Insufficient phosphorylation of STAT5 in Tregs inhibits the expression of BLIMP-1 but not IRF4, reduction the proportion of Tregs in pediatric aplastic anemia.

Deficiency in regulatory T cells (Tregs) is an important mechanism underlying the pathogenesis of pediatric aplastic anemia, but its specific mechanism is unclear. In our study, we aimed to investigate whether IL-2/STAT5 can regulate the proliferation of Tregs in aplastic anemia (AA) by regulating their expression of B lymphocyte-induced mature protein-1 (BLIMP-1) or interferon regulatory factor 4 (IRF4). Through clinical research and animal experiments, we found that poor activation of the IL-2/STAT5 signaling pathway may leads to low expression of BLIMP-1 in Tregs of children with AA, which leads to defects in the differentiation and proliferation of Tregs in AA. In AA model mice, treatment with IL-2c reversed the decrease in Treg proportions and reduction in Blimp-1 expression in Tregs by increasing the phosphorylation of Stat5 in Tregs. In AA, deficiency of IRF4 expression in Tregs is closely related to the deficiency of Tregs, but is not regulated by the IL-2/STAT5 pathway.

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.

Maresin 1 Activates LGR6 to Alleviate Neuroinflammation via the CREB/JMJD3/IRF4 Pathway in a Rat Model of Subarachnoid Hemorrhage.

Neuroinflammation is an early event of brain injury after subarachnoid hemorrhage (SAH). Whether the macrophage mediators in resolving inflammation 1 (MaR1) is involved in SAH pathogenesis is unknown. In this study, 205 male Sprague-Dawley rats were subjected to SAH via endovascular perforation in the experimental and control groups. MaR1 was dosed intranasally at 1 h after SAH, with LGR6 siRNA and KG-501, GSK-J4 administered to determine the signaling pathway. Neurobehavioral, histological and biochemical data were obtained from the animal groups with designated treatments. The results showed: (i) The leucine-rich repeat containing G protein-coupled receptor 6 (LGR6) was decreased after SAH and reached to the lowest level at 24 h after SAH. Jumonji d3 (JMJD3) protein levels tended to increase and peaked at 24 h after SAH. LGR6 and JMJD3 expression were co-localized with microglia. (ii) MaR1 administration mitigated short-term neurological deficits, brain edema and long-term neurobehavioral performance after SAH, and attenuated microglial activation and neutrophil infiltration. (iii) Knockdown of LGR6, inhibition of CREB phosphorylation or JMJD3 activity abolished the anti-neuroinflammatory effect of MaR1 on the expression of CREB, CBP, JMJD3, IRF4, IRF5, IL-1ß, IL-6 and IL-10, thus prevented microglial activation and neutrophil infiltration. Together, the results show that MaR1 can activate LGR6 and affect CREB/JMJD3/IRF4 signaling to attenuate neuroinflammation after SAH, pointing to a potential pharmacological utility in this disorder.

IRF4 affects the protective effect of regulatory T cells on the pulmonary vasculature of a bronchopulmonary dysplasia mouse model by regulating FOXP3.

BACKGROUND: Bronchopulmonary dysplasia (BPD) is a common chronic lung disease in preterm infants, characterised by compromised alveolar development and pulmonary vascular abnormalities. Emerging evidence suggests that regulatory T cells (Tregs) may confer protective effects on the vasculature. Knockdown of their transcription factor, interferon regulatory factor 4 (IRF4), has been shown to promote vascular endothelial hyperplasia. However, the involvement of Tregs and IRF4 in the BPD pathogenesis remains unclear. This study aimed to investigate the regulation of Tregs by IRF4 and elucidate its potential role in pulmonary vasculature development in a BPD mouse model. METHODS: The BPD model was established using 85% hyperoxia exposure, with air exposure as the normal control. Lung tissues were collected after 7 or 14 days of air or hyperoxia exposure, respectively. Haematoxylin-eosin staining was performed to assess lung tissue pathology. Immunohistochemistry was used to measure platelet endothelial cell adhesion molecule-1 (PECAM-1) level, flow cytometry to quantify Treg numbers, and Western blot to assess vascular endothelial growth factor (VEGFA), angiopoietin-1 (Ang-1), forkhead box protein P3 (FOXP3), and IRF4 protein levels. We also examined the co-expression of IRF4 and FOXP3 proteins using immunoprecipitation and immunofluorescence double staining. Furthermore, we employed CRISPR/Cas9 technology to knock down the IRF4 gene and observed changes in the aforementioned indicators to validate its effect on pulmonary vasculature development in mice. RESULTS: Elevated IRF4 levels in BPD model mice led to FOXP3 downregulation, reduced Treg numbers, and impaired pulmonary vascular development. Knockdown of IRF4 resulted in improved pulmonary vascular development and upregulated FOXP3 level. CONCLUSION: IRF4 may affect the protective role of Tregs in the proliferation of pulmonary vascular endothelial cells and pulmonary vascular development in BPD model mice by inhibiting the FOXP3 level.

Roles of interferon regulatory factor 4 in the AKI-CKD transition, glomerular diseases and kidney allograft rejection.

Interferon regulatory factor 4 (IRF4) is expressed in immune cells and is a member of the interferon regulatory factor family. Recently, it has been found that IRF4 plays important roles in the acute kidney injury (AKI)-chronic kidney disease (CKD) transition, glomerular diseases and kidney allograft rejection. In particular, the relationship between IRF4 and the AKI-CKD transition has attracted widespread attention. Furthermore, it was also found that the deficiency of IRF4 hindered the transition from AKI to CKD through the suppression of macrophage-to-fibroblast conversion, inhibition of M1-M2 macrophage polarization, and reduction in neutrophil inward flow. Additionally, an examination of the crucial role of IRF4 in glomerular disease was conducted. It was reported that inhibiting IRF4 could alleviate the progression of glomerular disease, and potential physiopathology mechanisms associated with IRF4 were postulated. Lastly, IRF4 was found to have detrimental effects on the development of antibody-mediated rejection (ABMR) and T-cell-mediated rejection (TCMR).

MUM1/IRF4 immunolabeling of neoplastic Langerhans histiocytes in a putative case of canine Langerhans cell histiocytosis.

Langerhans cell histiocytosis is a systemic histiocytic proliferative disease with cutaneous manifestations which is well described in human medical literature and has relatively recently been reclassified as a neoplastic disorder. The diagnosis of canine Langerhans cell histiocytosis has been proposed in the veterinary literature to refer to a histiocytic proliferative disease in the dog with clinical and histopathologic features that mirror the human disease. However, reports that invoke this diagnosis are rare and often lack complete diagnostic characterization. This case report presents an extensive diagnostic investigation of a putative case of Langerhans cell histiocytosis in a 3-year-old male castrated Golden Retriever dog, including gross, cytologic, histopathologic, and immunohistochemical findings. Furthermore, we document that canine LCH may have positive immunolabeling for the transcription factor multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4), which is classically used for the diagnosis of canine plasma cell neoplasms.

IRF4 is required for migration of CD4+ T cells to the intestine but not for Th2 and Th17 cell maintenance.

The transcription factor Interferon Regulatory Factor 4 (IRF4) is central in control of T cell activation and differentiation. Deficiency of IRF4 results in severe immune deficiency and affects maturation and function of most if not all T cell subsets. Here we use mouse infection models for Citrobacter rodentium and Strongyloides ratti to analyze the function of IRF4 in T helper (Th) 17 and Th2 cell responses, respectively. IRF4 deficient mice were impaired in the control of both pathogens, failed to mount Th17 and Th2 cell responses and showed impaired recruitment of T helper cells to the intestine, the infection site of both pathogens. Compromised intestinal migration was associated with reduced expression of the intestinal homing receptors α4ß7 integrin, CCR9 and GPR15. Identification of IRF4 binding sites in the gene loci of these receptors suggests a direct control of their expression by IRF4. Competitive T cell transfer assays further demonstrated that loss of one functional Irf4 allele already affected intestinal accumulation and Th2 and Th17 cell generation, indicating that lower IRF4 levels are of disadvantage for Th2 and Th17 cell differentiation as well as their migration to the intestine. Conversion of peripheral CD4+ T cells from an Irf4 wildtype to an Irf4 heterozygous or from an Irf4 heterozygous to a homozygous mutant genotype after C. rodentium or S. ratti infection did not reduce their capacity to produce Th17 or Th2 cytokines and only partially affected their persistence in the intestine, revealing that IRF4 is not essential for maintenance of the Th2 and Th17 phenotype and for survival of these T helper cells in the intestine. In conclusion, we demonstrate that the expression levels of IRF4 determine Th2 and Th17 cell differentiation and their intestinal accumulation but that IRF4 expression is not crucial for Th2 and Th17 cell survival.

Inhibition of IL-4/STAT6/IRF4 signaling reduces the epithelial-mesenchymal transition in eosinophilic chronic rhinosinusitis with nasal polyps.

BACKGROUND: Previous studies have shown that epithelial-to-mesenchymal transition (EMT) in nasal epithelial cells is critical for tissue remodeling of chronic rhinosinusitis with nasal polyps (CRSwNP). However, the precise mechanism underlying the EMT remains poorly understood. This study aimed to investigate the role of interleukin-4 (IL-4)/signal transducer and activator of transcription 6 (STAT6)/interferon regulatory factor 4 (IRF4) signaling pathway on EMT in eosinophilic CRSwNP. METHODS: We performed quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescent staining, and Western blotting to evaluate the expression of STAT6, IRF4, and EMT markers in sinonasal mucosal samples. Effects of IL-4-induced EMT were determined using primary human nasal epithelial cells (hNECs) from patients with eosinophilic CRSwNP. Wound scratch assay, cell morphology, Western blotting, and immunofluorescence cytochemistry were performed to evaluate EMT, and EMT-related markers. Next, human THP-1 monocytic cells were stimulated by phorbolate-12-myristate-13-acetate to differentiate into M0 and were subsequently polarized into M1 with lipopolysaccharide and interferon-γ, M2 with IL-4. The markers of the macrophage phenotype were assessed by Western blotting. The co-culture system was built to explore the interaction between macrophages (THP-1 cells) and hNECs. After co-culture with M2 macrophages, EMT-related markers of primary hNECs were evaluated by immunofluorescence cytochemistry and Western blotting. Enzymelinked immunosorbent assays were used to detect transforming growth factor beta 1 (TGF-ß1) in THP-1-derived supernatants. RESULTS: STAT6 and IRF4 mRNA and protein expression were significantly upregulated in both eosinophilic and noneosinophilic nasal polyps compared with control tissues. The expression of STAT6 and IRF4 in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps. STAT6 and IRF4 were not only expressed in epithelial cells but also in macrophages. The number of STAT6+CD68+ cells and IRF4+CD68+ cells in eosinophilic nasal polyps was higher than those in noneosinophilic nasal polyps and control tissues. EMT was enhanced in eosinophilic CRSwNP compared to the healthy controls and noneosinophilic CRSwNP. IL-4-stimulated human nasal epithelial cells exhibited EMT characteristics. The hNECs co-cultured with M2 macrophages demonstrated high levels of EMT-related markers. The TGF-ß1 level was significantly induced by IL-4 and elevated (M2) rather than control macrophages. The inhibition of STAT6 by AS1517499 reduced the expression of IRF4 in epithelial cells and macrophages and counteracted IL-4-induced EMT in epithelial cells. CONCLUSION: In eosinophilic nasal polyps, IL-4 induces STAT6 signaling to upregulate IRF4 expression in epithelial cells and macrophages. IL-4 promotes EMT of hNECs through the STAT6/IRF4 signaling pathway. IL-4-induced M2 macrophages enhanced EMT of hNECs. Inhibition of STAT6 can downregulate the expression of IRF4 and suppress the EMT process, thus providing a new strategy for the treatment of nasal polyps.

Animal models for large vessel vasculitis - The unmet need.

Our understanding of the pathogenesis of large vessel vasculitis (LVV) are mainly achieved by studying the arteries taken from temporal artery biopsy in giant cell arteries (GCA) or surgical or autopsy specimens in Takayasu arteritis (TAK). These artery specimens provide invaluable information about pathological changes in these conditions that GCA and TAK are similar but are distinctly different in immune cell infiltrate and distribution of inflammatory cells in anatomical locations. However, these specimens of established arteritis do not provide information of the arteritis initiation and early events which are impossible to obtain in human artery specimens. Animal models for LVV are needed but not available. Here, several approaches are proposed for experimentation to generate animal models to aid in delineating the interaction of immune reaction with arterial wall components.

Meta-Analysis of MS-Based Proteomics Studies Indicates Interferon Regulatory Factor 4 and Nucleobindin1 as Potential Prognostic and Drug Resistance Biomarkers in Diffuse Large B Cell Lymphoma.

The prognosis of diffuse large B cell lymphoma (DLBCL) is inaccurately predicted using clinical features and immunohistochemistry (IHC) algorithms. Nomination of a panel of molecules as the target for therapy and predicting prognosis in DLBCL is challenging because of the divergences in the results of molecular studies. Mass spectrometry (MS)-based proteomics in the clinic represents an analytical tool with the potential to improve DLBCL diagnosis and prognosis. Previous proteomics studies using MS-based proteomics identified a wide range of proteins. To achieve a consensus, we reviewed MS-based proteomics studies and extracted the most consistently significantly dysregulated proteins. These proteins were then further explored by analyzing data from other omics fields. Among all significantly regulated proteins, interferon regulatory factor 4 (IRF4) was identified as a potential target by proteomics, genomics, and IHC. Moreover, annexinA5 (ANXA5) and nucleobindin1 (NUCB1) were two of the most up-regulated proteins identified in MS studies. Functional enrichment analysis identified the light zone reactions of the germinal center (LZ-GC) together with cytoskeleton locomotion functions as enriched based on consistent, significantly dysregulated proteins. In this study, we suggest IRF4 and NUCB1 proteins as potential biomarkers that deserve further investigation in the field of DLBCL sub-classification and prognosis.

Mutually exclusive genetic interactions and gene essentiality shape the genomic landscape of primary melanoma.

Melanoma is a heterogenous malignancy with an unpredictable clinical course. Most patients who present in the clinic are diagnosed with primary melanoma, yet large-scale sequencing efforts have focused primarily on metastatic disease. In this study we sequence-profiled 524 American Joint Committee on Cancer Stage I-III primary tumours. Our analysis of these data reveals recurrent driver mutations, mutually exclusive genetic interactions, where two genes were never or rarely co-mutated, and an absence of co-occurring genetic events. Further, we intersected copy number calls from our primary melanoma data with whole-genome CRISPR screening data to identify the transcription factor interferon regulatory factor 4 (IRF4) as a melanoma-associated dependency. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Expression and clinical significance of interferon regulatory factor 4 and soluble suppression of tumorigenesis 2 in conjunctival epithelial cells and tears of patients with dry eye / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To investigate the expression and clinical significance of interferon regulatory factor 4(IRF4)and soluble suppression of tumorigenesis 2(sST2)in conjunctival epithelial cells and tears of patients with dry eye.METHODS: A total of 94 patients with dry eye who admitted to our hospital from January 2019 to December 2021 were selected as the dry eye group, and 97 physical examiners who underwent ophthalmic examination were selected as the control group at the same time. The conjunctival epithelial cells and tears of the subjects were collected, and the clinical indicators, including tear film break-up time(BUT), corneal fluorescein staining(CFS)score, and Schirmer Ⅰ test(SⅠt)were recorded. The levels of IRF4 and sST2 in conjunctival epithelial cells were detected by quantitative real-time polymerase chain reaction(qRT-PCR), and the levels of IRF4 and sST2 in tears were detected by enzyme-linked immunosorbent assay(ELISA). Pearson method was used to analyze the correlation between IRF4 and sST2 levels in conjunctival epithelial cells and tears and clinical indicators of dry eye patients.RESULTS: The levels of IRF4 and sST2 in conjunctival epithelial cells and tears in dry eye group before treatment were significantly higher than those in control group(P<0.001). The levels of IRF4 and sST2 in conjunctival epithelial cells and tears of dry eye patients at 4wk after treatment were significantly lower than those before treatment(P<0.001). The BUT and SⅠt of dry eye patients increased significantly at 4wk after treatment, and the CFS score decreased significantly(P<0.001). The levels of IRF4 and sST2 in conjunctival epithelial cells and tears of dry eye patients before treatment were positively correlated with CFS score before treatment and negatively correlated with BUT and SⅠt before treatment(P<0.001).CONCLUSION: The levels of IRF4 and sST2 in conjunctival epithelial cells and tears of patients with dry eye are increased, and are significantly correlated with BUT, SⅠt and CFS scores, which has potential to become a new therapeutic target for dry eye.

Construction and validation of an IRF4 risk score to predict prognosis and response to immunotherapy in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) remains a global health challenge due to high recurrence and metastasis rates. The interferon regulatory factor (IRF) family plays an essential role in the tumour immune microenvironment. However, an IRF family-based score that can predict prognosis and response to immunotherapy in HCC patients has not been adequately investigated. Here, we comprehensively evaluated the expression landscape and prognostic significance of IRF family genes as well as their relationship with the immune microenvironment. We further screened IRF4-associated genes to construct a signature and explored their biological features. Then, we established an IRF4 risk score consisting of nine IRF4-associated genes. Importantly, we demonstrated significant differences in the prognostic stratification and immune characteristics of HCC patients with different IRF4 risk scores. The predictive capability of the IRF4 risk score was validated in different HCC subgroups and independent HCC cohorts. Moreover, immunohistochemical analysis of our HCC cohort revealed a positive correlation between IRF4 and PD-1 expression. In vitro experiments demonstrated that the overexpression of IRF4 inhibited the proliferation and migration capacity of HCC cells by restricting the JAK2/STAT3 signalling pathway and epithelial-mesenchymal transition. Overall, our study identified a novel IRF4 risk score that could serve as a robust prognostic biomarker and provide therapeutic benefits for immunotherapy in HCC patients, which may be helpful for clinical decision-making for HCC patients.

Alvocidib inhibits IRF4 expression via super-enhancer suppression and adult T-cell leukemia/lymphoma cell growth.

Adult T-cell leukemia/lymphoma (ATL) is an intractable hematological malignancy with extremely poor prognosis. Recent studies have revealed that super-enhancers (SE) play important roles in controlling tumor-specific gene expression and are potential therapeutic targets for neoplastic diseases including ATL. Cyclin-dependent protein kinase (CDK) 9 is a component of a complex comprising transcription factors (TFs) that bind the SE region. Alvocidib is a CDK9 inhibitor that exerts antitumor activity by inhibiting RNA polymerase (Pol) II phosphorylation and suppressing SE-mediated, tumor-specific gene expression. The present study demonstrated that alvocidib inhibited the proliferation of ATL cell lines and tumor cells from patients with ATL. RNA sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) disclosed that SE regulated IRF4 in the ATL cell lines. Previous studies showed that IRF4 suppression inhibited ATL cell proliferation. Hence, IRF4 is a putative alvocidib target in ATL therapy. The present study revealed that SE-mediated IRF4 downregulation is a possible mechanism by which alvocidib inhibits ATL proliferation. Alvocidib also suppressed ATL in a mouse xenograft model. Hence, the present work demonstrated that alvocidib has therapeutic efficacy against ATL and partially elucidated its mode of action. It also showed that alvocidib is promising for the clinical treatment of ATL and perhaps other malignancies and neoplasms as well.

Expression levels of cellular inhibitor of apoptosis proteins and colitogenic cytokines are inversely correlated with the activation of interferon regulatory factor 4.

Cellular inhibitors of apoptosis proteins 1 (cIAP1) and 2 (cIAP2) are involved in signaling pathways mediated by Toll-like receptors (TLRs) and tumor necrosis factor (TNF)-α. Excessive activation of TLRs and TNF-α underlies the immunopathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). However, the roles played by cIAP1 and cIAP2 in the development of CD and UC remain poorly understood. In this study, we attempted to clarify the molecular link between cIAP1/cIAP2 and colonic inflammation. Human monocyte-derived dendritic cells (DCs) treated with siRNAs specific for cIAP1 or cIAP2 exhibited reduced pro-inflammatory cytokine responses upon stimulation with TLR ligands. Expression of cIAP1 and cIAP2 in human DCs was suppressed in the presence of interferon regulatory factor 4 (IRF4). This effect was associated with inhibition of cIAP1 and cIAP2 polyubiquitination. To verify these in vitro findings, we created mice overexpressing IRF4 in DCs and showed that these mice were resistant to trinitrobenzene sulfonic acid-induced colitis as compared with wild-type mice; these effects were accompanied by reduced expression levels of cIAP1 and cIAP2. Pro-inflammatory cytokine production by mesenteric lymph node cells upon stimulation with TLR ligands was reduced in mice with DC-specific IRF4 overexpression as compared with that in wild-type mice. Finally, in clinical samples of the colonic mucosa from patients with CD, there was a negative relationship between the percentage of IRF4+ DCs and percentages of cIAP1+ or cIAP2+ lamina propria mononuclear cells. These data suggest that the colitogenic roles of cIAP1 and cIAP2 are negatively regulated by IRF4.

Molecular identification and functional characterization of IRF4 from common carp (Cyprinus carpio. L) in immune response: a negative regulator in the IFN and NF-κB signalling pathways.

BACKGROUND: The interferon (IFN) regulatory factors (IRFs) were originally identified as transcription factors playing critical roles in the regulation of IFN-related genes in the signal pathway. In mammals, IRF4 plays a vital role in both the innate and adaptive immune system. This study aims to reveal the molecular characterization, phylogenetic analysis, expression profiles and the regulatory role in the IFN and NF-κB signalling pathways of IRF4 in common carp (Cyprinus carpio. L) (abbreviation, ccIRF4). RESULTS: Here, ccIRF4 was identified and characterized, it contained a DNA binding domain (DBD) which possess five tryptophans and an IRF-associated domain (IAD). The predicted protein sequence of the ccIRF4 showed higher identities with grass carp (Ctenopharyngodon idella) and zebrafish (Danio rerio). Phylogenetic analysis suggested that ccIRF4 has the closest relationship with zebrafish IRF4. Quantitative real-time PCR analysis showed that ccIRF4 was constitutively expressed in all investigated tissues with the highest expression level in the gonad. Polyinosinic:polycytidylic acid (poly I:C) stimulation up-regulated the ccIRF4 expressions in the liver, spleen, head kidney, skin, foregut and hindgut. Upon Aeromonas hydrophila injection, the expression level of ccIRF4 was up-regulated in all tissues with the exception of spleen. In addition, ccIRF4 was induced by lipopolysaccharide (LPS), peptidoglycan (PGN) and Flagellin in head kidney leukocytes (HKLs). Overexpression of the ccIRF4 gene in epithelioma papulosum cyprini cells (EPC) down regulated the expressions of IFN-related genes and proinflammatory factors. Dual-luciferase reporter assay revealed that ccIRF4 decreased the activation of NF-κB through MyD88. CONCLUSIONS: These results indicate that ccIRF4 participates in both antiviral and antibacterial immune response and negatively regulates the IFN and NF-κB response. Overall, our study on ccIRF4 provides more new insights into the innate immune system of common carp as well as a theoretical basis for investigating the pathogenesis and prevention of fish disease.

Modulation of fish immune response by interferon regulatory factor 4 in redlip mullet (Liza haematocheilus): Delineation through expression profiling, antiviral assay, and macrophage polarization analysis.

Interferon regulatory factor 4 (IRF4) is a crucial member of IRF family, which acts as an imperative transcription factor in the development and maturation of multiple lineages of blood cells and also plays a pivotal role in host defense against microbial infections. In the present study, we aimed to investigate the detailed structural and functional aspects of a redlip mullet IRF4 homolog (LhIRF4). The LhIRF4 open reading frame consists of 1347 base pairs encoding 449 amino acids, with the DNA-binding domain sharing significant homology with that of other vertebrate IRF4 homologs. The highest transcription levels of LhIRF4 were observed in the mullet intestine and spleen under normal physiological conditions. Furthermore, a time-dependent upregulation of LhIRF4 transcription was observed in the spleen and head kidney tissues upon pathogenic challenges. When overexpressed in mullet cells, LhIRF4 was localized to the nucleus and significantly stimulated the transcription of several host antiviral genes. Moreover, the overexpression of LhIRF4 strongly inhibited the replication of viral hemorrhagic septicemia virus (VHSV) in vitro. The function of LhIRF4 in regulation of macrophage M2 polarization has also been evidently demonstrated in RAW 264.7 cells. Taken together, our findings indicate the profound role of LhIRF4 in modulating immune responses against microbial infections in redlip mullet.

The molecular basis for the development of adult T-cell leukemia/lymphoma in patients with an IRF4K59R mutation.

Interferon regulatory factor 4 (IRF4) is an essential regulator in the development of many immune cells, including B- and T-cells and has been implicated directly in numerous hematological malignancies, including adult T-cell leukemia/lymphoma (ATLL). Recently, an activating mutation in the DNA-binding domain of IRF4 (IRF4K59R ) was found as a recurrent somatic mutation in ATLL patients. However, it remains unknown how this mutation gives rise to the observed oncogenic effect. To understand the mode of IRF4K59R -mediated gain of function in ATLL pathogenesis, we have determined the structural and affinity basis of IRF4K59R /DNA homodimer complex using X-ray crystallography and surface plasmon resonance. Our study shows that arginine substitution (R59) results in the reorientation of the side chain, enabling the guanidium group to interact with the phosphate backbone of the DNA helix. This markedly contrasts with the IRF4WT wherein the K59 interacts exclusively with DNA bases. Further, the arginine mutation causes enhanced DNA bending, enabling the IRF4K59R to interact more robustly with known DNA targets, as evidenced by increased binding affinity of the protein-DNA complex. Together, we demonstrate how key structural features underpin the basis for this activating mutation, thereby providing a molecular rationale for IRF4K59R -mediated ATLL development.

Pirfenidone inhibits the polarization of M2 macrophages by down-regulating IRF4 to reduce the occurrence and development of radiation-induced lung fibrosis / 中华放射肿瘤学杂志

Objective:To explore the preventive and therapeutic effect of pirfenidone (PFD) on radiation-induced lung fibrosis (RILF) and its mechanism.Methods:40 female C57/BL6 mice were randomly divided into 4 groups: negative control group (NC), PFD treatment group (PFD), radiation treatment group (RT) and radiation plus PFD treatment group (RT+ PFD). Mice in RT and RT+ PFD groups received a single whole lung X-ray consisting of a 50 Gy dose of radiation, delivered by small animal radiation research platform (SARRP). PFD at a dose of 300 mg/kg was administered orally 2 h before irradiation for 150 d. HE and Masson staining were used to detect the infiltration of inflammatory cells and the degree of pulmonary fibrosis. Quantitative real-time PCR (qPCR) and Western blotting (WB) were adopted to detect the expression levels of M1/M2 macrophage phenotypic markers. The expression levels of arginase-1(ARG-1), chitinase 3-like protein 3(YM-1) and interferon regulatory factor-4(IRF4) of macrophages stimulated with IL-4 and IL-13 were detected by WB. In addition, immunofluorescence staining was used to detect the expression and translocation of IRF4 in macrophages among different treatment groups.Results:HE and Masson staining showed that PFD could significantly inhibit radiation-induced infiltration of inflammatory cells and fibrosis in lung tissues. The M2 macrophages and expression levels of ARG-1 and YM-1 were down-regulated in the RT+ PFD group. Cell experiments further confirmed that PFD could significantly inhibit the polarization of macrophages to M2 induced by IL-4+ IL-13, which was mainly related to the down-regulation of IRF4.Conclusion:PFD has a preventive and therapeutic effect on RILF by inhibiting IRF4 and reducing the polarization of macrophages to M2.

Expression of interferon regulatory factor 4 and inflammation in secondary injury of intracerebral haemorrhage.

The aim of this study was to investigate the expression of interferon regulatory factor 4 (IRF4) and the inflammatory response in secondary injury of intracerebral haemorrhage (ICH). Twelve SD rats were randomly divided into a sham group and an ICH group, with 6 rats in each group. A rat model of ICH was established by injecting collagenase type IV into the right striatum of rats. The expression of IRF4 was measured by western blot and immunohistochemistry 48 h after ICH. In addition, 15 mm of hemin-induced PC12 cell injury was used to simulate an in vitro ICH model. IRF4 expression was detected by immunofluorescence (IF). Moreover, the inflammatory cytokines (IL-1b and IL-6) were measured by ELISA. The behavioural score of ICH rats was the lowest at 48 h after operation. The expression of IRF4 was significantly higher in the striatal tissue of ICH rats compared with the sham group (p < 0.05). Meanwhile, IF results showed that hemin induced the upregulation of IRF4 expression in rat pheochromocytoma cells PC12. In addition, IL-1b and IL-6 levels were significantly increased in the serum of ICH rats and in the supernatant of hemin-induced PC12 cells (p < 0.01). The inflammation in ICH is related to the increase of IRF4. It provides a theoretical basis for the clinical treatment of ICH.