RESUMO
The signaling organelles of the innate immune system consist of oligomeric protein complexes known as supramolecular organizing centers (SMOCs). Examples of SMOCs include myddosomes and inflammasomes, which respectively induce transcription-dependent and -independent inflammatory responses. The common use of oligomeric structures as signaling platforms suggests multifunctionality, but each SMOC has a singular biochemically defined function. Here, we report that the myddosome is a multifunctional organizing center. In addition to promoting inflammatory transcription factor activation, the myddosome drives the rapid induction of glycolysis. We identify the kinase TBK1 as a myddosome component that promotes glycolysis, but not nuclear factor κB (NF-κB) activation. Synthetic immunology approaches further diversified SMOC activities, as we created interferon- or necroptosis-inducing myddosomes, inflammasomes that induce interferon responses instead of pyroptosis, and a SMOC-like nanomachine that induces interferon expression in response to a chemical ligand. These discoveries demonstrate the flexibility of immune signaling organelles, which permits the design of user-defined innate immune responses.
Assuntos
Imunidade Inata/imunologia , Imunidade Inata/fisiologia , Transdução de Sinais/imunologia , Animais , Glicólise/imunologia , Inflamassomos , Camundongos , Camundongos Endogâmicos C57BL , Enzimas Multifuncionais/imunologia , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Organelas/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Toll-LikeRESUMO
Intestinal intraepithelial lymphocytes (IELs) are located at the critical interface between the intestinal lumen, which is chronically exposed to food and microbes, and the core of the body. Using high-resolution microscopy techniques and intersectional genetic tools, we investigated the nature of IEL responses to luminal microbes. We observed that TCRγδ IELs exhibit unique microbiota-dependent location and movement patterns in the epithelial compartment. This behavioral pattern quickly changes upon exposure to different enteric pathogens, resulting in increased interepithelial cell (EC) scanning, expression of antimicrobial genes, and glycolysis. Both dynamic and metabolic changes to γδ IEL depend on pathogen sensing by ECs. Direct modulation of glycolysis is sufficient to change γδ IEL behavior and susceptibility to early pathogen invasion. Our results uncover a coordinated EC-IEL response to enteric infections that modulates lymphocyte energy utilization and dynamics and supports maintenance of the intestinal epithelial barrier. VIDEO ABSTRACT.
Assuntos
Intestinos/citologia , Intestinos/imunologia , Infecções por Salmonella/imunologia , Linfócitos T/imunologia , Animais , Células Epiteliais/metabolismo , Vigilância Imunológica , Mucosa Intestinal/imunologia , Camundongos , Infecções por Salmonella/microbiologia , Salmonella typhimurium/fisiologiaRESUMO
Signal transduction proteins containing a pLxIS motif induce interferon (IFN) responses central to antiviral immunity. Apart from their established roles in activating the IFN regulator factor (IRF) transcription factors, the existence of additional pathways and functions associated with the pLxIS motif is unknown. Using a synthetic biology-based platform, we identified two orphan pLxIS-containing proteins that stimulate IFN responses independent of all known pattern-recognition receptor pathways. We further uncovered a diversity of pLxIS signaling mechanisms, where the pLxIS motif represents one component of a multi-motif signaling entity, which has variable functions in activating IRF3, the TRAF6 ubiquitin ligase, IκB kinases, mitogen-activated protein kinases, and metabolic activities. The most diverse pLxIS signaling mechanisms were associated with the highest antiviral activities in human cells. The flexibility of domains that regulate IFN signaling may explain their prevalence in nature.
Assuntos
Fator Regulador 3 de Interferon , Interferons , Transdução de Sinais , Fator 6 Associado a Receptor de TNF , Humanos , Interferons/metabolismo , Células HEK293 , Fator Regulador 3 de Interferon/metabolismo , Fator Regulador 3 de Interferon/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Domínios Proteicos , Animais , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Motivos de Aminoácidos , Proteínas Quinases Ativadas por Mitógeno/metabolismoRESUMO
The steep rise in food allergy (FA) has evoked environmental factors involved in disease pathogenesis, including the gut microbiota, diet, and their metabolites. Early introduction of solid foods synchronizes with the "weaning reaction," a time during which the microbiota imprints durable oral tolerance. Recent work has shown that children with FA manifest an early onset dysbiosis with the loss of Clostridiales species, which promotes the differentiation of ROR-γt+ regulatory T cells to suppress FA. This process can be reversed in pre-clinical mouse models by targeted bacteriotherapy. Here, we review the dominant tolerance mechanisms enforced by the microbiota to suppress FA and discuss therapeutic intervention strategies that act to recapitulate the early life window of opportunity in stemming the FA epidemic.
Assuntos
Dieta , Disbiose/microbiologia , Hipersensibilidade Alimentar/imunologia , Microbioma Gastrointestinal/fisiologia , Animais , Clostridiales/isolamento & purificação , Dessensibilização Imunológica/métodos , Humanos , Tolerância Imunológica/imunologia , Imunoglobulina E/imunologia , Camundongos , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologiaRESUMO
Epithelial barrier defects are implicated in the pathogenesis of inflammatory bowel disease (IBD); however, the role of microbiome dysbiosis and the cytokine networks orchestrating chronic intestinal inflammation in response to barrier impairment remain poorly understood. Here, we showed that altered Schaedler flora (ASF), a benign minimal microbiota, was sufficient to trigger colitis in a mouse model of intestinal barrier impairment. Colitis development required myeloid-cell-specific adaptor protein MyD88 signaling and was orchestrated by the cytokines IL-12, IL-23, and IFN-γ. Colon inflammation was driven by IL-12 during the early stages of the disease, but as the mice aged, the pathology shifted toward an IL-23-dependent inflammatory response driving disease chronicity. These findings reveal that IL-12 and IL-23 act in a temporally distinct, biphasic manner to induce microbiota-driven chronic intestinal inflammation. Similar mechanisms might contribute to the pathogenesis of IBD particularly in patients with underlying intestinal barrier defects.
Assuntos
Colite/imunologia , Doenças Inflamatórias Intestinais/imunologia , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Mucosa Intestinal/patologia , Microbiota/imunologia , Animais , Doença Crônica , Modelos Animais de Doenças , Humanos , Inflamação , Interferon gama/genética , Interferon gama/metabolismo , Interleucina-12/genética , Interleucina-23/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Quimeras de TransplanteRESUMO
B cells can present antigens to CD4+ T cells, but it is thought that dendritic cells (DCs) are the primary initiators of naive CD4+ T cell responses. Nanoparticles, including virus-like particles (VLPs), are attractive candidates as carriers for vaccines and drug delivery. Using RNA phage Qß-derived VLP (Qß-VLP) as a model antigen, we found that antigen-specific B cells were the dominant antigen-presenting cells that initiated naive CD4+ T cell activation. B cells were sufficient to induce T follicular helper cell development in the absence of DCs. Qß-specific B cells promoted CD4+ T cell proliferation and differentiation via cognate interactions and through Toll-like receptor signaling-mediated cytokine production. Antigen-specific B cells were also involved in initiating CD4+ T cell responses during immunization with inactivated influenza virus. These findings have implications for the rational design of nanoparticles as vaccine candidates, particularly for therapeutic vaccines that aim to break immune tolerance.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Imunização/métodos , Vacinas contra Influenza/imunologia , Animais , Apresentação de Antígeno/imunologia , Antígenos Virais/química , Antígenos Virais/imunologia , Diferenciação Celular/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Vírus da Influenza A Subtipo H1N1/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Nanopartículas/química , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Receptores Toll-Like/imunologia , Vacinas de Produtos Inativados/imunologiaRESUMO
Activation of Toll-like receptor 7 (TLR7) can induce lupus in mice, whereas activation of TLR9 can prevent it, even though both receptors interact with myeloid differentiation primary response gene 88 (MyD88) for downstream signaling. How TLR9 triggers anti-inflammatory responses in autoimmunity is unclear. Leibler et al. recently reported that TLR9 initiates anti-inflammatory signaling and inhibits lupus pathogenesis in a MyD88-independent but ligand-dependent manner.
Assuntos
Fator 88 de Diferenciação Mieloide , Receptor Toll-Like 9 , Camundongos , Animais , Receptor Toll-Like 9/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Camundongos Knockout , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismo , Anti-InflamatóriosRESUMO
Mice carrying a hypomorphic point mutation in the Ptpn6 gene (Ptpn6spin mice) develop an inflammatory skin disease that resembles neutrophilic dermatosis in humans. Here, we demonstrated that interleukin-1α (IL-1α) signaling through IL-1R and MyD88 in both stromal and immune cells drive inflammation in Ptpn6spin mice. We further identified SYK as a critical kinase that phosphorylates MyD88, promoted MyD88-dependent signaling and mediates dermatosis in Ptpn6spin mice. Our studies further demonstrated that SHP1 encoded by Ptpn6 binds and suppresses SYK activation to inhibit MyD88 phosphorylation. Downstream of SHP1 and SYK-dependent counterregulation of MyD88 tyrosine phosphorylation, we have demonstrated that the scaffolding function of receptor interacting protein kinase 1 (RIPK1) and tumor growth factor-ß activated kinase 1 (TAK1)-mediating signaling were required to spur inflammatory disease. Overall, these studies identify SHP1 and SYK crosstalk as a critical regulator of MyD88 post-translational modifications and IL-1-driven inflammation.
Assuntos
Inflamação/imunologia , Interleucina-1alfa/imunologia , Fator 88 de Diferenciação Mieloide/imunologia , Dermatopatias/imunologia , Quinase Syk/imunologia , Animais , Citometria de Fluxo , Células HEK293 , Humanos , Immunoblotting , Inflamação/genética , Inflamação/metabolismo , Interleucina-1alfa/genética , Interleucina-1alfa/metabolismo , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/imunologia , MAP Quinase Quinase Quinases/metabolismo , Camundongos Knockout , Modelos Imunológicos , Mutação , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/imunologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores de Interleucina-1/imunologia , Receptores de Interleucina-1/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Dermatopatias/genética , Dermatopatias/metabolismo , Quinase Syk/genética , Quinase Syk/metabolismoRESUMO
B cell participation in early embryo/fetal development and the underlying molecular pathways have not been explored. To understand whether maternal B cell absence or impaired signaling interferes with placental and fetal growth, we paired CD19-deficient (CD19-/-) mice, females with B cell-specific MyD88 (BMyD88-/-) or IL10 (BIL10-/-) deficiency as well as wild-type and MyD88-/- controls on C57Bl/6 background with BALB/c males. Pregnancies were followed by ultrasound and Doppler measurements. Implantation number was reduced in BMyD88-/- and MyD88-/- mice. Loss of MyD88 or B cell-specific deletion of MyD88 or IL10 resulted in decreased implantation areas at gestational day (gd) 5, gd8 and gd10, accompanied by reduced placental thickness, diameter and areas at gd10. Uterine artery resistance was enhanced in BIL10-/- dams at gd10. Challenge with 0.4â mg lipopolysaccharide/kg bodyweight at gd16 revealed that BMyD88-/-, BIL10-/- and CD19-/- mothers delivered preterm, whereas controls maintained their pregnancy. B cell-specific MyD88 and IL10 expression is essential for appropriate in utero development. IL10+B cells are involved in uterine blood flow regulation during pregnancy. Finally, B cell-specific CD19, MyD88 and IL10 expression influences susceptibility towards preterm birth.
Assuntos
Linfócitos B/metabolismo , Desenvolvimento Fetal , Feto/embriologia , Transdução de Sinais , Artéria Uterina/metabolismo , Útero , Resistência Vascular , Animais , Antígenos CD19/genética , Antígenos CD19/metabolismo , Feminino , Interleucina-10/deficiência , Interleucina-10/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/metabolismo , Gravidez , Útero/irrigação sanguínea , Útero/metabolismoRESUMO
The globally reemerging respiratory pathogen enterovirus D68 (EV-D68) is implicated in outbreaks of severe respiratory illness and associated with acute flaccid myelitis. However, there remains a lack of effective treatments for EV-D68 infection. In this work, we found that the host Toll-like receptor 7 (TLR7) proteins, which function as powerful innate immune sensors, were selectively elevated in expression in response to EV-D68 infection. Subsequently, we investigated the impact of Vesatolimod (GS-9620), a Toll-like receptor 7 agonist, on EV-D68 replication. Our findings revealed that EV-D68 infection resulted in increased mRNA levels of TLR7. Treatment with Vesatolimod significantly inhibited EV-D68 replication [half maximal effective concentration (EC50) = 0.1427 µM] without inducing significant cytotoxicity at virucidal concentrations. Although Vesatolimod exhibited limited impact on EV-D68 attachment, it suppressed RNA replication and viral protein synthesis after virus entry. Vesatolimod broadly inhibited the replication of circulating isolated strains of EV-D68. Furthermore, our findings demonstrated that treatment with Vesatolimod conferred resistance to both respiratory and neural cells against EV-D68 infection. Overall, these results present a promising strategy for drug development by pharmacologically activating TLR7 to initiate an antiviral state in EV-D68-infected cells selectively.IMPORTANCEConventional strategies for antiviral drug development primarily focus on directly targeting viral proteases or key components, as well as host proteins involved in viral replication. In this study, based on our intriguing discovery that enterovirus D68 (EV-D68) infection specifically upregulates the expression of immune sensor Toll-like receptor 7 (TLR7) protein, which is either absent or expressed at low levels in respiratory cells, we propose a potential antiviral approach utilizing TLR7 agonists to activate EV-D68-infected cells into an anti-viral defense state. Notably, our findings demonstrate that pharmacological activation of TLR7 effectively suppresses EV-D68 replication in respiratory tract cells through a TLR7/MyD88-dependent mechanism. This study not only presents a promising drug candidate and target against EV-D68 dissemination but also highlights the potential to exploit unique alterations in cellular innate immune responses induced by viral infections, selectively inducing a defensive state in infected cells while safeguarding uninfected normal cells from potential adverse effects associated with therapeutic interventions.
Assuntos
Antivirais , Enterovirus Humano D , Receptor 7 Toll-Like , Replicação Viral , Receptor 7 Toll-Like/agonistas , Receptor 7 Toll-Like/metabolismo , Humanos , Replicação Viral/efeitos dos fármacos , Enterovirus Humano D/efeitos dos fármacos , Antivirais/farmacologia , Indóis/farmacologia , Infecções por Enterovirus/virologia , Imunidade Inata/efeitos dos fármacos , Linhagem Celular , Internalização do Vírus/efeitos dos fármacos , PteridinasRESUMO
Intracellular compartmentalization of ligands, receptors and signaling molecules has been recognized as an important regulator of inflammation. The toll-like receptor (TLR) 2 pathway utilizes the trafficking molecule adaptor protein 3 (AP-3) to activate interleukin (IL)-6 signaling from within phagosomal compartments. To better understand the vesicular pathways that may contribute to intracellular signaling and cooperate with AP-3, we performed a vesicular siRNA screen. We identified Rab8 and Rab11 GTPases as important in IL-6 induction upon stimulation with the TLR2 ligand Pam3 CSK4 or the pathogen, Borrelia burgdorferi (Bb), the causative agent of Lyme disease. These Rabs were recruited to late and lysosomal stage phagosomes and co-transported with TLR2 signaling adaptors and effectors, such as MyD88, TRAM and TAK1, in an AP-3-dependent manner. Our data support a model where AP-3 mediates the recruitment of recycling and secretory vesicles and the assembly of signaling complexes at the phagosome.
Assuntos
Borrelia burgdorferi , Doença de Lyme , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Borrelia burgdorferi/metabolismo , Ligantes , Doença de Lyme/genética , Doença de Lyme/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Fagossomos/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Proteínas rab de Ligação ao GTP , Animais , CamundongosRESUMO
Interleukin-1ß is one of the most potent inducers of beta cell inflammation in the lead-up to type 1 diabetes. We have previously reported that IL1ß-stimulated pancreatic islets from mice with genetic ablation of stress-induced pseudokinase TRB3(TRB3KO) show attenuated activation kinetics for the MAP3K MLK3 and JNK stress kinases. However, JNK signaling constitutes only a portion of the cytokine-induced inflammatory response. Here we report that TRB3KO islets also show a decrease in amplitude and duration of IL1ß-induced phosphorylation of TAK1 and IKK, kinases that drive the potent NF-κB proinflammatory signaling pathway. We observed that TRB3KO islets display decreased cytokine-induced beta cell death, preceded by a decrease in select downstream NF-κB targets, including iNOS/NOS2 (inducible nitric oxide synthase), a mediator of beta cell dysfunction and death. Thus, loss of TRB3 attenuates both pathways required for a cytokine-inducible, proapoptotic response in beta cells. In order to better understand the molecular basis of TRB3-enhanced, post-receptor IL1ß signaling, we interrogated the TRB3 interactome using coimmunoprecipitation followed by mass spectrometry to identify immunomodulatory protein Flightless homolog 1 (Fli1) as a novel, TRB3-interacting protein. We show that TRB3 binds and disrupts Fli1-dependent sequestration of MyD88, thereby increasing availability of this most proximal adaptor required for IL1ß receptor-dependent signaling. Fli1 sequesters MyD88 in a multiprotein complex resulting in a brake on the assembly of downstream signaling complexes. By interacting with Fli1, we propose that TRB3 lifts the brake on IL1ß signaling to augment the proinflammatory response in beta cells.
Assuntos
Proteínas de Ciclo Celular , Interleucina-1beta , Transdução de Sinais , Animais , Camundongos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais/genética , Inibidores Enzimáticos/farmacologia , Apoptose/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Ativação Transcricional/genéticaRESUMO
This study retrospectively investigated the impact of interleukin-1 receptor-associated kinase-3 (IRAK-3/IRAK-M) silencing by methylation on the likelihood of multiple sclerosis (MS) activity. This cross-sectional study included 90 patients with MS: 45 with active disease (Group 1), 45 in remission (Group 2), and 45 healthy controls. The study included quantitation of IRAK-3 methylation index (MI%), IRAK-3 mRNA, and myeloid differentiation factor88 (MyD88) and assessment of NF-κB activity. IRAK-3 MI% was significantly higher in group 1 compared to group 2, accompanied by lower IRAK-3 mRNA expression, elevated circulating MyD88, and increased NF-κB activity. IRAK-3 MI% correlated negatively with its transcript and positively with MyD88 and NF-κB activity. A logistic regression model was created to predict active demyelination. The C-index was 0.924, which indicates a very strong prediction model. Within the limitations of current work, IRAK-3 methylation level seems to be a promising candidate biomarker for identifying MS patients at risk of relapse.
RESUMO
Exacerbated expression of TLR4 protein (foremost pattern recognition receptor) during obesity could trigger NF-κB/iNOS signaling through linker protein (MyD88), predisposed to an indispensable inflammatory response. The induction of this detrimental cascade leads to myocardial and vascular abnormalities. Molecular docking was studied for protein-ligand interaction between these potential targets and resveratrol. The pre-treatment of resveratrol (20 mg/kg/p.o/per day for ten weeks) was given to investigate the therapeutic effect against HFD-induced obesity and associated vascular endothelial dysfunction (VED) and myocardial infarction (MI) in Wistar rats. In addition to accessing the levels of serum biomarkers for VED and MI, oxidative stress, inflammatory cytokines, and histopathology of these tissues were investigated. Lipopolysaccharide (for receptor activation) and protein expression analysis were introduced to explore the mechanistic involvement of TLR4/MyD88/NF-κB/iNOS signaling. Assessment of in-silico analysis showed significant interaction between protein and ligand. The involvement of this proposed signaling (TLR4/MyD88/NF-κB/iNOS) was further endorsed by the impact of lipopolysaccharide and protein expression analysis in obese and treated rats. Moreover, resveratrol pre-treated rats showed significantly lowered cardio and vascular damage measured by the distinct down expression of the TLR4/MyD88/NF-κB/iNOS pathway by resveratrol treatment endorses its ameliorative effect against VED and MI.
Assuntos
Infarto do Miocárdio , Estilbenos , Ratos , Animais , NF-kappa B/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/metabolismo , Resveratrol/farmacologia , Estilbenos/farmacologia , Estilbenos/uso terapêutico , Lipopolissacarídeos/farmacologia , Ligantes , Simulação de Acoplamento Molecular , Ratos Wistar , Infarto do Miocárdio/tratamento farmacológico , DietaRESUMO
Osteoarthritis (OA) is one of the most common diseases of the skeleton. Long non-coding RNAs (lncRNAs) are emerging as key players in OA pathogenesis. This work sets out to determine the function of lncRNA MALAT1 in OA and the mechanisms by which it does so. Mesenchymal stem cells isolated from the human synovial membrane are called hSMSCs. The hSMSCs' surface markers were studied using flow cytometry. To determine whether or not hSMSC might differentiate, researchers used a number of different culture settings and labeling techniques. The expression levels of associated genes and proteins were determined using quantitative real-time polymerase chain reaction (RT-qPCR), western blotting (WB), and immunostaining. A dual luciferase reporter experiment and RNA immunoprecipitation (RIP) test demonstrated the direct association between miR-212-5p and MALAT1 or MyD88. MALAT1 was downregulated during the chondrogenic differentiation of hSMSCs, and underexpression of MALAT1 promotes chondrogenesis in hSMSCs. Using dual luciferase reporter and RIP assays facilitated the identification of MALAT1 as a competitive endogenous RNA (ceRNA) that sequesters miR-212-5p. Additionally, the expression of MYD88 was regulated by MALAT1 through direct binding with miR-212-5p. Significantly, the effects of MALAT1 on the chondrogenic differentiation of hSMSCs were counteracted by miR-212-5p/MYD88. Furthermore, our in vivo investigation revealed that the inhibition of MALAT1 mitigated osteoarthritis progression in rat models. In conclusion, the promotion of chondrogenic differentiation in hSMSCs and the protective effect on cartilage tissue in OA can be achieved by suppressing MALAT1, which regulates the miR-212-5p/MyD88 axis.
Assuntos
Células-Tronco Mesenquimais , MicroRNAs , Osteoartrite do Joelho , RNA Longo não Codificante , Animais , Humanos , Ratos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Condrogênese , Luciferases/metabolismo , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Inflammation and fibrosis play important roles in diabetic kidney disease (DKD). Previous studies have shown that glucagon-like peptide-1 receptor (GLP-1R) agonists had renal protective effects. However, the mechanisms are not clear. The present study explored the effect of liraglutide, a GLP-1R agonist, on the downregulation of glomerular inflammation and fibrosis in DKD by regulating the TLR4/MyD88/NF-kappaB signaling pathway in mesangial cells (MCs). In vitro, rat MCs were cultured in high glucose (HG). We found that liraglutide treatment significantly reduced the HG mediated activation of the TLR4/MYD88/NF-κB signaling pathway, extracellular matrix (ECM)-related proteins and inflammatory factors. A combination of TLR4 inhibitor (TAK242) and liraglutide did not synergistically inhibited inflammatory factors and ECM proteins. Furthermore, in the presence of TLR4 siRNA, liraglutide significantly blunted HG-induced expression of fibronectin protein and inflammatory factors. Importantly, TLR4 selective agonist - LPS or TLR4 overexpression eliminated the improvement effects of liraglutide on the HG-induced response. In vivo, administration of liraglutide for 8 weeks significantly improved the glomerular damage in streptozotocin-induced diabetic mice, and reduced the expression of TLR4/MYD88/NF-κB signaling proteins, ECM protein, and inflammatory factors in renal cortex. TLR4-/- diabetic mice showed significant amelioration in urine protein excretion rate, glomerular pathological damage, inflammation and fibrosis. Liraglutide attenuated glomerular hypertrophy, renal fibrosis and inflammatory response in TLR4-/- diabetic mice. Taken together, our findings suggest that TLR4/MYD88/NF-κB signaling is involved in the regulation of inflammatory response and ECM protein proliferation in DKD. Liraglutide alleviates inflammation and fibrosis by downregulating the TLR4/MYD88/NF-κB signaling pathway in MCs.
RESUMO
Acute lung injury (ALI) remains a significant clinical challenge due to the absence of effective treatment alternatives. This study presents a new method that employs a screening platform focusing on MyD88 affinity, anti-inflammatory properties, and toxicity. This platform was used to evaluate a 300-compound library known for its anti-inflammatory potential. Among the screened compounds, Bicyclol emerged as a standout, exhibiting MyD88 binding and a significant reduction in LPS-stimulated pro-inflammatory factors production in mouse primary peritoneal macrophages. By targeting MyD88, Bicyclol disrupts the MyD88/TLR4 complex and MyD88 polymer formation, thereby mitigating the MAPKs and NF-κB signaling pathways. In vivo experiments further confirmed Bicyclol's efficacy, demonstrating alleviated ALI symptoms, decreased inflammatory cytokines level, and reduced inflammatory cells presence in lung tissues. These findings were associated with a decrease in mortality in LPS-challenged mice. Overall, Bicyclol represents a promising treatment option for ALI by specifically targeting MyD88 and limiting inflammatory responses.
Assuntos
Lesão Pulmonar Aguda , Compostos de Bifenilo , Lipopolissacarídeos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide , Animais , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/prevenção & controle , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/patologia , Lipopolissacarídeos/toxicidade , Fator 88 de Diferenciação Mieloide/metabolismo , Camundongos , Masculino , Compostos de Bifenilo/farmacologia , Anti-Inflamatórios/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , NF-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Citocinas/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/patologia , Pulmão/metabolismoRESUMO
Gastric ulcer (GU) is a serious upper gastrointestinal tract disorder that affects people worldwide. The drugs now available for GU treatment have a high rate of relapses and drug interactions, as well as mild to severe side effects. As a result, new natural therapeutic medications for treating GU with fewer negative side effects are desperately needed. Because of quercetin's (QCT) diverse pharmacological effects and unique structural features, we decided to semi-synthesize new QCT derivatives and test them for antiulcer activity. Docking assays were performed on the synthesized compounds to determine their affinity for TLR-4/MD-2, MyD88/TIR, and NF-κB domains, an important inflammatory pathway involved in GU development and progression. Mice were given oral famotidine (40 mg/kg/day), QCT, QCT pentamethyl (QPM), or QCT pentaacetyl (QPA) (50 mg/kg/day) for 5 days before GU induction by a single intraperitoneal injection of indomethacin (INDO; 18 mg/kg). QPM and QPA have a stronger binding affinity for TLR-4/MD-2, MyD88/TIR and NF-κB domains than QCT. In comparison, they demonstrated the greatest reduction in ulcer score and index, gastric MDA and nitric oxide (NO) contents, MyD88 and NF-κB expressions, and gastric TLR-4 immunostaining. They also enhanced the levels of GSH, CAT, COX-1, and COX-2 in the gastric mucosa, as well as HO-1 and Nrf2 expression, with histological regression in gastric mucosal lesions, with QPA-treated mice demonstrating the best GU healing. QPA is safe against all of the target organs and adverse pathways studied, with good ADME properties. However, further in vitro experiments are necessary to demonstrate the inhibitory effects of QPM and QPA on the protein targets of interest. In addition, preclinical research on its bioavailability and safety is essential before clinical management can be undertaken. Overall, the new QPA derivative could one day serve as the basis for a new class of potential antiulcer drugs.
Assuntos
Indometacina , Úlcera Gástrica , Humanos , Camundongos , Animais , Indometacina/toxicidade , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/tratamento farmacológico , Úlcera Gástrica/patologia , Quercetina/farmacologia , Quercetina/uso terapêutico , Simulação de Acoplamento Molecular , Úlcera/metabolismo , Úlcera/patologia , NF-kappa B/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/metabolismo , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologiaRESUMO
BACKGROUND: Circular RNAs (CircRNAs) have been associated with acute lung injury (ALI), but their molecular mechanisms remain unclear. METHODS: This study developed a rat model of lipopolysaccharide (LPS)-induced ALI and evaluated the modeling effect by hematoxylin and eosin staining, Masson's trichrome staining, lung wet-to-dry weight ratio, terminal deoxynucleotidyl transferase UTP nick end labeling (TUNEL), and enzyme-linked immunosorbent assay (ELISA) detection of inflammatory factors (interleukin-1ß, tumor necrosis factor alpha, and interleukin-6). Using lung tissues from a rat model of LPS-induced ALI, we then conducted circRNA sequencing, mRNA sequencing, and bioinformatics analysis to obtain differential circRNA and mRNA expression profiles as well as potential ceRNA networks. Furthermore, we performed quantitative real-time polymerase chain reaction (qRT-PCR) assays to screen for circ-Phkb in ALI rat lung tissues, alveolar macrophages, and LPS-induced NR8383 cells. We conducted induction with or without LPS with circ-Phkb siRNA and overexpression lentivirus in NR8383. Cell Counting Kit-8, C5-Ethynyl-2'-deoxyuridine (Edu), TUNEL, and cytometry were used to identify proliferation and apoptosis, respectively. We detected inflammatory factors using ELISA. Finally, we used Western blot to detect the apoptosis-related proteins and TLR4/MyD88/NF-kB/CCL2 pathway activation. RESULTS: Our results revealed that both circRNA and mRNA profiles are different from those of the Sham group. We observed a significant circ-Phkb upregulation in NR8383 cells and LPS-exposed rats. Apoptosis and inflammation were greatly reduced when circ-Phkb expression was reduced in NR8383 cells, cell proliferation was increased, and circ-Phkb overexpression was decreased. CONCLUSIONS: In terms of mechanism, circ-Phkb suppression inhibits CCL2 expression via the TLR4/MyD88/NF-kB pathway in LPS-induced alveolar macrophages.
Assuntos
Lesão Pulmonar Aguda , MicroRNAs , Ratos , Animais , NF-kappa B/metabolismo , Macrófagos Alveolares/metabolismo , Lipopolissacarídeos/toxicidade , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 4 Toll-Like/genética , RNA Circular/genética , Apoptose , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Inflamação/metabolismo , RNA Mensageiro/metabolismo , MicroRNAs/genéticaRESUMO
Inflammation is the body's response to injuries, which depends on numerous regulatory factors. Among them, miRNAs have gained much attention for their role in regulating inflammatory gene expression at multiple levels. In particular, miR-21 is up-regulated during the inflammatory response and reported to be involved in the resolution of inflammation by down-regulating pro-inflammatory mediators, including MyD88. Herein, we evaluated the regulatory effects of miR-21 on the TLR-4/MyD88 pathway in an in vitro model of 6-mer HA oligosaccharides-induced inflammation in human chondrocytes. The exposition of chondrocytes to 6-mer HA induced the activation of the TLR4/MyD88 pathway, which culminates in NF-kB activation. Changes in miR-21, TLR-4, MyD88, NLRP3 inflammasome, IL-29, Caspase1, MMP-9, iNOS, and COX-2 mRNA expression of 6-mer HA-stimulated chondrocytes were examined by qRT-PCR. Protein amounts of TLR-4, MyD88, NLRP3 inflammasome, p-ERK1/2, p-AKT, IL-29, caspase1, MMP-9, p-NK-kB p65 subunit, and IKB-a have been evaluated by ELISA kits. NO and PGE2 levels have been assayed by colorimetric and ELISA kits, respectively. HA oligosaccharides induced a significant increase in the expression of the above parameters, including NF-kB activity. The use of a miR-21 mimic attenuated MyD88 expression levels and the downstream effectors. On the contrary, treatment with a miR-21 inhibitor induced opposite effects. Interestingly, the use of a MyD88 siRNA confirmed MyD88 as the target of miR-21 action. Our results suggest that miR-21 expression could increase in an attempt to reduce the inflammatory response, targeting MyD88.