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1.
Mediators Inflamm ; 2023: 3732315, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36654880

RESUMO

LIGHT is a member of the TNF superfamily and a proinflammatory cytokine involved in liver pathogenesis. Many liver diseases involve activation of Toll-like receptor 3 (TLR3), which is activated by double-stranded RNA (dsRNA). However, the involvement of LIGHT in TLR3 implicated liver diseases is not clear. In this study, we investigated the role of LIGHT in TLR3 involved liver pathogenesis by using a mouse model of TLR3 agonist poly(I:C)-induced hepatitis. We found LIGHT expression at both protein and mRNA level in liver tissues is dramatically increased during the course of poly(I:C)-induced liver injury. This induction depends on NF-κB activation as pretreating the mice with a NF-κB inhibitor abrogates LIGHT upregulation. Importantly, blockade of the LIGHT signaling pathway with the recombinant LIGHT receptor HVEM protein ameliorates liver injury in poly(I:C)-induced hepatitis. Conclusions. These results indicate that LIGHT amplification by NF-κB plays a significant role in TLR3 involved hepatitis and points LIGHT to be a potential drug target for liver disease therapy.


Assuntos
Hepatite , NF-kappa B , Receptor 3 Toll-Like , Citocinas , Hepatite/genética , Hepatite/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Poli I-C/farmacologia , Transdução de Sinais , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/genética , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/metabolismo , Animais , Camundongos , Modelos Animais de Doenças , Doença Aguda
2.
Nat Commun ; 14(1): 164, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36631495

RESUMO

Toll-like receptor 3 (TLR3) is a member of the TLR family, which plays an important role in the innate immune system and is responsible for recognizing viral double-stranded RNA (dsRNA). Previous biochemical and structural studies have revealed that a minimum length of approximately 40-50 base pairs of dsRNA is necessary for TLR3 binding and dimerization. However, efficient TLR3 activation requires longer dsRNA and the molecular mechanism underlying its dsRNA length-dependent activation remains unknown. Here, we report cryo-electron microscopy analyses of TLR3 complexed with longer dsRNA. TLR3 dimers laterally form a higher multimeric complex along dsRNA, providing the basis for cooperative binding and efficient signal transduction.


Assuntos
RNA de Cadeia Dupla , Receptor 3 Toll-Like , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Microscopia Crioeletrônica , Transdução de Sinais , Dimerização
3.
Behav Brain Res ; 438: 114200, 2023 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-36334783

RESUMO

There are many unanswered questions about the interaction between the immune system and behavior change, including the contributions of individual differences. The present study modeled individual differences in the immune system by comparing inbred Lewis rats, which have dysregulated stress and immune systems, to their genetically diverse parent strain, Wistar rats. The objective was to examine the consequences of an immune challenge on behavior and neuroimmune signaling in both strains. Peripheral administration of the toll-like receptor 3 (TLR3) agonist and viral memetic polyinosinic-polycytidylic acid (poly(I:C)) induced behavior changes in both strains, reducing locomotor activity and increasing avoidance behavior (time on the dark side of the light-dark box). Furthermore, poly(I:C) induced hyperarousal and increased avoidance behavior more in female Lewis than female Wistar rats. Baseline strain differences were also observed: Lewis rats had higher avoidance behavior and lower startle response than Wistars. Lewis rats also had lower levels of peripheral inflammation, as measured by spleen weight. Finally, poly(I:C) increased expression of genes in the TLR3 pathway, cytokine genes, and CD11b, a gene associated with proinflammatory actions of microglia, in the prelimbic cortex and central amygdala, with greater expression of cytokine genes in male rats. Lewis rats had lower baseline expression of some neuroimmune genes, particularly CD11b. Overall, we found constitutive strain differences in immune profiles and baseline differences in behavior, yet poly(I:C) generally induced similar behavior changes in males while hyperarousal and avoidance behavior were heightened in female Lewis rats.


Assuntos
Poli I-C , Receptor 3 Toll-Like , Animais , Feminino , Masculino , Ratos , Citocinas/metabolismo , Poli I-C/farmacologia , Ratos Endogâmicos Lew , Ratos Wistar , Receptor 3 Toll-Like/metabolismo
4.
Int J Mol Sci ; 23(24)2022 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36555168

RESUMO

Cardiac dysfunction is a life-threatening complication in sepsis. Upon infection and cardiac stress, the cardiac macrophage population expands. Recruited macrophages exhibit a predominantly proinflammatory phenotype and release danger-associated molecular patterns (DAMPs) that contribute to cardiac dysfunction. However, the underlying pathomechanisms are highly complex and not fully understood. Here, we utilized an indirect macrophage-cardiomyocyte co-culture model to study the effects of proinflammatory macrophages on the activation of different cardiac receptors (TLR3, TLR4, and TNFR) and their role in cardiac inflammation and caspase-3/7 activation. The stimulation of cardiomyocytes with conditioned medium of LPS-stimulated macrophages resulted in elevated IL-6 protein concentrations and relative IL-6 and TNFα mRNA levels. Conditioned medium from LPS-stimulated macrophages also induced NFκB translocation and increased caspase-3/7 activation in cardiomyocytes. Analyzing the role of different cardiac receptors, we found that TLR4 and TNFR inhibition reduces cardiac inflammation and that the inhibition of TNFR prevents NFκB translocation into the nuclei of cardiomyocytes, induced by exposure to conditioned medium of proinflammatory macrophages. Moreover, we demonstrated that TLR3 inhibition reduces macrophage-mediated caspase-3/7 activation. Our results suggest that the immune response of macrophages under inflammatory conditions leads to the release of DAMPs, such as eRNA and cytokines, which in turn induce cardiomyocyte dysfunction. Thus, the data obtained in this study contribute to a better understanding of the pathophysiological mechanisms of cardiac dysfunction.


Assuntos
Cardiopatias , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , Receptor 4 Toll-Like/metabolismo , Caspase 3/metabolismo , Interleucina-6/metabolismo , Receptor 3 Toll-Like/metabolismo , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Meios de Cultivo Condicionados/farmacologia , Meios de Cultivo Condicionados/metabolismo , Macrófagos/metabolismo , Inflamação/metabolismo , NF-kappa B/metabolismo , Cardiopatias/metabolismo
5.
Sci Rep ; 12(1): 21779, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36526691

RESUMO

Elevated serum cytokine production in COVID-19 patients is associated with disease progression and severity. However, the stimuli that initiate cytokine production in patients remain to be fully revealed. Virus-infected cells release virus-associated exosomes, extracellular vesicles of endocytic origin, into the blood to deliver viral cargoes able to regulate immune responses. Here, we report that plasma exosomes of COVID-19 patients contain SARS-CoV-2 double stranded RNA (dsRNA) and stimulate robust production of interleukin-6 (IL-6), IL-8, tumor necrosis factor-α (TNF-α), and other inflammatory cytokines and chemokines by human peripheral mononuclear cells. Exosome depletion abolished these stimulated responses. COVID-19 plasma exosomes induced proinflammatory responses in CD4+ T cells, CD8+ T cells, and CD14+ monocytes but not significantly in regulatory T cells, Th17 T cells, or central memory T cells. COVID-19 plasma exosomes protect the SARS-CoV-2 dsRNA cargo from RNase and deliver the dsRNA into recipient cells. These exosomes significantly increase expression of endosomal toll-like receptor 3 (TLR3), TLR7, TLR8, and TLR9 in peripheral T cells and monocytes. A pharmacological inhibitor of TLR3 considerably reduced cytokine and chemokine production by CD4+ and CD8+ T cells but not by CD14+ monocytes, highlighting divergent signaling pathways of immune cells in response to COVID-19 plasma exosomes. Our results identify a novel model of intercellular crosstalk following SARS-CoV-2 infection that evoke immune responses positioned to contribute to elevated cytokine production associated with COVID-19 progression, severity, and long-haul symptoms.


Assuntos
COVID-19 , Exossomos , Humanos , Exossomos/metabolismo , Receptor 3 Toll-Like/metabolismo , Leucócitos Mononucleares/metabolismo , Linfócitos T CD8-Positivos/metabolismo , SARS-CoV-2/metabolismo , COVID-19/metabolismo , Citocinas/metabolismo , RNA de Cadeia Dupla/metabolismo , Imunidade
6.
Cells ; 11(24)2022 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-36552771

RESUMO

Steroid receptor RNA activator gene (SRA1) emerges as a player in pathophysiological responses of adipose tissue (AT) in metabolic disorders such as obesity and type 2 diabetes (T2D). We previously showed association of the AT SRA1 expression with inflammatory cytokines/chemokines involved in metabolic derangement. However, the relationship between altered adipose expression of SRA1 and the innate immune Toll-like receptors (TLRs) as players in nutrient sensing and metabolic inflammation as well as their downstream signaling partners, including interferon regulatory factors (IRFs), remains elusive. Herein, we investigated the association of AT SRA1 expression with TLRs, IRFs, and other TLR-downstream signaling mediators in a cohort of 108 individuals, classified based on their body mass index (BMI) as persons with normal-weight (N = 12), overweight (N = 32), and obesity (N = 64), including 55 with and 53 without T2D. The gene expression of SRA1, TLRs-2,3,4,7,8,9,10 and their downstream signaling mediators including IRFs-3,4,5, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1), and nuclear factor-κB (NF-κB) were determined using qRT-PCR and SRA1 protein expression was determined by immunohistochemistry. AT SRA1 transcripts' expression was significantly correlated with TLRs-3,4,7, MyD88, NF-κB, and IRF5 expression in individuals with T2D, while it associated with TLR9 and TRAF6 expression in all individuals, with/without T2D. SRA1 expression associated with TLR2, IRAK1, and IRF3 expression only in individuals with obesity, regardless of diabetes status. Furthermore, TLR3/TLR7/IRAK1 and TLR3/TLR9 were identified as independent predictors of AT SRA1 expression in individuals with obesity and T2D, respectively. Overall, our data demonstrate a direct association between the AT SRA1 expression and the TLRs together with their downstream signaling partners and IRFs in individuals with obesity and/or T2D.


Assuntos
Diabetes Mellitus Tipo 2 , Receptor 3 Toll-Like , Humanos , Tecido Adiposo/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Fatores Reguladores de Interferon/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Obesidade/genética , Obesidade/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
7.
Nat Commun ; 13(1): 6876, 2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36371424

RESUMO

Toll-like Receptor 3 (TLR3) initiates a potent anti-viral immune response by binding to double-stranded RNA ligands. Previous crystallographic studies showed that TLR3 forms a homodimer when bound to a 46-base pair RNA ligand. However, this short RNA fails to initiate a robust immune response. To obtain structural insights into the length dependency of TLR3 ligands, we determine the cryo-electron microscopy structure of full-length TLR3 in a complex with a synthetic RNA ligand with an average length of ~400 base pairs. In the structure, the dimeric TLR3 units are clustered along the double-stranded RNA helix in a highly organized and cooperative fashion with a uniform inter-dimer spacing of 103 angstroms. The intracellular and transmembrane domains are dispensable for the clustering because their deletion does not interfere with the cluster formation. Our structural observation suggests that ligand-induced clustering of TLR3 dimers triggers the ordered assembly of intracellular signaling adaptors and initiates a robust innate immune response.


Assuntos
Poli I-C , Receptor 3 Toll-Like , Ligantes , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Poli I-C/farmacologia , RNA de Cadeia Dupla/genética , Sítios de Ligação , Microscopia Crioeletrônica
8.
Front Immunol ; 13: 1025796, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36341332

RESUMO

Dysregulated innate and adaptive immune response to rhinoviral infection plays an important role in the exacerbation or progressive course of chronic rhinosinusitis (CRS). However, few studies have evaluated whether rhinovirus-induced production of anti-viral interferon is deficient or delayed in inflammatory epithelial cells of patients with CRS with nasal polyps. The aim of the present study is to investigate the replication rates of rhinovirus 16 (RV 16), RV16-induced antiviral interferon secretion, and the expression levels of pattern recognition receptors after RV 16 infection or TLR3 stimulation with poly (I: C) in normal and inflammatory epithelial cells. Inflammatory epithelial cells were obtained from CRS patients with nasal polyps and normal epithelial cells were derived from ethmoid sinus mucosa during endoscopic reduction of blowout fracture or uncinate process mucosa of patients with septal deviation. Cultured cells were infected with RV 16 or treated with poly (I: C) for 24, 48, and 72 h. Cells and media were harvested at each time point and used to evaluate RV16 replication rates, the secretion of IFN-ß, -λ1, -λ2, viperin, Mx, and OAS, and the expression levels of TRL3, RIG-I, MDA5, phospho-NFκB, and phospho-IRF3. RV replication rates reached peak levels 48 h after inoculation in both normal and inflammatory epithelial cells and showed no difference between both groups of epithelial cells at any time point. The release of IFN-ß, -λ1, and -λ2 in normal and inflammatory epithelial cells was also strongly induced 48 h after RV16 inoculation but reached peak levels 24 h after poly (I: C) treatment. The expression levels of viperin, Mx, OAS, TLR3, RIG-I, MDA5, phospho-NFκB, and phospho-IRF3 showed similar patterns in both groups of epithelial cells. These results suggest that the production of RV16-induced antiviral interferons is not deficient or delayed in inflammatory epithelial cells from CRS patients with nasal polyps.


Assuntos
Pólipos Nasais , Sinusite , Humanos , Rhinovirus , Pólipos Nasais/metabolismo , Receptor 3 Toll-Like/metabolismo , Antivirais/metabolismo , Sinusite/metabolismo , Células Epiteliais , Interferons/metabolismo , Interferon beta/metabolismo , Doença Crônica
9.
J Integr Neurosci ; 21(6): 150, 2022 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-36424741

RESUMO

BACKGROUND: Current data indicates the incidence of neuropathic pain after surgical nerve injury is as high as 50%, thus representing a major problem for patients and for the medical system. Triptolide, a traditional Chinese herb, has anti-inflammatory effects on various neurodegenerative and neuroinflammatory diseases. This agent also reduces peripheral nerve injury-induced neuropathic pain, although the mechanism underlying this effect is still unknown. MATERIALS AND METHODS: The effects of triptolide on spinal nerve ligation (SNL) injury-induced neuropathic pain was studied in an animal model using behavioral, morphological and molecular biological methods. RESULTS: Repeated administration of intrathecal triptolide was found to alleviate SNL- or Poly(I:C) (toll-like receptor 3 agonist) injection-induced mechanical allodynia without any motor impairment. The mechanism by which triptolide reduces SNL- and Poly(I:C) injection-induced microglial activation appears to be via the inhibition of OX42 expression, which is a microglial-specific marker. Intrathecal triptolide also suppressed SNL- and Poly(I:C) injection-induced expression of spinal TRIF. TRIF transmits signals from activated TLR3 and is the downstream adaptor of TLR3 in microglia. In addition, intrathecal triptolide inhibited the expression of spinal pro-inflammatory IL-1 ß following SNL or Poly(I:C) injection. CONCLUSIONS: Intrathecal triptolide can suppress the TLR3/TRIF/IL-1 ß pathway in spinal microglia following SNL. This could be the underlying mechanism by which triptolide alleviate neuropathic pain induced by peripheral nerve injury.


Assuntos
Neuralgia , Traumatismos dos Nervos Periféricos , Ratos , Animais , Microglia , Receptor 3 Toll-Like/metabolismo , Interleucina-1beta/metabolismo , Ratos Sprague-Dawley , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/farmacologia
10.
Front Cell Infect Microbiol ; 12: 926036, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36310878

RESUMO

Dengue virus (DENV) infection is the most prevalent arthropod-borne virus disease and is endemic in more than 100 countries. Several DENV proteins have been shown to target crucial human host proteins to evade innate immune responses and establish a productive infection. Here we report that the DENV NS3 protein targets RIPK1 (Receptor Interacting Protein Kinase I), a central mediator of inflammation and cell death, and decreases intracellular RIPK1 levels during DENV infection. The interaction of NS3 with RIPK1 results in the inhibition of NF-κB activation in response to TNFR or TLR3 stimulation. Also, we observed that the effects of NS3 on RIPK1 were independent of NS3 protease activity. Our data demonstrate a novel mechanism by which DENV suppresses normal cellular functions to evade host innate immune responses.


Assuntos
Vírus da Dengue , Receptores Tipo I de Fatores de Necrose Tumoral , Humanos , NF-kappa B/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo
11.
J Am Heart Assoc ; 11(20): e026076, 2022 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-36216458

RESUMO

Background Spinal cord ischemia (SCI) remains a devastating complication after aortic dissection or repair. A primary hypoxic damage is followed by a secondary damage resulting in further cellular loss via apoptosis. Affected patients have a poor prognosis and limited therapeutic options. Shock wave therapy (SWT) improves functional outcome, neuronal degeneration and survival in murine spinal cord injury. In this first-in-human study we treated 5 patients with spinal cord ischemia with SWT aiming to prove safety and feasibility. Methods and Results Human neurons were subjected to ischemic injury with subsequent SWT. Reactive oxygen species and cellular apoptosis were quantified using flow cytometry. Signaling of the antioxidative transcription factor NRF2 (nuclear factor erythroid 2-related factor 2) and immune receptor Toll-like receptor 3 (TLR3) were analyzed. To assess whether SWT act via a conserved mechanism, transgenic tlr3-/- zebrafish created via CRISPR/Cas9 were subjected to spinal cord injury. To translate our findings into a clinical setting, 5 patients with SCI underwent SWT. Baseline analysis and follow-up (6 months) included assessment of American Spinal Cord Injury Association (ASIA) impairment scale, evaluation of Spinal Cord Independence Measure score and World Health Organization Quality of Life questionnaire. SWT reduced the number of reactive oxygen species positive cells and apoptosis upon ischemia via induction of the antioxidative factor nuclear factor erythroid 2-related factor 2. Inhibition or deletion of tlr3 impaired axonal growth after spinal cord lesion in zebrafish, whereas tlr3 stimulation enhanced spinal regeneration. In a first-in-human study, we treated 5 patients with SCI using SWT (mean age, 65.3 years). Four patients presented with acute aortic dissection (80%), 2 of them exhibited preoperative neurological symptoms (40%). Impairment was ASIA A in 1 patient (20%), ASIA B in 3 patients (60%), and ASIA D in 1 patient (20%) at baseline. At follow-up, 2 patients were graded as ASIA A (40%) and 3 patients as ASIA B (60%). Spinal cord independence measure score showed significant improvement. Examination of World Health Organization Quality of Life questionnaires revealed increased scores at follow-up. Conclusions SWT reduces oxidative damage upon SCI via immune receptor TLR3. The first-in-human application proved safety and feasibility in patients with SCI. SWT could therefore become a powerful regenerative treatment option for this devastating injury.


Assuntos
Tratamento por Ondas de Choque Extracorpóreas , Traumatismos da Medula Espinal , Isquemia do Cordão Espinal , Humanos , Camundongos , Animais , Idoso , Receptor 3 Toll-Like/metabolismo , Receptor 3 Toll-Like/uso terapêutico , Fator 2 Relacionado a NF-E2 , Peixe-Zebra , Estudos de Viabilidade , Espécies Reativas de Oxigênio , Qualidade de Vida , Isquemia do Cordão Espinal/etiologia , Isquemia do Cordão Espinal/prevenção & controle , Isquemia do Cordão Espinal/patologia , Traumatismos da Medula Espinal/terapia , Traumatismos da Medula Espinal/patologia , Medula Espinal/metabolismo , Estresse Oxidativo , Isquemia , /patologia
12.
Genes (Basel) ; 13(10)2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36292749

RESUMO

Toll-like receptor 3 (SpTLR3) from Schizothorax prenanti (S. prenanti) was cloned and identified, and the tissue distribution of the SpTLR3 gene was examined in this study. Moreover, the relative mRNA expression levels of myeloid differentiation factor 88 gene (SpMyD88) and seven TLR genes (SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3) from S. prenanti after lipopolysaccharide (LPS) challenge were analyzed through quantitative real-time polymerase chain reaction (qRT-PCR). The full length of SpTLR3 gene is 3097 bp, and complete coding sequence (CDS) is 2715 bp, which encodes 904 amino acids. The SpTLR3 amino acid sequence shared 43.94-100% identity with TLR3 sequences from other vertebrates; SpTLR3 was expressed in all eight tissues examined; and the highest level appeared in the liver, which was significantly higher than in all other tissues (p < 0.05), followed by the levels in the heart and muscles. LPS significantly up-regulated all eight genes in the S. prenanti tissues at 12 or 24 h (p < 0.05). Compared with the PBS control group, no significant transcripts changes were found in SpTLR2 or SpTLR3 at 12 h after LPS induction, but they were significantly up-regulated at 24 h (p < 0.001). The most abundant transcripts were found in the head kidney SpTLR22 genes after 24 h LPS induction, with high to low levels, which were SpTLR22-1 (564-fold), SpTLR22-3 (508-fold) and SpTLR22-2 (351-fold). Among these eight genes, the expression level of SpTLR4 was the least up-regulated. Overall, SpTLR4 in the head kidney was involved in the antibacterial immune response earlier, and the level was increased at 12 h with extreme significance after LPS stimulation (p < 0.001), while the other seven genes were the most significantly up-regulated at 24 h post injection. Taken together, the results suggest that SpMyD88, SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3 participate in an innate immune response stimulated by LPS, and the response intensity of the genes was organ-specific, with differing kinetics. Our findings will contribute to a more complete understanding of the roles of these TLR genes in antibacterial immunity.


Assuntos
Cyprinidae , Lipopolissacarídeos , Animais , Lipopolissacarídeos/farmacologia , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Cyprinidae/genética , Clonagem Molecular , RNA Mensageiro/genética , Antibacterianos , Aminoácidos/genética
13.
Cytokine ; 160: 156047, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36183616

RESUMO

BACKGROUND: Corneal transparency may be compromised by viral infections causing corneal scarring, edema, and neovascularization. Ocular injury results from collateral damage induced by exacerbated immune response in corneal stroma. Myofibroblasts play a key role in this process by producing a disorganized extracellular matrix and inflammatory mediators. However, the immune response profile of myofibroblasts during viral infections is still under study. The aim of this work was to analyze the cytokine profile of human limbal myfibroblasts (HLMs) stimulated with the double-stranded RNA analog polyinosinic:polycytidylic acid (poly I:C) and to identify their signaling pathways. METHODS: HLMs were isolated from cadaveric sclera-corneal rims and stimulated with poly I:C (10 µg/ml) for 12 h. The secretion of 36 cytokines was measured using the Human Cytokine Array Panel A. The secretion of IFN-ß was quantified by ELISA. The expression of pattern recognition receptors (PRRs) such as TLR3, RIG-1 and MDA5 were analyzed by western blot assays. Furthermore, translocation of the nuclear factors NF-κB, IRF3, and IRF7 was assessed by fluorescence staining. In addition, the differentially expressed cytokines were analyzed using the Core Analysis Tool of the Ingenuity Pathway Analysis IPA software. RESULTS: HLMs stimulated with poly I:C increased (fold change > 2) the secretion of G-CSF, sTREM-1, CXCL1, CCL1, CXCL8, CXCL10, CXCL11, CCL2, CCL5, IL-13, IL-6, IL-1ra, and IFN-ß compared with HLMs under basal conditions. Poly I:C stimulation also induced the expression of RIG-1 (p < 0.001), but the expression of TLR3 and MDA5 was unmodified. Finally, HLMs increased nuclear translocation of NF-κB, IRF3, and IRF7 after poly I:C stimulation. Bioinformatic analysis identified canonical signaling pathways associated with cell adhesion and diapedesis, chemokine signaling, and activation of IRFs by cytosolic pattern recognition receptors. CONCLUSIONS: These results demonstrate that HLMs secrete cytokines involved in immune cell activation and chemotaxis. The data suggest a key role for HLMs during viral infections in cornea and extend our knowledge about the signaling pathways they trigger.


Assuntos
NF-kappa B , Viroses , Antivirais/farmacologia , Córnea , Citocinas/metabolismo , Fator Estimulador de Colônias de Granulócitos/genética , Humanos , Interferon beta/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/genética , Interleucina-13/genética , Interleucina-6/genética , Miofibroblastos/metabolismo , NF-kappa B/metabolismo , Poli I-C/farmacologia , RNA de Cadeia Dupla , Receptores de Reconhecimento de Padrão , Receptor 3 Toll-Like/metabolismo
14.
Eur Cytokine Netw ; 33(2): 19-29, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-36266987

RESUMO

IL-36γ, a pro-inflammatory member of the IL-1 cytokine superfamily, can be induced and secreted by normal human foreskin keratinocytes (HFKs) in response to pathogenic stimuli, however, the mechanisms underlying the secretion are unknown. In this study, we demonstrate that stimulation with the TLR3 agonist, poly (I:C), led to a delayed secretion of IL-36γ compared to stimulation with the TLR5 agonist, flagellin, despite equal levels of the cytokine (p = 0.006). IL-36γ was shown to be released from HFKs in its inactive, uncleaved form, based on western blotting. Moreover, recombinant IL-36γ in its activated, cleaved form induced endogenous IL-36γ 10-fold (p = 0.004) and CXCL8 five-fold (p = 0.003) over baseline levels compared to unactivated full-length recombinant IL-36γ. The ratio of LC3b-II/LC3b-I was significantly higher in poly(I:C)-treated cells compared to flagellin-treated and unstimulated controls without a change in SQSTM1/p62 after 24 hours of stimulation (p = 0.043). Under fluorescence microscopy, poly(I:C) led to a two-fold increase at eight hours and four-fold increase at 24 hours in accumulated autophagosomes post-stimulation (p = 0.032). In contrast, autophagosomes were unchanged relative to baseline in response to flagellin. Bafilomycin A1 treatment enhanced poly(I:C)-mediated IL-36γ secretion (p = 0.044) while rapamycin led to a noticeable, but non-significant, increase in flagellin-mediated IL-36γ secretion, indicating that interrupting autophagic flux can alter IL-3γ grelease from HFKs. Finally, we show that, compared to clinically normal laryngeal tissue, there were significantly higher levels of LC3b-II in HPV-infected respiratory papilloma tissue, indicating a higher number of autophagosomes; a signature of disrupted autophagic flux.


Assuntos
Flagelina , Interleucina-1 , Humanos , Flagelina/farmacologia , Interleucina-1/farmacologia , Interleucina-1/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 5 Toll-Like , Proteína Sequestossoma-1 , Queratinócitos/metabolismo , Poli I-C/farmacologia , Citocinas , Autofagia , Sirolimo/farmacologia
15.
J Immunol Res ; 2022: 1800401, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36213326

RESUMO

Respiratory syncytial virus (RSV) infection can deteriorate asthma by inducing persistent airway inflammation. Increasing evidence elucidated that pyroptosis plays a pivotal role in asthma. Conciliatory anti-allergic decoction (CAD) exhibits an anti-inflammatory effect in ovalbumin (OVA)-induced asthma; however, the effects and mechanisms of CAD in RSV-infected asthmatic mice have not yet been elucidated. The RSV-infected asthmatic mice model and lipopolysaccharide (LPS)-induced 16HBE cell pyroptosis model were established, respectively. Pulmonary function, ELISA, and histopathologic analysis were performed to assess the airway inflammation and remodeling in mice with CAD treatment. Furthermore, ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) was conducted to identify the chemical compounds of high-dose CAD (30 g/kg). Cell viability and apoptosis of 16HBE cells were assessed by CCK-8 and flow cytometry assays, respectively. Finally, the expression levels of apoptosis-, pyroptosis-, and TLR3/NLRP3/NF-κB/IRF3 signaling-related genes were measured with qRT-PCR or western blotting, respectively. Pulmonary function tests showed that CAD significantly ameliorated respiratory dysfunction, airway hyperresponsiveness, inflammation cell recruitment in BALF, pulmonary inflammation, collagen deposition, and cell death in lung tissues. CAD significantly decreased the content of TNF-α, IL-13, IL-4, IL-1ß and IL-5 in the bronchoalveolar lavage fluid (BALF), IL-17, IL-6, and OVA-specific IgE in serum and increased serum IFN-γ in asthma mice. The results of UPLC-Q-TOF/MS showed that high-dose CAD had 88 kinds of chemical components. In vitro, CAD-contained serum significantly suppressed LPS-induced 16HBE cell apoptosis. Additionally, CAD and CAD-contained serum attenuated the up-regulated expressions of Bax, Cleaved caspase-3, NLRP3, ASC, Cleaved caspase-1, GSDMD-N, IL-18, IL-1ß, TLR3, p-P65, p-IκBα, and IRF3 but increased Bcl-1 and GSDMD levels in the asthma mice and LPS-induced 16HBE cells, respectively. These results illustrated that CAD may have a potential role in improving airway inflammation and pyroptosis through inhibition of the TLR3/NLRP3/NF-κB/IRF3 signaling pathway.


Assuntos
Antialérgicos , Asma , Animais , Antialérgicos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Asma/metabolismo , Líquido da Lavagem Broncoalveolar/química , Caspase 3/metabolismo , Imunoglobulina E , Inflamação/metabolismo , Fator Regulador 3 de Interferon , Interleucina-13/metabolismo , Interleucina-17/metabolismo , Interleucina-18/metabolismo , Interleucina-4/metabolismo , Interleucina-5 , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ovalbumina/efeitos adversos , Piroptose , Transdução de Sinais , Sincalida/metabolismo , Sincalida/farmacologia , Sincalida/uso terapêutico , Receptor 3 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína X Associada a bcl-2/metabolismo
16.
J Virol ; 96(18): e0093022, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36069544

RESUMO

Duck Tembusu virus (DTMUV) is an emerging pathogenic flavivirus that mainly causes a decrease in egg production in infected waterfowl. Similar to other members of the Flaviviridae family, it can proliferate in most mammalian cells and may also pose a potential threat to nonavian animals. In previous studies, we found that DTMUV infection can upregulate suppressor of cytokine signaling 1 (SOCS1) to inhibit type I interferon (IFN) production and promote virus replication, but the specific mechanism is unclear. Furthermore, little is known about the regulatory role of ubiquitination during flavivirus infection. In this study, we found that activation of Toll-like receptor 3 (TLR3) signaling rather than type I IFN stimulation led to the upregulation of SOCS1 during DTMUV infection. Further studies revealed that JOSD1 stabilized SOCS1 expression by binding to the SH2 domain of SOCS1 and mediating its deubiquitination. In addition, JOSD1 also inhibited type I IFN production through SOCS1. Finally, SOCS1 acts as an E3 ubiquitin ligase that binds to IFN regulatory factor 7 (IRF7) through its SH2 domain and mediates K48-linked ubiquitination and proteasomal degradation of IRF7, ultimately inhibiting type I IFN production mediated by IRF7 and promoting viral proliferation. These results will enrich and deepen our understanding of the mechanism by which DTMUV antagonizes the host interferon system. IMPORTANCE DTMUV is a newly discovered flavivirus that seriously harms the poultry industry. In recent years, there have been numerous studies on the involvement of ubiquitination in the regulation of innate immunity. However, little is known about the involvement of ubiquitination in the regulation of flavivirus-induced type I IFN signaling. In this study, we found that SOCS1 was induced by TLR3 signaling during DTMUV infection. Furthermore, we found for the first time that duck SOCS1 protein was also modified by K48-linked polyubiquitination, whereas our previous study found that SOCS1 was upregulated during DTMUV infection. Further studies showed that JOSD1 stabilized SOCS1 expression by mediating the deubiquitination of SOCS1. While SOCS1 acts as a negative regulator of cytokines, we found that DTMUV utilized SOCS1 to mediate the ubiquitination and proteasomal degradation of IRF7 and ultimately inhibit type I IFN production, thereby promoting its proliferation.


Assuntos
Infecções por Flavivirus , Flavivirus , Interações entre Hospedeiro e Microrganismos , Interferon Tipo I , Doenças das Aves Domésticas , Animais , Patos , Endopeptidases/genética , Endopeptidases/metabolismo , Retroalimentação Fisiológica , Flavivirus/metabolismo , Infecções por Flavivirus/imunologia , Infecções por Flavivirus/virologia , Interações entre Hospedeiro e Microrganismos/imunologia , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Receptor 3 Toll-Like/metabolismo , Ubiquitina-Proteína Ligases , Regulação para Cima
17.
Fish Shellfish Immunol ; 129: 182-190, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36058437

RESUMO

Salmonid alphavirus (SAV) infection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) causes pancreas disease (PD) with typical inflammatory responses, such as necrosis of the exocrine pancreas, cardiomyopathy and skeletal myopathy. However, the pathogenic mechanism underlying SAV infection is still unclear. Inflammation may cause damage to the body, but it is a defense response against infection by pathogenic microorganisms, of which nuclear factor-kappa B (NF-κB) is the main regulator. This study revealed that SAV can activate NF-κB, of which the viral nonstructural protein Nsp2 is the major activating protein. SAV activates the NF-κB signaling pathway by simultaneously up-regulating TLR3, 7, 8 and then the expression of the signaling molecule myeloid differentiation factor 88 (Myd88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). We found that Nsp2 can induce IκB degradation and p65 phosphorylation and transnucleation, and activate NF-κB downstream inflammatory cytokines. Nsp2 may simultaneously activate NF-κB through TLR3,7,8-dependent signaling pathways. Overexpression of Nsp2 can up-regulate mitochondrial antiviral signaling protein (MAVS) and then promote the expression of IFNa1 and antiviral protein Mx, which inhibits viral replication. This study shows that Nsp2 acts as a key activator protein for the NF-κB signaling pathway, which induces inflammation post-SAV infection. This study systematically analyzes the molecular mechanism of SAV activation of the NF-κB signaling pathway, and provides a theoretical basis for revealing the mechanism of innate immune response and inflammatory injury caused by SAV.


Assuntos
Infecções por Alphavirus , Alphavirus , Doenças dos Peixes , Oncorhynchus mykiss , Salmo salar , Alphavirus/fisiologia , Infecções por Alphavirus/veterinária , Animais , Antivirais , Citocinas/metabolismo , Inflamação/veterinária , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas de Resistência a Myxovirus/metabolismo , NF-kappa B/metabolismo , Oncorhynchus mykiss/metabolismo , Salmo salar/genética , Salmo salar/metabolismo , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/metabolismo , Receptor 3 Toll-Like/metabolismo , Proteínas não Estruturais Virais
18.
J Immunol ; 209(7): 1359-1369, 2022 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-36165200

RESUMO

Donor-specific HLA Abs contribute to Ab-mediated rejection (AMR) by binding to HLA molecules on endothelial cells (ECs) and triggering intracellular signaling, leading to EC activation and leukocyte recruitment. The molecular mechanisms involving donor-specific HLA Ab-mediated EC activation and leukocyte recruitment remain incompletely understood. In this study, we determined whether TLRs act as coreceptors for HLA class I (HLA I) in ECs. We found that human aortic ECs express TLR3, TLR4, TLR6, and TLR10, but only TLR4 was detected on the EC surface. Consequently, we performed coimmunoprecipitation experiments to examine complex formation between HLA I and TLR4. Stimulation of human ECs with HLA Ab increased the amount of complex formation between HLA I and TLR4. Reciprocal coimmunoprecipitation with a TLR4 Ab confirmed that the crosslinking of HLA I increased complex formation between TLR4 and HLA I. Knockdown of TLR4 or MyD88 with small interfering RNAs inhibited HLA I Ab-stimulated P-selectin expression, von Willebrand factor release, and monocyte recruitment on ECs. Our results show that TLR4 is a novel coreceptor for HLA I to stimulate monocyte recruitment on activated ECs. Taken together with our previous published results, we propose that HLA I molecules form two separate signaling complexes at the EC surface, that is, with TLR4 to upregulate P-selectin surface expression and capture of monocytes to human ECs and integrin ß4 to induce mTOR-dependent firm monocyte adhesion via ICAM-1 clustering on ECs, two processes implicated in Ab-mediated rejection.


Assuntos
Células Endoteliais , Molécula 1 de Adesão Intercelular , Células Cultivadas , Endotélio Vascular/metabolismo , Antígenos HLA/metabolismo , Humanos , Integrina beta4/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Monócitos , Fator 88 de Diferenciação Mieloide/metabolismo , Selectina-P/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 6 Toll-Like/metabolismo , Fator de von Willebrand/metabolismo
19.
Front Endocrinol (Lausanne) ; 13: 966455, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36093086

RESUMO

Endometriosis is characterized by the presence of inflamed and fibrotic endometrial tissue outside the uterine cavity. Previously, we found decreased SERPINA1 (alpha-1 antitrypsin) expression in endometriosis-like lesions in a mouse model of endometriosis, suggesting that it exacerbated inflammation in these lesions. However, the molecular mechanism(s) by which SERPINA1 affects expression of inflammatory factors and development of endometriotic lesions have not been fully characterized. To investigate the role of intracellular SERPINA1 in endometrial stromal cells (ESCs), we performed RNA sequence analysis using RNA extracted from ESCs in which SERPINA1 was knocked down. The analysis identified several toll-like receptor (TLR)-related factors as being upregulated. Silencing of SERPINA1 increased expression of TLR3 and TLR4 in ESCs, as well as several TLR signaling pathway components, including MYD88, IRAK1/4, interleukin (IL)-1ß, and interferon (IFN)-ß. TLR3 or TLR4 agonists increased expression of inflammatory factors in SERPINA1-knockdown ESCs, whereas TLR3 or TLR4 inhibitors decreased expression. In addition, treatment with recombinant IL-1ß or IFN-ß increased expression of MYD88 and inflammatory factors in ESCs. Immunohistochemical analysis of endometriotic tissues showed that TLR3, TLR4, and MYD88 were localized in endometriosis lesions. Taken together, the data suggest that reduced expression of SERPINA1 induces expression of inflammatory factors by ESCs, which in turn are associated with TLR3/4, IL-1ß, and IFN-ß signaling. Regulation of intracellular SERPINA1 levels in ESCs may be a strategy to inhibit inflammatory responses in endometriotic lesions.


Assuntos
Citocinas , Endometriose , Proteínas Adaptadoras de Transdução de Sinal , Animais , Citocinas/metabolismo , Endometriose/genética , Endometriose/metabolismo , Feminino , Humanos , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Células Estromais/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo , alfa 1-Antitripsina/genética , alfa 1-Antitripsina/metabolismo
20.
J Virol ; 96(18): e0121222, 2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36069553

RESUMO

The mitochondrial apoptosis pathway has the function to kill the cell, but recent work shows that this pathway can also be activated to a sublethal level, where signal transduction can be observed but the cell survives. Intriguingly, this signaling has been shown to contribute to inflammatory activity of epithelial cells upon infection with numerous agents. This suggests that microbial recognition can generate sublethal activity in the mitochondrial apoptosis pathway. Because this recognition is achieved by pattern recognition receptors (PRRs), it also implies that PRR signals are linked to the mitochondrial apoptosis apparatus. We here test this hypothesis during infection of epithelial cells with modified vaccinia virus Ankara (MVA). MVA recognition is achieved through receptors specific for nucleic acids, and we present evidence that the three receptors, Toll-like receptor 3 (TLR3), RIG-I/MDA5, and cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING), are involved in this signaling. When stimulated directly by specific ligands, all three receptors could trigger sublethal apoptosis signals. During infection with MVA, sublethal apoptosis signals were unmasked in X-linked IAP (XIAP)-deficient cells, where apoptosis induction was observed. Deletion of any of the three signaling adapters, TRIF, MAVS, and STING, reduced the DNA damage response, a sensitive measure of sublethal apoptosis signals. Our results suggest that PRRs signal via mitochondria, where they generate sublethal signals through the BCL-2-family, which may contribute to the response to infectious agents. IMPORTANCE A contribution of the mitochondrial apoptosis apparatus, in the absence of cell death, to the reaction of nonprofessional immune cells to viruses is suggested to play a role as a broad alert system of an infected cell: the apoptosis system can be activated by many upstream signals and could therefore act as a central coordinator of viral recognition. The proapoptotic activity of PRRs has been documented in multiple situations, but this activity seems too low to be meaningful, and a physiological significance of such activity is not immediately obvious. This work suggests the alternative interpretation that PRRs do not have the primary function to induce apoptosis but to trigger sublethal signals in the apoptosis system. A number of lines of recent research suggest that mitochondria contribute to cellular reactions, and this pathway may be a way of triggering an early host response.


Assuntos
Apoptose , Mitocôndrias , Ácidos Nucleicos , Receptores de Reconhecimento de Padrão , Viroses , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Humanos , Imunidade Inata , Mitocôndrias/imunologia , Nucleotidiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Receptor 3 Toll-Like/metabolismo , Vírus Vaccinia , Viroses/imunologia
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