators of pathways downstream of viral RNA and DNA sensors, and the inflammasome. Given their involvement in these pathways, we propose that TRIM proteins contribute to the development and pathology of autoimmune and autoinflammatory conditions, thus making them potential novel targets for therapeutic manipulation. [ABSTRACT FROM AUTHOR]

Ro52 is a member of the TRIM family of single-protein E3 ligases and is also a target for autoantibody production in systemic lupus erythematosus and Sjögren's syndrome. We previously demonstrated a novel function of Ro52 in the ubiquitination and proteasomal degradation of IRF3 following TLR3/4 stimulation. We now present evidence that Ro52 has a similar role in regulating the stability and activity of IRF7. Endogenous immunoprecipitation of Ro52-bound proteins revealed that IRF7 associates with Ro52, an effect which increases following TLR7 and TLR9 stimulation, suggesting that Ro52 interacts with IRF7 post-pathogen recognition. Furthermore, we show that Ro52 ubiquitinates IRF7 in a dose-dependent manner, resulting in a decrease in total IRF7 expression and a subsequent decrease in IFN-a production. IRF7 stability was increased in bone marrow-derived macrophages from Ro52-deficient mice stimulated with imiquimod or CpG-B, consistent with a role for Ro52 in the negative regulation of IRF7 signalling. Taken together, these results suggest that Ro52-mediated ubiquitination promotes the degradation of IRF7 following TLR7 and TLR9 stimulation. As Ro52 is known to be IFN-inducible, this system constitutes a negative-feedback loop that acts to protect the host from the prolonged activation of the immune response. [ABSTRACT FROM AUTHOR]