Physical and functional interaction among Irf8 enhancers during dendritic cell differentiation.

The production of type 1 conventional dendritic cells (cDC1s) requires high expression of the transcription factor IRF8. Three enhancers at the Irf8 3' region function in a differentiation stage-specific manner. However, whether and how these enhancers interact physically and functionally remains unclear. Here, we show that the Irf8 3' enhancers directly interact with each other and contact the Irf8 gene body during cDC1 differentiation. The +56 kb enhancer, which functions from multipotent progenitor stages, activates the other 3' enhancers through an IRF8-dependent transcription factor program, that is, in trans. Then, the +32 kb enhancer, which operates in cDC1-committed cells, reversely acts in cis on the other 3' enhancers to maintain the high expression of Irf8. Indeed, mice with compound heterozygous deletion of the +56 and +32 kb enhancers are unable to generate cDC1s. These results illustrate how multiple enhancers cooperate to induce a lineage-determining transcription factor gene during cell differentiation.

Nicorandil-Pretreated Mesenchymal Stem Cell-Derived Exosomes Facilitate Cardiac Repair After Myocardial Infarction via Promoting Macrophage M2 Polarization by Targeting miR-125a-5p/TRAF6/IRF5 Signaling Pathway.

Background: Exosomes derived from bone marrow mesenchymal stem cells (MSC-exo) have been considered as a promising cell-free therapeutic strategy for ischemic heart disease. Cardioprotective drug pretreatment could be an effective approach to improve the efficacy of MSC-exo. Nicorandil has long been used in clinical practice for cardioprotection. This study aimed to investigate whether the effects of exosomes derived from nicorandil pretreated MSC (MSCNIC-exo) could be enhanced in facilitating cardiac repair after acute myocardial infarction (AMI). Methods: MSCNIC-exo and MSC-exo were collected and injected into the border zone of infarcted hearts 30 minutes after coronary ligation in rats. Macrophage polarization was detected 3 days post-infarction, cardiac function as well as histological pathology were measured on the 28th day after AMI. Macrophages were separated from the bone marrow of rats for in vitro model. Exosomal miRNA sequencing was conducted to identify differentially expressed miRNAs between MSCNIC-exo and MSC-exo. MiRNA mimics and inhibitors were transfected to MSCs or macrophages to explore the specific mechanism. Results: Compared to MSC-exo, MSCNIC-exo showed superior therapeutic effects on cardiac functional and structural recovery after AMI and markedly elevated the ratio of CD68+ CD206+/ CD68+cells in infarcted hearts 3 days post-infarction. The notable ability of MSCNIC-exo to promote macrophage M2 polarization was also confirmed in vitro. Exosomal miRNA sequencing and both in vivo and in vitro experiments identified and verified that miR-125a-5p was an effector of the roles of MSCNIC-exo in vivo and in vitro. Furthermore, we found miR-125a-5p promoted macrophage M2 polarization by inhibiting TRAF6/IRF5 signaling pathway. Conclusion: This study suggested that MSCNIC-exo could markedly facilitate cardiac repair post-infarction by promoting macrophage M2 polarization by upregulating miR-125a-5p targeting TRAF6/IRF5 signaling pathway, which has great potential for clinical translation.

Biophysical and functional characterization of N-terminal domain of Human Interferon Regulatory Factor 6.

BACKGROUND: Interferon regulatory factor 6 (IRF6) has a key function in palate fusion during palatogenesis during embryonic development, and mutations in IRF6 cause orofacial clefting disorders. METHODS AND RESULTS: The in silico analysis of IRF6 is done to obtain leads for the domain boundaries and subsequently the sub-cloning of the N-terminal domain of IRF6 into the pGEX-2TK expression vector and successfully optimized the overexpression and purification of recombinant glutathione S-transferase-fused NTD-IRF6 protein under native conditions. After cleavage of the GST tag, NTD-IRF6 was subjected to protein folding studies employing Circular Dichroism and Intrinsic fluorescence spectroscopy at variable pH, temperature, and denaturant. CD studies showed predominantly alpha-helical content and the highest stability of NTD-IRF6 at pH 9.0. A comparison of native and renatured protein depicts loss in the secondary structural content. Intrinsic fluorescence and quenching studies have identified that tryptophan residues are majorly present in the buried areas of the protein and a small fraction was on or near the protein surface. Upon the protein unfolding with a higher concentration of denaturant urea, the peak of fluorescence intensity decreased and red shifted, confirming that tryptophan residues are majorly present in a more polar environment. While regulating IFNß gene expression during viral infection, the N-terminal domain binds to the promoter region of Virus Response Element-Interferon beta (VRE-IFNß). Along with the protein folding analysis, this study also aimed to identify the DNA-binding activity and determine the binding affinities of NTD-IRF6 with the VRE-IFNß promoter region. The protein-DNA interaction is specific as demonstrated by gel retardation assay and the kinetics of molecular interactions as quantified by Biolayer Interferometry showed a strong affinity with an affinity constant (KD) value of 7.96 × 10-10 M. CONCLUSION: NTD-IRF6 consists of a mix of α-helix and ß-sheets that show temperature-dependent cooperative unfolding between 40 °C and 55 °C. Urea-induced unfolding shows moderate tolerance to urea as the mid-transition concentration of urea (Cm) is 3.2 M. The tryptophan residues are majorly buried as depicted by fluorescence quenching studies. NTD-IRF6 has a specific and high affinity toward the promoter region of VRE-IFNß.

Type I interferon associated epistasis may contribute to early disease-onset and high disease activity in juvenile-onset lupus.

Pathologic type I interferon (T1IFN) expression is a key feature in systemic lupus erythematosus (SLE) that associates with disease activity. When compared to adult-onset disease, juvenile-onset (j)SLE is characterized by increased disease activity and damage, which likely relates to increased genetic burden. To identify T1IFN-associated gene polymorphisms (TLR7, IRAK1, miR-3142/miR-146a, IRF5, IRF7, IFIH1, IRF8, TYK2, STAT4), identify long-range linkage disequilibrium and gene:gene interrelations, 319 jSLE patients were genotyped using panel sequencing. Coupling phenotypic quantitative trait loci (QTL) analysis identified 10 jSLE QTL that associated with young age at onset (<12 years; IRAK1 [rs1059702], TLR7 [rs3853839], IFIH1 [rs11891191, rs1990760, rs3747517], STAT4 [rs3021866], TYK2 [rs280501], IRF8 [rs1568391, rs6638]), global disease activity (SLEDAI-2 K >10; IFIH1 [rs1990760], STAT4 [rs3021866], IRF8 [rs903202, rs1568391, rs6638]), and mucocutaneous involvement (TLR7 [rs3853839], IFIH1 [rs11891191, rs1990760]). This study suggests T1IFN-associated polymorphisms and gene:gene interrelations in jSLE. Genotyping of jSLE patients may allow for individualized treatment and care.

Interferon Regulatory Factor 4 (IRF4) Plays a Key Role in Osteoblast Differentiation of Postmenopausal Osteoporosis.

BACKGROUND: Postmenopausal osteoporosis (PMOP) is a prevalent disease, which features decreased bone mass, bone weakness and deteriorated bone microstructure in postmenopausal women. Although many factors have been revealed to contribute to the occurrence of PMOP, its mechanism remains undefined. This work aimed to identify significant changes in gene expression during PMOP formation and to examine the most valuable differential genes in postmenopausal osteoporosis versus the control group. METHODS: The GSE68303 dataset that contains 12 ovariectomize (OVX) experimental and 11 sham groups was downloaded and analyzed. The results indicated that interferon regulatory factor 4 (IRF4) might be a hub gene in the development of postmenopausal osteoporosis. Western blot and immunohistochemistry were carried out to evaluate IRF4 levels in thoracic vertebra extracts from OVX and Sham mice. To assess IRF4's impact on osteogenic differentiation in postmenopausal bone marrow mesenchymal stem cells (BM-MSCs), IRF4 overexpression (OV-IRF4) and knockdown (Sh-IRF4) plasmids were constructed. RESULTS: The results showed that comparing with the sham group, bone samples from the OVX group showed higher IRF4 expression. Alkaline phosphatase (ALP) staining revealed that IRF4 overexpression significantly inhibited ALP activity, while IRF4 knockdown promoted ALP activity in BM-MSCs. Simvastatin-treated OVX mice showed increased total bone volume/total tissue volume (BV/TV) and elevated Runx2 expression by immunohistochemical staining compared with the OVX group. CONCLUSIONS: This study demonstrated that IRF4 is associated with OVX induced osteoporosis, it can regulate bone stability by inhibiting the osteogenic differentiation BM-MSCs. This study may help enhance our understanding of the molecular mechanism of PMOP formation, providing new insights into estrogen defiance induced osteoporosis.

Review on the Role of IRF6 in the Pathogenesis of Non-syndromic Orofacial Clefts.

Non-syndromic orofacial clefts (NSOCs) are the most common craniofacial malformation. In the complex aetiology and pathogenesis of NSOCs, genetic factors play a crucial role and IRF6, located at chromosome 1q32.2, is the best documented NSOC susceptibility gene. IRF6 is a key factor in oral maxillofacial development and known to contribute the most in NSOCs. It is essential to conduct a complete review of the existing results on IRF6 to further understand its role in the pathogenesis of NSOCs. Thus, the present authors summarised the research progress on the mechanism of IRF6 in NSOCs from both genetic and functional perspectives in this review.

The immunostimulatory activity of sea spray aerosols: bacteria and endotoxins activate TLR4, TLR2/6, NF-κB and IRF in human cells.

Frequent exposure to sea spray aerosols (SSA) containing marine microorganisms and bioactive compounds may influence human health. However, little is known about potential immunostimulation by SSA exposure. This study focuses on the effects of marine bacteria and endotoxins in SSA on several receptors and transcription factors known to play a key role in the human innate immune system. SSA samples were collected in the field (Ostend, Belgium) or generated in the lab using a marine aerosol reference tank (MART). Samples were characterized by their sodium contents, total bacterial counts, and endotoxin concentrations. Human reporter cells were exposed to SSA to investigate the activation of toll-like receptor 4 (TLR4) in HEK-Blue hTLR4 cells and TLR2/6 in HEK-Blue hTLR2/6 cells, as well as the activation of nuclear factor kappa B (NF-κB) and interferon regulatory factors (IRF) in THP1-Dual monocytes. These responses were then correlated to the total bacterial counts and endotoxin concentrations to explore dose-effect relationships. Field SSA contained from 3.0 × 103 to 6.0 × 105 bacteria/m3 air (averaging 2.0 ± 1.9 × 105 bacteria/m3 air) and an endotoxin concentration ranging from 7 to 1217 EU/m3 air (averaging 389 ± 434 EU/m3 air). In contrast, MART SSA exhibited elevated levels of total bacterial count (from 2.0 × 105 to 2.4 × 106, averaging 7.3 ± 5.5 × 105 cells/m3 air) and endotoxin concentration from 536 to 2191 (averaging 1310 ± 513 EU/m3 air). SSA samples differentially activated TLR4, TLR2/6, NF-κB and IRF. These immune responses correlated dose-dependently with the total bacterial counts, endotoxin levels, or both. This study sheds light on the immunostimulatory potential of SSA and its underlying mechanisms, highlighting the need for further research to deepen our understanding of the health implications of SSA exposure.

IRF4-mediated Treg phenotype switching can aggravate hyperoxia-induced alveolar epithelial cell injury.

Bronchopulmonary dysplasia (BPD) is characterized by alveolar dysplasia, and evidence indicates that interferon regulatory factor 4 (IRF4) is involved in the pathogenesis of various inflammatory lung diseases. Nonetheless, the significance and mechanism of IRF4 in BPD remain unelucidated. Consequently, we established a mouse model of BPD through hyperoxia exposure, and ELISA was employed to measure interleukin-17 A (IL-17 A) and interleukin-6 (IL-6) expression levels in lung tissues. Western blotting was adopted to determine the expression of IRF4, surfactant protein C (SP-C), and podoplanin (T1α) in lung tissues. Flow cytometry was utilized for analyzing the percentages of FOXP3+ regulatory T cells (Tregs) and FOXP3+RORγt+ Tregs in CD4+ T cells in lung tissues to clarify the underlying mechanism. Our findings revealed that BPD mice exhibited disordered lung tissue structure, elevated IRF4 expression, decreased SP-C and T1α expression, increased IL-17 A and IL-6 levels, reduced proportion of FOXP3+ Tregs, and increased proportion of FOXP3+RORγt+ Tregs. For the purpose of further elucidating the effect of IRF4 on Treg phenotype switching induced by hyperoxia in lung tissues, we exposed neonatal mice with IRF4 knockout to hyperoxia. These mice exhibited regular lung tissue structure, increased proportion of FOXP3+ Tregs, reduced proportion of FOXP3+RORγt+ Tregs, elevated SP-C and T1α expression, and decreased IL-17 A and IL-6 levels. In conclusion, our findings demonstrate that IRF4-mediated Treg phenotype switching in lung tissues exacerbates alveolar epithelial cell injury under hyperoxia exposure.

Identification and validation of IRF6 related to ovarian cancer and biological function and prognostic value.

BACKGROUND: Ovarian cancer (OC) is a severe gynecological malignancy with significant diagnostic and therapeutic challenges. The discovery of reliable cancer biomarkers can be used to adjust diagnosis and improve patient care. However, serous OC lacks effective biomarkers. We aimed to identify novel biomarkers for OC and their pathogenic causes. METHODS: The present study used the differentially expressed genes (DEGs) obtained from the "Limma" package and WGCNA modules for intersection analysis to obtain DEGs in OC. Three hub genes were identified-claudin 3 (CLDN3), interferon regulatory factor 6 (IRF6), and prostasin (PRSS8)-by searching for hub genes through the PPI network and verifying them in GSE14407, GSE18520, GSE66957, and TCGA + GTEx databases. The correlation between IRF6 and the prognosis of OC patients was further confirmed in Kaplan-Miller Plotter. RT-qPCR and IHC confirmed the RNA and protein levels of IRF6 in the OC samples. The effect of IRF6 on OC was explored using transwell invasion and scratch wound assays. Finally, we constructed a ceRNA network of hub genes and used bioinformatics tools to predict drug sensitivity. RESULTS: The joint analysis results of TCGA, GTEx, and GEO databases indicated that IRF6 RNA and protein levels were significantly upregulated in serous OC and were associated with OS and PFS. Cell function experiments revealed that IRF6 knockdown inhibited SKOV3 cell proliferation, migration and invasion. CONCLUSION: IRF6 is closely correlated with OC development and progression and could be considered a novel biomarker and therapeutic target for OC patients.

WTAP Mediated N6-methyladenosine RNA Modification of ELF3 Drives Cellular Senescence by Upregulating IRF8.

N6-methyladenosine (m6A), the most prevalent posttranscriptional RNA modification, involved in various diseases and cellular processes. However, the underlying mechanisms of m6A regulation in skin aging are still not fully understood. In this study, proteomics analysis revealed a significant correlation between Wilms' tumor 1-associating protein (WTAP) expression and cellular senescence. Next, upregulated WTAP was detected in aging skin tissues and senescent human dermal fibroblasts (HDFs). Functionally, overexpressed WTAP induced senescence and knockdown of WTAP rescued senescence of HDFs. Mechanistically, WTAP directly targeted ELF3 and promoted its expression in an m6A-dependent manner. Exogenous-ELF3 overexpression evidently reversed shWTAP-suppressed fibroblast senescence. Furthermore, ELF3 induced IRF8-mediated senescence-associated secretory phenotype (SASP) by binding to the (-817 to -804) site of the IRF8 promoter directly. In vivo, overexpression of WTAP evidently increased senescence cells in skin and induced skin aging. In summary, these findings revealed the critical role of WTAP-mediated m6A modification in skin aging and identified ELF3 as an important target of m6A modification in HDFs senescence, providing a new idea for delaying the aging process.

Assessing Interferon Regulatory Factor 4 Complex Formation: Differential Behavior of Homocomplexes Versus Heterocomplexes Induced by Mutations.

Interferon regulatory factor 4 (IRF4) is a crucial transcription factor that plays a vital role in lymphocyte development, including in the fate-determining steps in terminal differentiation. It is also implicated in the development of lymphoid tumors such as multiple myeloma and adult T-cell leukemia. IRF4 can form a homodimer and multiple heterocomplexes with other transcription factors such as purine-rich box1 and activator protein 1. Each protein complex binds to specific DNA sequences to regulate a distinct set of genes. However, the precise relationship among these complex formations remains unclear. Herein, we investigated the abilities of IRF4 proteins with functional mutations in the IRF-association domain and autoinhibitory region to form complexes using luciferase reporter assays. The assays allowed us to selectively assess the activity of each complex. Our results revealed that certain IRF-association domain mutants, previously known to have impaired heterocomplex formation, maintained or even enhanced homodimer activity. This discrepancy suggests that the mutated amino acid residues selectively influence homodimer activity. Conversely, a phosphomimetic serine mutation in the autoinhibitory region displayed strong activating effects in all complexes. Furthermore, we observed that partner proteins involved in heterocomplex formation could disrupt the activity of the homodimer, suggesting a potential competition between homocomplexes and heterocomplexes. Our findings provide new insights into the mechanistic function of IRF4.

Murine IRF8 Mutation Offers New Insight into Osteoclast and Root Resorption.

Interferon regulatory factor 8 (IRF8), a transcription factor expressed in immune cells, functions as a negative regulator of osteoclasts and helps maintain dental and skeletal homeostasis. Previously, we reported that a novel mutation in the IRF8 gene increases susceptibility to multiple idiopathic cervical root resorption (MICRR), a form of tooth root resorption mediated by increased osteoclast activity. The IRF8 G388S variant in the highly conserved C-terminal motif is predicted to alter the protein structure, likely impairing IRF8 function. To investigate the molecular basis of MICRR and IRF8 function in osteoclastogenesis, we generated Irf8 knock-in (KI) mice using CRISPR/Cas9 technique modeling the human IRF8G388S mutation. The heterozygous (Het) and homozygous (Homo) Irf8 KI mice showed no gross morphological defects, and the development of hematopoietic cells was unaffected and similar to wild-type (WT) mice. The Irf8 KI Het and Homo mice showed no difference in macrophage gene signatures important for antimicrobial defenses and inflammatory cytokine production. Consistent with the phenotype observed in MICRR patients, Irf8 KI Het and Homo mice demonstrated significantly increased osteoclast formation and resorption activity in vivo and in vitro when compared to WT mice. The oral ligature-inserted Het and Homo mice displayed significantly increased root resorption and osteoclast-mediated alveolar bone loss compared to WT mice. The increased osteoclastogenesis noted in KI mice is due to the inability of IRF8G388S mutation to inhibit NFATc1-dependent transcriptional activation and downstream osteoclast specific transcripts, as well as its impact on autophagy-related pathways of osteoclast differentiation. This translational study delineates the IRF8 domain important for osteoclast function and provides novel insights into the IRF8 mutation associated with MICRR. IRF8G388S mutation mainly affects osteoclastogenesis while sparing immune cell development and function. These insights extend beyond oral health and significantly advance our understanding of skeletal disorders mediated by increased osteoclast activity and IRF8's role in osteoclastogenesis.

Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions.

Many protein-protein interactions involve the binding of short protein segments to peptide-binding domains. Usually, such interactions require the recognition of linear motifs with variable conservation. The combination of highly conserved and more variable regions in the same ligands often contributes to the multispecificity of binding, a common property of enzymes and cell signaling proteins. Characterization of amino acid preferences of peptide-binding domains is important for the design of mediators of protein-protein interactions (PPIs). Computational methods are an efficient alternative to the often costly and cumbersome experimental techniques, enabling the design of potential mediators that can be later validated in downstream experiments. Here, we described a methodology using the Pepspec application of the Rosetta molecular modeling package to predict the amino acid preferences of peptide-binding domains. This methodology is useful when the structure of the receptor protein and the nature of the peptide ligand are both known or can be inferred. The methodology starts with a well-characterized anchor from the ligand, which is extended by randomly adding amino acid residues. The binding affinity of peptides generated this way is then evaluated by flexible-backbone peptide docking in order to select the peptides with the best predicted binding scores. These peptides are then used to calculate amino acid preferences and to optionally compute a position-weight matrix (PWM) that can be used in further studies. To illustrate the application of this methodology, we used the interaction between subunits of human interferon regulatory factor 5 (IRF5), previously known to be multispecific but globally guided by a short conserved motif called pLxIS. The estimated amino acid preferences were consistent with previous knowledge about the IRF5 binding surface. Positions occupied by phosphorylatable serine residues exhibited a high frequency of aspartate and glutamate, likely because their negatively charged side chains are similar to phosphoserine.

The IRAK1/IRF5 axis initiates IL-12 response by dendritic cells and control of Toxoplasma gondii infection.

Activation of endosomal Toll-like receptor (TLR) 7, TLR9, and TLR11/12 is a key event in the resistance against the parasite Toxoplasma gondii. Endosomal TLR engagement leads to expression of interleukin (IL)-12 via the myddosome, a protein complex containing MyD88 and IL-1 receptor-associated kinase (IRAK) 4 in addition to IRAK1 or IRAK2. In murine macrophages, IRAK2 is essential for IL-12 production via endosomal TLRs but, surprisingly, Irak2-/- mice are only slightly susceptible to T. gondii infection, similar to Irak1-/- mice. Here, we report that upon T. gondii infection IL-12 production by different cell populations requires either IRAK1 or IRAK2, with conventional dendritic cells (DCs) requiring IRAK1 and monocyte-derived DCs (MO-DCs) requiring IRAK2. In both populations, we identify interferon regulatory factor 5 as the main transcription factor driving the myddosome-dependent IL-12 production during T. gondii infection. Consistent with a redundant role of DCs and MO-DCs, mutations that affect IL-12 production in both cell populations show high susceptibility to infection in vivo.

Interferon regulatory factors inhibit TiLV replication by activating interferon-a3 in tilapia (Oreochromis niloticus).

Tilapia lake virus (TiLV) is an emerging virus that seriously threatens the tilapia industries worldwide. Interferon regulatory factors (IRFs), which are the crucial mediators regulating the response of interferon (IFN) to combat invading viruses, have not yet been reported in tilapia during TiLV infection. Here, six IRF (IRF1, IRF2, IRF4, IRF7, IRF8, and IRF9) homologs from tilapia were characterized and analyzed. These IRFs typically shared the conserved domains and phylogenetic relationship with IRF homologs of other species. Tissue distribution analysis showed that all six IRF genes were expressed in various tissues, with the highest expression in immune-related tissues. Furthermore, overexpression of IRFs in tilapia brain (TiB) cells significantly inhibited TiLV propagation, as evidenced by decreased viral segment 8 gene transcripts and copy numbers of viral segment 1. More importantly, all six IRF genes significantly enhanced the promoter activity of type I interferon-a3 (IFNa3) in TiB cells, suggesting that tilapia IRF genes serve as positive regulators in activating IFNa3. Surprisingly, the promoter activity of IFNa3 mediated by IRF genes was markedly inhibited post-TiLV infection, indicating that TiLV antagonized IRF-mediated IFN immune response. Taken together, six IRF genes of tilapia are highly conserved transcription factors that inhibit TiLV infection by activating the promoter of IFNa3, which is in turn restrained by TiLV. These findings broaden our knowledge about the functionality of IRF-mediated antiviral immunity in tilapia against TiLV infection and host-TiLV interaction, which lays a foundation for developing antiviral strategies in tilapia cultural industries.

UBE2J1 promotes ALV-A proviral DNA synthesis through the STAT3/IRF1 signaling pathway.

The ubiquitin-binding enzyme E2J1 is located on the endoplasmic reticulum membrane. It plays a role in transport throughout the process of ubiquitination. In mammals, UBE2J1 can promote RNA virus replication. However, the biological function of chicken UBE2J1 is unclear. In this study, chicken UBE2J1 was cloned for the first time, and UBE2J1 overexpression and shRNA knockdown plasmids were constructed. In chicken embryo fibroblasts, overexpression of UBE2J1 promoted the replication of subtype A avian leukosis virus, while knockdown of UBE2J1 inhibited the replication of ALV-A virus. In addition, we divided virus replication into virus adsorption and invasion into DF-1 cells, synthesis of proviral DNA, and release of viral particles. UBE2J1 promoted the replication of ALV-A virus by promoting the synthesis of proviral DNA. This result was caused by UBE2J1 inhibiting the production of interferon by inhibiting the STAT3/IRF1 pathway. We mutated ser at position 184 of UBE2J1 to Gly and found that this site plays a role as the phosphorylation site of UBE2J1. We confirmed that UBE2J1 promotes ALV-A replication in chicken embryo fibroblasts by inhibiting the STAT3/IRF1 pathway. This study provides new ideas and insights into ubiquitin-related proteins and antiviral immunity.

DNA hypomethylation ameliorates erosive inflammatory arthritis by modulating interferon regulatory factor-8.

Epigenetic regulation plays a crucial role in the pathogenesis of autoimmune diseases such as inflammatory arthritis. DNA hypomethylating agents, such as decitabine (DAC), have been shown to dampen inflammation and restore immune homeostasis. In the present study, we demonstrate that DAC elicits potent anti-inflammatory effects and attenuates disease symptoms in several animal models of arthritis. Transcriptomic and epigenomic profiling show that DAC-mediated hypomethylation regulates a wide range of cell types in arthritis, altering the differentiation trajectories of anti-inflammatory macrophage populations, regulatory T cells, and tissue-protective synovial fibroblasts (SFs). Mechanistically, DAC-mediated demethylation of intragenic 5'-Cytosine phosphate Guanine-3' (CpG) islands of the transcription factor Irf8 (interferon regulatory factor 8) induced its re-expression and promoted its repressor activity. As a result, DAC restored joint homeostasis by resetting the transcriptomic signature of negative regulators of inflammation in synovial macrophages (MerTK, Trem2, and Cx3cr1), TREGs (Foxp3), and SFs (Pdpn and Fapα). In conclusion, we found that Irf8 is necessary for the inhibitory effect of DAC in murine arthritis and that direct expression of Irf8 is sufficient to significantly mitigate arthritis.

[Correlation between methylation of interferon regulatory factor 6 gene promoter in renal tissues and overall survival of patients with Kidney renal clear cell carcinoma].

OBJECTIVE: To assess the prognostic value of methylation of interferon regulatory factor 6 (IRF6) gene promoter in patients diagnosed with Kidney renal clear cell carcinoma (KIRC). METHODS: The primary lesions of fifty KIRC patients who were diagnosed at the First Affiliated Hospital of Nanjing Medical University from January 2016 to January 2020 were collected. The expression of IRF6 protein was determined with an immunohistochemical method. The correlation between the level of IRF6 expression and survival and/or metastasis status was analyzed. The mRNA and protein levels of the IRF6 in KIRC and normal renal tissues were compared by using bioinformatic tools. The difference in the methylation rate of the IRF6 gene promoter between tumor and adjacent tissues was analyzed by searching the online databases. Statistical analysis was carried out for the methylation status of the IRF6 gene promoter region to select those negatively correlated with the overall survival (OS) among the patients. In vitro experiments were conducted with cell lines to verify the correlation between the status of promoter methylation and transcription level of the IRF6 gene. RESULTS: The mRNA and protein levels of the IRF6 gene in KIRC tissues were significantly lower than those of the normal controls, and this was more prominent in patients who had died or developed metastasis. The extent of IRF6 gene promoter methylation in the KIRC tissues was much higher compared with that of the adjacent normal renal tissues. There was a significant negative correlation between the methylation of the IRF6 gene promoter and mRNA level of the IRF6 (R = -0.52). The higher methylation degree in the IRF6 gene promoter regions cg12034118 and cg16030177, the shorter the OS and worse prognosis in the patients. Only twenty CpG sites in cg12034118 were confirmed to be highly methylated in KIRC cell lines. The transcription level of the IRF6 gene was upregulated in a time- and dose-dependent manner after the treatment with demethylation reagent 5-azadeoxycytidine. CONCLUSION: The methylation of IRF6 gene promoter in the renal tissues of KIRC patients is closely correlated with the OS. Cg12034118 may provide a promising biomarker for laboratory detection, and its high methylation rate has certain reference value for the prognosis.

IRF4 induces M1 macrophage polarization and aggravates ulcerative colitis progression by the Bcl6-dependent STAT3 pathway.

Ulcerative colitis (UC) is an idiopathic chronic intestinal inflammation. An increasing body of evidence shows that macrophages play an important role in the pathogenesis of UC. Interferon regulatory factor 4 (IRF4) is crucial for the development of autoimmune diseases via regulating immune cells. This research was designed to explore the function of IRF4 in UC and its association with macrophage polarization. The in vitro model of UC was established by stimulating colonic epithelial cells with tumor necrosis factor α (TNF-α). A mouse model of UC was constructed by injecting C57BL/6 mice with dextran sulfate sodium salt. Flow cytometry was used to assess percentage of CD11b+ CD86+ and CD11b+ CD206+ cells in bone marrow macrophages. Occult blood tests were used to detect hematochezia. Hematoxylin and eosin staining assay was used to assess colon pathological changes. Enzyme-linked immunosorbent assay (ELISA) was used to detect concentrations of inflammatory cytokines. The interaction of IRF4 and B-cell lymphoma 6 (Bcl6) was confirmed using GST pull-down and coimmunoprecipitation assays. Our findings revealed that IRF4 promoted cell apoptosis and stimulated M1 macrophage polarization in vitro. Furthermore, IRF4 aggravated symptoms of the mouse model of UC and aggravated M1 macrophage polarization in vivo. IRF4 negatively regulated Bcl6 expression. Downregulation of Bcl6 promoted apoptosis and M1 macrophage polarization in the presence of IRF4 in vitro and in vivo. Moreover, Bcl6 positively mediated the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. In conclusion, IRF4 aggravated UC progression through promoting M1 macrophage polarization via Bcl6/JAK2/STAT3 pathway. These findings suggested that IRF4 might be a good target to competitively inhibit or to treat with UC.

GWAS for systemic sclerosis identifies six novel susceptibility loci including one in the Fcγ receptor region.

Here we report the largest Asian genome-wide association study (GWAS) for systemic sclerosis performed to date, based on data from Japanese subjects and comprising of 1428 cases and 112,599 controls. The lead SNP is in the FCGR/FCRL region, which shows a penetrating association in the Asian population, while a complete linkage disequilibrium SNP, rs10917688, is found in a cis-regulatory element for IRF8. IRF8 is also a significant locus in European GWAS for systemic sclerosis, but rs10917688 only shows an association in the presence of the risk allele of IRF8 in the Japanese population. Further analysis shows that rs10917688 is marked with H3K4me1 in primary B cells. A meta-analysis with a European GWAS detects 30 additional significant loci. Polygenic risk scores constructed with the effect sizes of the meta-analysis suggest the potential portability of genetic associations beyond populations. Prioritizing the top 5% of SNPs of IRF8 binding sites in B cells improves the fitting of the polygenic risk scores, underscoring the roles of B cells and IRF8 in the development of systemic sclerosis. The results also suggest that systemic sclerosis shares a common genetic architecture across populations.