Interferon alpha promotes caspase-8 dependent ultraviolet light-mediated keratinocyte apoptosis via interferon regulatory factor 1.

Introduction: Ultraviolet (UV) light is a known trigger of both cutaneous and systemic disease manifestations in lupus patients. Lupus skin has elevated expression of type I interferons (IFNs) that promote increased keratinocyte (KC) death after UV exposure. The mechanisms by which KC cell death is increased by type I IFNs are unknown. Methods: Here, we examine the specific cell death pathways that are activated in KCs by type I IFN priming and UVB exposure using a variety of pharmacological and genetic approaches. Mice that overexpress Ifnk in the epidermis were exposed to UVB light and cell death was measured. RNA-sequencing from IFN-treated KCs was analyzed to identify candidate genes for further analysis that could drive enhanced cell death responses after UVB exposure. Results: We identify enhanced activation of caspase-8 dependent apoptosis, but not other cell death pathways, in type I IFN and UVB-exposed KCs. In vivo, overexpression of epidermal Ifnk resulted in increased apoptosis in murine skin after UVB treatment. This increase in KC apoptosis was not dependent on known death ligands but rather dependent on type I IFN-upregulation of interferon regulatory factor 1 (IRF1). Discussion: These data suggest that enhanced sensitivity to UV light exhibited by lupus patients results from type I IFN priming of KCs that drives IRF1 expression resulting in caspase-8 activation and increased apoptosis after minimal exposures to UVB.

Transcriptomics confirms IRF1 as a key regulator of pyroptosis in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a retinal microvascular complication caused by hyperglycemia, which can lead to visual impairment or blindness. Pyroptosis is a type of inflammation-related programmed cell death, activated by caspase-1, resulting in the maturation of IL-1ß and IL-18 and the rupture of the cell membrane. RNA sequencing (RNA-seq) is a high-throughput sequencing technique that reveals the presence and quantity of RNA in the genome at a specific time point, i.e., the transcriptome. RNA-seq can analyze gene expression levels, splicing variants, mutations, fusions, editing and other post-transcriptional modifications, as well as gene expression differences between different samples or conditions. It has been widely used in biological and medical research, clinical diagnosis and new drug development. This study aimed to establish an in vitro model of diabetic retinopathy by culturing human retinal endothelial cells (HREC) with high glucose (30 mmol/L), and to detect their transcriptome expression by RNA-seq, screen for key genes related to pyroptosis, and validate the sequencing results by subsequent experiments. METHODS: We used RNA-seq to detect the transcriptome expression differences between HREC cells cultured with high glucose and control group, and identified differentially expressed genes by GO/KEGG analysis. We constructed a PPI network and determined the key genes by Cytoscape software and CytoHubba plugin. We validated the expression of related factors by Western Blot, qPCR and ELISA. RESULTS: We performed GO and KEGG analysis on the RNA-seq data and found differentially expressed genes. We used Cytoscape and CytoHubba plugin to screen out IRF1 as the key gene, and then detected the expression of IRF1 in HREC under high glucose and control group by Western Blot and qPCR. We found that the expression of Caspase-1, GSDMD and IL-1ß proteins in HREC under high glucose increased, while the expression of these proteins decreased after the inhibition of IRF1 by siRNA. ELISA showed that the secretion of IL-1ß in HREC under high glucose increased, while the inhibition of IRF1 reduced the secretion of IL-1ß. These results indicate that IRF1 plays an important role in DR, and provides a new target and strategy for the prevention and treatment of this disease.

Mefloquine enhances the efficacy of anti-PD-1 immunotherapy via IFN-γ-STAT1-IRF1-LPCAT3-induced ferroptosis in tumors.

BACKGROUND: Ferroptosis plays an important role in enhancing the efficacy of anti-programmed cell death 1 (PD-1) immunotherapy; however, the molecular mechanisms by which tumor ferroptosis sensitizes melanoma and lung cancer to anti-PD-1 immunotherapy have not been elucidated. METHODS: Cytotoxicity assays, colony formation assays, flow cytometry and animal experiments were used to evaluate the effects of mefloquine (Mef) on survival and ferroptosis in melanoma and lung cancer. RNA sequencing, Real-time quantitative PCR (qRT-PCR), western blotting, chromatin immunoprecipitation-qPCR and flow cytometry were used to determine the molecular mechanisms by which Mef regulates lysophosphatidylcholine acyltransferase 3 (LPCAT3). The relationship between LPCAT3 and the efficacy of anti-PD-1 immunotherapy was verified via a clinical database and single-cell RNA sequencing (ScRNA-Seq). RESULTS: In this study, we discovered that Mef induces ferroptosis. Furthermore, treatment with Mef in combination with T-cell-derived interferon-γ (IFN-γ) enhanced tumor ferroptosis and sensitized melanoma and lung cancer cells to anti-PD-1 immunotherapy. Mechanistically, Mef upregulated the expression of LPCAT3, a key gene involved in lipid peroxidation, by activating IFN-γ-induced STAT1-IRF1 signaling, and knocking down LPCAT3 impaired the induction of ferroptosis by Mef+IFN-γ. Clinically, analysis of the transcriptome and single-cell sequencing results in patients with melanoma showed that LPCAT3 expression was significantly lower in patients with melanoma than in control individuals, and LPCAT3 expression was positively correlated with the efficacy of anti-PD-1 immunotherapy. CONCLUSIONS: In conclusion, our study demonstrated a novel mechanism by which LPCAT3 is regulated, and demonstrated that Mef is a highly promising new target that can be utilized to enhance the efficacy of anti-PD-1 immunotherapy.

Cediranib enhances the transcription of MHC-I by upregulating IRF-1.

Diminished or lost Major Histocompatibility Complex class I (MHC-I) expression is frequently observed in tumors, which obstructs the immune recognition of tumor cells by cytotoxic T cells. Restoring MHC-I expression by promoting its transcription and improving protein stability have been promising strategies for reestablishing anti-tumor immune responses. Here, through cell-based screening models, we found that cediranib significantly upregulated MHC-I expression in tumor cells. This finding was confirmed in various non-small cell lung cancer (NSCLC) cell lines and primary patient-derived lung cancer cells. Furthermore, we discovered cediranib achieved MHC-I upregulation through transcriptional regulation. interferon regulatory factor 1 (IRF-1) was required for cediranib induced MHC-I transcription and the absence of IRF-1 eliminated this effect. Continuing our research, we found cediranib triggered STAT1 phosphorylation and promoted IRF-1 transcription subsequently, thus enhancing downstream MHC-I transcription. In vivo study, we further confirmed that cediranib increased MHC-I expression, enhanced CD8+ T cell infiltration, and improved the efficacy of anti-PD-L1 therapy. Collectively, our study demonstrated that cediranib could elevate MHC-I expression and enhance responsiveness to immune therapy, thereby providing a theoretical foundation for its potential clinical trials in combination with immunotherapy.

TNF and IFNγ-induced cell death requires IRF1 and ELAVL1 to promote CASP8 expression.

TNFα and IFNγ (TNF/IFNγ) synergistically induce caspase-8 activation and cancer cell death. However, the mechanism of IFNγ in promoting TNF-initiated caspase-8 activation in cancer cells is poorly understood. Here, we found that in addition to CASP8, CYLD is transcriptionally upregulated by IFNγ-induced transcription factor IRF1. IRF1-mediated CASP8 and CYLD upregulation additively mediates TNF/IFNγ-induced cancer cell death. Clinically, the expression levels of TNF, IFNγ, CYLD, and CASP8 in melanoma tumors are increased in patients responsive to immune checkpoint blockade (ICB) therapy after anti-PD-1 treatment. Accordingly, our genetic screen revealed that ELAVL1 (HuR) is required for TNF/IFNγ-induced caspase-8 activation. Mechanistically, ELAVL1 binds CASP8 mRNA and extends its stability to sustain caspase-8 expression both in IFNγ-stimulated and in basal conditions. Consequently, ELAVL1 determines death receptors-initiated caspase-8-dependent cell death triggered from stimuli including TNF and TRAIL by regulating basal/stimulated caspase-8 levels. As caspase-8 is a master regulator in cell death and inflammation, these results provide valuable clues for tumor immunotherapy and inflammatory diseases.

Roles of Interferon Regulatory Factor 1 in Tumor Progression and Regression: Two Sides of a Coin.

IRF1 is a transcription factor well known for its role in IFN signaling. Although IRF1 was initially identified for its involvement in inflammatory processes, there is now evidence that it provides a function in carcinogenesis as well. IRF1 has been shown to affect several important antitumor mechanisms, such as induction of apoptosis, cell cycle arrest, remodeling of tumor immune microenvironment, suppression of telomerase activity, suppression of angiogenesis and others. Nevertheless, the opposite effects of IRF1 on tumor growth have also been demonstrated. In particular, the "immune checkpoint" molecule PD-L1, which is responsible for tumor immune evasion, has IRF1 as a major transcriptional regulator. These and several other properties of IRF1, including its proposed association with response and resistance to immunotherapy and several chemotherapeutic drugs, make it a promising object for further research. Numerous mechanisms of IRF1 regulation in cancer have been identified, including genetic, epigenetic, transcriptional, post-transcriptional, and post-translational mechanisms, although their significance for tumor progression remains to be explored. This review will focus on the established tumor-suppressive and tumor-promoting functions of IRF1, as well as the molecular mechanisms of IRF1 regulation identified in various cancers.

Identification of IRF1 as a Novel Pyroptosis-Related Prognostic Biomarker of Atopic Dermatitis.

Purpose: The aim of this study was to characterize key biomarkers associated with pyroptosis in atopic dermatitis (AD). Materials and methods: To identify the differentially expressed pyroptosis-related genes (DEPRGs), the gene expression profiles GSE16161 and GSE32924 from the Gene Expression Omnibus (GEO) database were utilized. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to determine the potential biological functions and involved pathways. Furthermore, protein-protein interaction network analyses were performed to identify hub genes. The types and proportions of infiltrating immune cells were detected by immune filtration analysis using CIBERSORT. A 12-axis competing endogenous RNA (ceRNA) network was constructed utilizing the miRNet database. Immunohistochemistry (IHC) further validated the differential expression of a key gene IRF1 in the skin tissues collected from AD patients. The collection of skin tissue from human subjects in this study were reviewed and approved by the IRB of Yueyang Integrated Chinese and Western Medicine Hospital (KYSKSB2020-125). Results: The study identified a total of 76 DEPRGs, which were enriched in genes associated with the inflammatory response and immune regulation. There was a higher percentage of activated dendritic cells and a lower percentage of resting mast cells in AD samples. PVT1 expression was associated with upregulation of hub genes including CXCL8, IRF1, MKI67, and TP53 in the ceRNA network and was correlated with activated dendritic cells in AD. As a transcription factor, IRF1 could regulate the production of downstream inflammatory factors. The IHC study revealed that IRF1 was overexpressed in the skin tissues of AD patients, which were consistent with the results of the bioinformatic study. Conclusions: IRF1 and its related genes were identified as key pyroptosis-related biomarkers in AD, which is a crucial pathway in the pathogenesis of AD.

PARP7-mediated ADP-ribosylation of FRA1 promotes cancer cell growth by repressing IRF1- and IRF3-dependent apoptosis.

PARP7 was reported to promote tumor growth in a cell-autonomous manner and by repressing the antitumor immune response. Nevertheless, the molecular mechanism of how PARP7-mediated ADP-ribosylation exerts these effects in cancer cells remains elusive. Here, we identified PARP7 as a nuclear and cysteine-specific mono-ADP-ribosyltransferase that modifies targets critical for regulating transcription, including the AP-1 transcription factor FRA1. Loss of FRA1 ADP-ribosylation via PARP7 inhibition by RBN-2397 or mutation of the ADP-ribosylation site C97 increased FRA1 degradation by the proteasome via PSMC3. The reduction in FRA1 protein levels promoted IRF1- and IRF3-dependent cytokine as well as proapoptotic gene expression, culminating in CASP8-mediated apoptosis. Furthermore, high PARP7 expression was indicative of the PARP7 inhibitor response in FRA1-positive lung and breast cancer cells. Collectively, our findings highlight the connected roles of PARP7 and FRA1 and emphasize the clinical potential of PARP7 inhibitors for FRA1-driven cancers.

IRF1 regulates self-renewal and stress responsiveness to support hematopoietic stem cell maintenance.

Hematopoietic stem cells (HSCs) are tightly controlled to maintain a balance between blood cell production and self-renewal. While inflammation-related signaling is a critical regulator of HSC activity, the underlying mechanisms and the precise functions of specific factors under steady-state and stress conditions remain incompletely understood. We investigated the role of interferon regulatory factor 1 (IRF1), a transcription factor that is affected by multiple inflammatory stimuli, in HSC regulation. Our findings demonstrate that the loss of IRF1 from mouse HSCs significantly impairs self-renewal, increases stress-induced proliferation, and confers resistance to apoptosis. In addition, given the frequent abnormal expression of IRF1 in leukemia, we explored the potential of IRF1 expression level as a stratification marker for human acute myeloid leukemia. We show that IRF1-based stratification identifies distinct cancer-related signatures in patient subgroups. These findings establish IRF1 as a pivotal HSC controller and provide previously unknown insights into HSC regulation, with potential implications to IRF1 functions in the context of leukemia.

Quantifying dynamic pro-inflammatory gene expression and heterogeneity in single macrophage cells.

Macrophages must respond appropriately to pathogens and other pro-inflammatory stimuli in order to perform their roles in fighting infection. One way in which inflammatory stimuli can vary is in their dynamics-that is, the amplitude and duration of stimulus experienced by the cell. In this study, we performed long-term live cell imaging in a microfluidic device to investigate how the pro-inflammatory genes IRF1, CXCL10, and CXCL9 respond to dynamic interferon-gamma (IFNγ) stimulation. We found that IRF1 responds to low concentration or short duration IFNγ stimulation, whereas CXCL10 and CXCL9 require longer or higherconcentration stimulation to be expressed. We also investigated the heterogeneity in the expression of each gene and found that CXCL10 and CXCL9 have substantial cell-to-cell variability. In particular, the expression of CXCL10 appears to be largely stochastic with a subpopulation of nonresponding cells across all the stimulation conditions tested. We developed both deterministic and stochastic models for the expression of each gene. Our modeling analysis revealed that the heterogeneity in CXCL10 can be attributed to a slow chromatin-opening step that is on a similar timescale to that of adaptation of the upstream signal. In this way, CXCL10 expression in individual cells can remain stochastic in response to each pulse of repeated stimulation, which we also validated by experiments. Together, we conclude that pro-inflammatory genes in the same signaling pathway can respond to dynamic IFNγ stimulus with very different response features and that upstream signal adaptation can contribute to shaping heterogeneous gene expression.

Correlative analysis of transcriptome and proteome in Penaeus vannamei reveals key signaling pathways are involved in IFN-like antiviral regulation mediated by interferon regulatory factor (PvIRF).

Interferon regulatory factors (IRFs) are crucial transcription factors that regulate interferon (IFN) induction in response to pathogen invasion. The regulatory mechanism of IRF has been well studied in vertebrates, but little has been known in arthropods. Therefore, in order to obtain new insights into the potential molecular mechanism of Peneaus vannamei IRF (PvIRF) in response to viral infection, comprehensive comparative analysis of the transcriptome and proteome profiles in shrimp infected with WSSV after knocking down PvIRF was conducted by using RNA sequencing (RNA-seq) and isobaric tags for relative and absolute quantification (iTRAQ). The sequence characterization, molecular functional evolution and 3D spatial structure of PvIRF were analyzed by using bioinformatics methods. PvIRF share the higher homology with different species in N-terminal end (containing DNA binding domain (DBD) including DNA sequence recognition sites and metal binding site) than that in C-terminal end. Within 4 IRF subfamilies of vertebrates, PvIRF had closer relationship with IRF1 subfamily. The DBD of PvIRF and C. gigas IRF1a were composed of α-helices and ß-folds which was similar with the DBD structure of M. musculus IRF2. Interestingly, different from the five Tryptophan repeats highly homologous in the DBD of vertebrate IRF, the first and fifth tryptophans of PvIRF mutate to Phenylalanine and Leucine respectively, while the mutations were conserved among shrimp IRFs. RNAi knockdown of PvIRF gene by double-strand RNA could obviously promote the in vivo propagation of WSSV in shrimp and increase the mortality of WSSV-infected shrimp. It suggested that PvIRF was involved in inhibiting the replication of WSSV in shrimp. A total of 8787 transcripts and 2846 proteins were identified with significantly differential abundances in WSSV-infected shrimp after PvIRF knockdown, among which several immune-related members were identified and categorized into 10 groups according to their possible functions. Furthermore, the variation of expression profile from members of key signaling pathways involving JAK/STAT and Toll signaling pathway implied that they might participate IRF-mediated IFN-like regulation in shrimp. Correlative analyses indicated that 722 differentially expressed proteins (DEPs) shared the same expression profiles with their corresponding transcripts, including recognition-related proteins (CTLs and ITGs), chitin-binding proteins (peritrophin), and effectors (ALFs and SWD), while 401 DEPs with the opposite expression profiles across the two levels emphasized the critical role of post-transcriptional and post-translational modification. The results provide candidate signaling pathway including pivotal genes and proteins involved in the regulatory mechanism of interferon mediated by IRF on shrimp antiviral response. This is the first report in crustacean to explore the IFN-like antiviral regulation pathway mediated by IRF on the basis of transcriptome and proteomics correlative analysis, and will provide new ideas for further research on innate immune and defense mechanisms of crustacean.

RSV replication modifies the XBP1s binding complex on the IRF1 upstream enhancer to potentiate the mucosal anti-viral response.

Introduction: The unfolded protein response (UPR) has emerged as an important signaling pathway mediating anti-viral defenses to Respiratory Syncytial Virus (RSV) infection. Earlier we found that RSV replication predominantly activates the evolutionarily conserved Inositol Requiring Enzyme 1α (IRE1α)-X-Box Binding Protein 1 spliced (XBP1s) arm of the Unfolded Protein Response (UPR) producing inflammation, metabolic adaptation and cellular plasticity, yet the mechanisms how the UPR potentiates inflammation are not well understood. Methods: To understand this process better, we examined the genomic response integrating RNA-seq and Cleavage Under Targets and Release Using Nuclease (CUT&RUN) analyses. These data were integrated with an RNA-seq analysis conducted on RSV-infected small airway cells ± an IRE1α RNAse inhibitor. Results: We identified RSV induced expression changes in ~3.2K genes; of these, 279 required IRE1α and were enriched in IL-10/cytokine signaling pathways. From this data set, we identify those genes directly under XBP1s control by CUT&RUN. Although XBP1s binds to ~4.2 K high-confidence genomic binding sites, surprisingly only a small subset of IL10/cytokine signaling genes are directly bound. We further apply CUT&RUN to find that RSV infection enhances XBP1s loading on 786 genomic sites enriched in AP1/Fra-1, RELA and SP1 binding sites. These control a subset of cytokine regulatory factor genes including IFN response factor 1 (IRF1), CSF2, NFKB1A and DUSP10. Focusing on the downstream role of IRF1, selective knockdown (KD) and overexpression experiments demonstrate IRF1 induction controls type I and -III interferon (IFN) and IFN-stimulated gene (ISG) expression, demonstrating that ISG are indirectly regulated by XBP1 through IRF1 transactivation. Examining the mechanism of IRF1 activation, we observe that XBP1s directly binds a 5' enhancer sequence whose XBP1s loading is increased by RSV. The functional requirement for the enhancer is demonstrated by targeting a dCas9-KRAB silencer, reducing IRF1 activation. Chromatin immunoprecipitation shows that XBP1 is required, but not sufficient, for RSV-induced recruitment of activated phospho-Ser2 Pol II to the enhancer. Discussion: We conclude that XBP1s is a direct activator of a core subset of IFN and cytokine regulatory genes in response to RSV. Of these IRF1 is upstream of the type III IFN and ISG response. We find that RSV modulates the XBP1s binding complex on the IRF1 5' enhancer whose activation is required for IRF1 expression. These findings provide novel insight into how the IRE1α-XBP1s pathway potentiates airway mucosal anti-viral responses.

SPOP targets the immune transcription factor IRF1 for proteasomal degradation.

Adaptation of the functional proteome is essential to counter pathogens during infection, yet precisely timed degradation of these response proteins after pathogen clearance is likewise key to preventing autoimmunity. Interferon regulatory factor 1 (IRF1) plays an essential role as a transcription factor in driving the expression of immune response genes during infection. The striking difference in functional output with other IRFs is that IRF1 also drives the expression of various cell cycle inhibiting factors, making it an important tumor suppressor. Thus, it is critical to regulate the abundance of IRF1 to achieve a 'Goldilocks' zone in which there is sufficient IRF1 to prevent tumorigenesis, yet not too much which could drive excessive immune activation. Using genetic screening, we identified the E3 ligase receptor speckle type BTB/POZ protein (SPOP) to mediate IRF1 proteasomal turnover in human and mouse cells. We identified S/T-rich degrons in IRF1 required for its SPOP MATH domain-dependent turnover. In the absence of SPOP, elevated IRF1 protein levels functionally increased IRF1-dependent cellular responses, underpinning the biological significance of SPOP in curtailing IRF1 protein abundance.

Immune regulator IRF1 contributes to ZBP1-, AIM2-, RIPK1-, and NLRP12-PANoptosome activation and inflammatory cell death (PANoptosis).

The innate immune system provides the first line of defense against pathogens and cellular insults and is activated by pattern recognition receptors sensing pathogen- or damage-associated molecular patterns. This activation can result in inflammation via cytokine release as well as the induction of lytic regulated cell death (RCD). Innate immune signaling can also induce the expression of interferon regulatory factor 1 (IRF1), an important molecule in regulating downstream inflammation and cell death. While IRF1 has been shown to modulate some RCD pathways, a comprehensive evaluation of its role in inflammatory cell death pathways is lacking. Here, we examined the role of IRF1 in cell death during inflammasome and PANoptosome activation using live cell imaging, Western blotting, and ELISA in primary murine macrophages. IRF1 contributed to the induction of ZBP1- (Z-DNA binding protein 1), AIM2- (absent in melanoma-2), RIPK1- (receptor interacting protein kinase 1), and NLRP12 (NOD-like receptor family, pyrin domain-containing 12)-PANoptosome activation and PANoptosis. Furthermore, IRF1 regulated the cell death under conditions where inflammasomes, along with caspase-8 and RIPK3, act as integral components of PANoptosomes to drive PANoptosis. However, it was dispensable for other inflammasomes that form independent of the PANoptosome to drive pyroptosis. Overall, these findings define IRF1 as an upstream regulator of PANoptosis and suggest that modulating the activation of molecules in the IRF1 pathway could be used as a strategy to treat inflammatory and infectious diseases associated with aberrant inflammatory cell death.

IRF1 suppresses colon cancer proliferation by reducing SPI1-mediated transcriptional activation of GPX4 and promoting ferroptosis.

IRF1 is a tumor suppressor gene in colon cancer. This study aimed to explore the potential regulation of IRF1 on the ferroptosis of colon cancer and the mechanisms underlying its regulation of GPX4 transcription. IRF1 interacting transcription factors regulating GPX4 transcription were predicted and validated. The role of the IRF1/SPI1-GPX4 axis on the ferroptosis of colon cancer cells was explored. Results showed that IRF1 overexpression reduced GPX4 transcription, increased reactive oxygen species (ROS) and lipid ROS accumulation, and enhanced erastin-induced colon cancer cell growth in vitro and in vivo. SPI1 could directly bind to the GPX4 promoter (-414 to -409) and activate its transcription. IRF1 could bind to SPI1 and suppress its transcriptional activating effects on GPX4 expression. SPI1 overexpression reduced ROS and lipid ROS accumulation and increased colon cancer cell viability and colony formation upon erastin induction. These trends were reversed by IRF1 overexpression. In conclusion, this study revealed a novel oncogenic mechanism of SPI1 by reducing erastin-induced ferroptosis in colon cancer. IRF1 interacts with SPI1 and suppresses its transcriptional activating effect on GPX4 expression. Through this mechanism, IRF1 can enhance erastin-induced ferroptosis of colon cancer. The IRF1/SPI1-GPX4 axis might play a crucial role in modulating ferroptosis in colon cancer and might serve as a potential therapeutic target in the future.

ABCA1 deficiency contributes to podocyte pyroptosis priming via the APE1/IRF1 axis in diabetic kidney disease.

Decreased ATP Binding Cassette Transporter A1 (ABCA1) expression and caspase-4-mediated noncanonical inflammasome contribution have been described in podocytes in diabetic kidney disease (DKD). To investigate a link between these pathways, we evaluated pyroptosis-related mediators in human podocytes with stable knockdown of ABCA1 (siABCA1) and found that mRNA levels of IRF1, caspase-4, GSDMD, caspase-1 and IL1ß were significantly increased in siABCA1 compared to control podocytes and that protein levels of caspase-4, GSDMD and IL1ß were equally increased. IRF1 knockdown in siABCA1 podocytes prevented increases in caspase-4, GSDMD and IL1ß. Whereas TLR4 inhibition did not decrease mRNA levels of IRF1 and caspase-4, APE1 protein expression increased in siABCA1 podocytes and an APE1 redox inhibitor abrogated siABCA1-induced expression of IRF1 and caspase-4. RELA knockdown also offset the pyroptosis priming, but ChIP did not demonstrate increased binding of NFκB to IRF1 promoter in siABCA1 podocytes. Finally, the APE1/IRF1/Casp1 axis was investigated in vivo. APE1 IF staining and mRNA levels of IRF1 and caspase 11 were increased in glomeruli of BTBR ob/ob compared to wildtype. In conclusion, ABCA1 deficiency in podocytes caused APE1 accumulation, which reduces transcription factors to increase the expression of IRF1 and IRF1 target inflammasome-related genes, leading to pyroptosispriming.

The underlying mechanism of transcription factor IRF1, PRDM1, and ZNF263 involved in the regulation of NPPB rs3753581 on pulse pressure hypertension.

To investigate the correlation between NPPB gene variants and pulse pressure hypertension and the underlying regulatory mechanisms and try to confirm that NPPB may be a potential molecular target of gene therapy for pulse pressure hypertension. A total of 898 participants were recruited from the First Affiliated Hospital of Fujian Medical University and the plasmids with differential expression of NPPB were constructed. Genotype distribution of NPPB(rs3753581, rs198388, and rs198389)was analyzed and the expression of N-terminal pro-B-type natriuretic peptide(NT-proBNP) and renin-angiotensin -aldosterone system(RAAS) related indicators were identified in the groups studied. According to a genotype analysis, there was a significant difference in the genotype distribution of NPPB rs3753581 among the groups (P = 0.034). In logistic regression analysis, NPPB rs3753581 TT was associated with a 1.8-fold greater risk of pulse pressure hypertension than NPPB rs3753581 GG (odds ratio = 1.801; 95% confidence interval: 1.070-3.032; P = 0.027). The expression of NT-proBNP and RAAS related indicators in clinical and laboratory samples showed striking differences. The activity of firefly and Renilla luciferase in pGL-3-NPPB-luc (-1299G) was higher than pGL-3-NPPBmut-luc(-1299 T)(P < 0.05). The binding of NPPB gene promoter rs3753581 (-1299G) with transcription factors IRF1, PRDM1, and ZNF263 was predicted and validated by the bioinformatics software TESS and chromatin immunoprecipitation(P < 0.05). NPPB rs3753581 was correlated with genetic susceptibility to pulse pressure hypertension and the transcription factors IRF1, PRDM1, and ZNF263 may be involved in the regulation of NPPB rs3753581 promoter (-1299G) on the expression of NT-proBNP/RAAS.

A robust gene prognostic index composed of GZMB, IRF1, and TP63 can stratify the risk of two metastatic urothelial carcinoma cohorts based on immune checkpoint blockade therapy.

BACKGROUND: Immune checkpoint blockade (ICB) therapy has become a first-line treatment option for metastatic urothelial carcinoma (mUC) patients who do not meet the criteria of cisplatin. Still, only a few people can benefit from it, so useful predictive markers are needed. METHODS: Download the ICB-based mUC and chemotherapy-based bladder cancer cohorts, and extract the expression data of pyroptosis-related genes (PRG). The LASSO algorithm was used to construct the PRG prognostic index (PRGPI) in the mUC cohort, and we verified the prognostic ability of PRGPI in two mUC and two bladder cancer cohorts. RESULTS: Most of the PRG in the mUC cohort were immune-activated genes, and a few were immunosuppressive genes. The PRGPI composed of GZMB, IRF1, and TP63 can stratify the risk of mUC. In IMvigor210 and GSE176307 cohorts, the P-values of Kaplan Meier analysis was < 0.01 and 0.002, respectively. PRGPI could also predict ICB response, and the chi-square test of the two cohorts had P-values of 0.002 and 0.046, respectively. In addition, PRGPI can also predict the prognosis of two bladder cancer cohorts without ICB therapy. The PRGPI and the expression of PDCD1/CD274 had a high degree of synergistic correlation. The Low PRGPI group showed prominent characteristics of immune infiltration and was enriched in the immune signal activation pathway. CONCLUSION: The PRGPI we constructed can effectively predict the treatment response and overall survival rate of mUC patients treated with ICB. The PRGPI can help mUC patients achieve individualized and accurate treatment in the future.

Exosomal IRF1-loaded rat adipose-derived stem cell sheet contributes to wound healing in the diabetic foot ulcers.

BACKGROUND: Cell-based therapy has been recognized as a novel technique for the management of diabetic foot ulcers, and cell-sheet engineering leads to improved efficacy in cell transplantation. This study aims to explore the possible molecular mechanism of the rat adipose-derived stem cell (ASC) sheet loaded with exosomal interferon regulatory factor 1 (IRF1) in foot wound healing. METHODS: Rats were rendered diabetic with streptozotocin, followed by measurement of miR-16-5p expression in wound tissues. Relationship between IRF1, microRNA (miR)-16-5p, and trans-acting transcription factor 5 (SP5) was analyzed using luciferase activity, RNA pull-down, and chromatin immunoprecipitation assays. IRF1 was overexpressed in rat ASCs (rASCs) or loaded onto the rASC sheet, and then exosomes were extracted from rASCs. Accordingly, we assessed the effects of IRF1-exosome or IRF1-rASC sheet on the proliferation and migration of the fibroblasts along with endothelial cell angiogenesis. RESULTS: miR-16-5p was poorly expressed in the wound tissues of diabetic rats. Overexpression of miR-16-5p promoted fibroblast proliferation and migration as well as endothelial cell angiogenesis, thus expediting wound healing. IRF1 was an upstream transcription factor that could bind to the miR-16-5p promoter and increase its expression. In addition, SP5 was a downstream target gene of miR-16-5p. IRF1-exosome from rASCs or the IRF1-rASC sheet facilitated the foot wound healing in diabetic rats through miR-16-5p-dependent inhibition of SP5. CONCLUSION: The present study demonstrates that exosomal IRF1-loaded rASC sheet regulates miR-16-5p/SP5 axis to facilitate wound healing in diabetic rats, which aids in development of stem cell-based therapeutic strategies for diabetic foot wounds.

CSNK2B modulates IRF1 binding to functional DNA elements and promotes basal and agonist-induced antiviral signaling.

Interferon regulatory factor 1 (IRF1) is a critical component of cell-intrinsic innate immunity that regulates both constitutive and induced antiviral defenses. Due to its short half-life, IRF1 function is generally considered to be regulated by its synthesis. However, how IRF1 activity is controlled post-translationally has remained poorly characterized. Here, we employed a proteomics approach to identify proteins interacting with IRF1, and found that CSNK2B, a regulatory subunit of casein kinase 2, interacts directly with IRF1 and constitutively modulates its transcriptional activity. Genome-wide CUT&RUN analysis of IRF1 binding loci revealed that CSNK2B acts generally to enhance the binding of IRF1 to chromatin, thereby enhancing transcription of key antiviral genes, such as PLAAT4 (also known as RARRES3/RIG1/TIG3). On the other hand, depleting CSNK2B triggered abnormal accumulation of IRF1 at AFAP1 loci, thereby down-regulating transcription of AFAP1, revealing contrary effects of CSNK2B on IRF1 binding at different loci. AFAP1 encodes an actin crosslinking factor that mediates Src activation. Importantly, CSNK2B was also found to mediate phosphorylation-dependent activation of AFAP1-Src signaling and exert suppressive effects against flaviviruses, including dengue virus. These findings reveal a previously unappreciated mode of IRF1 regulation and identify important effector genes mediating multiple cellular functions governed by CSNK2B and IRF1.