Interferon Regulatory Factor Research Articles

Integrin β8 Facilitates Macrophage Infiltration and Polarization by Regulating CCL5 to Promote LUAD Progression.

The tumor microenvironment (TME) influences cancer progression and metastasis. Integrin β8 (ITGβ8), a member of the integrin family, is upregulated in various cancers. In this study, it is determined as a key factor that mediates the interaction between lung adenocarcinoma (LUAD) cells and macrophages. Increased expression levels of ITGβ8 are associated with increased numbers of CD163+ macrophages and poor prognosis in LUAD patients. The overexpression of ITGβ8 in LUAD cells promotes the polarization of THP-1 macrophages toward the M2 phenotype. In contrast, TCM (conditioned medium from the co-culture system) from THP-1 macrophages and ITGβ8-overexpressing A549 cells promoted the proliferation and invasion of A549 cells. Mechanistically, chemokine (C-C motif) ligand 5 (CCL5) plays an important role in mediating ITGβ8-induced macrophage polarization, and the phosphoinositide 3-kinase (PI3K)/AKT serine/threonine kinase (AKT)/interferon regulatory factor 9 (IRF9) pathway is involved in this process. Moreover, interleukin 8 (IL8) and interleukin 10 (IL10) produced by M2-like macrophages regulate the expression of ITGβ8 in LUAD cells through the spi-1 proto-oncogene (SPI1). This study elucidates the feedback mechanism of ITGβ8 between LUAD cells and macrophages.

Panobinostat Attenuates Experimental Autoimmune Encephalomyelitis in Mice via Suppressing Oxidative Stress-Related Neuroinflammation and Mitochondrial Dysfunction

Multiple sclerosis (MS) is an autoimmune disease mediated by T helper cells, which is characterized by neuroinflammation, axonal or neuronal loss, demyelination, and astrocytic gliosis. Histone deacetylase inhibitors (HDACis) are noted for their roles in easing inflammatory conditions and suppressing the immune response. Panobinostat, an HDACi, is now being used in treating multiple myeloma. Nevertheless, the effect of panobinostat on autoimmune diseases remains largely unclear. Thus, our research endeavored to determine if the administration of panobinostat could prevent experimental autoimmune encephalomyelitis (EAE) in mice, one of the most commonly used animal models of MS, and further explored the underlying mechanisms. The EAE mice were generated and then administered continuously with panobinostat at a dosage of 30 mg/kg for 16 days. The results indicated that panobinostat markedly alleviated the clinical symptoms of EAE mice, inhibiting demyelination and loss of oligodendrocytes in the central nervous system (CNS). Moreover, panobinostat decreased inflammation and the activation of microglia and astrocytes in the spinal cords of EAE mice. Mechanistically, treatment with panobinosat significantly suppressed M1 microglial polarization by blocking the activation of toll-like receptor 2 (TLR2)/myeloid differentiation factor 88 (MyD88)/interferon regulatory factor 5 (IRF5) pathway. Additionally, panobinostat inhibited mitochondrial dysfunction and reduced oxidative stress in the spinal cords of EAE mice. In conclusion, our findings reveal that panobinostat significantly ameliorates experimental autoimmune encephalomyelitis in mice by inhibiting oxidative stress-linked neuroinflammation and mitochondrial dysfunction.

METTL3 facilitates kidney injury through promoting IRF4-mediated plasma cell infiltration via an m6A-dependent manner in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown cause. N6-methyladenosine (m6A) is the most common mRNA modification and participates in various immune processes such as interferon production and immune cell regulation. However, the role of m6A in dysregulated immune response of SLE remains unknown. PBMCs from SLE patients were collected to compare the m6A modification profile by methylated RNA immunoprecipitation sequencing (MeRIP-seq). Interferon regulatory factor 4 (IRF4) was identified by combination with MeRIP-seq and RNA-Seq. IRF4 and methyltransferase 3 (METTL3) were detected using qRT-PCR and WB. Clinical significance of IRF4 in SLE patients was explored subsequently. IRF4 expression in B cell subsets of female MRL/lpr mice was detected by flow cytometry. Adeno-associated viruses (AAV) including AAV9-METTL3-OE and/or AAV9-IRF4-sh were treated with female MRL/lpr mice. Autoantibody levels and kidney injury were tested by ELISA, pathological staining, and immunofluorescence. m6A level of IRF4 was detected by MeRIP-qPCR. The downstream effectors of IRF4 contributing to renal pathology were explored by RNA-seq and verified by qRT-PCR. m6A methylation features were obviously aberrant in SLE patients, and IRF4 was the upregulated gene modified by m6A. METTL3 and IRF4 expressions were elevated in SLE patients and kidney of MRL/lpr mice. Clinical analysis indicated that SLE patients with high IRF4 level were more prone to kidney damage. IRF4 expression was especially increased in plasma cells of MRL/lpr mice. METTL3 induced renal IRF4 expression, plasma creatinine, ANA and urine ALB levels, IgG and C3 deposition, and renal damage and plasma cell infiltration were aggravated in MRL/lpr mice. However, IRF4 depletion could partially reduce METTL3-induced kidney damage. Meanwhile, m6A level of IRF4 elevated with METTL3 overexpression. Also, the expression of Cxcl1, Bcl3, and Fos mRNA were significantly reduced after knockdown of IRF4, which were mainly involved in TNF signaling pathway. Our study confirmed that upregulated METTL3 promoting IRF4 expression in an m6A-dependent manner, thus causing plasma cell infiltration-mediated kidney damage of SLE. This provides new evidence for the role of m6A in SLE kidney injury.

How Does African Swine Fever Virus Evade the cGAS-STING Pathway?

African swine fever (ASF), a highly infectious and devastating disease affecting both domestic pigs and wild boars, is caused by the African swine fever virus (ASFV). ASF has resulted in rapid global spread of the disease, leading to significant economic losses within the swine industry. A significant obstacle to the creation of safe and effective ASF vaccines is the existing knowledge gap regarding the pathogenesis of ASFV and its mechanisms of immune evasion. The cyclic GMP–AMP synthase (cGAS)–stimulator of interferon genes (STING) pathway is a major pathway mediating type I interferon (IFN) antiviral immune response against infections by diverse classes of pathogens that contain DNA or generate DNA in their life cycles. To evade the host’s innate immune response, ASFV encodes many proteins that inhibit the production of type I IFN by antagonizing the cGAS-STING signaling pathway. Multiple proteins of ASFV are involved in promoting viral replication by protein–protein interaction during ASFV infection. The protein QP383R could impair the function of cGAS. The proteins EP364R, C129R and B175L could disturb the function of cyclic guanosine monophosphate-adenosine monophosphate (cGAMP). The proteins E248R, L83L, MGF505-11R, MGF505-7R, H240R, CD2v, E184L, B175L and p17 could interfere with the function of STING. The proteins MGF360-11L, MGF505-7R, I215L, DP96R, A151R and S273R could affect the function of TANK Binding Kinase 1 (TBK1) and IκB kinase ε (IKKε). The proteins MGF360-14L, M1249L, E120R, S273R, D129L, E301R, DP96R, MGF505-7R and I226R could inhibit the function of Interferon Regulatory Factor 3 (IRF3). The proteins MGF360-12L, MGF505-7R/A528R, UBCv1 and A238L could inhibit the function of nuclear factor kappa B (NF-Κb).

Pan-cancer analysis of IRF1 focusing on prognostic and immunological roles in non-small cell lung cancer

Interferon regulatory factor 1 (IRF1) significantly affects tumour occurrence and development. This study aimed to analyse its function as a pan-cancer prognostic indicator. We compared IRF1 expression and prognostic significance in normal and tumour samples from different databases. Accordingly, we performed in vitro experiments and immunohistochemistry (IHC) to investigate the role of IRF1 in non-small cell lung cancer (NSCLC). Our findings indicate that IRF1 expression is significantly correlated with prognosis, the tumour microenvironment, and immune cell infiltration. Furthermore, receiver operating characteristic (ROC) analysis revealed that IRF1 had high accuracy in distinguishing cancerous tissues from normal ones. Notably, IRF1 expression was linked to immune-related and immune checkpoint genes. Cell proliferation, invasion, and migration were significantly related to IRF1 expression. IHC indicated that IRF1 was downregulated in NSCLC tissues. Our study provides comprehensive bioinformatic analysis and experimental verification of IRF1, suggesting its potential as a prognostic biomarker in cancer.

Regulatory mechanism of TRIM21 in sepsis-induced acute lung injury by promoting IRF1 ubiquitination.

Sepsis-induced acute lung injury (ALI) is characterized by inflammatory damage to pulmonary endothelial and epithelial cells. The aim of this study is to probe the significance and mechanism of tripartite motif-containing protein 21 (TRIM21) in sepsis-induced ALI. The sepsis-induced ALI mouse model was established by cecum ligation and puncture. The mice were infected with lentivirus and treated with proteasome inhibitor MG132. The lung respiratory damage, levels of interleukin-6 (IL-6), tumour necrosis factor α (TNF-α), IL-10 and pathological changes were observed. The expression levels of TRIM21, interferon regulatory factors 1 (IRF1) and triggering receptor expressed on myeloid cells 2 (TREM2) were measured and their interactions were analysed. The ubiquitination level of IRF1 was detected. TRIM21 and TREM2 were downregulated and IRF1 was upregulated in sepsis-induced ALI mice. TRIM21 overexpression eased inflammation and lung injury. TRIM21 promoted IRF1 degradation via ubiquitination modification. IRF1 bonded to the TREM2 promoter to inhibit its transcription. Overexpression of IRF1 or silencing TREM2 reversed the improvement of TRIM21 overexpression on lung injury in mice. In conclusion, TRIM21 reduced IRF1 expression by ubiquitination to improve TREM2 expression and ameliorate sepsis-induced ALI.

Single-cell transcriptomics reveals IRF7 regulation of the tumor microenvironment in isocitrate dehydrogenase wild-type glioma.

Mutations in isocitrate dehydrogenase (IDH) are important markers of glioma prognosis. However, few studies have examined the gene expression regulatory network (GRN) in IDH-mutant and wild-type gliomas. In this study, single-cell RNA sequencing and spatial transcriptome sequencing were used to analyze the GRN of cell subsets in patients with IDH-mutant and wild-type gliomas. Through gene transcriptional regulation analysis, we identified the M4 module, whose transcription factor activity is highly expressed in IDH wild-type gliomas compared to IDH-mutants. Enrichment analysis revealed that these genes were predominantly expressed in microglia and macrophages, with significant enrichment in interferon-related signaling pathways. Interferon regulatory factor 7 (IRF7), a transcription factor within this pathway, showed the highest percentage of enrichment and was primarily localized in the core region of wild-type IDH tumors. A machine-learning prognostic model identified novel subgroups within the wild-type IDH population. Additionally, IRF7 was shown to promote the proliferation and migration of T98G and U251 cells in vitro, and its knockdown affected glioma cell proliferation in vivo. This study systematically established the regulatory mechanism of IDH transcriptional activity in gliomas at the single-cell level and drew a corresponding cell map. The study presents a transcriptional regulatory activity map for IDH wild-type gliomas, involving single-cell RNA sequencing and spatial transcriptomics to identify gene regulatory networks, machine learning models for IDH subtyping, and experimental validation, highlighting the role of IRF7 in glioma progression.

Nuclear factor kappa-B cell (NF-κB), interferon regulatory Factor, and glucocorticoid receptor pathway activation in major depressive Disorder: The role of cytomegalovirus infection

Altered activity of major immunoregulatory pathways has been reported in major depressive disorder (MDD) and is thought to underlie the elevations in circulating inflammatory mediators present in a subgroup of patients. However, the drivers of these changes in gene expression remain unclear. One potential modulator of immune function is viral infection. Here we examined the relationship between cytomegalovirus (CMV), a common herpesvirus, that has been shown to be a pathological cofactor in inflammatory disorders, and activity of key coordinators of the innate inflammatory response in MDD. We used RNAseq to characterize gene expression differences in in 79 unmedicated individuals with MDD and 80 healthy controls (HCs). A well-established bioinformatic strategy was used to quantify transcription control pathway activity based on the relative prevalence of pre-specified transcription factor-binding motifs in the promoters of differentially expressed genes. The main aim was to characterize diagnostic differences in immunoregulatory pathway activity and determine if these were related to CMV serostatus or antibody titer (viral reactivation). Significantly increased activity of interferon regulatory factor 1 (IRF1) and nuclear factor kappa-B cell (NF-κB) pathways was observed in the MDD group compared with HCs. Transcript Origin Analyses using cell-specific reference transcriptomes indicated that the MDD-associated transcriptome changes derived primarily from myeloid lineage immune cells (classical and non-classical monocytes). A more modest MDD-associated upregulation of glucocorticoid receptor (GR) pathway activity was also present. CMV infection/activity across the combined MDD and HC groups was weakly related to GR pathway activation but not to IRF1 and NF-κB activity; the most salient signature of CMV was activation and/or expansion of the CD8+ T-cell population. The elevated MDD-associated NF-κB (but not IRF1) activity was markedly attenuated after controlling for CMV antibody titer or for CD8+ T-cell prevalence. At least some of the NF-κB signal in MDD may be attributable to the cellular immune response to CMV, suggesting that CMV infection may be one of several pathways contributing to inflammation in depression. The pronounced activation of the antiviral IRF-1 pathway in MDD suggests the contribution of viral processes although this specific antiviral effect was not specific to CMV.CMV may indirectly drive interferon responses by impairing T-cell control of other viral infections.

Loss of interferon regulatory factor-1 prevents lung fibrosis by upregulation of pon1 expression

BackgroundInterferon regulatory factor-1 (IRF1) is a transcription factor that plays a significant role in various biological processes, including inflammatory injury, viral infection, cell death, and immune responses, and it has been extensively studied in the context of different lung diseases. However, the mechanism underlying its involvement in lung fibrosis remains largely unknown.MethodsWild type (WT) mice, IRF1 global-null mice (Irf1−/−) were subjected to a bleomycin-induced lung fibrosis model to enable examination of the role of IRF1 in lung fibrosis. Proteomic analysis of lung tissue from WT and Irf1−/− mice treated with saline or bleomycin was performed to explore the mechanism of IRF1 in regulating lung fibrosis.ResultsIn the bleomycin-induced fibrosis mouse model, increased expression of IRF1 was observed. Irf1 knockout mice displayed decreased lung fibrosis relative to WT mice following treatment with bleomycin. The protein expression of fibronectin, as assessed by the Western blot analysis of lung tissues, was downregulated in Irf1−/− mice. We observed a similar reduction in collagen content using hydroxyproline detection. Histologically, there was less collagen deposition in the lungs of Irf1−/− mice compared with WT mice. Proteomics data revealed that IRF1 may be involved in lung fibrosis via the regulation of ferroptosis. We determined that paraoxonase 1(PON1), a poorly characterized protein in lung fibrosis, was upregulated in Irf1−/− mice following exposure to bleomycin. In vitro experiments revealed that IRF1 could regulate the level of GSH and MDA through PON1. We also determined that PON1 levels were lower in the plasma of IPF patients compared with healthy controls.ConclusionOur data highlight the importance of IRF1 in the fibrotic process, and PON1 may be a potential mediator of IRF1 in the progression of lung fibrosis.

Interferon Regulatory Factors (IRF1, IRF4, IRF5, IRF7 and IRF9) in Sichuan taimen (Hucho bleekeri): Identification and Functional Characterization

Background/Objectives: Interferon regulatory factors (IRFs) are multifunctional transcription factors that play important roles in the transcriptional regulation of interferons and in the immune response to pathogens. Therefore, studying the interferon system in fish is highly relevant in the prevention and treatment of viral diseases. Methods: In this study, five IRF genes (IRF1, IRF4, IRF5, IRF7 and IRF9) were identified and characterized in Hucho bleekeri, and their expression profiles were determined after LPS and Poly(I:C) treatment. Results: These IRFs have typical DNA-binding domains and IRF-association domains. Amino acid sequence comparison revealed high homology between these IRFs and those of other vertebrates, with the highest homology being with other salmonid fish. Phylogenetic analysis revealed that these IRFs are divided into four subfamilies (IRF1, IRF3, IRF4 and IRF5), with both IRF4 and IRF9 belonging to the IRF4 subfamily. IRF genes were widely expressed in all of the tested tissues, with IRF1, IRF4 and IRF9 being highly expressed in the spleen and kidney and IRF5 and IRF7 highly expressed in the gonads. IRF1, IRF4 and IRF5 expression was induced at different time points post-LPS challenge. IRF7 and IRF9 expression in the spleen and head kidney was not significantly altered by LPS induction. Poly(I:C) treatment altered IRF expression more significantly than LPS treatment. Poly(I:C) significantly altered the spleen and head kidney expression of all five IRFs. Conclusions: These findings reveal the potential role of IRFs in the antiviral response of H. bleekeri and provide a reference for examining signal transduction pathways in the interferon system in fish.

Co-delivery of Interferon Regulatory Factor 5 (IRF5) siRNA and dasatinib by a disulfide bond bearing polymeric carrier for enhanced anti-inflammatory effects

Co-delivery of chemical drugs and nucleic acids has attracted a great interest recently for treatment of inflammatory diseases. Dasatinib (DB), a tyrosine kinase inhibitor with anti-cancer effects, and Interferon Regulatory Factor 5 (IRF5) siRNA have shown anti-inflammatory effects. In the present study, a novel redox-responsive polymeric micelle was designed for co-delivery of DB and IRF5 siRNA-expressing plasmid (psiRF5) to enhance anti-inflammatory effects on macrophages. psiRF5 was condensed efficiently to redox-responsive micelles of DB-conjugated chitosan (CN) composed of disulfide bond, from different molecular weights of CN to form CN-SS-DB/psiRF5 micelles. The micelles with optimum N/P ratios had particle sizes of 287.8 and 245.4 nm and positive zeta potentials. The disulfide bond bearing micelles showed a redox-responsive drug release, protected the plasmid from being dissociated or degraded in exposure with heparin, serum and DNase I, and significantly enhanced the transfection efficiency and IRF5-gene silencing compared to naked psiRF5. The optimum micelles exhibited a dramatic reduction in IRF5 expression and revealed a notably higher anti-inflammatory effect than either DB or psiRF5, as indicated by more IL-10 and less IL-6 and TNF-α production by LPS-stimulated RAW264.7 macrophages incubated with the co-delivery system. The resultant nanocarriers might be promising for more effective treatment of inflammatory diseases.

IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating ubiquitylation and SUMOylation of MFN1/2

Osteoarthritis (OA) is an age-related cartilage-degenerating joint disease. Mitochondrial dysfunction has been reported to promote the development of OA. Poly (ADP-ribose) polymerase family member 12 (PARP12) is a key regulator of mitochondrial function, protein translation, and inflammation. However, the role of PARP12 in OA-based cartilage degradation and the underlying mechanisms are relatively unknown. Here, we first demonstrated that PARP12 inhibits mitophagy and promotes OA progression in human OA cartilage and a monosodium iodoacetate-induced rat OA model. Using mass spectrometry and co-immunoprecipitation assay, PARP12 was shown to interact with ISG15, upregulate mitofusin 1 and 2 (MFN1/2) ISGylation, which downregulated MFN1/2 ubiquitination and SUMOylation, thereby inhibiting PINK1/Parkin-dependent chondrocyte mitophagy and promoting cartilage degradation. Moreover, inflammatory cytokine-induced interferon regulatory factor 1 (IRF1) activation was required for the upregulation of PARP12 expression, and it directly bound to the PARP12 promoter to activate transcription. XAV-939 inhibited PARP12 expression and suppressed OA pathogenesis in vitro and in vivo. Clinically, PARP12 can be used to predict the severity of OA; thus, it represents a new target for the study of mitophagy and OA progression. In brief, the IRF1-mediated upregulation of PARP12 promoted cartilage degradation by inhibiting PINK1/Parkin-dependent mitophagy via ISG15-based attenuation of MFN1/2 ubiquitylation and SUMOylation. Our data provide new insights into the molecular mechanisms underlying PARP12-based regulation of mitophagy and can facilitate the development of therapeutic strategies for the treatment of OA.

CircPCNXL2 promotes preeclampsia progression by suppressing trophoblast cell proliferation and invasion via miR-487a-3p/interferon regulatory factor 2 axis.

Preeclampsia (PE) has culminated in maternal and perinatal sickness and death across the world, affecting approximately 4.6% of pregnancies. Circular RNAs (circRNAs) have been linked to the biology of numerous pathologies, including PE. Here, we investigated the functional role of circPCNXL2 in the progression of PE. We employed the GEO database to get the expression profile of circPCNXL2 in patients with PE. This was followed by the detection of the expression of circPCNXL2 and miR-326 by qRT-PCR. The role of circPCNXL2 on trophoblast cell proliferation, migration, and invasion was confirmed with cell viability assays, the transwell assay, and the colony formation assay. Further, we employed dual luciferase, FISH, RNA pull-down assay and Western blot analysis to determine the interaction between the expression of circPCNXL2, miR-487a-3p, and IRF2. Findings from this study revealed that proliferation and migration of trophoblast cells were significantly increased in the HTR-8/SVneo cells after silencing circPCNXL2. Additionally, knockdown of circPCNXL2 remarkably increased miR-487a-3p expression, while IRF2 expression was remarkably reduced (P < 0.05), indicating the presence of complementary binding sequence on miR-487a-3p with which they sequester circPCNXL2. Rescue experiments revealed that interaction occurs between circPCNXL2, miR-487a-3p, and the IRF2 protein, indicating that circPCNXL2 expression elicits suppression of migration and proliferation of trophoblast cells via the miR-487a-3p/IRF2 pathway. We demonstrated that circPCNXL2 upregulation promotes pre-eclampsia by inhibiting proliferation and migration of trophoblast cells via the miR-487a-3p/IRF2 pathway or axis.

Diesel exhaust particles activate macrophages and IRF5 expression in atherosclerosis

Abstract Introduction Over 20% of global deaths related to cardiovascular disease have been attributed in part to air pollution, particularly fine particulate matter (PM2.5) (1). In highly trafficked urban areas, smaller ultrafine diesel exhaust particles (DEP) represent a significant proportion of PM2.5. DEP is associated with the progression of atherosclerosis and increased plaque susceptibility to rupture (2). We have previously shown that interferon regulatory factor 5 (IRF5) has a key role in necrotic core formation, a distinguishing feature of vulnerable atherosclerotic plaques, by rendering macrophages defective at efferocytosis (3). Pronounced elevation of IRF5 is also seen in symptomatic carotid plaques, particularly in the rupture-prone shoulder regions (4). Purpose We examined the effect of DEP on macrophage phenotype and IRF5 expression in atherosclerosis. Methods Bone marrow-derived macrophages (BMDMs) from C57BL/6 mice, differentiated with GM-CSF (to mimic inflammatory macrophages) or M-CSF (mimicking homeostatic resident macrophages) were incubated with PBS or DEP (10 µg/mL) in vitro for 18 hours. Expression of inflammatory cytokines (IL-6, IL-12α), macrophage polarisation markers CD11c (itgax, inflammatory) and CD206 (mrc1, resident anti-inflammatory) and IRF5 was quantified by real-time PCR. In addition, high fat-fed ApoE-/- mice were exposed to saline or DEP (35 µL at 1 mg/mL) twice weekly for 5 weeks by pulmonary administration. The pan-macrophage marker CD64, CD206 and IRF5 were quantified in paraffin-embedded sections of brachiocephalic artery lesions by immunohistochemistry. Results In M-CSF-differentiated murine macrophages incubated with DEP, significant fold increases in gene expression (relative to PBS control) were observed: IRF5 (1.4-fold), IL-6 (31.5-fold) and IL-12α (5.8-fold) while CD206 (0.8-fold) decreased (Figure 1a, n = 5, p&amp;lt;0.05, two-tailed paired t-tests). CD11c expression was unchanged. No statistically significant differences were detected in GM-CSF-differentiated macrophages. In the brachiocephalic arteries of DEP-exposed ApoE-/- mice we observed increases in CD64+ macrophages (28.2±7.4 vs 15.5±5.6) and IRF5 (16.6±3.6 vs 10.1±3.4) expression, while CD206 expression decreased (4.1±0.9 vs 11.0±1.2) in comparison to saline-exposed mice (Figure 1b mean±SD, n = 5, p&amp;lt;0.05, two-tailed unpaired t-tests, data expressed as % of intima area). Conclusion DEP promotes CD64+ macrophages and IRF5 expression in murine atherosclerosis, while inflammation and IRF5 is induced in vitro, but only in M-CSF-differentiated murine BMDMs. This suggests that DEP has no impact on inflammatory macrophages but could switch homeostatic resident macrophages to a more inflammatory phenotype via the activation of IRF5. Therefore, IRF5 is a promising candidate for further investigation into the initial macrophage response to inhaled particles that leads to the exacerbation of vascular disease.

Macrophage-T cell hybrid membrane-camouflaged biomimetic nanoparticles ameliorate autoimmune myocarditis via suppressing macrophage pyroptosis by target gene silencing

Abstract Background Current management of myocarditis mainly relies on conventional approaches and immunosuppressive therapies. However, these strategies often yield limited efficacy. Our previous research revealed the pivotal role of macrophage pyroptosis in myocarditis pathogenesis. Therefore, targeted intervention to inhibit macrophage pyroptosis may provide new insights into myocarditis treatment. Purpose We previously identified interferon regulatory factor 1 (IRF1) as the key modulator of macrophage pyroptosis in myocarditis. In this study, we synthesized a nano-delivery platform consisting of a macrophage-T cell hybrid membrane-coated zeolitic imidazolate framework-8 (ZIF-8) encapsulating IRF1-small interfering RNA (siRNA@ZIF@HM). We aimed to evaluate its targeting capability and therapeutic efficacy for macrophage pyroptosis in myocarditis. Methods The fabrication of siRNA@ZIF@HM was validated using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Cellular uptake, cytotoxicity, endolysosome escape, IRF1 silencing, and anti-pyroptotic effect were assessed using mouse bone marrow-derived macrophages and the mouse macrophage cell line J774A.1 and RAW264.7. An experimental autoimmune myocarditis (EAM) mouse model was induced in Balb/c mice for in vivo investigations. Pharmacokinetics and targeting capability were determined with ex vivo fluorescence imaging and immunofluorescence staining. Subsequently, mice were randomly allocated into control, EAM + siRNA, EAM + siRNA@ZIF, and EAM + siRNA@ZIF@HM groups to assess the therapeutic efficacy. Additionally, the biodistribution and biosafety of siRNA@ZIF@HM were also evaluated. Results TEM, DLS, and element mapping confirmed the successful fabrication of siRNA@ZIF@HM, with a diameter of approximately 130 nm. The nanoparticle exhibited pH responsiveness and membrane markers of macrophage and T lymphocytes (Figure 1). It demonstrated high targeting specificity for pro-inflammatory macrophages and myocarditis. Immunofluorescence microscopy revealed successful endolysosome escape of IRF1-siRNA in macrophages. Consequently, the nanoparticle significantly silenced IRF1 protein expression and suppressed macrophage pyroptosis both in vivo and in vitro, resulting in the amelioration of autoimmune myocarditis (Figure 2). Furthermore, the nanoparticle showed good biocompatibility with no significant changes observed in other organs. Conclusions The pH-responsive, macrophage-T cell hybrid membrane-coated biomimetic nanoparticle demonstrates promising capability for targeted siRNA delivery to myocardial inflammation sites, particularly pro-inflammatory macrophages. Additionally, targeting IRF1-mediated macrophage pyroptosis represents a potential therapeutic strategy for myocarditis treatment.Figure 1Figure 2

Postoperative Innate Immune Dysregulation, Proteomic, and Monocyte Epigenomic Changes After Colorectal Surgery: A Substudy of a Randomized Controlled Trial.

Colorectal surgery is associated with moderate-to-severe postoperative complications in over 25% of patients, predominantly infections. Monocyte epigenetic alterations leading to immune tolerance could explain postoperative increased susceptibility to infections. This research explores whether changes in monocyte DNA accessibility contribute to postoperative innate immune dysregulation. Damage-associated molecular patterns (DAMPs) and ex vivo cytokine production capacity were measured in a randomized controlled trial (n = 100) in colorectal surgery patients, with additional exploratory subgroup proteomic (proximity extension assay; Olink) and epigenomic analyses (Assay for Transposase-Accessible Chromatin [ATAC sequencing]). Monocytes of healthy volunteers were used to study the effect of high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70) on cytokine production capacity in vitro. Plasma DAMPs were increased after surgery. HMGB1 showed a mean 235% increase from before- (preop) to the end of surgery (95% confidence interval [CI] [166 - 305], P < .0001) and 90% increase (95% CI [63-118], P = .0004) preop to postoperative day 1 (POD1). HSP70 increased by a mean 12% from preop to the end of surgery (95% CI [3-21], not significant) and 30% to POD1 (95% CI [18-41], P < .0001). Nuclear deoxyribonucleic acid (nDNA) increases by 66% (95% CI [40-92], P < .0001) at the end of surgery and 94% on POD1 (95% CI [60-127], P < .0001). Mitochondrial DNA (mtDNA) increases by 370% at the end of surgery (95% CI [225-515], P < .0001) and by 503% on POD1 (95% CI [332-673], P < .0001). In vitro incubation of monocytes with HSP70 decreased cytokine production capacity of tumor necrosis factor (TNF) by 46% (95% CI [29-64], P < .0001), IL-6 by 22% (95% CI [12-32], P = .0004) and IL-10 by 19% (95% CI [12-26], P = .0015). In vitro incubation with HMGB1 decreased cytokine production capacity of TNF by 34% (95% CI [3-65], P = .0003), interleukin 1β (IL-1β) by 24% (95% CI [16-32], P < .0001), and IL-10 by 40% (95% CI [21-58], P = .0009). Analysis of the inflammatory proteome alongside epigenetic shifts in monocytes indicated significant changes in gene accessibility, particularly in inflammatory markers such as CXCL8 (IL-8), IL-6, and interferon-gamma (IFN-γ). A significant enrichment of interferon regulatory factors (IRFs) was found in loci exhibiting decreased accessibility, whereas enrichment of activating protein-1 (AP-1) family motifs was found in loci with increased accessibility. These findings illuminate the complex epigenetic modulation influencing monocytes' response to surgical stress, shedding light on potential biomarkers for immune dysregulation. Our results advocate for further research into the role of anesthesia in these molecular pathways and the development of personalized interventions to mitigate immune dysfunction after surgery.

Super-Enhancer-Driven IRF2BP2 is Activated by Master Transcription Factors and Sustains T-ALL Cell Growth and Survival.

Super enhancers (SEs) are large clusters of transcriptional enhancers driving the expression of genes crucial for defining cell identity. In cancer, tumor-specific SEs activate key oncogenes, leading to tumorigenesis. Identifying SE-driven oncogenes in tumors and understanding their functional mechanisms is of significant importance. In this study, a previously unreported SE region is identified in T-cell acute lymphoblastic leukemia (T-ALL) patient samples and cell lines. This SE activates the expression of interferon regulatory factor 2 binding protein 2 (IRF2BP2) and is regulated by T-ALL master transcription factors (TFs) such as ETS transcription factor ERG (ERG), E74 like ETS transcription factor 1 (ELF1), and ETS proto-oncogene 1, transcription factor (ETS1). Hematopoietic system-specific IRF2BP2 conditional knockout mice is generated and showed that IRF2BP2 has minimal impact on normal T cell development. However, in vitro and in vivo experiments demonstrated that IRF2BP2 is crucial for T-ALL cell growth and survival. Loss of IRF2BP2 affects the MYC and E2F pathways in T-ALL cells. Cleavage under targets and tagmentation (CUT&Tag) assays and immunoprecipitation revealed that IRF2BP2 cooperates with the master TFs of T-ALL cells, targeting the enhancer of the T-ALL susceptibility gene recombination activating 1 (RAG1) and modulating its expression. These findings provide new insights into the regulatory network within T-ALL cells, identifying potential new targets for therapeutic intervention.

Essential role of interferon-regulatory factor 4 in regulating diabetogenic CD4+ T and innate immune cells in autoimmune diabetes in NOD mice.

Haploinsufficiency of the transcription factor interferon-regulatory factor 4 (IRF4) prevents the onset of spontaneous diabetes in NOD mice. However, the immunological mechanisms of the IRF4-mediated disease regulation remain unclear. This study aims to investigate the role of IRF4 in the pathogenesis of autoimmune diabetes by conducting adoptive transfer experiments using donor IRF4 gene-deficient CD4+ T cells from BDC2.5-transgenic (Tg) NOD mice and recipient Rag1-knockout NOD mice, respectively. Through this approach, we analyzed both clinical and immunological phenotypes of the recipient mice. Additionally, IRF4-deficient BDC2.5 CD4+ T cells were stimulated to assess their immunological and metabolic phenotypes in vitro. The findings revealed that diabetes was completely prevented in the recipients with Irf4-/- T cells and was approximately 50% lower in those with Irf4+/- T cells than in wild type (WT) controls, whereas Irf4-/- recipients with WT T cells only showed a delayed onset of diabetes. Islet-infiltrating T cells isolated from recipients with Irf4+/- T cells exhibited significantly lower proliferation and IFN-γ/IL-17 double-positive cell fraction rates compared with those in WT controls. Irf4-/- BDC2.5 CD4+ T cells stimulated in vitro showed a reduced number of cell divisions, decreased antigen-specific T-cell markers, and impairment of glycolytic capacity compared with those observed in WT controls. We concluded that IRF4 predominantly regulates the diabetogenic potential in a dose-dependent manner by mediating the proliferation and differentiation of islet-infiltrating T cells while playing an adjunctive role in the innate immune responses toward diabetes progression in NOD mice.

TBK1 is involved in M-CSF-induced macrophage polarization through mediating the IRF5/IRF4 axis.

TANK binding kinase 1 (TBK1) is an important kinase that is involved in innate immunity and tumor development. Macrophage colony-stimulating factor (M-CSF) regulates the differentiation and function of macrophages towards the immunosuppressive M2 phenotype in the glioblastoma multiforme microenvironment. The role of TBK1 in macrophages, especially in regulating macrophage polarization in response to M-CSF stimulation, remains unclear. Here, we found high TBK1 expression in human glioma-infiltrating myeloid cells and that phosphorylated TBK1 was highly expressed in M-CSF-stimulated macrophages but not in granulocyte-macrophage CSF-induced macrophages (granulocyte-macrophage-CSF is involved in the polarization of M1 macrophages). Conditional deletion of TBK1 in myeloid cells induced M-CSF-stimulated bone marrow-derived macrophages to exhibit a proinflammatory M1-like phenotype with increased protein expression of CD86, interleukin-1β and tumor necrosis factor-α, as well as decreased expression of arginase 1. Mechanistically, TBK1 deletion or inhibition by amlexanox or GSK8612 reduced the expression of the transcription factor interferon-regulatory factor (IRF)4 and increased the level of IRF5 activation in macrophages stimulated with M-CSF, leading to an M1-like profile with highly proinflammatory factors. IRF5 deletion reversed the effect of TBK1 inhibition on M-CSF-mediated macrophage polarization. Our findings suggest that TBK1 contributes to the regulation of macrophage polarization in response to M-CSF stimulation partly through the IRF5/IRF4 axis.

JunB is required for CD8+ T cell responses to acute infections.

Basic-leucine zipper transcription factor ATF-like (BATF) and interferon regulatory factor 4 (IRF4) are crucial transcription factors for generation of cytotoxic effector and memory CD8+ T cells. JunB is required for expression of genes controlled by BATF and IRF4 in CD4+ T cell responses, but the role of JunB in CD8+ T cells remains unknown. Here, we demonstrate that JunB is essential for cytotoxic CD8+ T cell responses. JunB expression is transiently induced, depending on T cell receptor (TCR) signal strength. JunB deficiency severely impairs clonal expansion of effector CD8+ T cells in response to acute infection with Listeria monocytogenes. Junb-deficient CD8+ T cells fail to control transcription and chromatin accessibility of a specific set of genes regulated by BATF and IRF4, resulting in impaired cell survival, glycolysis, and cytotoxic CD8+ T cell differentiation. Furthermore, JunB deficiency enhances expression of coinhibitory receptors, including programmed death receptor 1 (PD-1) and T-cell immunoglobulin mucin-3 (TIM3) upon activation of naïve CD8+ T cells. These results indicate that JunB, in collaboration with BATF and IRF4, promotes multiple key events in the early stage of cytotoxic CD8+ T cell responses.