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Abstract Background Patients with lung adenocarcinoma (LUAD) have a low response rate to immune checkpoint blockade. It is highly important to explore the tumor immune escape mechanism of LUAD patients and expand the population of patients who may benefit from immunotherapy. Methods Based on 954 bulk RNA-seq data of LUAD patients and 15 single-cell RNA-seq data, the relationships between tumor immune dysfunction and exclusion (TIDE) scores and survival prognosis in each patient were calculated and evaluated, and the immune escape mechanism affecting the independent prognosis of LUAD patients was identified. Functional enrichment analysis explored the antitumour immune response and biological behavior of tumor cells among different LUAD groups. Single-cell annotation and pseudotemporal analysis were used to explore the target molecules and immune escape mechanisms of LUAD. Results Myeloid-derived suppressor cells (MDSCs) and IRF8 were identified as risk and protective factors for the independent prognosis of LUAD patients, respectively. In the tumor microenvironment of patients with high infiltration of MDSCs, the antitumor immune response is significantly suppressed, while tumor cell division, proliferation, and distant metastasis are significantly enhanced. Single-cell RNA-seq analysis revealed that IRF8 is an important regulator of MDSC differentiation in LUAD myeloid cells. In addition, IRF8 may regulate the differentiation of MDSCs through the IL6-JAK-STAT3 signalling pathway. Conclusions IRF8 deficiency impairs the normal development of LUAD myeloid cells and induces their differentiation into MDSCs, thereby accelerating the immune escape of LUAD cells. IRF8-targeted activation to inhibit the formation of MDSCs may be a new target for immunotherapy in LUAD.

Abstract Background Nonspecific Orbital Inflammation (NSOI) represents a persistent and idiopathic proliferative inflammatory disorder, characterized by polymorphous lymphoid infiltration within the orbit. The transcription factor Interferon Regulatory Factor 8 (IRF8), integral to the IRF protein family, was initially identified as a pivotal element for the commitment and differentiation of myeloid cell lineage. Serving as a central regulator of innate immune receptor signaling, IRF8 orchestrates a myriad of functions in hematopoietic cell development. However, the intricate mechanisms underlying IRF8 production remain to be elucidated, and its potential role as a biomarker for NSOI is yet to be resolved. Methods IRF8 was extracted from the intersection analysis of common DEGs of GSE58331 and GSE105149 from the GEO and immune- related gene lists in the ImmPort database using The Lasso regression and SVM-RFE analysis. We performed GSEA and GSVA with gene sets coexpressed with IRF8, and observed that gene sets positively related to IRF8 were enriched in immune-related pathways. To further explore the correlation between IRF8 and immune-related biological process, the CIBERSORT algorithm and ESTIMATE method were employed to evaluate TME characteristics of each sample and confirmed that high IRF8 expression might give rise to high immune cell infiltration. Finally, the GSE58331 was utilized to confirm the levels of expression of IRF8. Results Among the 314 differentially expressed genes (DEGs), some DEGs were found to be significantly different. With LASSO and SVM-RFE algorithms, we obtained 15 hub genes. For biological function analysis in IRF8, leukocyte mediated immunity, leukocyte cell-cell adhesion, negative regulation of immune system process were emphasized. B cells naive, Macrophages M0, Macrophages M1, T cells CD4 memory activated, T cells CD4 memory resting, T cells CD4 naive, and T cells gamma delta were shown to be positively associated with IRF8. While, Mast cells resting, Monocytes, NK cells activated, Plasma cells, T cells CD8, and T cells regulatory (Tregs) were shown to be negatively linked with IRF8. The diagnostic ability of the IRF8 in differentiating NSOI exhibited a good value. Conclusions This study discovered IRF8 that are linked to NSOI. IRF8 shed light on potential new biomarkers for NSOI and tracking its progression.

Atherosclerosis, a lipid‐driven chronic inflammatory disorder, is a significant global health concern associated with high rates of morbidity and mortality, imposing a substantial societal burden. The purpose of this study is to investigate the possible molecular mechanisms of atherosclerosis and identify potential therapeutic targets. We conducted an integrated bioinformatics analysis using data from peripheral blood mononuclear cell and TISSUE databases obtained from the Gene Expression Omnibus, to identify key genes associated with the progression of atherosclerosis. Here, IRF8 was found to be a key gene in atherosclerosis patients. Silencing IRF8 with small interfering RNA reduced inflammation in endothelial cells. This suggests IRF8 is a crucial biomarker for immune infiltration in atherosclerosis advance. [ABSTRACT FROM AUTHOR]

Background: Patients with lung adenocarcinoma (LUAD) have a low response rate to immune checkpoint blockade. It is highly important to explore the tumor immune escape mechanism of LUAD patients and expand the population of patients who may benefit from immunotherapy. Methods: Based on 954 bulk RNA-seq data of LUAD patients and 15 single-cell RNA-seq data, the relationships between tumor immune dysfunction and exclusion (TIDE) scores and survival prognosis in each patient were calculated and evaluated, and the immune escape mechanism affecting the independent prognosis of LUAD patients was identified. Functional enrichment analysis explored the antitumour immune response and biological behavior of tumor cells among different LUAD groups. Single-cell annotation and pseudotemporal analysis were used to explore the target molecules and immune escape mechanisms of LUAD. Results: Myeloid-derived suppressor cells (MDSCs) and IRF8 were identified as risk and protective factors for the independent prognosis of LUAD patients, respectively. In the tumor microenvironment of patients with high infiltration of MDSCs, the antitumor immune response is significantly suppressed, while tumor cell division, proliferation, and distant metastasis are significantly enhanced. Single-cell RNA-seq analysis revealed that IRF8 is an important regulator of MDSC differentiation in LUAD myeloid cells. In addition, IRF8 may regulate the differentiation of MDSCs through the IL6-JAK-STAT3 signalling pathway. Conclusions: IRF8 deficiency impairs the normal development of LUAD myeloid cells and induces their differentiation into MDSCs, thereby accelerating the immune escape of LUAD cells. IRF8-targeted activation to inhibit the formation of MDSCs may be a new target for immunotherapy in LUAD. [ABSTRACT FROM AUTHOR]

Although microwave ablation (MWA) is an important curative therapy in colorectal cancer liver metastasis, recurrence still occurs clinically. Our previous studies have shown that the expression of programmed cell death 1 ligand 1 (PD‐L1) is upregulated following MWA, suggesting that MWA combined with anti‐PD‐L1 treatment can serve as a promising clinical therapeutic strategy against cancer. Using MWA‐treated preclinical mice models, MWA combined with αPD‐L1 treatment decreased tumor growth and prolonged overall survival (OS). Furthermore, through flow cytometry and single‐cell RNA sequencing analysis, we determined that the MWA plus αPD‐L1 therapy significantly suppressed CD8+ T cell exhaustion and enhanced their effector function. A significant increase in γ‐interferon (IFN‐γ) stimulated transcription factors, specifically Irf8, was observed. This enhancement facilitated the polarization of tumor‐associated macrophages (TAM1s and TAM2s) through the nuclear factor‐κB/JAK‐STAT1 signaling pathway. Furthermore, the combination therapy stimulated the production of CXC motif chemokine ligand (CXCL9) by TAM1s and tumor cells, potentially increasing the chemotaxis of CD8 T cells and Th1 cells. Knocking out Cxcl9 in MC38 tumor cells or using CXCL9 blockade enhanced tumor growth of untreated tumors and shortened OS. Taken together, our study showed that blocking the IFN‐γ‐Cxcl9‐CD8+ T axis promoted tumor progression and discovered a potential involvement of IRF8‐regulated TAMs in preventing T cell exhaustion. Collectively, we identified that the combination of MWA with anti‐PD‐L1 treatment holds promise as a therapeutic strategy to rejuvenate the immune response against tumors. This merits further exploration in clinical studies. [ABSTRACT FROM AUTHOR]

Background: Nonspecific Orbital Inflammation (NSOI) represents a persistent and idiopathic proliferative inflammatory disorder, characterized by polymorphous lymphoid infiltration within the orbit. The transcription factor Interferon Regulatory Factor 8 (IRF8), integral to the IRF protein family, was initially identified as a pivotal element for the commitment and differentiation of myeloid cell lineage. Serving as a central regulator of innate immune receptor signaling, IRF8 orchestrates a myriad of functions in hematopoietic cell development. However, the intricate mechanisms underlying IRF8 production remain to be elucidated, and its potential role as a biomarker for NSOI is yet to be resolved. Methods: IRF8 was extracted from the intersection analysis of common DEGs of GSE58331 and GSE105149 from the GEO and immune- related gene lists in the ImmPort database using The Lasso regression and SVM-RFE analysis. We performed GSEA and GSVA with gene sets coexpressed with IRF8, and observed that gene sets positively related to IRF8 were enriched in immune-related pathways. To further explore the correlation between IRF8 and immune-related biological process, the CIBERSORT algorithm and ESTIMATE method were employed to evaluate TME characteristics of each sample and confirmed that high IRF8 expression might give rise to high immune cell infiltration. Finally, the GSE58331 was utilized to confirm the levels of expression of IRF8. Results: Among the 314 differentially expressed genes (DEGs), some DEGs were found to be significantly different. With LASSO and SVM-RFE algorithms, we obtained 15 hub genes. For biological function analysis in IRF8, leukocyte mediated immunity, leukocyte cell-cell adhesion, negative regulation of immune system process were emphasized. B cells naive, Macrophages M0, Macrophages M1, T cells CD4 memory activated, T cells CD4 memory resting, T cells CD4 naive, and T cells gamma delta were shown to be positively associated with IRF8. While, Mast cells resting, Monocytes, NK cells activated, Plasma cells, T cells CD8, and T cells regulatory (Tregs) were shown to be negatively linked with IRF8. The diagnostic ability of the IRF8 in differentiating NSOI exhibited a good value. Conclusions: This study discovered IRF8 that are linked to NSOI. IRF8 shed light on potential new biomarkers for NSOI and tracking its progression. [ABSTRACT FROM AUTHOR]

Bladder cancer (BCa) is a significant health issue and poses a healthcare burden on patients, highlighting the importance of an effective detection method. Here, we developed a urine DNA methylation diagnostic panel for distinguishing between BCa and non-BCa. In the discovery stage, an analysis of the TCGA database was conducted to identify BCa-specific DNA hypermethylation markers. In the validation phase, DNA methylation levels of urine samples were measured with real-time quantitative methylation-specific PCR (qMSP). Comparative analysis of the methylation levels between BCa and non-BCa, along with the receiver operating characteristic (ROC) analyses with machine learning algorithms (logistic regression and decision tree methods) were conducted to develop practical diagnostic panels. The performance evaluation of the panel shows that the individual biomarkers of ZNF671, OTX1, and IRF8 achieved AUCs of 0.86, 0.82, and 0.81, respectively, while the combined yielded an AUC of 0.91. The diagnostic panel using the decision tree algorithm attained an accuracy, sensitivity, and specificity of 82.6%, 75.0%, and 90.9%, respectively. Our results show that the urine-based DNA methylation diagnostic panel provides a sensitive and specific method for detecting and stratifying BCa, showing promise as a standard test that could enhance the diagnosis and prognosis of BCa in clinical settings. [ABSTRACT FROM AUTHOR]

Aim To investigate the molecular mechanism of interaction between rheumatoid arthritis (RA) and atherosclerosis (As) based on bioinformatics analysis. Methods The gene expression profiles of As and RA were downloaded from GEO database, differentially expressed genes between RA and As were identified through the test sets, the biological function of common differentially expressed genes was studied by enrichment analysis. Cytoscape software was used to construct the differentially expressed gene protein-protein interaction network and screen the hub genes. Transcriptional regulatory relationship revealed by the TRRUST database predicts transcription factors. Transcription factors were validated by test sets, and hub genes were validated by validation sets and blood samples. Results A total of 198 differentially expressed genes were identified. Functional enrichment analysis showed that differentially expressed genes were mainly concentrated in signaling pathways regulated by cytokines, leukocyte migration, positive regulation of leukocytes, and interaction between cytokines and cytokine receptors. Cytoscape demonstrated the differentially expressed genes and gene clustering modules, obtained the hub genes CCL5, CCR1, CCR2, CCR5, IRF8, ITGAM, ITGB2, LCP2, NCF2 and PTPRC, and the results of validation sets showed that the genes were reliable. qPCR results showed that the expression levels of CCR1 and IRF8 in patients with As combined with RA were significantly higher than those in healthy people. Conclusion The regulatory effect of CCR1 and IRF8 is likely to be the hub factor of RA merging with As. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 8 (IRF8) is a transcription factor necessary for the maturation of microglia, as well as other peripheral immune cells. It also regulates the transition of microglia and other immune cells to a pro-inflammatory phenotype. Irf8 is also a known risk gene for multiple sclerosis and lupus, and it has recently been shown to be downregulated in schizophrenia. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. Here, we show by RNAseq from Irf8 -/- male and female mouse brains that several genes involved in regulation of neural activity are dysregulated. We then show that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to lethal seizures in male and female Irf8 -/- mice. Finally, we identify that TNF-α is elevated specifically in microglia in the CNS, and genetic or acute pharmacological blockade of TNF-α in the Irf8 -/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. Our data further suggest that neuronal function is impacted by loss of IRF8, a factor involved in neuropsychiatric and neurodegenerative diseases.SIGNIFICANCE STATEMENT Here, we identify a previously unknown and key role for interferon regulator factor 8 (IRF8) in regulating neural excitability and seizures. We further determine that these effects on neural circuits are through elevated TNF-α in the CNS. As IRF8 has most widely been studied in the context of regulating the development and inflammatory signaling in microglia and other immune cells, we have uncovered a novel function. Further, IRF8 is a risk gene for multiple sclerosis and lupus, IRF8 is dysregulated in schizophrenia, and elevated TNF-α has been identified in a multitude of neurologic conditions. Thus, elucidating these IRF8 and TNF-α-dependent effects on brain circuit function has profound implications for understanding underlying, therapeutically relevant mechanisms of disease.

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. [ABSTRACT FROM AUTHOR]

Several single cell profiling studies of human hematopoietic stem and progenitor cells indicate the existence of circulating CD123+CD127+ lymphoid progenitors that express IRF8 and which are dendritic cell (DC)- and lymphoid [B and T cell/innate lymphoid cell (ILC)]-transcriptionally primed. IRF8 -expressing CD123+CD127+ lymphoid progenitors are also found in tissues that support ILC, DC, and T cell development, suggesting that downstream lineage specification and commitment are modulated by the specific tissue microenvironment. Expression of IRF8 unlocks DC lineage potential in these human lymphoid progenitors but is differentially regulated in downstream steps of DC and B/T/ILC differentiation. CD123 expression (a surrogate cell surface marker for IRF8) does not preclude hematopoietic progenitors from harboring lymphoid potential. The abundance of CD123+CD127+ lymphoid progenitors in peripheral blood is affected by stem cell mobilizers. This might impact lymphoid reconstitution following transplantation with hematopoietic stem cell-mobilized peripheral blood. The hematopoietic model proposed here highlights an underappreciated role for transcription factor interferon regulatory factor 8 in unlocking human dendritic cell, B and T cell, and innate lymphoid cell potential in CD123+CD127+ lymphoid progenitors. Understanding the development and differentiation of CD123+CD127+ lymphoid progenitors across tissues has important basic and translational implications. Lymphopoiesis is the process in which B and T cells, and innate lymphoid cells (ILCs) develop from hematopoietic progenitors that exhibit early lymphoid priming. The branching points where lymphoid-primed human progenitors are further specified to B/T/ILC differentiation trajectories remain unclear. Here, we discuss the emerging role of interferon regulatory factor (IRF)8 as a key factor to bridge human lymphoid and dendritic cell (DC) differentiation, and the current evidence for the existence of circulating and tissue-resident CD123+CD127+ lymphoid progenitors. We propose a model whereby DC/B/T/ILC lineage programs in circulating CD123+CD127+ lymphoid progenitors are expressed in balance. Upon tissue seeding, the tissue microenvironment tilts this molecular balance towards a specific lineage, thereby determining in vivo lineage fates. Finally, we discuss the translational implication of these lymphoid precursors. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 8 (IRF8) is a key regulator of innate immune receptor signaling that resists pathogen invasion by regulating cell growth and differentiation. Porcine epidemic diarrhea virus (PEDV) targets the intestine and damages the mucosal barrier. However, whether IRF8 regulates PEDV replication remains unclear. We revealed that PEDV infection activated IRF8 expression. Moreover, IRF8 deletion drastically promoted PEDV replication and invasion, increasing the virus copies and titers. Hypomethylation enrichment of activating protein (AP)-2α was significantly negatively correlated with high IRF8 expression, and AP-2α directly targeted the IRF8 promoter to regulate PEDV replication. Furthermore, IRF8 overexpression decreased the cellular reactive oxygen species levels and mitochondrial membrane potential and increased the antioxidant enzyme activities to alleviate PEDV-induced oxidative damage. IRF8 overexpression suppressed apoptotic gene expression, thereby inhibiting apoptosis in response to PEDV stimulation. Taken together, this study demonstrates that AP-2a is involved in PEDV-induced epigenetic modification of IRF8 to reduce cell apoptosis and oxidative stress and facilitate host resistance to PEDV in the intestinal epithelium. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are sentinel immune cells that form a critical bridge linking the innate and adaptive immune systems. Extensive research addressing the cellular origin and heterogeneity of the DC network has revealed the essential role played by the spatiotemporal activity of key transcription factors. In response to environmental signals DC mature but it is only following the sensing of environmental signals that DC can induce an antigen specific T cell response. Thus, whilst the coordinate action of transcription factors governs DC differentiation, sensing of environmental signals by DC is instrumental in shaping their functional properties. In this review, we provide an overview that focuses on recent advances in understanding the transcriptional networks that regulate the development of the reported DC subsets, shedding light on the function of different DC subsets. Specifically, we discuss the emerging knowledge on the heterogeneity of cDC2s, the ontogeny of pDCs, and the newly described DC subset, DC3. Additionally, we examine critical transcription factors such as IRF8, PU.1, and E2-2 and their regulatory mechanisms and downstream targets. We highlight the complex interplay between these transcription factors, which shape the DC transcriptome and influence their function in response to environmental stimuli. The information presented in this review provides essential insights into the regulation of DC development and function, which might have implications for developing novel therapeutic strategies for immune-related diseases. [ABSTRACT FROM AUTHOR]

We highlight the utility of interferon regulatory factor 8 (IRF8), a novel marker of monocytic and dendritic cell lineages, in the diagnosis of a case of blastic plasmacytoid dendritic cell neoplasm (BPDCN) presenting initially in the skin. A 60‐year‐old male with a previous history of myelodysplastic syndrome presented with cutaneous nodules on chest and scalp. A punch biopsy specimen of a skin nodule showed a diffuse dermal infiltrate of atypical mononuclear cells. The neoplastic cells expressed CD4, CD56, CD43, and TdT but showed minimal reaction for TCL‐1 and CD123, and were negative for CD34, CD117, and MPO, confounding the diagnosis. IRF8 performed in retrospect was strongly positive. A new punch biopsy specimen of a chest nodule showed the blastoid tumor cells were positive for TCL‐1, CD4, and CD56, but dim CD123. Subsequent bone marrow involvement showed blastoid tumor cells with intense positivity for CD123, CD4, and CD56, which was supportive of the BPDCN diagnosis. BPDCN cases with weak or variable CD123 and TCL‐1 expression represent a potential diagnostic pitfall. In a recent study, 15 cases of BPDCN showed uniformly strong staining for IRF8, while CD123 was dim or negative in 4 of these 15 cases. We suggest IRF8 may be a useful marker for BPDCN, especially in cases with weak or variable expression of CD123 and TCL1. [ABSTRACT FROM AUTHOR]

The transcription factor interferon regulatory factor 8 (IRF8), as a member of the IRF family, is essential for myeloid cell differentiation. However, the precise role of IRF8 in the pathogenesis of acute myeloid leukemia (AML) remains unknown. By using multivariate analysis, we discovered that high IRF8 expression was an independent poor predictor of overall survival (OS) in AML patients from our clinical follow-up study. The proliferation of three AML cell lines was significantly inhibited by shRNA-mediated knockdown of IRF8, owing to cell cycle S-phase arrest. Furthermore, we demonstrated that knocking down IRF8 could suppress the expression of CyclinA and CyclinB1, resulting in a shift in cell cycle distribution. Loss of IRF8 in AML cells decreased the expression of STAT3 and phosphor-STAT3 (pSTAT3), which are key factors in JAK/STAT signal pathway and are important for AML progression. Using a xenograft mouse model, we discovered the antiproliferative effect of losing IRF8 in vivo. In conclusion, this study found that IRF8 may play a prognostic factor and therapeutic target in AML. [ABSTRACT FROM AUTHOR]

The transcription factor Interferon regulatory factor 8 (IRF8) is involved in maintaining B cell identity. However, how IRF8 regulates T cell independent B cell responses are not fully characterized. Here, an in vivo CRISPR/Cas9 system was optimized to generate Irf8-deficient murine B cells and used to determine the role of IRF8 in B cells responding to LPS stimulation. Irf8-deficient B cells more readily formed CD138+ plasmablasts in response to LPS with the principal dysregulation occurring at the activated B cell stage. Transcriptional profiling revealed an upregulation of plasma cell associated genes prematurely in activated B cells and a failure to repress the gene expression programs of IRF1 and IRF7 in Irf8-deficient cells. These data expand on the known roles of IRF8 in regulating B cell identity by preventing premature plasma cell formation and highlight how IRF8 helps evolve TLR responses away from the initial activation towards those driving humoral immunity.

Bladder cancer (BCa) is a significant health issue and poses a healthcare burden on patients, highlighting the importance of an effective detection method. Here, we developed a urine DNA methylation diagnostic panel for distinguishing between BCa and non-BCa. In the discovery stage, an analysis of the TCGA database was conducted to identify BCa-specific DNA hypermethylation markers. In the validation phase, DNA methylation levels of urine samples were measured with real-time quantitative methylation-specific PCR (qMSP). Comparative analysis of the methylation levels between BCa and non-BCa, along with the receiver operating characteristic (ROC) analyses with machine learning algorithms (logistic regression and decision tree methods) were conducted to develop practical diagnostic panels. The performance evaluation of the panel shows that the individual biomarkers of ZNF671, OTX1, and IRF8 achieved AUCs of 0.86, 0.82, and 0.81, respectively, while the combined yielded an AUC of 0.91. The diagnostic panel using the decision tree algorithm attained an accuracy, sensitivity, and specificity of 82.6%, 75.0%, and 90.9%, respectively. Our results show that the urine-based DNA methylation diagnostic panel provides a sensitive and specific method for detecting and stratifying BCa, showing promise as a standard test that could enhance the diagnosis and prognosis of BCa in clinical settings.

T‐cell acute lymphoblastic leukemia (T‐ALL) is an aggressive hematological malignancy with a poor prognosis, urging for novel therapeutic targets and treatment strategies. N6‐methyladenosine (m6A) is a crucial methylation modification that affects the pathogenesis of leukemia by regulating the mRNA of key genes. Interferon regulatory factor 8 (IRF8) is a crucial transcription factor for hematological lineage commitment, but its role in T‐ALL is unclear. Here, IRF8 is shown to suppress T‐ALL. The expression of IRF8 is abnormally silenced in patients with T‐ALL. Knockout of Irf8 significantly hastens the progression of Notch1‐induced T‐ALL in vivo. Overexpression of IRF8 suppresses the proliferation and invasion of T‐ALL cells by inhibiting the phosphatidylinositol 3‐kinase/AKT signaling pathway. The fat mass‐ and obesity‐associated protein (FTO), an m6A demethylase, is responsible for directly binding to m6A sites in 3′ untranslated region of IRF8 messenger RNA (mRNA) and inducing mRNA degradation via m6A modification. Targeting the FTO‐IRF8 axis is used as a proof of concept therapy; inhibition of FTO's demethylase activity drastically alleviates the proliferation of leukemic cells and prolongs the survival of T‐ALL mice by restoring IRF8 expression. This study elucidates the pathogenesis of T‐ALL from the perspective of epitranscriptomics and provides new insight into the genetic mechanisms and targeted therapy of T‐ALL. [ABSTRACT FROM AUTHOR]

Ferroptosis has emerged as a cytotoxic T lymphocyte (CTL)-induced tumor cell death pathway. The regulation of tumor cell sensitivity to ferroptosis is incompletely understood. Here, we report that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified the ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells acquire resistance to intrinsic ferroptosis induction and IRF8-deficient tumor cells also exhibit decreased ferroptosis in response to tumor-specific CTLs. Irf8 deletion increased p53 expression in tumor cells and knocking out p53 in IRF8.KO tumor cells restored tumor cell sensitivity to intrinsic ferroptosis induction. Furthermore, IRF8.KO tumor cells grew significantly faster than WT tumor cells in immune-competent mice. To restore IRF8 expression in tumor cells, we designed and synthesized codon usage-optimized IRF8-encoding DNA to generate IRF8-encoding plasmid NTC9385R-mIRF8. Restoring IRF8 expression via a lipid nanoparticle-encapsulated NTC9385R-mIRF8 plasmid therapy suppressed established tumor growth in vivo. In human cancer patients, nivolumab responders have a significantly higher IRF8 expression level in their tumor cells as compared to the non-responders. Our data determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis. [ABSTRACT FROM AUTHOR]

Objective: To assess the effects of nebulized inhaled Mycobacterium vaccae on allergic airway inflammation, airway hyperresponsiveness, and Th1/Th2 cell imbalance in mice with ovalbumin (OVA)-induced asthma. Methods: Mice received OVA sensitization and challenge for establishment of the asthmatic model. For intervention, mice received Mycobacterium vaccae nebulization once every other day from the first day of sensitization to the day before challenge. After challenge, pulmonary histological analysis and airway responsiveness measurement were performed. In addition, Th1/Th2 cytokines and OVA-specific IgE levels in bronchoalveolar lavage fluid were measured by ELISA. Th1/Th2 subset ratios and the expression of interferon-regulatory factor 4 (IRF4), IRF8 and Toll-like receptor 4 (TLR4) in dendritic cells were evaluated by flow cytometry. Results: Severe inflammatory infiltration and airway hyperresponsiveness were observed in OVA-induced asthmatic mice. Asthmatic mice showed higher Th2 cytokine concentration and increased percentage of Th2 cells, along with lower Th1 cytokine concentration and reduced percentage of Th1 cells compared with the normal control. Moreover, an imbalance of IRF4+ and IRF8+ in dendritic cells was found in asthmatic mice. Nebulized inhaled Mycobacterium vaccae reduced airway hyperresponsiveness and inflammation in OVA-induced asthmatic mice. In addition, nebulized inhaled Mycobacterium vaccae enhanced TLR4 and IRF8 expression, and alleviated the imbalance of Th1/Th2 as well as IRF4+ and IRF8+ in dendritic cells. Conclusions: Nebulized inhaled Mycobacterium vaccae protects against asthma by alleviating the imbalance of Th1/Th2 and IRF4/IRF8 in OVA-induced asthmatic mice. [ABSTRACT FROM AUTHOR]

Abstract Background Interferon regulatory factor-8 (IRF8) and nuclear factor-activated T cells c1 (NFATc1) are two transcription factors that have an important role in osteoclast differentiation. Thanks to ChIP-seq technology, scientists can now estimate potential genome-wide target genes of IRF8 and NFATc1. However, finding target genes that are consistently up-regulated or down-regulated across different studies is hard because it requires analysis of a large number of high-throughput expression studies from a comparable context. Method We have developed a machine learning based method, called, Cohort-based TF target prediction system (cTAP) to overcome this problem. This method assumes that the pathway involving the transcription factors of interest is featured with multiple “functional groups” of marker genes pertaining to the concerned biological process. It uses two notions, Gene-Present Sufficiently (GP) and Gene-Absent Insufficiently (GA), in addition to log2 fold changes of differentially expressed genes for the prediction. Target prediction is made by applying multiple machine-learning models, which learn the patterns of GP and GA from log2 fold changes and four types of Z scores from the normalized cohort’s gene expression data. The learned patterns are then associated with the putative transcription factor targets to identify genes that consistently exhibit Up/Down gene regulation patterns within the cohort. We applied this method to 11 publicly available GEO data sets related to osteoclastgenesis. Result Our experiment identified a small number of Up/Down IRF8 and NFATc1 target genes as relevant to osteoclast differentiation. The machine learning models using GP and GA produced NFATc1 and IRF8 target genes different than simply using a log2 fold change alone. Our literature survey revealed that all predicted target genes have known roles in bone remodeling, specifically related to the immune system and osteoclast formation and functions, suggesting confidence and validity in our method. Conclusion cTAP was motivated by recognizing that biologists tend to use Z score values present in data sets for the analysis. However, using cTAP effectively presupposes assembling a sizable cohort of gene expression data sets within a comparable context. As public gene expression data repositories grow, the need to use cohort-based analysis method like cTAP will become increasingly important.

Simple Summary: Triple-negative breast cancers (TBNCs) represent 10–20% of all breast cancers. TNBCs are more frequent in younger women, and present more aggressive features and poorer prognosis. Few specific therapeutics are available for TNBC treatment and it is crucial to better characterize TNBC biology in order to discover new therapeutic targets. In this study, we focused on the immune tumor microenvironment, particularly on macrophages that have been less studied in the context of TNBC. Macrophages are very plastic cells; their phenotype and function can change depending upon environmental conditions. Therefore, we used four macrophage markers to quantify the macrophage infiltrate (CD68, IRF8, CD163, and CD206) in tumors from 285 patients with TNBC. We demonstrated for the first time that a population of macrophages, defined by CD206 expression, delineates a subgroup of TNBCs that may have a better prognosis. These results could help to refine the patients' prognosis and develop new therapeutic strategies. Background: Triple-negative breast cancers (TNBCs) have a worse prognosis, but might respond to immunotherapies. Macrophages are plastic cells that can adopt various phenotypes and functions. Although they are a major immune population in TNBCs, the relationship between tumor-associated macrophages (TAMs) and TNBC progression has been rarely explored, with controversial results. Methods: We evaluated the prognostic impact of TAMs, quantified by immunohistochemistry with anti-CD68, -IRF8, -CD163, and -CD206 antibodies, in a well-described cohort of 285 patients with non-metastatic TNBC. Results: CD68 (p = 0.008), IRF8 (p = 0.001), and CD163 (p < 0.001) expression positively correlated with higher tumor grade, while CD206 was associated with smaller tumor size (p < 0.001). All macrophage markers were associated with higher tumor-infiltrating lymphocyte numbers and PD-L1 expression. Univariate survival analyses reported a significant positive correlation between CD163+ or CD206+ TAMs and relapse-free survival (respectively: HR = 0.52 [0.28–0.97], p = 0.027, and HR = 0.51 [0.31–0.82], p = 0.005), and between CD206+ TAMs and overall survival (HR = 0.54 [0.35–0.83], p = 0.005). In multivariate analysis, there was a trend for an association between CD206+ TAMs and relapse-free survival (HR = 0.63 [0.33–1.04], p = 0.073). Conclusions: These data suggest that CD206 expression defines a TAM subpopulation potentially associated with favorable outcomes in patients with TNBC. CD206 expression might identify an immune TNBC subgroup with specific therapeutic options. [ABSTRACT FROM AUTHOR]

Anti‐CD20 monoclonal antibodies such as Rituximab, Ofatumumab, and Obinutuzumab are widely used to treat lymphomas and autoimmune diseases. They act by depleting B cells, mainly through Fc‐dependent effectors functions. Some patients develop resistance to treatment but the underlying mechanisms are poorly understood. Here, we performed a genome‐wide CRISPR/Cas9 screen to identify genes regulating the efficacy of anti‐CD20 antibodies. We used as a model the killing of RAJI B cells by Rituximab through complement‐dependent‐cytotoxicity (CDC). As expected, the screen identified MS4A1, encoding CD20, the target of Rituximab. Among other identified genes, the role of Interferon Regulatory Factor 8 (IRF8) was validated in two B‐cell lines. IRF8 KO also decreased the efficacy of antibody‐dependent cellular cytotoxicity and phagocytosis (ADCC and ADCP) induced by anti‐CD20 antibodies. We further show that IRF8 is necessary for efficient CD20 transcription. Levels of IRF8 and CD20 RNA or proteins correlated in normal B cells and in hundreds of malignant B cells. Therefore, IRF8 regulates CD20 expression and controls the depleting capacity of anti‐CD20 antibodies. Our results bring novel insights into the pathways underlying resistance to CD20‐targeting immunotherapies. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 8 (IRF8) is a transcription factor of the IRF protein family. IRF8 was originally identified as an essentialfactor for myeloid cell lineage commitment and differentiation. Deletion of Irf8 leads to massive accumulation of CD11b+Gr1+ immature myeloid cells (IMCs), particularly the CD11b+Ly6Chi/+Ly6G− polymorphonuclear myeloid-derived suppressor cell-like cells (PMN-MDSCs). Under pathological conditions such as cancer, Irf8 is silenced by its promoter DNA hypermethylation, resulting in accumulation of PMN-MDSCs and CD11b+ Ly6G+Ly6Clo monocytic MDSCs (M-MDSCs) in mice. IRF8 is often silenced in MDSCs in human cancer patients. MDSCs are heterogeneous populations of immune suppressive cells that suppress T and NK cell activity to promote tumor immune evasion and produce growth factors to exert direct tumor-promoting activity. Emerging experimental data reveals that IRF8 is also expressed in non-hematopoietic cells. Epithelial cell-expressed IRF8 regulates apoptosis and represses Osteopontin (OPN). Human tumor cells may use the IRF8 promoter DNA methylation as a mechanism to repress IRF8 expression to advance cancer through acquiring apoptosis resistance and OPN up-regulation. Elevated OPN engages CD44 to suppress T cell activation and promote tumor cell stemness to advance cancer. IRF8 thus is a transcription factor that regulates both the immune and non-immune components in human health and diseases. [ABSTRACT FROM AUTHOR]

Abstract Background Microglia cells represent the resident innate immune cells of the retina and are important for retinal development and tissue homeostasis. However, dysfunctional microglia can have a negative impact on the structural and functional integrity of the retina under native and pathological conditions. Methods In this study, we examined interferon-regulatory factor 8 (Irf8)–deficient mice to determine the transcriptional profile, morphology, and temporospatial distribution of microglia lacking Irf8 and to explore the effects on retinal development, tissue homeostasis, and formation of choroidal neovascularisation (CNV). Results Our study shows that Irf8-deficient MG exhibit a considerable loss of microglial signature genes accompanied by a severely altered MG morphology. An in-depth characterisation by fundus photography, fluorescein angiography, optical coherence tomography and electroretinography revealed no major retinal abnormalities during steady state. However, in the laser-induced CNV model, Irf8-deficient microglia showed an increased activity of biological processes critical for inflammation and cell adhesion and a reduced MG cell density near the lesions, which was associated with significantly increased CNV lesion size. Conclusions Our results suggest that loss of Irf8 in microglia has negligible effects on retinal homeostasis in the steady state. However, under pathological conditions, Irf8 is crucial for the transformation of resident microglia into a reactive phenotype and thus for the suppression of retinal inflammation and CNV formation.

Objectives: Interferon regulatory factor 8 (IRF8) is a new biomarker shown to be positive in monocytic leukemias as well as in B cells. As a transcription factor, it plays a critical role in pre-B-cell differentiation and induction of tolerance pathways, among other functions. Given the frequent diagnostic dilemma in CD30-positive large cell lymphomas that could resemble both Hodgkin lymphoma and anaplastic large cell lymphoma (ALCL), we sought to determine whether IRF8 can be useful in distinguishing between these neoplasms that require different treatment strategies.Methods: In this retrospective study, 74 cases of classic Hodgkin lymphoma (CHL) and 7 cases of nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) on a tissue microarray (TMA), as well as 15 individual cases of ALK-negative ALCL and 4 cases of ALK-positive ALCL, were stained for IRF8. Paired box 5 (PAX5) immunostaining of the TMA was also performed and compared alongside since that marker is occasionally the only marker to help clinically differentiate between T- and B-cell lymphomas with anaplastic/Hodgkin-like cytology.Results: None (0%) of the ALCLs were positive for IRF8 while all (100%) of the NLPHLs and 85% of the CHLs were positive for IRF8. Six (8%) cases of CHL were PAX5 negative but IRF8 positive. Conversely, seven (10%) cases of CHL were PAX5 positive but IRF8 negative. Four (6%) cases of CHL were negative for both PAX5 and IRF8.Conclusions: There is significant morphologic and immunophenotypic (CD30 positive and CD45 and CD20 negative) overlap between CHL and ALCL. Since many ALCLs show downregulation of lineage-specific T-cell markers or are "null cell" type, only PAX5 has been a reliable marker to differentiate between borderline cases. This is further confounded by positivity of PAX5 in some ALCLs due to amplification of PAX5. On the basis of recent discoveries of IRF8 function as well as performance as an immunostain, we tested this marker in human lymphoma samples and found that it aids in the discrimination between these tumors. [ABSTRACT FROM AUTHOR]

A higher ratio of M1/M2 macrophages and an elevated chemerin level are both related to increased risk of preeclampsia. However, the crosstalk between these two events and their collective contribution to preeclampsia are not well understood. In this study, we assessed the impacts of chemerin chemokine-like receptor 1 (CMKLR1)/p-Akt/CEBPα axis in regulating macrophage polarization and mediating the pathogenic effects of chemerin on preeclampsia. We showed that chemerin, in a dose- and time-dependent manner, stimulated M1 macrophage polarization, inhibited macrophage-induced trophoblast invasion and migration, and suppressed macrophage-mediated angiogenesis. All these chemerin-induced phenotypes are essentially mediated by sequentially CMKLR1, Akt activation, and CEBPα. Mechanistically, CEBPα acted as a transcriptional activator for both IRF8 and chemerin. In vivo, chemerin aggravated preeclampsia, while α-NETA, an inhibitor for CMKLR1, significantly suppressed M1 macrophage polarization and alleviated preeclampsia. In summary, chemerin, by activating CMKLR1/Akt/CEBPα axis, forms a positive feedback loop, promotes M1 macrophage polarization, suppresses trophoblast migration/invasion and angiogenesis, and contributes to preeclampsia. Therefore, targeting chemerin signaling may benefit the prevention and/or treatment of preeclampsia. [ABSTRACT FROM AUTHOR]

Background: Autosomal recessive (AR) complete IRF8 deficiency is a rare severe inborn error of immunity underlying an absence of blood myeloid mononuclear cells, intracerebral calcifications, and multiple infections. Only three unrelated patients have been reported. Materials and Methods: We studied an Argentinian child with multiple infectious diseases and severe pulmonary alveolar proteinosis (PAP). We performed whole-exome sequencing (WES) and characterized his condition by genetic, immunological, and clinical means. Results: The patient was born and lived in Argentina. He had a history of viral pulmonary diseases, disseminated disease due to bacillus Calmette-Guérin (BCG), PAP, and cerebral calcifications. He died at the age of 10 months from refractory PAP. WES identified two compound heterozygous variants in IRF8: c.55del and p.R111*. In an overexpression system, the p.R111* cDNA was loss-of-expression, whereas the c.55del cDNA yielded a protein with a slightly lower molecular weight than the wild-type protein. The mutagenesis of methionine residues downstream from c.55del revealed a re-initiation of translation. However, both variants were loss-of-function in a luciferase assay, suggesting that the patient had AR complete IRF8 deficiency. The patient had no blood monocytes or dendritic cells, associated with neutrophilia, and normal counts of NK and other lymphoid cell subsets. Conclusion: We describe the fourth patient with AR complete IRF8 deficiency. This diagnosis should be considered in children with PAP, which is probably due to the defective development or function of alveolar macrophages. [ABSTRACT FROM AUTHOR]

The morbidity and mortality of myeloproliferative neoplasms (MPNs) are primarily caused by arterial and venous complications, progression to myelofibrosis, and transformation to acute leukemia. However, identifying molecular-based biomarkers for risk stratification of patients with MPNs remains a challenge. We have previously shown that interferon regulatory factor-8 (IRF8) and IRF4 serve as tumor suppressors in myeloid cells. In this study, we evaluated the expression of IRF4 and IRF8 and the JAK2V617F mutant allele burden in patients with MPNs. Patients with decreased IRF4 expression were correlated with a more developed MPN phenotype in myelofibrosis (MF) and secondary AML (sAML) transformed from MPNs versus essential thrombocythemia (ET). Negative correlations between the JAK2V617F allele burden and the expression of IRF8 (P < 0.05) and IRF4 (P < 0.001) and between white blood cell (WBC) count and IRF4 expression (P < 0.05) were found in ET patients. IRF8 expression was negatively correlated with the JAK2V617F allele burden (P < 0.05) in polycythemia vera patients. Complete response (CR), partial response (PR), and no response (NR) were observed in 67.5%,10%, and 22.5% of ET patients treated with hydroxyurea (HU), respectively, in 12 months. At 3 months, patients in the CR group showed high IRF4 and IRF8 expression compared with patients in the PR and NR groups. In the 12-month therapy period, low IRF4 and IRF8 expression were independently associated with the unfavorable response to HU and high WBC count. Our data indicate that the expression of IRF4 and IRF8 was associated with the MPN phenotype, which may serve as biomarkers for the response to HU in ET. [ABSTRACT FROM AUTHOR]

Abstract Background Characterization of breast cancer (BC) through the determination of conventional markers such as ER, PR, HER2, and Ki67 has been useful as a predictive and therapeutic tool. Also, assessment of tumor-infiltrating lymphocytes has been proposed as an important prognostic aspect to be considered in certain BC subtypes. However, there is still a need to identify additional biomarkers that could add precision in distinguishing therapeutic response of individual patients. To this end, we focused in the expression of interferon regulatory factor 8 (IRF8) in BC cells. IRF8 is a transcription factor which plays a well-determined role in myeloid cells and that seems to have multiple antitumoral roles: it has tumor suppressor functions; it acts downstream IFN/STAT1, required for the success of some therapeutic regimes, and its expression in neoplastic cells seems to depend on a cross talk between the immune contexture and the tumor cells. The goal of the present study was to examine the relationship between IRF8 with the therapeutic response and the immune contexture in BC, since its clinical significance in this type of cancer has not been thoroughly addressed. Methods We identified the relationship between IRF8 expression and the clinical outcome of BC patients and validated IRF8 as predictive biomarker by using public databases and then performed in silico analysis. To correlate the expression of IRF8 with the immune infiltrate in BC samples, we performed quantitative multiplex immunohistochemistry. Results IRF8 expression can precisely predict the complete pathological response to monoclonal antibody therapy or to select combinations of chemotherapy such as FAC (fluorouracil, adriamycin, and cytoxan) in ER-negative BC subtypes. Analysis of immune cell infiltration indicates there is a strong correlation between activated and effector CD8+ T cell infiltration and tumoral IRF8 expression. Conclusions We propose IRF8 expression as a potent biomarker not only for prognosis, but also for predicting therapy response in ER-negative BC phenotypes. Its expression in neoplastic cells also correlates with CD8+ T cell activation and infiltration. Therefore, our results justify new efforts towards understanding mechanisms regulating IRF8 expression and how they can be therapeutically manipulated.

Functional and phenotypic heterogeneity of dendritic cells (DCs) play crucial roles in facilitating the development of diverse immune responses essential for host protection. Here, we report that KDM5C, a histone lysine demethylase, regulates conventional or classical DC (cDC) and plasmacytoid DC (pDC) population heterogeneity and function. Mice deficient in KDM5C in DCs have increased proportions of cDC2Bs and cDC1s, which is partly dependent on type I interferon (IFN) and pDCs. Loss of KDM5C results in an increase in Ly6C− pDCs, which, compared to Ly6C+ pDCs, have limited ability to produce type I IFN and more efficiently stimulate antigen-specific CD8 T cells. KDM5C-deficient DCs have increased expression of inflammatory genes, altered expression of lineage-specific genes, and decreased function. In response to Listeria infection, KDM5C-deficient mice mount reduced CD8 T cell responses due to decreased antigen presentation by cDC1s. Thus, KDM5C is a key regulator of DC heterogeneity and critical driver of the functional properties of DCs. [Display omitted] • KDM5C regulates pDC and cDC heterogeneity • Ly6C− pDCs are poor producers of IFN-α but can present antigens to CD8 T cells • KDM5C antagonizes IRF-driven gene expression, increasing baseline activation of pDC and cDC1 • KDM5C is required for functional responses of pDCs and cDC1s Guak et al. report that the histone lysine demethylase KDM5C fine-tunes lineage-specific gene expression and regulates the composition of DC populations. Loss of KDM5C leads to increased IRF-driven inflammatory gene expression at steady state, resulting in diminished type I IFN production and antigen presentation when stimulated. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 8 (IRF8) is an important transcriptional regulatory factor involving in multiple biological process, such as the antiviral immune response, immune cell proliferation and differentiation. In the present study, the involvement of a previously identified IRF8 homologue (Cg IRF8) in regulating haemocyte proliferation of oyster were further investigated. Cg IRF8 mRNA transcripts were detectable in all the stages of C. gigas larvae with the highest level in D-veliger (1.76-fold of that in zygote, p < 0.05). Its mRNA transcripts were also detected in all the three haemocyte subpopulations of adult oysters with the highest expression in granulocytes (2.79-fold of that in agranulocytes, p < 0.01). After LPS stimulation, the mRNA transcripts of Cg IRF8 in haemocytes significantly increased at 12 h and 48 h, which were 2.04-fold and 1.65-fold (p < 0.05) of that in control group, respectively. Meanwhile, the abundance of Cg IRF8 protein in the haemocytes increased significantly at 12 h after LPS stimulation (1.71-fold of that in seawater, p < 0.05). The immunofluorescence assay and Western blot showed that LPS stimulation induced an obvious nucleus translocation of Cg IRF8 protein in haemocytes. After the expression of Cg IRF8 was inhibited by the injection of Cg IRF8 siRNA, the percentage of EdU positive haemocytes, the proportion of granulocytes, and the mRNA expression levels of Cg GATA and Cg SCL all declined significantly at 12 h after LPS stimulation, which was 0.64-fold (p < 0.05), 0.7-fold (p < 0.05), 0.31-fold and 0.54-fold (p < 0.001) of that in the NC group, respectively. While the expression level of cell proliferation-related protein Cg CDK2, Cg CDC6, Cg CDC45 and Cg PCNA were significantly increased (1.99-fold, and 2.41-fold, 3.76-fold and 4.79-fold compared to that in the NC group respectively, p < 0.001). Dual luciferase reporter assay demonstrated that Cg IRF8 was able to activate the Cg GATA promoter in HEK293T cells after transfection of Cg GATA and Cg IRF8. These results collectively indicated that Cg IRF8 promoted haemocyte proliferation by regulating the expression of Cg GATA and other related genes in the immune response of oyster. • Cg IRF8 showed increased expression in oyster haemocytes after LPS stimulation. • The percentage of EdU positive haemocytes, and proportion of granulocytes decreased in Cg IRF8-RNAi oysters. • Cg IRF8 was able to bind the promoter of Cg GATA, and regulated transcription of cyclins. [ABSTRACT FROM AUTHOR]

Neuroinflammation associated with microglial activation plays a role in the development of Parkinson's disease (PD). The upregulation of interferon regulatory factor 8 (IRF8) in microglia following peripheral nerve injury has been observed to induce microglial activation. This suggests the potential therapeutic significance of IRF8 in PD. This research aims to explore the effects of IRF8 on the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and lipopolysaccharide (LPS)-induced neuroinflammation, along with its underlying mechanisms. The study examines the differential expression of IRF8 and its effects on neuropathological changes using a PD mouse model and a PD model established from BV2 cells in vitro. IRF8 was found to be prominently expressed in the substantia nigra pars compacta (SNpc) region of PD mice and LPS-stimulated BV2 cells, while the expression of tyrosine hydroxylase (TH) and dopamine (DA) content in the SNpc region of PD mice was notably reduced. MPTP treatment and LPS stimulation intensified microglial activation, inflammation, and activation of the AMPK/mTOR signaling pathway in vivo and in vitro , respectively. Upon IRF8 silencing in the PD mouse and cell models, the knockdown of IRF8 ameliorated MPTP-induced behavioral deficits, increased the counts of TH and Nissl-positive neurons and DA content, reduced the number of Iba-1-positive microglia, and reduced the content of inflammatory factors, possibly by inhibiting the AMPK/mTOR signaling pathway. Similar outcomes were observed in the PD cell model. In conclusion, the suppression of IRF8 alleviates neuroinflammation through regulating microglial activation in PD models in vivo and in vitro by the AMPK/mTOR signaling pathway. • Knockdown of IRF8 alleviates neuroinflammation through regulating microglial activation in Parkinson's disease. • Knockdown of IRF8 potently protects dopaminergic neurons via the AMPK/mTOR pathway in a Parkinson's disease model. • IRF8 may be a promising target for clinical diagnosis or therapy to ameliorate with Parkinson's disease. [ABSTRACT FROM AUTHOR]

Age‐associated B cells (ABCs) have emerged as critical components of immune responses. Their inappropriate expansion and differentiation have increasingly been linked to the pathogenesis of autoimmune disorders, aging‐associated diseases, and infections. ABCs exhibit a distinctive phenotype and, in addition to classical B cell markers, often express the transcription factor T‐bet and myeloid markers like CD11c; hence, these cells are also commonly known as CD11c+T‐bet+ B cells. Formation of ABCs is promoted by distinctive combinations of innate and adaptive signals. In addition to producing antibodies, these cells display antigen‐presenting and proinflammatory capabilities. It is becoming increasingly appreciated that the ABC compartment exhibits a high degree of heterogeneity, plasticity, and sex‐specific regulation and that ABCs can differentiate into effector progeny via several routes particularly in autoimmune settings. In this review, we will discuss the initial insights that have been obtained on the molecular machinery that controls ABCs and we will highlight some of the unique aspects of this control system that may enable ABCs to fulfill their distinctive role in immune responses. Given the expanding array of autoimmune disorders and pathophysiological settings in which ABCs are being implicated, a deeper understanding of this machinery could have important and broad therapeutic implications for the successful, albeit daunting, task of targeting these cells. [ABSTRACT FROM AUTHOR]

Mediator of IRF3 activation (MITA) is a significant signal adaptor in the retinoic acid-inducible gene-I like receptor (RLR) signaling pathway and plays an important role in the innate immune system. As a transcription factor, nuclear factor kappa B (NF-κB) can be available in many signaling pathways including the RLR signaling pathway and relative to biological processes like immune responses. In this study, it is determined that IRF4b and IRF8 can have a negative effect on NF-κB signaling pathway mediated by MITA in fish. Firstly, it is found that IRF4b and IRF8 have an inhibitory function on MITA-mediated NF-κB signaling pathway. It is interesting that IRF4b and IRF8 have similar functions to achieve precise downregulated and the degradation of MITA through the ubiquitin-proteasome pathway. IRF is taken as the core domain of IRF4b or IRF8 for the downregulation to MITA. This study provides data on MITA-mediated NF-κB signaling pathway in teleost fish and provides new insights into the regulatory mechanism in fish immune system. [ABSTRACT FROM AUTHOR]

Abstract Background Retinal degeneration is often accompanied by microglia-mediated neuroinflammation. Ferulic acid (FA), an active ingredient of traditional Chinese medicines (TCMs), has been reported to have anti-inflammatory effects. This study explores the impact of FA on microglia-mediated neuroinflammation and associated retinal degeneration in rd10 mice. Methods Rd10 mice received different concentrations of FA every day from postnatal day (P)4 to P24. On P25, the visual function of the mice was evaluated by electroretinogram, and retinae were collected for further investigation. Microglial activation and the expression of relevant cytokines in the retina were evaluated by qPCR, western blotting and immunofluorescence staining. Retinal structure was assessed by haematoxylin and eosin (HE) staining. Results Supplementation with 50 mg/kg FA provided optimal protection against retinal degeneration, with treated mice exhibiting more photoreceptor nuclei as well as greater wave amplitude amplification on electroretinogram than untreated mice. FA suppressed microglial activation both in vivo and in vitro, and inhibited the expression of pro-inflammatory factors Tnfα, IL1β, and Ccl2 in the retinae of rd10 mice. Furthermore, FA suppressed the activation of STAT1 and subsequently inhibited IRF8 expression, potentially highlighting a role for these pathways in FA-mediated immunomodulatory activity. Conclusions Attenuation of neuroinflammation by FA may be beneficial for retarding retinal degeneration.

The functions and profiles of lncRNAs during infectious bursal disease virus (IBDV) infection have not been determined, yet. The objectives of this study were to determine the antiviral action of loc107051710 lncRNA during IBDV infection by investigating the relationship between loc107051710 and IRF8, Type I IFN, STATs, and ISGs. DF-1 cells were either left untreated as non-infected controls (n = 1) or infected with IBDV (n = 3). RNA sequencing was applied for analysis of mRNAs and lncRNAs expression. Differentially expressed genes were verified by RT-qPCR. Then identification, of 230 significantly different expressed genes (182 mRNAs and 48 lncRNA) by pairwise comparison of the infected and control groups, was carried out. The functions of differentially expressed lncRNAs were investigated by selection of lncRNAs and mRNAs significantly enriched in the aforementioned biological processes and signaling pathways for construction of lncRNA-mRNA co-expression networks. The techniques of gene ontology and Kyoto Encyclopedia of Genes and Genomes pathways were applied. It was suggested that these differentially expressed genes were involved in the interaction between the host and IBDV. Loc107051710 was found to have potential antiviral effects. RT-qPCR and western blot were applied and revealed that loc107051710 was required for induction of IRF8, type I IFN, STAT, and ISG expression, and its knockdown promoted IBDV replication. By fluorescence in situ hybridization, it was found that loc107051710 was translocated from the nucleus to the cytoplasm after infection with IBDV. Overall, loc107051710 promoted the production of IFN-α and IFN-β by regulating IRF8, thereby promoting the antiviral activity of ISGs.

Mediator of IRF3 activation (MITA) is a significant signal adaptor in the retinoic acid-inducible gene-I like receptor (RLR) signaling pathway and plays an important role in the innate immune system. As a transcription factor, nuclear factor kappa B (NF-κB) can be available in many signaling pathways including the RLR signaling pathway and relative to biological processes like immune responses. In this study, it is determined that IRF4b and IRF8 can have a negative effect on NF-κB signaling pathway mediated by MITA in fish. Firstly, it is found that IRF4b and IRF8 have an inhibitory function on MITA-mediated NF-κB signaling pathway. It is interesting that IRF4b and IRF8 have similar functions to achieve precise downregulated and the degradation of MITA through the ubiquitin-proteasome pathway. IRF is taken as the core domain of IRF4b or IRF8 for the downregulation to MITA. This study provides data on MITA-mediated NF-κB signaling pathway in teleost fish and provides new insights into the regulatory mechanism in fish immune system.

Background: Interferon regulatory factor-8 (IRF8) and nuclear factor-activated T cells c1 (NFATc1) are two transcription factors that have an important role in osteoclast differentiation. Thanks to ChIP-seq technology, scientists can now estimate potential genome-wide target genes of IRF8 and NFATc1. However, finding target genes that are consistently up-regulated or down-regulated across different studies is hard because it requires analysis of a large number of high-throughput expression studies from a comparable context. Method: We have developed a machine learning based method, called, Cohort-based TF target prediction system (cTAP) to overcome this problem. This method assumes that the pathway involving the transcription factors of interest is featured with multiple "functional groups" of marker genes pertaining to the concerned biological process. It uses two notions, Gene-Present Sufficiently (GP) and Gene-Absent Insufficiently (GA), in addition to log2 fold changes of differentially expressed genes for the prediction. Target prediction is made by applying multiple machine-learning models, which learn the patterns of GP and GA from log2 fold changes and four types of Z scores from the normalized cohort's gene expression data. The learned patterns are then associated with the putative transcription factor targets to identify genes that consistently exhibit Up/Down gene regulation patterns within the cohort. We applied this method to 11 publicly available GEO data sets related to osteoclastgenesis. Result: Our experiment identified a small number of Up/Down IRF8 and NFATc1 target genes as relevant to osteoclast differentiation. The machine learning models using GP and GA produced NFATc1 and IRF8 target genes different than simply using a log2 fold change alone. Our literature survey revealed that all predicted target genes have known roles in bone remodeling, specifically related to the immune system and osteoclast formation and functions, suggesting confidence and validity in our method. Conclusion: cTAP was motivated by recognizing that biologists tend to use Z score values present in data sets for the analysis. However, using cTAP effectively presupposes assembling a sizable cohort of gene expression data sets within a comparable context. As public gene expression data repositories grow, the need to use cohort-based analysis method like cTAP will become increasingly important. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 8 (IRF8) is involved in the pathogenesis of neuropathic pain. However, whether and how IRF8 can regulate the nicotine withdrawal (NTW)-induced hyperalgesia has not been clarified.

ABSTRACT: Interferon regulatory factor 8 (IRF8) is also known as interferon (IFN) consensus sequence binding protein (ICSBP), which plays an important role in IFN signal transduction. In this study, we cloned the full-length coding sequence of Cherry Valley duck IRF8 (duIRF8) and analyzed its structure. In addition, we tested the distribution of IRF8 in the tissues of healthy Cherry Valley ducks, and the changes in IRF8 expression levels in the tissues after virus infection. The results show that the open reading frame (ORF) of IRF8 is 1293 bp, encodes 430 amino acids, and have 3 conserved domains: the N-terminal DBD domain, the C-terminal IAD domain, and the NLS domain. Besides, from the analysis of the phylogenetic tree, it can be known that the duIRF8 has the highest homology with the anser cygnoides, and has less homology with the fish. Analyzing the distribution level of IRF8 in the tissues, it is found that the expression level of IRF8 in the liver of Cherry Valley duck is the highest. However, after infection with duck Tambusu virus, novel duck reovirus, and duck plague virus, the expression of IRF8 in the spleen and brain all showed up-regulation. These data indicate that IRF8 is involved in the host's innate immune response against virus in Cherry Valley duck.

Ferroptosis has emerged as a cytotoxic T lymphocyte (CTL)-induced tumor cell death pathway. The regulation of tumor cell sensitivity to ferroptosis is incompletely understood. Here, we report that interferon regulatory factor 8 (IRF8) functions as a regulator of tumor cell intrinsic ferroptosis. Genome-wide gene expression profiling identified the ferroptosis pathway as an IRF8-regulated pathway in tumor cells. IRF8.KO tumor cells acquire resistance to intrinsic ferroptosis induction and IRF8-deficient tumor cells also exhibit decreased ferroptosis in response to tumor-specific CTLs. Irf8 deletion increased p53 expression in tumor cells and knocking out p53 in IRF8.KO tumor cells restored tumor cell sensitivity to intrinsic ferroptosis induction. Furthermore, IRF8.KO tumor cells grew significantly faster than WT tumor cells in immune-competent mice. To restore IRF8 expression in tumor cells, we designed and synthesized codon usage-optimized IRF8-encoding DNA to generate IRF8-encoding plasmid NTC9385R-mIRF8. Restoring IRF8 expression via a lipid nanoparticle-encapsulated NTC9385R-mIRF8 plasmid therapy suppressed established tumor growth in vivo. In human cancer patients, nivolumab responders have a significantly higher IRF8 expression level in their tumor cells as compared to the non-responders. Our data determine that IRF8 represses p53 expression to maintain tumor cell sensitivity to intrinsic ferroptosis.

Background: Glioblastoma (GBM) has a highly immunosuppressive tumor immune microenvironment (TIME), largely mediated by myeloid-derived suppressor cells (MDSCs). Here, we utilized a retroviral replicating vector (RRV) to deliver Interferon Regulatory Factor 8 (IRF8), a master regulator of type 1 conventional dendritic cell (cDC1) development, in a syngeneic murine GBM model. We hypothesized that RRV-mediated delivery of IRF8 could "reprogram" intratumoral MDSCs into antigen-presenting cells (APCs) and thereby restore T-cell responses., Methods: Effects of RRV-IRF8 on survival and tumor growth kinetics were examined in the SB28 murine GBM model. Immunophenotype was analyzed by flow cytometry and gene expression assays. We assayed functional immunosuppression and antigen presentation by ex vivo T-cell-myeloid co-culture., Results: Intratumoral injection of RRV-IRF8 in mice bearing intracerebral SB28 glioma significantly suppressed the tumor growth and prolonged survival. RRV-IRF8 treated tumors exhibited significant enrichment of cDC1s and CD8+ T-cells. Additionally, myeloid cells derived from RRV-IRF8 tumors showed decreased expression of the immunosuppressive markers Arg1 and IDO1 and demonstrated reduced suppression of naïve T-cell proliferation in ex vivo co-culture, compared to controls. Furthermore, DCs from RRV-IRF8 tumors showed increased antigen presentation compared to those from control tumors. In vivo treatment with azidothymidine (AZT), a viral replication inhibitor, showed that IRF8 transduction in both tumor and non-tumor cells is necessary for survival benefit, associated with a reprogrammed, cDC1- and CD8 T-cell-enriched TIME., Conclusions: Our results indicate that reprogramming of glioma-infiltrating myeloid cells by in vivo expression of IRF8 may reduce immunosuppression and enhance antigen presentation, achieving improved tumor control., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Microglia are innate immune cells in the brain. Transcription factor IRF8 is highly expressed in microglia. However, its role in postnatal microglia development is unknown. We demonstrate that IRF8 binds stepwise to enhancer regions of postnatal microglia along with Sall1 and PU.1, reaching a maximum after day 14. IRF8 binding correlated with a stepwise increase in chromatin accessibility, which preceded the initiation of microglia-specific transcriptome. Constitutive and postnatal Irf8 deletion led to a loss of microglia identity and gain of disease-associated microglia-like genes. Combined analysis of scRNA-seq and scATAC-seq revealed a correlation between chromatin accessibility and transcriptome at a single-cell level. IRF8 was also required for microglia-specific DNA methylation patterns. Lastly, in the 5xFAD model, constitutive and postnatal Irf8 deletion reduced the interaction of microglia with Aβ plaques and the size of plaques, lessening neuronal loss. Together, IRF8 sets the epigenetic landscape, which is required for postnatal microglia gene expression., Competing Interests: Disclosure: There is no conflict of interest.

Strict control of B lymphocyte development is required for the ability to mount humoral immune responses to diverse foreign antigens while remaining self-tolerant. In the bone marrow, B lineage cells transit through several developmental stages in which they assemble a functional B cell receptor in a stepwise manner. The immunoglobulin heavy chain gene is rearranged at the pro-B stage. At the large pre-B stage, cells with a functional heavy chain expand in response to signals from IL-7 and the pre-BCR. Cells then cease proliferation at the small pre-B stage and rearrange the immunoglobulin light chain gene. The fully formed BCR is subsequently expressed on the surface of immature B cells and autoreactive cells are culled by central tolerance mechanisms. Once in the periphery, transitional B cells develop into mature B cell subsets such as marginal zone and follicular B cells. These developmental processes are controlled by transcription factor networks, central to which are IRF4 and IRF8. These were thought to act redundantly during B cell development in the bone marrow, with their functions diverging in the periphery where IRF4 limits the number of marginal zone B cells and is required for germinal center responses and plasma cell differentiation. Because of IRF4's unique role in mature B cells, we hypothesized that it may also have functions earlier in B cell development that cannot be compensated for by IRF8. Indeed, we find that IRF4 has a unique role in upregulating the pre-B cell marker CD25, limiting IL-7 responsiveness, and promoting migration to CXCR4 such that IRF4-deficient mice have a partial block at the pre-B cell stage. We also find that IRF4 acts in early transitional B cells to restrict marginal zone B cell development, as deletion of IRF4 in mature B cells with CD21-cre impairs plasma cell differentiation but has no effect on marginal zone B cell numbers. These studies highlight IRF4 as the dominant IRF family member in early B lymphopoiesis. [ABSTRACT FROM AUTHOR]

Interferon regulatory factor 8 (IRF8), a myeloid lineage transcription factor, emerges as an essential regulator for microglial activation. However, the precise role of IRF8 during Japanese encephalitis virus (JEV) infection in the brain remains elusive. Here, we report that JEV infection enhances IRF8 expression in the infected mouse brain. Comparative transcriptional profiling of whole-brain RNA analysis and validation by quantitative reverse transcription-PCR (qRTPCR) reveals an impaired interferon gamma (IFN-γ) and related gene expression in Irf8 knockout (Irf8-/-)-infected mice. Further, Ifnγ knockout (Ifnγ-/-) mice exhibit a reduced level of Irf8. Both Ifnγ-/- and Irf8-/- mice exhibit significantly reduced levels of activated (CD11b+ CD45hi, CD11b+ CD45lo, Cd68, and CD86) and infiltrating immune cells (Ly6C+, CD4, and CD8) in the infected brain compared to those of wild-type (WT) mice. However, a higher level of granulocyte cell (Ly6G+) infiltration is evident in Irf8-/- mice as well as the increased concentration of tumor necrosis factor alpha (TNF-a), interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP1) levels in the brain. Interestingly, neither the Irf8-/- nor the Ifnγ-/- conferred protection against lethal JEV challenge to mice and exhibit augmentation in JEV replication in the brain. The gain of function of Irf8 by overexpressing functional IRF8 in an IRF8-deficient cell line attenuates viral replication and enhances IFN-γ production. Overall, we summarize that in the murine model of JEV encephalitis, IRF8 modulation affects JEV replication. We also show that lack of Irf8 affects immune cell abundance in circulation and the infected brain, leading to a reduction in IFN-γ level and increased viral load in the brain. IMPORTANCE Microglial cells, the resident macrophages in the brain, play a vital role in Japanese encephalitis virus (JEV) pathogenesis. The deregulated activity of microglia can be lethal for the brain. Therefore, it is crucial to understand the regulators that drive microglia phenotype changes and induce inflammation in the brain. Interferon regulatory factor 8 (IRF8) is a myeloid lineage transcription factor involved in microglial activation. However, the impact of IRF8 modulation on JEV replication remains elusive. Moreover, the pathways regulated by IRF8 to initiate and amplify pathological neuroinflammation are not well understood. Here, we demonstrated the effect of IRF8 modulation on JEV replication, microglial activation, and immune cells infiltration in the brain. [ABSTRACT FROM AUTHOR]

IRF8 is a key regulator of innate immunity receptor signaling and plays diverse functions in the development of hematopoietic cells. The effects of IRF8 on hematopoietic stem cells (HSCs) are still unknown. Here, it is demonstrated that IRF8 deficiency results in a decreased number of long‐term HSCs (LT‐HSCs) in mice. However, the repopulation capacity of individual HSCs is significantly increased. Transcriptomic analysis shows that IFN‐γ and IFN‐α signaling is downregulated in IRF8‐deficient HSCs, while their response to proinflammatory cytokines is unchanged ex vivo. Further tests show that Irf8−/− HSCs can not respond to CpG, an agonist of Toll‐like receptor 9 (TLR9) in mice, while long‐term CpG stimulation increases wild‐type HSC abundance and decreases their bone marrow colony‐forming capacity. Mechanistically, as the primary producer of proinflammatory cytokines in response to CpG stimulation, dendritic cells has a blocked TLR9 signaling due to developmental defect in Irf8−/− mice. Macrophages remain functionally intact but severely reduce in Irf8−/− mice. In NK cells, IRF8 directly regulates the expression of Tlr9 and its deficiency leads to no increased IFNγ production upon CpG stimulation. These results indicate that IRF8 regulates HSCs, at least in part, through controlling TLR9 signaling in diverse innate immune cells. [ABSTRACT FROM AUTHOR]