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Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.
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Leucemia Aguda Bifenotípica/tratamento farmacológico , Leucemia Aguda Bifenotípica/metabolismo , Proteólise/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Interleucina-1/metabolismo , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Leucina Linfoide-Mieloide/metabolismo , Enzimas de Conjugação de UbiquitinaRESUMO
Toll-like receptors (TLRs) in mammalian systems are well known for their role in innate immunity. In addition, TLRs also fulfil crucial functions outside immunity, including the dorsoventral patterning function of the original Toll receptor in Drosophila and neurogenesis in mice. Recent discoveries in flies suggested key roles for TLRs in epithelial cells in patterning of junctional cytoskeletal activity. Here, we address the function of TLRs and the downstream key signal transduction component IRAK4 in human epithelial cells. Using differentiated human Caco-2 cells as a model for the intestinal epithelium, we show that these cells exhibit baseline TLR signalling, as revealed by p-IRAK4, and that blocking IRAK4 function leads to a loss of epithelial tightness involving key changes at tight and adherens junctions, such as a loss of epithelial tension and changes in junctional actomyosin. Changes upon IRAK-4 inhibition are conserved in human bronchial epithelial cells. Knockdown of IRAK4 and certain TLRs phenocopies the inhibitor treatment. These data suggest a model whereby TLR receptors near epithelial junctions might be involved in a continuous sensing of the epithelial state to promote epithelial tightness and integrity.
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Quinases Associadas a Receptores de Interleucina-1 , Receptores Toll-Like , Humanos , Células CACO-2 , Imunidade Inata , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Recent interest in understanding cardiomyocyte cell cycle has been driven by potential therapeutic applications in cardiomyopathy. However, despite recent advances, cardiomyocyte mitosis remains a poorly understood process. For example, it is unclear how sarcomeres are disassembled during mitosis to allow the abscission of daughter cardiomyocytes. METHODS: Here, we use a proteomics screen to identify adducin, an actin capping protein previously not studied in cardiomyocytes, as a regulator of sarcomere disassembly. We generated many adeno-associated viruses and cardiomyocyte-specific genetic gain-of-function models to examine the role of adducin in neonatal and adult cardiomyocytes in vitro and in vivo. RESULTS: We identify adducin as a regulator of sarcomere disassembly during mammalian cardiomyocyte mitosis. α/γ-adducins are selectively expressed in neonatal mitotic cardiomyocytes, and their levels decline precipitously thereafter. Cardiomyocyte-specific overexpression of various splice isoforms and phospho-isoforms of α-adducin in identified Thr445/Thr480 phosphorylation of a short isoform of α-adducin as a potent inducer of neonatal cardiomyocyte sarcomere disassembly. Concomitant overexpression of this α-adducin variant along with γ-adducin resulted in stabilization of the adducin complex and persistent sarcomere disassembly in adult mice, which is mediated by interaction with α-actinin. CONCLUSIONS: These results highlight an important mechanism for coordinating cytoskeletal morphological changes during cardiomyocyte mitosis.
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Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.
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PURPOSE: To report a case of a five-month-old Chinese infant who died of interleukin-1 receptor-associated kinase-4 (IRAK-4) deficiency presenting with rapid and progressive Pseudomonas aeruginosa sepsis. METHODS: The genetic etiology of IRAK-4 deficiency was confirmed through trio-whole exome sequencing and Sanger sequencing. Functional consequences were invested using an in vitro minigene splicing assay. RESULTS: Trio-whole exome sequencing of genomic DNA identified two novel compound heterozygous mutations, IRAK-4 (NM_016123.3): c.942-1G > A and c.644_651+ 6delTTGCAGCAGTAAGT in the proband, which originated from his symptom-free parents. These mutations were predicted to cause frameshifts and generate three truncated proteins without enzyme activity. CONCLUSIONS: Our findings expand the range of IRAK-4 mutations and provide functional support for the pathogenic effects of splice-site mutations. Additionally, this case highlights the importance of considering the underlying genetic defects of immunity when dealing with unusually overwhelming infections in previously healthy children and emphasizes the necessity for timely treatment with wide-spectrum antimicrobials.
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Quinases Associadas a Receptores de Interleucina-1 , Infecções por Pseudomonas , Pseudomonas aeruginosa , Sepse , Humanos , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/deficiência , Pseudomonas aeruginosa/genética , Infecções por Pseudomonas/genética , Masculino , Lactente , Sepse/genética , Sepse/microbiologia , Doenças da Imunodeficiência Primária/genética , Mutação com Perda de Função , Heterozigoto , Sequenciamento do Exoma , Síndromes de Imunodeficiência/genéticaRESUMO
Persistent and uncontrolled inflammation is the root cause of various debilitating diseases. Given that interleukin-1 receptor-associated kinase 4 (IRAK4) is a critical modulator of inflammation, inhibition of its activity with selective drug molecules (IRAK4 inhibitors) represents a promising therapeutic strategy for inflammatory disorders. To exploit the full potential of this treatment approach, drug carriers for efficient delivery of IRAK4 inhibitors to inflamed tissues are essential. Herein, the first nanoparticle-based platform for the targeted systemic delivery of a clinically tested IRAK4 inhibitor, PF-06650833, with limited aqueous solubility (57 µg mL-1 ) is presented. The developed nanocarriers increase the intrinsic aqueous dispersibility of this IRAK4 inhibitor by 40 times. A targeting peptide on the surface of nanocarriers significantly enhances their accumulation after intravenous injection in inflamed tissues of mice with induced paw edema and ulcerative colitis when compared to non-targeted counterparts. The delivered IRAK4 inhibitor markedly abates inflammation and dramatically suppresses paw edema, mitigates colitis symptoms, and reduces proinflammatory cytokine levels in the affected tissues. Importantly, repeated injections of IRAK4 inhibitor-loaded nanocarriers have no acute toxic effect on major organs of mice. Therefore, the developed nanocarriers have the potential to significantly improve the therapeutic efficacy of IRAK4 inhibitors for different inflammatory diseases.
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Colite , Quinases Associadas a Receptores de Interleucina-1 , Camundongos , Animais , Quinases Associadas a Receptores de Interleucina-1/química , Citocinas , Inflamação/tratamento farmacológico , EdemaRESUMO
Interleukin-1 receptor-associated kinase 4 (IRAK4) is a crucial serine/threonine protein kinase that belongs to the IRAK family and plays a pivotal role in Toll-like receptor (TLR) and Interleukin-1 receptor (IL-1R) signaling pathways. Due to IRAK4's significant role in immunity, inflammation, and malignancies, it has become an intriguing target for discovering and developing potent small-molecule inhibitors. Consequently, there is a pressing need for rapid and accurate prediction of IRAK4 inhibitor activity. Leveraging a comprehensive dataset encompassing activity data for 1628 IRAK4 inhibitors, we constructed a prediction model using the LightGBM algorithm and molecular fingerprints. This model achieved an R2 of 0.829, an MAE of 0.317, and an RMSE of 0.460 in independent testing. To further validate the model's generalization ability, we tested it on 90 IRAK4 inhibitors collected in 2023. Subsequently, we applied the model to predict the activity of 13,268 compounds with docking scores less than - 9.503 kcal/mol. These compounds were initially screened from a pool of 1.6 million molecules in the chemdiv database through high-throughput molecular docking. Among these, 259 compounds with predicted pIC50 values greater than or equal to 8.00 were identified. We then performed ADMET predictions on these selected compounds. Finally, through a rigorous screening process, we identified 34 compounds that adhere to the four complementary drug-likeness rules, making them promising candidates for further investigation. Additionally, molecular dynamics simulations confirmed the stable binding of the screened compounds to the IRAK4 protein. Overall, this work presents a machine learning model for accurate prediction of IRAK4 inhibitor activity and offers new insights for subsequent structure-guided design of novel IRAK4 inhibitors.
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Periapical lesions are common pathologies affecting the alveolar bone, often initiated by intraradicular lesions resulting from microbial exposure to dental pulp. These microorganisms trigger inflammatory and immune responses. When endodontic treatment fails to eliminate the infection, periapical lesions persist, leading to bone loss. The RANK/RANKL/OPG pathway plays a crucial role in both the formation and the destruction of the bone. In this study, the objective was to inhibit the RANK/RANKL pathway in vitro within exposed Thp-1 macrophages to endodontic microorganisms, specifically Enterococcus faecalis, which was isolated from root canals of 20 patients with endodontic secondary/persistent infection, symptomatic and asymptomatic, and utilizing an α-IRAK-4 inhibitor, we introduced endodontic microorganisms and/or lipoteichoic acid from Streptococcus spp. to cellular cultures in a culture plate, containing thp-1 cells and/or PBMC from patients with apical periodontitis. Subsequently, we assessed the percentages of RANK+, RANKL+, and OPG+ cells through flow cytometry and measured the levels of several inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-8, IL-10, and IL-12p70) in the cellular culture supernatant through a CBA kit and performed analysis by flow cytometry. A significant difference was observed in the percentages of RANK+RANKL+, OPG+ RANKL+ cells in thp-1 cells and PBMCs from patients with apical periodontitis. The findings revealed significant differences in the percentages of the evaluated cells, highlighting the novel role of the IRAK-4 inhibitor in addressing this oral pathology, apical periodontitis, where bone destruction is observed.
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Macrófagos , Periodontite Periapical , Ligante RANK , Receptor Ativador de Fator Nuclear kappa-B , Transdução de Sinais , Humanos , Ligante RANK/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Células THP-1 , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Periodontite Periapical/metabolismo , Periodontite Periapical/microbiologia , Periodontite Periapical/patologia , Citocinas/metabolismo , Enterococcus faecalis , Lipopolissacarídeos , Cavidade Pulpar/microbiologia , Cavidade Pulpar/metabolismo , Masculino , Osteoprotegerina/metabolismo , Adulto , Ácidos Teicoicos/farmacologiaRESUMO
IRAK4 is a critical mediator in NF-κB-regulated inflammatory signaling and has emerged as a promising therapeutic target for the treatment of autoimmune diseases; however, none of its inhibitors have received FDA approval. In this study, we identified a novel small-molecule IRAK4 kinase inhibitor, DW18134, with an IC50 value of 11.2 nM. DW18134 dose-dependently inhibited the phosphorylation of IRAK4 and IKK in primary peritoneal macrophages and RAW264.7 cells, inhibiting the secretion of TNF-α and IL-6 in both cell lines. The in vivo study demonstrated the efficacy of DW18134, significantly attenuating behavioral scores in an LPS-induced peritonitis model. Mechanistically, DW18134 reduced serum TNF-α and IL-6 levels and attenuated inflammatory tissue injury. By directly blocking IRAK4 activation, DW18134 diminished liver macrophage infiltration and the expression of related inflammatory cytokines in peritonitis mice. Additionally, in the DSS-induced colitis model, DW18134 significantly reduced the disease activity index (DAI) and normalized food and water intake and body weight. Furthermore, DW18134 restored intestinal damage and reduced inflammatory cytokine expression in mice by blocking the IRAK4 signaling pathway. Notably, DW18134 protected DSS-threatened intestinal barrier function by upregulating tight junction gene expression. In conclusion, our findings reported a novel IRAK4 inhibitor, DW18134, as a promising candidate for treating inflammatory diseases, including peritonitis and IBD.
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Doenças Inflamatórias Intestinais , Quinases Associadas a Receptores de Interleucina-1 , Peritonite , Animais , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Camundongos , Peritonite/tratamento farmacológico , Peritonite/induzido quimicamente , Células RAW 264.7 , Doenças Inflamatórias Intestinais/tratamento farmacológico , Doenças Inflamatórias Intestinais/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Modelos Animais de Doenças , Transdução de Sinais/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Humanos , Masculino , Fosforilação/efeitos dos fármacos , Citocinas/metabolismo , NF-kappa B/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Human herpesvirus-6 (HHV-6) infection can rarely cause life-threatening conditions, such as encephalitis, in otherwise healthy children, with unclear pathogenesis. We studied a child who presented with acute HHV-6 encephalitis at the age of 10 months and who was homozygous for a novel missense mutation in IRAK4, encoding interleukin-1 receptor-associated kinase 4, identified by whole-exome sequencing. We tested the damaging impact of this mutation in silico by molecular dynamics simulations and in vitro by biochemical and functional experiments utilizing cell lines and patient's cells. We found that the mutation is severely hypomorphic, impairing both the expression and function of IRAK-4. Patient's leukocytes had barely detectable levels of IRAK-4 and diminished anti-viral immune responses to various stimuli inducing different Toll-like receptors and cytosolic nucleic acid sensors. Overall, these findings suggest that acute HHV-6 encephalitis can result from inborn errors of immunity to virus. This study represents the first report of isolated acute HHV-6 infection causing encephalitis in an inherited primary immunodeficiency, notably autosomal recessive (AR) partial IRAK-4 deficiency, and the first report of AR IRAK-4 deficiency presenting with a severe viral disease, notably HHV-6 encephalitis upon an acute infection, thereby expanding the clinical spectrum of IRAK-4 deficiency.
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Encefalite Viral , Herpesvirus Humano 6 , Doenças da Imunodeficiência Primária , Infecções por Roseolovirus , Criança , Humanos , Lactente , Quinases Associadas a Receptores de Interleucina-1/genética , Receptores Toll-Like/metabolismo , Doenças da Imunodeficiência Primária/genética , Encefalite Viral/diagnóstico , Encefalite Viral/genética , Herpesvirus Humano 6/genética , Infecções por Roseolovirus/diagnóstico , Infecções por Roseolovirus/genéticaRESUMO
Small ubiquitin-like modifier (SUMO) regulates various biological processes, including the MyD88/TICAMs-IRAKs-TRAF6-NF-κB pathway, one of the core immune pathways. However, its functions are inconsistent between invertebrates and vertebrates and have rarely been investigated in lower chordates, including amphioxus and fishes. Here, we investigated the SUMOylation gene system in the amphioxus, a living basal chordate. We found that amphioxus has a SUMOylation system that has a complete set of genes and preserves several ancestral traits. We proceeded to study their molecular functions using the mammal cell lines. Both amphioxus SUMO1 and SUMO2 were shown to be able to attach to NF-κB Rel and to inhibit NF-κB activation by 50-75% in a dose-dependent fashion. The inhibition by SUMO2 could be further enhanced by the addition of the SUMO E2 ligase UBC9. In comparison, while human SUMO2 inhibited RelA, human SUMO1 slightly activated RelA. We also showed that, similar to human PIAS1-4, amphioxus PIAS could serve as a SUMO E3 ligase and promote its self-SUMOylation. This suggests that amphioxus PIAS is functionally compatible in human cells. Moreover, we showed that amphioxus PIAS is not only able to inhibit NF-κB activation induced by MyD88, TICAM-like, TRAF6 and IRAK4 but also able to suppress NF-κB Rel completely in the presence of SUMO1/2 in a dose-insensitive manner. This suggests that PIAS could effectively block Rel by promoting Rel SUMOylation. In comparison, in humans, only PIAS3, but not PIAS1/2/4, has been reported to promote NF-κB SUMOylation. Taken together, the findings from amphioxus, together with those from mammals and other species, not only offer insights into the functional volatility of the animal SUMO system, but also shed light on its evolutionary transitions from amphioxus to fish, and ultimately to humans.
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Anfioxos , NF-kappa B , Humanos , Animais , NF-kappa B/genética , NF-kappa B/metabolismo , Ubiquitina , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anfioxos/genética , Anfioxos/metabolismo , Mamíferos/metabolismo , Chaperonas Moleculares , Proteínas Inibidoras de STAT Ativados/genéticaRESUMO
MyD88-dependent pathway mediated by Toll-like receptor is one of the vital ways activating immune responses. In order to identify the role of MyD88-dependent signaling pathway in yellow catfish, the Pf_MyD88, Pf_IRAK4, Pf_IRAK1, Pf_TRAF6 and Pf_NFκB1 (p105) (Pf: abbreviation of Pelteobagrus fulvidraco) were cloned and characterized respectively. The Pf_MyD88, Pf_IRAK4, Pf_IRAK1 and Pf_TRAF6 were all highly conserved among species and showed the highest homology to that of Pangasianodon hypophthalmus. Pf_NFκB1 showed the highest homology to that of Ictalurus punetaus. All of the five genes showed similar expression patterns in various tissues, with the highest expression level in the liver. These genes also showed similar expression levels in different embryonic development stages, except Pf_IRAK4. The higher expression level was detected from fertilized eggs to 1 day post hatching (dph), lower expression from 3 dph to 30 dph. After stimulation of inactivated Aeromonas hydrophila, the mRNA expressions of Pf_MyD88, Pf_IRAK4, Pf_IRAK1, Pf_TRAF6 and Pf_NFκB1 were significantly increased at 24 h in the liver, spleen, head kidney and trunk kidney, suggesting that all the five genes were involved in the innate immune response of yellow catfish. These results showed that MyD88-dependent signaling pathway plays important roles for disease defensing in the innate immune response. Meanwhile, inactivated A. hydrophila can cause strong innate immune response, which provides theoretical bases for the application of inactivated vaccines in defense against bacterial diseases of teleost.
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Peixes-Gato , Doenças dos Peixes , Animais , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Aeromonas hydrophila/fisiologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Peixes/químicaRESUMO
The IRAK-4 kinase lies at a critical signaling node that drives cancer cell survival through multiple mechanisms, activation, and translocation of NF-κB mediated inflammatory responses and innate immune signaling through regulation of interferon-α/ß receptor (IFNα/ß). Inhibition, of IRAK-4, has consequently drawn a lot of attention in recent years to address indications ranging from oncology to autoimmune disorders to neurodegeneration, etc. However, the key stumbling block in targeting IRAK-4 is that despite the inhibition of the kinase activity using an inhibitor the target remains effective, reducing the potential of an inhibitor. This is due to the "scaffolding effect" because of which although regulation of downstream processes by IRAK-4 has been primarily linked with kinase function; however, still, various reports have suggested that IRAK-4 has a non-kinase function in a variety of cell types. This is attributed to the myddosome complex formed by IRAK-4 with myd88, IRAK-2, and IRAK-1 which by itself can cause the activation of downstream effector TRAF6 despite inhibition of the kinase domain of IRAK-4. With this challenge, several groups initiated the development of targeting protein degraders of IRAK-4 using Proteolysis-Targeting Chimeras (PROTACs) technology to completely remove the IRAK-4 from the cellular milieu. In this review, we will capture all these developments and the evolving science around this target.
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Quinases Associadas a Receptores de Interleucina-1 , Transdução de Sinais , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , NF-kappa B/metabolismo , Fosforilação , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Interleukin-1 receptor associated kinase-4 (IRAK4) has emerged as a therapeutic target for inflammatory and autoimmune diseases. Through reversing the amide of CA-4948 and computer aided structure-activity relationship (SAR) studies, a series of IRAK4 inhibitors with oxazolo[4,5-b]pyridine scaffold were identified. Compound 32 showed improved potency (IC50 = 43 nM) compared to CA-4948 (IC50 = 115 nM), but suffered from hERG inhibition (IC50 = 5.7 µM). Further optimization led to compound 42 with reduced inhibition of hERG (IC50 > 30 µM) and 13-fold higher activity (IC50 = 8.9 nM) than CA-4948. Importantly, compound 42 had favorable in vitro ADME and in vivo pharmacokinetic properties. Furthermore, compound 42 significantly reduced LPS-induced production of serum TNF-α and IL-6 cytokines in the mouse model. The overall profiles of compound 42 support it as a lead for the development of IRAK4 inhibitors for the treatment of inflammatory and autoimmune disorders.
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Citocinas , Quinases Associadas a Receptores de Interleucina-1 , Animais , Camundongos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Lipopolissacarídeos/farmacologia , Síndrome de Resposta Inflamatória Sistêmica , Relação Estrutura-AtividadeRESUMO
Escalated innate immunity plays a critical role in SARS-CoV-2 pathology; however, the molecular mechanism is incompletely understood. Thus, we aim to characterize the molecular mechanism by which SARS-CoV-2 Spike protein advances human macrophage (MÏ´) inflammatory and glycolytic phenotypes and uncover novel therapeutic strategies. We found that human MÏ´s exposed to Spike protein activate IRAK4 phosphorylation. Blockade of IRAK4 in Spike protein-stimulated MÏ´s nullifies signaling of IRAK4, AKT, and baseline p38 without affecting ERK and NF-κB activation. Intriguingly, IRAK4 inhibitor (IRAK4i) rescues the SARS-CoV-2-induced cytotoxic effect in ACE2+HEK 293 cells. Moreover, the inflammatory reprogramming of MÏ´s by Spike protein was blunted by IRAK4i through IRF5 and IRF7, along with the reduction of monokines, IL-6, IL-8, TNFα, and CCL2. Notably, in Spike protein-stimulated MÏ´s, suppression of the inflammatory markers by IRAK4i was coupled with the rebalancing of oxidative phosphorylation over metabolic activity. This metabolic adaptation promoted by IRAK4i in Spike protein-activated MÏ´s was shown to be in part through constraining PFKBF3, HIF1α, cMYC, LDHA, lactate expression, and reversal of citrate and succinate buildup. IRAK4 knockdown could comparably impair Spike protein-enhanced inflammatory and metabolic imprints in human MÏ´s as those treated with ACE2, TLR2, and TLR7 siRNA. Extending these results, in murine models, where human SARS-CoV-2 Spike protein was not recognized by mouse ACE2, TLRs were responsible for the inflammatory and glycolytic responses instigated by Spike protein and were dysregulated by IRAK4i therapy. In conclusion, IRAK4i may be a promising strategy for severe COVID-19 patients by counter-regulating ACE2 and TLR-mediated MÏ´ hyperactivation. IRAK4i therapy counteracts MÏ´ inflammatory and glycolytic reprogramming triggered by Spike protein. This study illustrates that SARS-CoV-2 Spike protein activates IRAK4 signaling via ACE2 as well as TLR2 and TLR7 sensing in human MÏ´s. Remarkably, IRAK4i treatment can dysregulate both ACE-dependent and independent (via TLR sensing) SARS-CoV-2 Spike protein-activated inflammatory and metabolic imprints.
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COVID-19 , Glicoproteína da Espícula de Coronavírus , Enzima de Conversão de Angiotensina 2 , Animais , Células HEK293 , Humanos , Fatores Reguladores de Interferon/metabolismo , Fatores Reguladores de Interferon/farmacologia , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Macrófagos/metabolismo , Camundongos , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Receptor 2 Toll-Like/metabolismo , Receptor 7 Toll-Like/metabolismoRESUMO
BACKGROUND: The biomaterial integration depends on its interaction with the host immune system. Monocyte-macrophage lineage cells are immediately recruited to the implant site, polarized into different phenotypes, and fused into multinucleated cells, thus playing roles in tissue regeneration. IL-1R-associated kinase 4 (IRAK4) inhibition was reported to antagonize inflammatory osteolysis and regulate osteoclasts and foreign body giant cells (FBGCs), which may be a potential target in implant osseointegration. METHODS: In in-vitro experiments, we established simulated physiological and inflammatory circumstances in which bone-marrow-derived macrophages were cultured on sand-blasted and acid-etched (SLA) titanium surfaces to evaluate the induced macrophage polarization, multinucleated cells formation, and biological behaviors in the presence or absence of IRAK4i. Then, bone marrow stromal stem cells (BMSCs) were cultured in the conditioned media collected from the aforementioned induced osteoclasts or FBGCs cultures to clarify the indirect coupling effect of multinucleated cells on BMSCs. We further established a rat implantation model, which integrates IRAK4i treatment with implant placement, to verify the positive effect of IRAK4 inhibition on the macrophage polarization, osteoclast differentiation, and ultimately the early peri-implant osseointegration in vivo. RESULTS: Under inflammatory conditions, by transforming the monocyte-macrophage lineage cells from M1 to M2, IRAK4i treatment could down-regulate the formation and activity of osteoclast and relieve the inhibition of FBGC generation, thus promoting osteogenic differentiation in BMSCs and improve the osseointegration. CONCLUSION: This study may improve our understanding of the function of multinucleated cells and offer IRAK4i as a therapeutic strategy to improve early implant osseointegration and help to eliminate the initial implant failure.
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Implantes Dentários , Quinases Associadas a Receptores de Interleucina-1 , Animais , Ratos , Monócitos , Osseointegração , OsteogêneseRESUMO
Introduction: To investigate the inhibitory effect of quercetin (QUE) on dendritic cells (DCs) through the toll-like receptor 4/interleukin-1 receptor-associated kinase 4/nuclear factor kappa-B (TLR4/IRAK4/NF-κB) signalling pathway. Material and methods: CCK-8 and apoptosis assays were performed to determine the optimal concentration and action time of QUE to inhibit DCs. Protein extracts from treated DCs were used for Western blotting experiments to determine the relative expression levels of TLR4, IRAK4, and NF-κB p65 proteins. Changes in the ratio of CD86 and CD11c positive cells on the DCs surface were detected using flow cytometry. The molecular docking technique was used to analyse the binding site and free energy of QUE and IRAK4. Results: CCK-8 and apoptosis assays suggested that QUE inhibited the activity and function of DCs in a time-dose-dependent manner. The results of Western blotting suggested that the relative expression levels of TLR4, IRAK4, and NF-κB p65 proteins were increased in the lipopolysaccharide (LPS) group compared with the normal control group, and the relative expression of the above proteins was decreased after treatment with QUE and IRAK4-IN-4. The results of flow cytometry suggested that LPS increased the expression of CD86 and CD11c on the surface of DCs, and QUE and IRAK4-IN-4 decreased the expression of CD86 and CD11c induced by LPS. Molecular docking results showed that the binding sites of QUE and IRAK4 were stable, with the minimum binding energies comparable to that of IRAK4-IN-4. Conclusions: Quercetin may inhibit the activity and function of DCs through the TLR4/IRAK4/NF-κB signalling pathway, and IRAK4 may be its target.
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Growing pieces of evidence show that the long noncoding RNAs (lncRNAs) as new regulators participate in the regulation of various physiological and pathological processes. The study of lncRNA in lower invertebrates is still unclear compared with that in mammals. Here, we identified a novel lncRNA, termed IRAK4-related lncRNA (IRL), as a key regulator for innate immunity in teleost fish. We find that miR-27c-3p inhibits IRAK4 expression and thus weakens the NF-κB-mediated signaling pathway. Furthermore, the Gram-negative bacterium Vibrio anguillarum and lipopolysaccharide significantly upregulated host lncRNA IRL expression. Results indicate that IRL functions as a competing endogenous RNA for miR-27c-3p to regulate protein abundance of IRAK4; thus, invading microorganisms are eliminated and immune responses are promoted. Our study also demonstrates the regulation mechanism that lncRNA IRL can competitively adsorb miRNA to regulate the miR-27c-3p/IRAK4 axis that is widespread in teleost fish.
Assuntos
Doenças dos Peixes/imunologia , Proteínas de Peixes/imunologia , Quinases Associadas a Receptores de Interleucina-1/imunologia , NF-kappa B/imunologia , Perciformes/imunologia , RNA Longo não Codificante/imunologia , Vibrioses/veterinária , Animais , Pareamento de Bases , Sequência de Bases , Mapeamento Cromossômico , Cromossomos/química , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Doenças dos Peixes/genética , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Imunidade Inata , Quinases Associadas a Receptores de Interleucina-1/genética , Intestinos/citologia , Intestinos/imunologia , Rim/citologia , Rim/imunologia , Lipopolissacarídeos/farmacologia , MicroRNAs/genética , MicroRNAs/imunologia , NF-kappa B/genética , Perciformes/genética , Perciformes/microbiologia , Cultura Primária de Células , RNA Longo não Codificante/genética , Transdução de Sinais , Vibrio/crescimento & desenvolvimento , Vibrio/patogenicidade , Vibrioses/genética , Vibrioses/imunologia , Vibrioses/microbiologiaRESUMO
Valency can be defined as the number of discrete interactions a biomolecule can engage in. Valency can be critical for function, such as determining whether a molecule acts as a scaffold for assembling large supramolecular complexes or forms a functional dimer. Here, we highlight the importance of the role of valency in regulating immune responses, with a focus on innate immunity. We discuss some of the ways in which valency itself is regulated through transcriptional, post-transcriptional, and post-translational modifications. Finally, we propose that the valency model can be applied at the whole cell level to study differences in individual cell responses with relevance to putative therapeutic applications.
Assuntos
Sistema Imunitário , Imunidade Inata/genética , Modelos Imunológicos , Animais , Variação Biológica Individual , Regulação da Expressão Gênica , Humanos , Imunomodulação , Mediadores da Inflamação/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Processamento de Proteína Pós-Traducional , Processamento Pós-Transcricional do RNARESUMO
Opioid addiction remains a severe health problem. While substantial insights underlying opioid addiction have been yielded from neuron-centric studies, the contribution of non-neuronal mechanisms to opioid-related behavioral adaptations has begun to be recognized. Toll-like receptor 4 (TLR4), a pattern recognition receptor, has been widely suggested in opioid-related behaviors. Interleukin-1 receptor-associated kinase 4 (IRAK4) is a kinase essential for TLR4 responses, However, the potential role of IRAK4 in opioid-related responses has not been examined. Here, we explored the role of IRAK4 in cue-induced opioid-seeking behavior in male rats. We found that morphine self-administration increased the phosphorylation level of IRAK4 in the nucleus accumbens (NAc) in rats; the IRAK4 signaling remained activated after morphine extinction and cue-induced reinstatement test. Both systemic and local inhibition of IRAK4 in the NAc core attenuated cue-induced morphine-seeking behavior without affecting the locomotor activity and cue-induced sucrose-seeking. In addition, inhibition of IRAK4 also reduced the cue-induced reinstatement of fentanyl-seeking. Our findings suggest an important role of IRAK4 in opioid relapse-like behaviors and provide novel evidence in the association between innate immunity and drug addiction.