IRF-1 mRNA Research Articles

Arsenic-induced mtDNA release promotes inflammatory responses through cGAS-STING signaling in chicken hepatocytes

Arsenic is a toxic element that can cause severe liver damage in humans and animals. Arsenic-based inorganic pesticides, such as lead arsenate, copper arsenate, and calcium arsenate, are widely used for insect control and can eventually affect human health through accumulation in the food chain. However, the relationship between arsenic trioxide (ATO)-induced hepatotoxicity and the cGAS-STING signaling pathway has not been reported. The aim of this study was to investigate the potential role of inflammatory response in ATO-induced hepatotoxicity in chickens. In this study, we found that ATO exposure resulted in mtDNA leakage into the cytoplasm of chicken hepatocytes, which activated the cGAS-STING pathway and significantly increased the cGAS, STING, TBK1, and IRF7 mRNA and protein expression levels. Moreover, type I interferon response was activated. Concurrently, STING triggered the activation of the traditional NF-κB signaling pathway and promoted the expression of pro-inflammatory cytokine genes, including TNF-α, IL-6, and IL-1β. Subsequently, we found that both mtDNA clearance with EtBr and inhibition of the cGAS-STING pathway with H-151 reversed the ATO-induced innate immune and inflammatory responses. In summary, the above findings indicate that chicken hepatocytes can induce innate immune responses and inflammatory responses via mtDNA-cGAS-STING under ATO-exposure conditions, which is of great significance for further studies on the toxicity mechanism of ATO.

SLC25A12 inhibits Japanese encephalitis virus replication by interacting with the NS1 and enhancing the type I interferon response

Japanese encephalitis virus (JEV) is a mosquito-borne, zoonotic orthoflavivirus causing human encephalitis and reproductive disorders in pigs. Cell-intrinsic antiviral restriction factors are the first line of defense that prevent a virus from establishing a productive infection, while the molecular mechanism of the virus-host interaction is still not fully understood. Our in vitro experiments demonstrated that the Solute Carrier Family 25 Member 12 (SLC25A12) interacted with the JEV nonstructural protein 1 (NS1) and inhibited JEV replication. Furthermore, we showed that knockdown or knockout of SLC25A12 promoted JEV replication, while overexpression of SLC25A12 repressed viral replication. Finally, we demonstrated that SLC25A12 increased IRF7 mRNA levels, which promoted IFN-β expression and subsequently induced antiviral effects. Collectively, our study revealed that SLC25A12 interacted with NS1, inhibiting viral RNA synthesis and transcription and enhancing type I interferon induction for antiviral effects.

The canonical antiviral protein oligoadenylate synthetase 1 elicits antibacterial functions by enhancing IRF1 translation

An important property of the host innate immune response during microbial infection is its ability to control the expression of antimicrobial effector proteins, but how this occurs post-transcriptionally is not well defined. Here, we describe a critical antibacterial role for the classic antiviral gene 2'-5'-oligoadenylate synthetase 1 (OAS1). Human OAS1 and its mouse ortholog, Oas1b, are induced by interferon-γ and protect against cytosolic bacterial pathogens such as Francisella novicida and Listeria monocytogenes invitro and invivo. Proteomic and transcriptomic analysis showed reduced IRF1 protein expression in OAS1-deficient cells. Mechanistically, OAS1 binds and localizes IRF1 mRNA to the rough endoplasmic reticulum (ER)-Golgi endomembranes, licensing effective translation of IRF1 mRNA without affecting its transcription or decay. OAS1-dependent translation of IRF1 leads to the enhanced expression of antibacterial effectors, such as GBPs, which restrict intracellular bacteria. These findings uncover a noncanonical function of OAS1 in antibacterial innate immunity.

Smoking-Mediated miR-301a/IRF1 Axis Controlling Immunotherapy Response in Lung Squamous Cell Carcinoma Revealed by Bioinformatic Analysis.

Smoking is an established risk factor for a variety of malignant tumors, the most well-known of which is lung cancer. Various molecular interactions are known to link tobacco smoke exposure to lung cancer, but new data are still emerging on the effects of smoking on lung cancer development, progression, and tumor response to therapy. In this study, we reveal in further detail the previously established association between smoking and hsa-mir-301a activity in lung squamous cell carcinoma, LUSC. Using different bioinformatic tools, we identified IRF1 as a key smoking-regulated target of hsa-mir-301a in LUSC. We further confirmed this relationship experimentally using clinical LUSC tissue samples and intact lung tissue samples. Thus, increased hsa-mir-301a levels, decreased IRF1 mRNA levels, and their negative correlation were shown in LUSC tumor samples. Additional bioinformatic investigation for potential pathways impacted by such a mechanism demonstrated IRF1's multifaceted role in controlling the antitumor immune response in LUSC. IRF1 was then shown to affect tumor immune infiltration, the expression of immune checkpoint molecules, and the efficacy of immune checkpoint blockade therapy. As a result, here we suggest a smoking-regulated mir301a/IRF1 molecular axis that could modulate the antitumor immune response and immunotherapy efficacy in LUSC, opening up novel opportunities for future research.

The prognostic impact of transforming growth factor beta 2 (TGFB2) mRNA levels, in conjunction with interferon-gamma receptor activation of interferon regulatory factor 5 (IRF5) and expression of CD276/B7-H3 in low-grade gliomas.

e14564 Background: Low-grade glioma (LGG) tumors are characterized by a low infiltration of immune cells, requiring therapeutic interventions to boost the immune response. Methods: We analyzed mRNA expression datasets from the UCSC Xena web platform to compare normal brain tissue with LGG tumor samples to screen for upregulated genes. We also used cBioportal to determine the relationship between mRNA expression levels of 513 LGG patients and their overall survival (OS) outcomes. Results: Tumor-associated macrophage (TAM) markers, MSR1/CD204, CD86, and CD68, exhibited a 6-fold (P<0.0001), 8.9-fold (P<0.0001), and 15.6-fold increases in mRNA expression levels, respectively in LGG tumors. Both TGFB1 (4.1-fold increase, P<0.0001) and TGFB2 (2.2-fold increase, P<0.0001) were upregulated in brain tumors, suggesting that TGFB ligands are pivotal in establishing an immunosuppressive TME. In addition, mRNA upregulation of interferon-gamma receptors, IFNGR1 and IFNGR2, and signaling molecules STAT1, IRF1, and IRF5, pointed to an essential role for IFN-γ mediated remodeling of the TME. A tumor-associated antigen, CD276/B7-H3, mRNA expression showed a significant (P<0.0001) 4.03-fold increase in tumor tissue. Multivariate Cox proportional hazards models investigating the interaction of TGFB2 and activation of IFNGR2, STAT1, IRF1, or IRF5 showed that the prognostic impact of high mRNA levels (25 percentile cut-off) of TGFB2high was independent of IFNGR2, STAT1, IRF1, or IRF5 mRNA levels (TGFB2high HR (95% CI) = 4.07 (2.35-7.06), 6 (3.62-10.11), 4.38 (2.67-7.17), and 4.48 (2.82-7.12) for models with IFNGR2, STAT1, IRF1, or IRF5 respectively). Patients with high levels of TGFB2 were overrepresented by LGG patients with IDH wild-type mutation status. The prognostic impact of TGFB2high and IDH wild-type observed by the increases in hazard ratios for TGFB2 (HR (95% CI range) = 2.02 (1.05-3.89)) and IDH wild-type (HR (95% CI range) = 4.44 (1.9-10.4)) were independent predictors of survival requiring risk stratification of patients identify LGG patients with IDH wild-type and TGFB2high in the design of clinical trials. The ratio of TGFB1:TGFB2 expression levels was also a prognostic indicator for OS, whereby patients who exhibited a 2.5-fold greater level of TGFB1 compared to TGFB2 mRNA levels experienced improved survival outcomes. A 1.8-fold higher TGFB2 levels, the mRNA expression corresponded to worse OS. Conclusions: The specific abrogation of TGFB2 and not TGFB1 by OT101, a TGFB2-specific synthetic phosphorothioate antisense oligodeoxynucleotide, is a prerequisite to employing a strategy to target TGFB pathways. IRF5 and CD276/B7-H3 as prognostic markers are potential targets for combination therapies with TGFB2 inhibitors.

Accelerated plasma-cell differentiation in Bach2-deficient mouse B cells is caused by altered IRF4 functions.

Transcription factors BACH2 and IRF4 are both essential for antibody class-switch recombination (CSR) in activated B lymphocytes, while they oppositely regulate the differentiation of plasma cells (PCs). Here, we investigated how BACH2 and IRF4 interact during CSR and plasma-cell differentiation. We found that BACH2 organizes heterochromatin formation of target gene loci in mouse splenic B cells, including targets of IRF4 activation such as Aicda, an inducer of CSR, and Prdm1, a master plasma-cell regulator. Release of these gene loci from heterochromatin in response to B-cell receptor stimulation was coupled to AKT-mTOR pathway activation. In Bach2-deficient B cells, PC genes' activation depended on IRF4 protein accumulation, without an increase in Irf4 mRNA. Mechanistically, a PU.1-IRF4 heterodimer in activated B cells promoted BACH2 function by inducing gene expression of Bach2 and Pten, a negative regulator of AKT signaling. Elevated AKT activity in Bach2-deficient B cells resulted in IRF4 protein accumulation. Thus, BACH2 and IRF4 mutually modulate the activity of each other, and BACH2 inhibits PC differentiation by both the repression of PC genes and the restriction of IRF4 protein accumulation.

Transforming Growth Factor Beta 2 (TGFB2) mRNA Levels, in Conjunction with Interferon-Gamma Receptor Activation of Interferon Regulatory Factor 5 (IRF5) and Expression of CD276/B7-H3, Are Therapeutically Targetable Negative Prognostic Markers in Low-Grade Gliomas.

LGG tumors are characterized by a low infiltration of immune cells, requiring therapeutic interventions to boost the immune response. We conducted a study analyzing mRNA expression datasets from the UCSC Xena web platform. To screen for upregulated genes, we sought to compare normal brain tissue with LGG tumor samples. We also used cBioportal to determine the relationship between mRNA expression levels of 513 LGG patients and their overall survival (OS) outcomes. Three tumor-associated macrophage (TAM) markers, MSR1/CD204, CD86, and CD68, exhibited a 6-fold (p < 0.0001), 8.9-fold (p < 0.0001), and 15.6-fold increase in mRNA expression levels, respectively, in LGG tumors. In addition, both TGFB1 (4.1-fold increase, p < 0.0001) and TGFB2 (2.2-fold increase, p < 0.0001) ligands were also upregulated in these tumors compared to normal brain tissue, suggesting that TGFB ligands are pivotal in establishing an immunosuppressive, angiogenic, and pro-tumorigenic TME in gliomas mediated through TAMs. In addition, mRNA upregulation of interferon-gamma receptors, IFNGR1 and IFNGR2, and the downstream signaling molecules STAT1, IRF1, and IRF5, pointed to an essential role for IFN-γ mediated remodeling of the TME. Interestingly, the mRNA expression of a tumor-associated antigen, CD276/B7-H3, showed a significant (p < 0.0001) 4.03-fold increase in tumor tissue, giving further insights into the roles of macrophages and tumor cells in supporting the immunosuppressive TME. Multivariate Cox proportional hazards models investigating the interaction of TGFB2 and activation of IFNGR2, STAT1, IRF1, or IRF5 showed that the prognostic impact of high mRNA levels (25th percentile cut-off) of TGFB2 was independent of IFNGR2, STAT1, IRF1, or IRF5 mRNA levels (TGFB2high HR (95% CI) = 4.07 (2.35-7.06), 6 (3.62-10.11), 4.38 (2.67-7.17), and 4.48 (2.82-7.12) for models with IFNGR2, STAT1, IRF1, or IRF5, respectively) and age at diagnosis. Patients with high levels of TGFB2 and IFNGR2 were over-represented by LGG patients with isocitrate dehydrogenase wild-type (IDHwt) mutation status. The prognostic impact of high levels of TGFB2 and IDH wild-type observed by the increases in hazard ratios for TGFB2 (HR (95% CI range) = 2.02 (1.05-3.89)) and IDH wild-type (HR (95% CI range) = 4.44 (1.9-10.4)) were independent predictors of survival, suggesting that risk stratification of patients identifies LGG patients with IDH wild-type and high levels of TGFB2 in the design of clinical trials. Furthermore, we have additional IRF5 and CD276/B7-H3 as prognostic markers that can also be targeted for combination therapies with TGFB2 inhibitors. In support of these findings, we demonstrated that low levels of gene methylation in TGFB2, IFNGR2, IRF1, IRF5, STAT1, and CD276 were associated with significantly worse overall survival (OS) outcomes. This suggests that potential mechanisms to increase the expression of these prognostic markers occur via the action of demethylation enzymes.

Genome editing of FTR42 improves zebrafish survival against virus infection by enhancing IFN immunity

Genome editing of FTR42 improves zebrafish survival against virus infection by enhancing IFN immunity

The mechanism of Langchuangding in treatment of systemic lupus erythematosus via modulating TLR7-IRF7-IFNα pathway

ObjectSystemic lupus erythematosus (SLE) is a complex autoimmune disease characterized by aberrant activity of the immune system. Plasmacytoid dendritic cells (pDCs) which the main producer of activated type I interferon, are related to SLE disease activity. To investigate the mechanism of Langchuangding (LCD) improving SLE based on TLR7-IRF7-IFNα pathway. MethodsSLE patients were randomly divided into Chinese medicine combined with western medicine (CWM) group and western medicine (WM) group, to observe the effect of LCD. The percent of pDCs in peripheral blood of SLE patients were detected by flow cytometry, and the influence of LCD on gene expression in SLE patients were detected by gene microarray. Mouse bone marrow cells were differentiated into dendritic like cells (DLC), then divided into Blank, immune complex (IC), LCD and dexamethasone (DXM) group. Employed RT-qPCR to detect MyD88, and IRF7 mRNA, and western blotting to determinate TLR7, MyD88, and p-IRF7 proteins. The IFNα in SLE patients were detected by enzyme-linked immunosorbent assay (ELISA). Employ dual luciferase to observe the interferon stimulated response element (ISRE) gene. ResultspDCs in WM group was higher than that of CWM group. The plasma IFNα in CWM group was significantly lower than that in WM group. The gene microarray showed that the gene expression of IFNα related signaling pathway in peripheral blood mononuclear cell (PBMC) and genes related to activation and proliferation of immune cells were down-regulated after LCD treatment. The DLCs MyD88, and IRF7 mRNA were down-regulated, TLR7, MyD88, and p-IRF7 proteins were significantly reduced, and the supernatant IFNα was significantly decreased in LCD group. LCD were mildly inhibited activation of ISRE in 293T cells. ConclusionsIn certain degree, LCD is beneficial to SLE patients. LCD therapy SLE may be through TLR7 signaling pathway, and IRF7 may be a promising therapeutic target for the treatment of SLE.

Mesenchymal stem cells (MSCs) from the mouse bone marrow show differential expression of interferon regulatory factors IRF-1 and IRF-2.

Interferon regulatory factors (IRF-1 and IRF-2) are transcription factors widely implicated in various cellular processes, including regulation of inflammatory responses to pathogens, cell proliferation, oncogenesis, differentiation, autophagy, and apoptosis. We have studied the expression of IRF-1, IRF-2 mRNAs by RT-PCR, cellular localization of the proteins by immunofluorescence, and expression of mRNAs of genes regulated by IRF-1, IRF-2 by RT-PCR in mouse bone marrow cells (BMCs) and mesenchymal stem cells (MSCs). Higher level of IRF-1 mRNA was observed in BMCs and MSCs compared to that of IRF-2. Similarly, differential expression of IRF-1 and IRF-2 proteins was observed in BMCs and MSCs. IRF-1 was predominantly localized in the cytoplasm, whereas IRF-2 was localized in the nuclei of BMCs. MSCs showed nucleo-cytoplasmic distribution of IRF-1 and nuclear localization of IRF-2. Constitutive expression of IRF-1 and IRF-2 target genes: monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), cyclooxygenase-2 (COX-2), matrix metalloproteinase-9 (MMP-9), and caspase-1was observed in both BMCs and MSCs. MSCs showed constitutive expression of the pluripotency-associated factors, Oct3/4 and Sox-2. Lipopolysaccharide (LPS)-treatment of MSCs induced prominent cellular localization of IRF-1 and IRF-2. Our results suggest that IRF-1 and IRF-2 exhibit differential expression of their mRNAs and subcellular localization of the proteins in BMCs and MSCs. These cells also show differential levels of constitutive expression of IRF-1 and IRF-2 target genes. This may regulate immune-responsive properties of BMCs and MSCs through IRF-1, IRF-2-dependent gene expression and protein-protein interaction. Regulating IRF-1 and IRF-2 may be helpful for immunomodulatory functions of MSCs for cell therapy and regenerative medicine.

The Exposure to Lead (Pb) Exacerbates Immunological Abnormalities in BTBR T+ Itpr3tf/J Mice through the Regulation of Signaling Pathways Relevant to T Cells.

Autism spectrum disorder (ASD) is a common neurodevelopmental illness characterized by abnormal social interactions, communication difficulties, and repetitive and limited behaviors or interests. The BTBR T+ Itpr3tf/J (BTBR) mice have been used extensively to research the ASD-like phenotype. Lead (Pb) is a hazardous chemical linked to organ damage in the human body. It is regarded as one of the most common metal exposure sources and has been connected to the development of neurological abnormalities. We used flow cytometry to investigate the molecular mechanism behind the effect of Pb exposure on subsets of CD4+ T cells in the spleen expressing IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Furthermore, using RT-PCR, we studied the effect of Pb on the expression of numerous genes in brain tissue, including IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Pb exposure increased the population of CD4+IFN-γ+, CD4+T-bet+, CD4+STAT1+, CD4+STAT4+, CD4+IL-9+, CD4+IRF4+, CD4+IL-22+, and CD4+AhR+ cells in BTBR mice. In contrast, CD4+IL-10+ and CD4+Foxp3+ cells were downregulated in the spleen cells of Pb-exposed BTBR mice compared to those treated with vehicle. Furthermore, Pb exposure led to a significant increase in IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, and AhR mRNA expression in BTBR mice. In contrast, IL-10 and Foxp3 mRNA expression was significantly lower in those treated with the vehicle. Our data suggest that Pb exposure exacerbates immunological dysfunctions associated with ASD. These data imply that Pb exposure may increase the risk of ASD.

EZH2 Inhibition Overcomes Immunomodulatory Drug (IMiD) Resistance in Multiple Myeloma Cell Lines in a Cereblon Pathway Dependent Manner

Introduction Immunomodulatory agents (IMiDs) are effective therapies for multiple myeloma (MM), approved for use at all stages of disease. IMiDs function as molecular glues at cereblon (CRBN), part of the CRL4 CRBN E3 ubiquitin ligase complex, designating neo-substrates for proteasomal degradation. In myeloma cells the B cell transcription factors Ikaros and Aiolos are degraded, leading to downregulation of IRF4 which is critical for myeloma cell survival. Novel CRBN ligase modulators (CELMoDs) are currently in early phase clinical trials and have demonstrated activity in patients refractory to IMiDs, but resistance inevitably develops over time. EZH2 is a histone methyltransferase which acts at histone 3 lysine 27 (H3K27) leading to trimethylation (H3K27me3). The aberrant expression of EZH2 leads to abnormal gene repression, contributing to tumour development and progression. EZH2 inhibitors (EZH2i) have been shown to overcome drug resistance in several cancers and we hypothesized that combining EZH2i with IMiDs/CELMoDS would overcome resistance to these agents in MM cell lines. Methods MM cell lines KMS11, RPMI and MOLP8 were used as models of IMiD/CELMoD resistance and MM.1S as a control model of sensitivity. The isogenic pair of KMS11 with CRBN knockout (KMS11 CRBN KO) was established using CRISPR-Cas9. Following concentration/duration optimisation, cell lines were treated with EZH2i (Tazemetostat) 0.25-1µM or DMSO control for 5 days alone, and then in combination with IMiD (Lenalidomide, Len, 0-20 µM, Pomalidomide, Pom, 0-8 µM) or CELMoD (Iberdomide, CC-220, 0-2 µM and Mezigdomide, CC-92480, 0-0.1uM) for a further 5 days. Cell viability was measured using CellTiter Blue® and the level of synergy assessed using SynergyFinder (Bliss model). The effect on apoptosis and cell cycle was examined using flow cytometry after AnnexinV-PI and/or DAPI staining respectively. To elucidate the mechanism of synergy, the effect of the single agents and combinations on key CRBN pathway effectors (CRBN, Ikaros/Aiolos, IRF4) were examined using WB and qPCR. ChIP-PCR was employed to assess the changes in enrichment of H3K27me3 and key MM transcription factors (Ikaros/Aiolos) within the IRF4 promoter region upon EZH2i treatment. Results MM.1S cells were sensitive to IMiDs (Len/Pom) and CELMoDS (CC-220/CC-92480) with GI50s of 0.279 µM, 0.08 µM, 2 nM and 0.3 nM respectively. KMS11, MOLP8 and RPMI cell lines were resistant at more than 10x the GI50 in MM.1S. In these models of resistance, the cells express functional CRBN protein, demonstrated by the degradation of neo-substrates Ikaros/Aiolos. However, whilst their degradation results in reduced IRF4 expression and cell death in sensitive cells (e.g. MM.1S), this does not occur in the resistant models suggesting an uncoupling of Ikaros/Aiolos control of IRF4 expression in the resistant setting. Combining EZH2i with IMiDs/CELMoDs led to a reduction in cell viability (e.g. CC-220 shown in Fig.1A) and demonstrated synergy across resistant cell lines (KMS11, MOLP8 and RPMI). An increase in apoptosis was seen using AnnexinV/PI staining, with no demonstrable change in cell cycle. WB and qPCR results showed a significant reduction in IRF4 mRNA and protein with the combination treatment. The combination of EZH2i and IMiD/CELMoDs did not reduce cell viability or IRF4 expression in the KMS11 CRBN KO cell line ( Fig.1A), indicating that the observed synergistic effect is CRBN-dependent. Compared to DMSO control, EZH2i increased IRF4 mRNA, with a reduction of H3K27me3 enrichment observed within the IRF4 promoter region by ChIP-PCR in KMS11 ( Fig.1B). In addition, EZH2i also enhanced Ikaros and Aiolos enrichment within the IRF4 promoter region ( Fig.1B). Conclusions Our results suggest that combining EZH2i with IMiDs/CELMoDs can overcome resistance to these agents in MM cell line models, with synergy that is CRBN-dependent. By examining the key components of the CRBN pathway we identified that EZH2i reduced H3K27me3 and increased Ikaros and Aiolos association at the IRF4 promoter, suggesting a possible re-coupling of Ikaros/Aiolos to IRF4 expression, which may be responsible for reinstating IMiD/CELMoD activity, driving the synergistic effect seen.

MAP kinase inhibitor PD98059 regulates Th1, Th9, Th17, and natural T regulatory cells in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis

Experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS), provides significant insights into the mechanisms that initiate and drive autoimmunity. MS is a chronic autoimmune disease of the central nervous system, characterized by inflammatory infiltration associated with demyelination. T lymphocyte cells play a crucial role in MS, whereas natural T regulatory (nTreg) cells prevent autoimmune inflammation by suppressing lymphocyte activity. This study sought to investigate the role of PD98059, a selective MAP kinase inhibitor, in Th1, Th9, Th17, and nTreg cells using the SJL/J mouse model of EAE. Following EAE development, the mice were intraperitoneally administered PD98059 (5 mg/kg for two weeks) daily. We evaluated the effects of PD98059 on Th1 (IFN-γ and T-bet), Th9 (IL-9 and IRF4), Th17 (IL-17A and RORγT), and nTreg (FoxP3 and Helios) cells in the spleen using flow cytometry. Moreover, we explored the effects of PD98059 on the IFN-γ, T-bet, IL-9, IRF4, IL-17A, RORγT, FoxP3, and Helios mRNA and protein levels in brain tissues using qRT-PCR and Western blot analyses. PD98059 treatment significantly decreased the proportion of CD4+IFN-γ+, CD4+T-bet+, CD4+IL-9+, CD4+IRF4+, CD4+IL-17A+, CD4+RORγT+, CD4+IL-17A+, and CD4+RORγT+ cells while increasing that of CD4+FoxP3+ and CD4+Helios+ cells. In addition, PD98059 administration decreased the mRNA and protein levels of IFN-γ, T-bet, IL-9, IRF4, IL-17A, and RORγT but increased those of FoxP3 and Helios in the brain tissue of EAE mice. Our findings suggest that PD98059 corrects immune dysfunction in EAE mice, which is concurrent with the modulation of multiple signaling pathways.

IRF4 as a novel target involved in malignant transformation of oral submucous fibrosis into oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) in the context of oral submucous fibrosis (OSF) has a high incidence owing to undefined pathogenesis. Identifying key genes and exploring the underlying molecular mechanisms involved in the conversion of OSF into OSCC are in urgent need. Differentially expressed genes (DEGs) between OSCC and OSF were dug from GEO databases and a total of 170 DEGs were acquired. Functional association of DEGs were analyzed by GO and KEGG. Protein–protein interactions (PPIs) analysis was carried out and candidate biomarkers were identified by Gene co-expression analysis and Cox analyses. Hub genes were confirmed by qRT-PCR in tissues and cell lines, of which we found that IRF4 mRNA was successively up-regulated from Normal to OSF and then to OSCC and associated with immune infiltrating levels. In addition, Immunohistochemical (IHC) and Immunofluorescence (IF) assays were conducted to validate the consistent upregulation of IRF4 and the oncogene role of IRF4 in OSF and OSCC at translation level. IRF4 may be indicative biomarker in transformation of OSF into OSCC. High IRF4 expression contribute to increased immune infiltration of OSCC and may provide a novel diagnostic marker for OSCC patients translated from OSF.

NSUN2-mediated m5C methylation of IRF3 mRNA negatively regulates type I interferon responses during various viral infections

ABSTRACT 5-Methylcytosine (m5C) is a widespread post-transcriptional RNA modification and is reported to be involved in manifold cellular responses and biological processes through regulating RNA metabolism. However, its regulatory role in antiviral innate immunity has not yet been elucidated. Here, we report that NSUN2, a typical m5C methyltransferase, negatively regulates type I interferon responses during various viral infections, including SARS-CoV-2. NSUN2 specifically mediates m5C methylation of IRF3 mRNA and accelerates its degradation, resulting in low levels of IRF3 and downstream IFN-β production. Knockout or knockdown of NSUN2 enhanced type I interferon and downstream ISGs during various viral infection in vitro. And in vivo, the antiviral innate response is more dramatically enhanced in Nsun2+/− mice than in Nsun2+/+ mice. The highly m5C methylated cytosines in IRF3 mRNA were identified, and their mutation enhanced cellular IRF3 mRNA levels. Moreover, infection with Sendai virus (SeV), vesicular stomatitis virus (VSV), herpes simplex virus 1 (HSV-1), or Zika virus (ZIKV) resulted in a reduction of endogenous NSUN2 levels. Especially, SARS-CoV-2 infection (WT strain and BA.1 omicron variant) also decreased endogenous levels of NSUN2 in COVID-19 patients and K18-hACE2 KI mice, further increasing type I interferon and downstream ISGs. Together, our findings reveal that NSUN2 serves as a negative regulator of interferon response by accelerating the fast turnover of IRF3 mRNA, while endogenous NSUN2 levels decrease during SARS-CoV-2 and various viral infections to boost antiviral responses for effective elimination of viruses.

Changes in Th9 and Th17 lymphocytes and functional cytokines and their relationship with thyroid-stimulating hormone receptor antibodies at different stages of graves' disease.

ObjectiveGraves’ disease (GD) is an organ-specific autoimmune disease characterized by the production of thyroid-stimulating antibodies (TSAb). The newly discovered CD4+ T helper cells, Th9 and Th17 lymphocytes, have been confirmed to be closely associated with a variety of immune diseases. However, relationships with the onset and development of GD remain unclear. The purpose of this study was to investigate the roles of Th9 and Th17 in the pathogenesis and prognosis of GD.PatientsWe recruited 26 patients with newly diagnosed GD, 45 patients with GD in remission, and 20 healthy individuals.MeasurementsThyroid function and autoantibodies were evaluated using chemiluminescence immunoassays. Th9 and Th17 cells were analyzed using flow cytometry. The expression of Foxo1, IRF-4, RORc, IL-9, and IL-17 mRNA was examined using real-time PCR, and IL-9 and IL-17 protein levels were measured using enzyme-linked immunosorbent assay.ResultsTh9, Th17, and characteristic cytokines IL-9 and IL-17 in the GD-untreated group were significantly higher than those in the control and remission groups. The above indexes significantly decreased in the remission group, with the levels in the TRAb− remission group being similar to those in the normal group, while in the TRAb+ remission group, levels were differentially increased. TRAb titer was positively correlated with the levels of Th9, Th17, and their functional cytokines.ConclusionsTh9 and Th17 cells may be involved in the pathogenesis and disease outcome of GD, which could provide a new direction for developing immunotherapy for patients with GD.

Nephropathogenic Infectious Bronchitis Virus Mediates Kidney Injury in Chickens via the TLR7/NF-κB Signaling Axis.

Nephropathogenic infectious bronchitis virus (NIBV) is one of the most important viral pathogens in the world poultry industry. Here, we used RT–qPCR, WB and immunofluorescence to explore the interaction between NIBV and the host innate immune system of the kidney. Multiple virions were found in the kidney tissues of the disease group under electron microscopy, and pathological changes such as structural damage of renal tubules and bleeding were observed by HE staining. In addition, we found that the mRNA levels of TLR7, TRAF6, and IKKβ were upregulated after NIBV infection. IRF7 mRNA levels decreased significantly at 5 dpi and increased significantly at 11 to 18 dpi. The NF-κB P65 mRNA level increased significantly at 5 to 18 dpi and decreased at 28 dpi. However, NIBV infection-induced NF-κB P65 protein levels were downregulated at multiple time points. Moreover, we demonstrated that the cytokine (IFN-γ, IL-8, and IL-6) mRNA and protein expression levels were increased significantly at multiple time points after NIBV infection. Furthermore, immunofluorescence analysis showed that NF-κB P65 and IFN-γ were mainly located in the nuclear or perinuclear region. The positive signal intensity of NF-κB P65 was significantly lower than that of the normal group at 1 to 5 dpi, and there was no significant change in the subsequent time period. The positive signal intensity of IFN-γ decreased significantly at 5 dpi, and increased significantly at 11 to 28 dpi. In conclusion, we found that NIBV promoted cytokine release through the TLR7/NF-κB signaling axis, thus causing kidney injury.

CCR1 antagonist ameliorates experimental autoimmune encephalomyelitis by inhibition of Th9/Th22-related markers in the brain and periphery

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system. The disease manifestation is associated with the proliferation and activation of lymphocytes and astrocytes, leading to demyelination and neuronal damage. Most of the current therapies are not completely effective, and few target the underlying pathophysiology of MS. T helper 9 (Th9)- and Th22-dominant cells have been proven to play a pathogenic role in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. The goal of the present study was to investigate the therapeutic efficacy of J-113863, a novel CCR1 chemokine receptor, on PLP139–151-induced EAE in SJL/J mice. Following induction of EAE, mice were treated with J-113863 (10 mg/kg) or saline intraperitoneally daily from day 14 until day 25, and the clinical score was evaluated. We further investigated the effect of J-113863 on IL-9, IRF4, IL-22, IFN-γ, STAT3, AhR, and IL-17A in CD3+, CD4+, CCR6+, and CCR8+ spleen cells using flow cytometry. We also analyzed the effect of J-113863 on IL-9, IRF4, IL-22, IFN-γ, STAT3, AhR, and IL-17A mRNA expression levels. Our results revealed that J-113863 treatment notably attenuated the severity of clinical scores in EAE mice. J-113863 treatment decreased the percentage expression of CD4+IL-9+, CCR8+IL-9+, CD4+IRF4+, CD3+IL-22+, CCR6+IL-22+, CD3+IFN-γ+, CCR6+IFN-γ+, CD3+STAT3+, CCR6+STAT3+, CD4+IL-17A+, and CCR6+IL-17A+, and increased the percentage of CD3+AhR+, and CCR6+AhR+ cells in the spleen. These results confirmed that J-113863 suppressed Th9/Th22 cells to reduce demyelination in EAE mice, suggesting its potential role as a novel drug candidate for MS treatment.

TBK1-METTL3 axis facilitates antiviral immunity.

mRNA m6A modification is heavily involved in modulation of immune responses. However, its function in antiviral immunity is controversial, and how immune responses regulate m6A modification remains elusive. We here find TBK1, a key kinase of antiviral pathways, phosphorylates the core m6A methyltransferase METTL3 at serine 67. The phosphorylated METTL3 interacts with the translational complex, which is required for enhancing protein translation, thus facilitating antiviral responses. TBK1 also promotes METTL3 activation and m6A modification to stabilize IRF3 mRNA. Type I interferon (IFN) induction is severely impaired in METTL3-deficient cells. Mettl3fl/fl-lyz2-Cre mice are more susceptible to influenza A virus (IAV)-induced lethality than control mice. Consistently, Ythdf1-/- mice show higher mortality than wild-type mice due to decreased IRF3 expression and subsequently attenuated IFN production. Together, we demonstrate that innate signals activate METTL3 via TBK1, and METTL3-mediated m6A modification secures antiviral immunity by promoting mRNA stability and protein translation.

M6A modifications regulate intestinal immunity and rotavirus infection.

N6-methyladenosine (m6A) is an abundant mRNA modification and affects many biological processes. However, how m6A levels are regulated during physiological or pathological processes such as virus infections, and the in vivo function of m6A in the intestinal immune defense against virus infections are largely unknown. Here, we uncover a novel antiviral function of m6A modification during rotavirus (RV) infection in small bowel intestinal epithelial cells (IECs). We found that rotavirus infection induced global m6A modifications on mRNA transcripts by down-regulating the m6a eraser ALKBH5. Mice lacking the m6A writer enzymes METTL3 in IECs (Mettl3ΔIEC) were resistant to RV infection and showed increased expression of interferons (IFNs) and IFN-stimulated genes (ISGs). Using RNA-sequencing and m6A RNA immuno-precipitation (RIP)-sequencing, we identified IRF7, a master regulator of IFN responses, as one of the primary m6A targets during virus infection. In the absence of METTL3, IECs showed increased Irf7 mRNA stability and enhanced type I and III IFN expression. Deficiency in IRF7 attenuated the elevated expression of IFNs and ISGs and restored susceptibility to RV infection in Mettl3ΔIEC mice. Moreover, the global m6A modification on mRNA transcripts declined with age in mice, with a significant drop from 2 weeks to 3 weeks post birth, which likely has broad implications for the development of intestinal immune system against enteric viruses early in life. Collectively, we demonstrated a novel host m6A-IRF7-IFN antiviral signaling cascade that restricts rotavirus infection in vivo.