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1.
PLoS One ; 15(10): e0235803, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33031374

RESUMO

Cystic Fibrosis (CF), caused by mutations affecting the CFTR gene, is characterised by viscid secretions in multiple organ systems. CF airways contain thick mucus, creating a gradient of hypoxia, which promotes the establishment of polymicrobial infection. Such inflammation predisposes to further infection, a self-perpetuating cycle in mediated by NF-κB. Anaerobic Gram-negative Prevotella spp. are found in sputum from healthy volunteers and CF patients and in CF lungs correlate with reduced levels of inflammation. Prevotella histicola (P. histicola) can suppress murine lung inflammation, however, no studies have examined the role of P. histicola in modulating infection and inflammation in the CF airways. We investigated innate immune signalling and NF-kB activation in CF epithelial cells CFBE41o- in response to clinical stains of P. histicola and Pseudomonas aeruginosa (P. aeruginosa). Toll-Like Receptor (TLR) expressing HEK-293 cells and siRNA assays for TLRs and IKKα were used to confirm signalling pathways. We show that P. histicola infection activated the alternative NF-kB signalling pathway in CF bronchial epithelial cells inducing HIF-1α protein. TLR5 signalling was responsible for the induction of the alternative NF-kB pathway through phosphorylation of IKKα. The induction of transcription factor HIF-1α was inversely associated with the induction of the alternative NF-kB pathway and knockdown of IKKα partially restored canonical NF-kB activation in response to P. histicola. This study demonstrates that different bacterial species in the respiratory microbiome can contribute differently to inflammation, either by activating inflammatory cascades (P. aeruginosa) or by muting the inflammatory response by modulating similar or related pathways (P. histicola). Further work is required to assess the complex interactions of the lung microbiome in response to mixed bacterial infections and their effects in people with CF.


Assuntos
Brônquios/imunologia , Fibrose Cística/imunologia , NF-kappa B/metabolismo , Prevotella/imunologia , Infecções por Pseudomonas/complicações , Pseudomonas aeruginosa/imunologia , Receptores Toll-Like/metabolismo , Brônquios/metabolismo , Brônquios/microbiologia , Brônquios/patologia , Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Fibrose Cística/patologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Humanos , Interleucina-8/metabolismo , NF-kappa B/genética , Prevotella/isolamento & purificação , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Transdução de Sinais , Receptores Toll-Like/imunologia
2.
PLoS One ; 15(10): e0240577, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33057358

RESUMO

The causative agent of severe acute respiratory syndrome (SARS) reported by the Chinese Center for Disease Control (China CDC) has been identified as a novel Betacoronavirus (SARS-CoV-2). A computational approach was adopted to identify multiepitope vaccine candidates against SARS-CoV-2 based on S, N and M proteins being able to elicit both humoral and cellular immune responses. In this study, the sequence of the virus was obtained from NCBI database and analyzed with in silico tools such as NetMHCpan, IEDB, BepiPred, NetCTL, Tap transport/proteasomal cleavage, Pa3P, GalexyPepDock, I-TASSER, Ellipro and ClusPro. To identify the most immunodominant regions, after analysis of population coverage and epitope conservancy, we proposed three different constructs based on linear B-cell, CTL and HTL epitopes. The 3D structure of constructs was assessed to find discontinuous B-cell epitopes. Among CTL predicted epitopes, S257-265, S603-611 and S360-368, and among HTL predicted epitopes, N167-181, S313-330 and S1110-1126 had better MHC binding rank. We found one putative CTL epitope, S360-368 related to receptor-binding domain (RBD) region for S protein. The predicted epitopes were non-allergen and showed a high quality of proteasomal cleavage and Tap transport efficiency and 100% conservancy within four different clades of SARS-CoV-2. For CTL and HTL epitopes, the highest population coverage of the world's population was calculated for S27-37 with 86.27% and for S196-231, S303-323, S313-330, S1009-1030 and N328-349 with 90.33%, respectively. We identified overall 10 discontinuous B-cell epitopes for three multiepitope constructs. All three constructs showed strong interactions with TLRs 2, 3 and 4 supporting the hypothesis of SARS-CoV-2 susceptibility to TLRs 2, 3 and 4 like other Coronaviridae families. These data demonstrated that the novel designed multiepitope constructs can contribute to develop SARS-CoV-2 peptide vaccine candidates. The in vivo studies are underway using several vaccination strategies.


Assuntos
Infecções por Coronavirus/prevenção & controle , Epitopos/imunologia , Proteínas do Nucleocapsídeo/imunologia , Pandemias/prevenção & controle , Pneumonia Viral/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas de Subunidades/imunologia , Proteínas da Matriz Viral/imunologia , Infecções por Coronavirus/imunologia , Epitopos/química , Antígenos HLA/química , Antígenos HLA/imunologia , Humanos , Simulação de Acoplamento Molecular , Proteínas do Nucleocapsídeo/química , Pneumonia Viral/imunologia , Glicoproteína da Espícula de Coronavírus/química , Receptores Toll-Like/química , Receptores Toll-Like/metabolismo , Vacinas de Subunidades/química , Proteínas da Matriz Viral/química
3.
Nat Commun ; 11(1): 4596, 2020 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-32929083

RESUMO

Earlier studies indicate that either the canonical or non-canonical pathways of inflammasome activation have a limited role on malaria pathogenesis. Here, we report that caspase-8 is a central mediator of systemic inflammation, septic shock in the Plasmodium chabaudi-infected mice and the P. berghei-induced experimental cerebral malaria (ECM). Importantly, our results indicate that the combined deficiencies of caspases-8/1/11 or caspase-8/gasdermin-D (GSDM-D) renders mice impaired to produce both TNFα and IL-1ß and highly resistant to lethality in these models, disclosing a complementary, but independent role of caspase-8 and caspases-1/11/GSDM-D in the pathogenesis of malaria. Further, we find that monocytes from malaria patients express active caspases-1, -4 and -8 suggesting that these inflammatory caspases may also play a role in the pathogenesis of human disease.


Assuntos
Caspase 8/metabolismo , Inflamação/patologia , Malária Cerebral/enzimologia , Animais , Encéfalo/patologia , Caspase 1/metabolismo , Células Dendríticas/metabolismo , Ativação Enzimática , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Interferon gama/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos , Malária Cerebral/genética , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Plasmodium chabaudi/fisiologia , Baço/metabolismo , Receptores Toll-Like/metabolismo
4.
PLoS One ; 15(9): e0239238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32941515

RESUMO

Zika virus (ZIKV) is a single-stranded RNA virus belonging to the family Flaviviridae. ZIKV predominantly enters cells using the TAM-family protein tyrosine kinase receptor AXL, which is expressed on a range of cell types, including neural progenitor cells, keratinocytes, dendritic cells, and osteoblasts. ZIKV infections have been associated with fetal brain damage, which prompted the World Health Organization to declare a public health emergency in 2016. ZIKV infection has also been linked to birth defects in other organs. Several studies have reported congenital heart defects (CHD) in ZIKV infected infants and cardiovascular complications in adults infected with ZIKV. To develop a better understanding of potential causes for these pathologies at a cellular level, we characterized ZIKV infection of human fetal cardiac mesenchymal stromal cells (fcMSCs), a cell type that is known to contribute to both embryological development as well as adult cardiac physiology. Total RNA, supernatants, and/or cells were collected at various time points post-infection to evaluate ZIKV replication, cell death, and antiviral responses. We found that ZIKV productively infected fcMSCs with peak (~70%) viral mRNA detected at 48 h. Use of an antibody blocking the AXL receptor decreased ZIKV infection (by ~50%), indicating that the receptor is responsible to a large extent for viral entry into the cell. ZIKV also altered protein expression of several mesenchymal cell markers, which suggests that ZIKV could affect fcMSCs' differentiation process. Gene expression analysis of fcMSCs exposed to ZIKV at 6, 12, and 24 h post-infection revealed up-regulation of genes/pathways associated with interferon-stimulated antiviral responses. Stimulation of TLR3 (using poly I:C) or TLR7 (using Imiquimod) prior to ZIKV infection suppressed viral replication in a dose-dependent manner. Overall, fcMSCs can be a target for ZIKV infection, potentially resulting in CHD during embryological development and/or cardiovascular issues in ZIKV infected adults.


Assuntos
Células-Tronco Embrionárias Humanas/virologia , Células-Tronco Mesenquimais/virologia , Miócitos Cardíacos/virologia , Replicação Viral , Infecção por Zika virus/virologia , Zika virus/fisiologia , Animais , Morte Celular , Células Cultivadas , Chlorocebus aethiops , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Interferons/genética , Interferons/metabolismo , Células-Tronco Mesenquimais/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Células Vero , Zika virus/patogenicidade , Infecção por Zika virus/metabolismo
5.
Proc Natl Acad Sci U S A ; 117(38): 23581-23587, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32900946

RESUMO

Proteolytic activation of phenoloxidase (PO) and the cytokine Spätzle during immune responses of insects is mediated by a network of hemolymph serine proteases (HPs) and noncatalytic serine protease homologs (SPHs) and inhibited by serpins. However, integration and conservation of the system and its control mechanisms are not fully understood. Here we present biochemical evidence that PO-catalyzed melanin formation, Spätzle-triggered Toll activation, and induced synthesis of antimicrobial peptides are stimulated via hemolymph (serine) protease 5 (HP5) in Manduca sexta Previous studies have demonstrated a protease cascade pathway in which HP14 activates proHP21; HP21 activates proPAP2 and proPAP3, which then activate proPO in the presence of a complex of SPH1 and SPH2. We found that both HP21 and PAP3 activate proHP5 by cleavage at ESDR176*IIGG. HP5 then cleaves proHP6 at a unique site of LDLH112*ILGG. HP6, an ortholog of Drosophila Persephone, activates both proHP8 and proPAP1. HP8 activates proSpätzle-1, whereas PAP1 cleaves and activates proPO. HP5 is inhibited by Manduca sexta serpin-4, serpin-1A, and serpin-1J to regulate its activity. In summary, we have elucidated the physiological roles of HP5, a CLIPB with unique cleavage specificity (cutting after His) that coordinates immune responses in the caterpillar.


Assuntos
Hemolinfa , Proteínas de Insetos , Manduca , Serina Proteases , Animais , Hemolinfa/enzimologia , Hemolinfa/imunologia , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , Manduca/enzimologia , Manduca/imunologia , Manduca/metabolismo , Serina Proteases/imunologia , Serina Proteases/metabolismo , Transdução de Sinais , Receptores Toll-Like/imunologia , Receptores Toll-Like/metabolismo
7.
Exp Parasitol ; 218: 108007, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32979344

RESUMO

Macrophages are the primary host cell for Leishmania parasites, by Toll like receptors (TLR-MyD88) that are central components of the innate and adaptive immunity against leishmania infection. The CD40/CD40L interaction has also been shown to be important in resistance to various protozoa. In this context, one of the most important properties of suppressors of cytokine signalling (SOCS) proteins, especially SOCS1 and SOCS3, is the regulation of macrophages cell for Leishmania parasites. In the present study we evaluated variants of molecules involved in activation and modulation of leishmanicidal signaling cascades and the possible associations between polymorphisms present in the TLR2, TLR4, MyD88, CD40, SOCS1, SOCS3 genes with susceptibility/resistent to Leishmania. The results suggest the absence of any association between TLR2 and TLR4 variants and susceptibility to Leishmaniasis. Analysis of the nucleotide sequence encoding the TIR recognition domain of the MyD88 molecule showed that it is highly conserved when compared to the reference sequences. In contrast, heterozygous rs 12953258, which reflects a decrease in the expression of SOCS3, suggesting that it may be involved in the leishmaniasis susceptibility. This study is a first advance in the analysis of polymorphisms of genes involved in the signaling pathway of the macrophage and their relationship with leishmaniases infection and disease progression.


Assuntos
Variação Genética , Leishmaniose Cutânea/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD40/genética , Antígenos CD40/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Doenças Endêmicas , Feminino , Frequência do Gene , Humanos , Leishmaniose Cutânea/etiologia , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , População Rural , Alinhamento de Sequência , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Venezuela , Adulto Jovem
8.
PLoS One ; 15(8): e0237034, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745117

RESUMO

Production of IFN-γ is a key innate immune mechanism that limits replication of intracellular bacteria such as Francisella tularensis (Ft) until adaptive immune responses develop. Previously, we demonstrated that the host cell types responsible for IFN-γ production in response to murine Francisella infection include not only natural killer (NK) and T cells, but also a variety of myeloid cells. However, production of IFN-γ by mouse dendritic cells (DC) is controversial. Here, we directly demonstrated substantial production of IFN-γ by DC, as well as hybrid NK-DC, from LVS-infected wild type C57BL/6 or Rag1 knockout mice. We demonstrated that the numbers of conventional DC producing IFN-γ increased progressively over the course of 8 days of LVS infection. In contrast, the numbers of conventional NK cells producing IFN-γ, which represented about 40% of non-B/T IFN-γ-producing cells, peaked at day 4 after LVS infection and declined thereafter. This pattern was similar to that of hybrid NK-DC. To further confirm IFN-γ production by infected cells, DC and neutrophils were sorted from naïve and LVS-infected mice and analyzed for gene expression. Quantification of LVS by PCR revealed the presence of Ft DNA not only in macrophages, but also in highly purified, IFN-γ producing DC and neutrophils. Finally, production of IFN-γ by infected DC was confirmed by immunohistochemistry and confocal microscopy. Notably, IFN-γ production patterns similar to those in wild type mice were observed in cells derived from LVS-infected TLR2, TLR4, and TLR2xTLR9 knockout (KO) mice, but not from MyD88 KO mice. Taken together, these studies demonstrate the pivotal roles of DC and MyD88 in IFN-γ production and in initiating innate immune responses to this intracellular bacterium.


Assuntos
Interferon gama/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Receptores Toll-Like/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Francisella tularensis/imunologia , Imunidade Inata/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Neutrófilos/metabolismo , Baço/metabolismo , Linfócitos T/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/imunologia , Tularemia/microbiologia
9.
Cells ; 9(9)2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32854433

RESUMO

Coronaviruses (CoVs) are a diverse family of the enveloped human and animal viruses reported as causative agents for respiratory and intestinal infections. The high pathogenic potential of human CoVs, including SARS-CoV, MERS-CoV and SARS-CoV-2, is closely related to the invasion mechanisms underlying the attachment and entry of viral particles to the host cells. There is increasing evidence that sialylated compounds of cellular glycocalyx can serve as an important factor in the mechanism of CoVs infection. Additionally, the sialic acid-mediated cross-reactivity with the host immune lectins is known to exert the immune response of different intensity in selected pathological stages. Here, we focus on the last findings in the field of glycobiology in the context of the role of sialic acid in tissue tropism, viral entry kinetics and immune regulation in the CoVs infections.


Assuntos
Betacoronavirus/fisiologia , Infecções por Coronavirus/imunologia , Citocinas/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/fisiologia , Ácido N-Acetilneuramínico/metabolismo , Pneumonia Viral/imunologia , Vírus da SARS/fisiologia , Síndrome Respiratória Aguda Grave/imunologia , Animais , Infecções por Coronavirus/virologia , Humanos , Camundongos , Pandemias , Pneumonia Viral/virologia , Receptores de Reconhecimento de Padrão/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Receptores Toll-Like/metabolismo , Internalização do Vírus
10.
Life Sci ; 259: 118250, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32791152

RESUMO

AIMS: Several microbial toll-like receptor (TLR) ligands, bacterial DNA and bacterial cell wall fragments have been identified in the synovium of rheumatoid arthritis (RA) patients, proving bacterial involvement in the pathogenesis of RA. The current study aimed to verify that low dose polymyxin B could prevent the development of chronic inflammatory arthritis. METHODS: Twelve days post adjuvant injection, Sprague-Dawley rats were treated twice weekly with methotrexate (0.5 mg/kg) or daily with polymyxin B (1 mg/kg) or with combination of both for 1 or 2 weeks. Arthritis progression was assessed by hind paw swelling, serum levels of tumor growth factor-1ß (TGF-1ß), tumor necrosis factor-alpha (TNF-α), high sensitivity C-reactive protein (HS-CRP) and nuclear factor kappa B (NF-κB) were measured using ELISA. Cyclooxygenase-1 (Cox-1) and Cox-2 activities, as well as mRNA expression of TLR-2 and TLR-4 were determined. Histopathological examination of the ankle joint was performed as well as immunohistochemistry for anti-TLR-4. Histopathological assessment of toxic effects on the kidney was performed. KEY FINDINGS: Adjuvant arthritis led to a significant swelling of the hind paw and alteration in all serum parameters, TLR-2 and TLR-4 expression, as well as Cox-2 activity. These alterations were associated with histopathological changes of the joints. Polymyxin B reduced significantly all biomarkers of inflammation, showing better effect of the combination in most of the studied parameters, with minimal signs of nephrotoxicity. SIGNIFICANCE: In conclusion, results showed that polymyxin B possesses significant anti-arthritic activity which may be attributed to inhibition of the TLR-4, NF-κB and Cox-2 signaling pathway.


Assuntos
Artrite Experimental/tratamento farmacológico , Polimixina B/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Artrite Experimental/fisiopatologia , Artrite Reumatoide/tratamento farmacológico , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/metabolismo , Ciclo-Oxigenase 2/fisiologia , Adjuvante de Freund/farmacologia , Inflamação/tratamento farmacológico , Masculino , NF-kappa B/metabolismo , Polimixina B/metabolismo , Polimixina B/uso terapêutico , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Membrana Sinovial/metabolismo , Receptores Toll-Like/metabolismo , Receptores Toll-Like/fisiologia , Fator de Necrose Tumoral alfa/metabolismo
11.
PLoS One ; 15(7): e0214497, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639963

RESUMO

The Bashbay sheep (Ovis aries), an indigenous breed of Xinjiang, China, has many excellent characteristics. It is resistant to Mycoplasma ovipneumoniae infection, the causative agent of mycoplasma ovipneumonia, a chronic respiratory disease that is harmful to the sheep industry. To date, knowledge regarding the mechanisms responsible for M. ovipneumoniae pathogenesis in scant. Herein, we report the results of transcriptome profiling of lung tissues from Bashbay sheep experimentally infected with an M. ovipneumoniae strain at 4 and 14 days post-infection, in comparison to mock-infected animals (0 d). Transcriptome profiling was performed by deep RNA sequencing, using the Illumina platform. The analysis of differentially expressed genes was performed to determine concomitant gene-specific temporal patterns of mRNA expression in the lungs after M. ovipneumoniae infection. We found 1048 differentially expressed genes (575 up-regulated, 473 down-regulated) when comparing transcriptomic data at 4 and 0 days post-infection, and 2823 (1362 up-regulated, 1461 down-regulated) when comparing 14 versus 0 days post-infection. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the differentially expressed genes at 4 and 14 versus 0 days post-infection were enriched in 245 and 287 pathways, respectively, and the Toll-like receptor (TLR) signaling pathway was considered most closely related to MO infection (p < 0.01). Two pathways (LAMP-TLR2/TLR6-MyD88-MKK6-AP1-IL1B and LAMP-TLR8MyD88-IRF5-RANTES) were identified based on the TLR signaling pathway from differentially expressed genes related M. ovipneumoniae infection. Gene Ontology analysis showed that differentially expressed genes in different groups were enriched for 1580 and 4561 terms, where those most closely related to M. ovipneumoniae infection are positive regulators of inflammatory responses (p < 0.01). These results could aid in understanding how M. ovipneumoniae infection progresses in the lungs and may provide useful information regarding key regulatory pathways.


Assuntos
Pulmão/metabolismo , Pneumonia por Mycoplasma/patologia , Análise de Sequência de RNA/métodos , Doenças dos Ovinos/patologia , Transcriptoma , Animais , Regulação para Baixo , Mycoplasma ovipneumoniae/isolamento & purificação , Mycoplasma ovipneumoniae/patogenicidade , Pneumonia por Mycoplasma/genética , Pneumonia por Mycoplasma/veterinária , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ovinos , Doenças dos Ovinos/genética , Doenças dos Ovinos/metabolismo , Transdução de Sinais/genética , Fatores de Tempo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Regulação para Cima
12.
PLoS Pathog ; 16(7): e1008622, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32634175

RESUMO

Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confined L. monocytogenes (Δhly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Δhly L. monocytogenes mutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion in lgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions in pgdA and oatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A ΔhlyΔpgdAΔoatA strain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confined L. monocytogenes was also dependent on TLR2 and Unc93b1. Co-administration of Δhly and ΔactA resulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response to L. monocytogenes infection in vitro is mostly TLR2-dependent and immune suppression by phagosome-confined bacteria in vivo is mostly dependent on endosomal TLRs.


Assuntos
Tolerância Imunológica/imunologia , Interleucina-10/metabolismo , Listeriose/imunologia , Receptores Toll-Like/imunologia , Animais , Endossomos/imunologia , Endossomos/metabolismo , Interleucina-10/imunologia , Listeria monocytogenes/imunologia , Listeriose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fagossomos/imunologia , Fagossomos/metabolismo , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Receptores Toll-Like/metabolismo
13.
Curr Probl Cardiol ; 45(9): 100648, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32703535

RESUMO

The exceptional outbreak of COVID-19 pandemic has let the scientific community to work closely and quickly learnt things in a very short period of time. This has let us recognize that thromboembolic complications are responsible for morbidity and mortality among the COVID-19 infected patients. Available data have suggested a possible multifactorial basis of these complications, and while efforts are being made to treat this infection, preventive measures with the use of systemic anticoagulation were quickly adopted to deal with this issue. Despite obvious benefits as appeared with the use of systemic anticoagulation, most of the emerged data were retrospective, hence raise questions on the possible interplay of the confounders as well as long-term benefits and safety of systemic anticoagulation.


Assuntos
Anticoagulantes/uso terapêutico , Transtornos da Coagulação Sanguínea/sangue , Infecções por Coronavirus/sangue , Pneumonia Viral/sangue , Tromboembolia/sangue , Betacoronavirus , Transtornos da Coagulação Sanguínea/tratamento farmacológico , Transtornos da Coagulação Sanguínea/etiologia , Infecções por Coronavirus/complicações , Infecções por Coronavirus/tratamento farmacológico , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/tratamento farmacológico , Coagulação Intravascular Disseminada/etiologia , Células Endoteliais , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Heparina/uso terapêutico , Heparina de Baixo Peso Molecular/uso terapêutico , Humanos , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/tratamento farmacológico , Tromboembolia/tratamento farmacológico , Tromboembolia/etiologia , Receptores Toll-Like/metabolismo , Fator de von Willebrand/metabolismo
14.
Nat Commun ; 11(1): 3147, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561720

RESUMO

Transposons are known to participate in tissue aging, but their effects on aged stem cells remain unclear. Here, we report that in the Drosophila ovarian germline stem cell (GSC) niche, aging-related reductions in expression of Piwi (a transposon silencer) derepress retrotransposons and cause GSC loss. Suppression of Piwi expression in the young niche mimics the aged niche, causing retrotransposon depression and coincident activation of Toll-mediated signaling, which promotes Glycogen synthase kinase 3 activity to degrade ß-catenin. Disruption of ß-catenin-E-cadherin-mediated GSC anchorage then results in GSC loss. Knocking down gypsy (a highly active retrotransposon) or toll, or inhibiting reverse transcription in the piwi-deficient niche, suppresses GSK3 activity and ß-catenin degradation, restoring GSC-niche attachment. This retrotransposon-mediated impairment of aged stem cell maintenance may have relevance in many tissues, and could represent a viable therapeutic target for aging-related tissue degeneration.


Assuntos
Proteínas Argonauta/metabolismo , Senescência Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Células Germinativas/metabolismo , Animais , Proteínas Argonauta/genética , Caderinas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Inativação Gênica , Quinase 3 da Glicogênio Sintase/metabolismo , Ovário/citologia , Ovário/metabolismo , Retroelementos/genética , Transdução de Sinais , Nicho de Células-Tronco/fisiologia , Células-Tronco/metabolismo , Receptores Toll-Like/metabolismo , beta Catenina/metabolismo
15.
DNA Cell Biol ; 39(7): 1313-1321, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32543891

RESUMO

Toll-like receptors (TLRs) play crucial roles in the recognition of invading pathogens and the immune system. However, the effect of TLRs in asthma is still not fully known. This study was performed to better understand the role of TLR signatures in asthma. Blood samples from case-control studies (study 1: 348 asthmas and 39 normal controls and validation study 2: 411 asthmas and 87 normal controls) were enrolled. The single-sample gene set enrichment analysis method was performed to quantify the abundance of 21 TLR signatures. Gene ontology analysis and pathway function analysis were conducted for functional analysis, and a protein-protein interaction network was constructed. The area under the curve (AUC) value was used to assess the diagnostic capacity. In this study, TLR2/TLR3/TLR4 pathway, MyD88-dependent/independent TLR pathway, positive regulation of TLR4 pathway, and TLR binding signatures were significantly higher in asthma. Functional analysis showed that biological processes and pathways were still involved in TLR cascades and TLR signaling pathway. Eleven hub TLR-related genes were identified, and further validation demonstrated that the combination of TLR-related genes was a good diagnostic biomarker for asthma (AUC = 0.8). Our study provided more insight into the underlying immune mechanism of how TLR signatures affected asthma. The use of the easy-to-apply TLR-related genes might represent a promising blood-based biomarker for early detection of asthma.


Assuntos
Asma/diagnóstico , Asma/metabolismo , Receptores Toll-Like/metabolismo , Asma/genética , Asma/terapia , Biologia Computacional , Regulação da Expressão Gênica , Humanos , Imunoterapia , Prognóstico , Mapeamento de Interação de Proteínas
16.
Nat Commun ; 11(1): 2816, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499527

RESUMO

The intense arms race between bacteria and phages has led to the development of diverse antiphage defense systems in bacteria. Unlike well-known restriction-modification and CRISPR-Cas systems, recently discovered systems are poorly characterized. One such system is the Thoeris defense system, which consists of two genes, thsA and thsB. Here, we report structural and functional analyses of ThsA and ThsB. ThsA exhibits robust NAD+ cleavage activity and a two-domain architecture containing sirtuin-like and SLOG-like domains. Mutation analysis suggests that NAD+ cleavage is linked to the antiphage function of Thoeris. ThsB exhibits a structural resemblance to TIR domain proteins such as nucleotide hydrolases and Toll-like receptors, but no enzymatic activity is detected in our in vitro assays. These results further our understanding of the molecular mechanism underlying the Thoeris defense system, highlighting a unique strategy for bacterial antiphage resistance via NAD+ degradation.


Assuntos
Bacteriófagos/genética , Escherichia coli/virologia , NAD/metabolismo , Bacillus cereus/metabolismo , Sistemas CRISPR-Cas , Clonagem Molecular , Cristalografia por Raios X , Análise Mutacional de DNA , Escherichia coli/metabolismo , Hidrolases/metabolismo , Cinética , Mutação , Domínios Proteicos , Estrutura Secundária de Proteína , Receptores Toll-Like/metabolismo
17.
Adv Exp Med Biol ; 1263: 145-173, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32588327

RESUMO

Toll-like receptors (TLRs) in the tumor microenvironment (TME) are expressed not only in innate and adaptive immune cells but also in stromal cells such as fibroblasts, endothelial cells (EC), and tumor cells. The role of TLR signaling in the TME is complex and controversial due to their wide expression within the TME. Moreover, TLR signaling may culminate in different outcomes depending on the type of tumor, the implicated TLR, the type of TLR ligands, and, most importantly, the main type of cell(s) that are targeted by TLR ligands. Understanding to what extent these complex TLR signals impact on tumor progression merits further investigation, as it can help improve existing anti-cancer treatments or unravel new ones. In most cases, TLR signaling in tumor cells and in immune cells is associated with pro-tumoral and anti-tumoral effects, respectively. A better understanding of the relationship between TLRs and the TME, especially in humans, is required to design better anti-cancer therapies, considering that most current TLR-involved treatments were disappointing in clinical trials.In this chapter, we will discuss the impact of TLR signaling on the hallmarks of cancer, by highlighting their effects in tumor, immune, and stromal cells within the TME. Furthermore, we will discuss how the understanding of the role of TLRs can pave the way to develop new anti-cancer treatments and even predict clinical outcome and chemotherapy efficacy.


Assuntos
Neoplasias/imunologia , Neoplasias/metabolismo , Receptores Toll-Like/metabolismo , Microambiente Tumoral , Humanos , Transdução de Sinais
18.
Postgrad Med ; 132(7): 604-613, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32496926

RESUMO

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading worldwide. Antiviral therapy is the most important treatment for COVID-19. Among the drugs under investigation, anti-malarials, chloroquine (CQ) and hydroxychloroquine (HCQ), are being repurposed as treatment for COVID-19. CQ/HCQ were shown to prevent receptor recognition by coronaviruses, inhibit endosome acidification, which interferes with membrane fusion, and exhibit immunomodulatory activity. These multiple mechanisms may work together to exert a therapeutic effect on COVID-19. A number of in vitro studies revealed inhibitory effects of CQ/HCQ on various coronaviruses, including SARS-CoV-2 although conflicting results exist. Several clinical studies showed that CQ/HCQ alone or in combination with a macrolide may alleviate the clinical symptoms of COVID-19, promote viral conversion, and delay disease progression, with less serious adverse effects. However, recent studies indicated that the use of CQ/HCQ, alone or in combination with a macrolide, did not show any favorable effect on patients with COVID-19. Adverse effects, including prolonged QT interval after taking CQ/HCQ, may develop in COVID-19 patients. Therefore, current data are not sufficient enough to support the use of CQ/HCQ as therapies for COVID-19 and increasing caution should be taken about the application of CQ/HCQ in COVID-19 before conclusive findings are obtained by well-designed, multi-center, randomized, controlled studies.


Assuntos
Antivirais/uso terapêutico , Cloroquina/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Hidroxicloroquina/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Antivirais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/metabolismo , Cloroquina/farmacologia , Coronavirus Humano 229E/efeitos dos fármacos , Citocinas/efeitos dos fármacos , Citocinas/metabolismo , Glicosilação , Humanos , Hidroxicloroquina/farmacologia , Imunidade Inata , Técnicas In Vitro , Síndrome do QT Longo/induzido quimicamente , Ativação Linfocitária/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Pandemias , Peptidil Dipeptidase A/efeitos dos fármacos , Peptidil Dipeptidase A/metabolismo , Espécies Reativas de Oxigênio , Vírus da SARS/efeitos dos fármacos , Transdução de Sinais , Linfócitos T , Receptores Toll-Like/efeitos dos fármacos , Receptores Toll-Like/metabolismo , Resultado do Tratamento , Internalização do Vírus/efeitos dos fármacos
19.
Pharmacol Res ; 157: 104820, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32360484

RESUMO

The Coronavirus Disease 2019 (COVID-19) pandemic has become a huge threaten to global health, which raise urgent demand of developing efficient therapeutic strategy. The aim of the present study is to dissect the chemical composition and the pharmacological mechanism of Qingfei Paidu Decoction (QFPD), a clinically used Chinese medicine for treating COVID-19 patients in China. Through comprehensive analysis by liquid chromatography coupled with high resolution mass spectrometry (MS), a total of 129 compounds of QFPD were putatively identified. We also constructed molecular networking of mass spectrometry data to classify these compounds into 14 main clusters, in which exhibited specific patterns of flavonoids (45 %), glycosides (15 %), carboxylic acids (10 %), and saponins (5 %). The target network model of QFPD, established by predicting and collecting the targets of identified compounds, indicated a pivotal role of Ma Xing Shi Gan Decoction (MXSG) in the therapeutic efficacy of QFPD. Supportively, through transcriptomic analysis of gene expression after MXSG administration in rat model of LPS-induced pneumonia, the thrombin and Toll-like receptor (TLR) signaling pathway were suggested to be essential pathways for MXSG mediated anti-inflammatory effects. Besides, changes in content of major compounds in MXSG during decoction were found by the chemical analysis. We also validate that one major compound in MXSG, i.e. glycyrrhizic acid, inhibited TLR agonists induced IL-6 production in macrophage. In conclusion, the integration of in silico and experimental results indicated that the therapeutic effects of QFPD against COVID-19 may be attributed to the anti-inflammatory effects of MXSG, which supports the rationality of the compatibility of TCM.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Medicamentos de Ervas Chinesas/análise , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Animais , Anti-Inflamatórios/análise , Anti-Inflamatórios/farmacologia , Células Cultivadas , Simulação por Computador , Infecções por Coronavirus/genética , Expressão Gênica/efeitos dos fármacos , Ácido Glicirrízico/farmacologia , Humanos , Interleucina-6/metabolismo , Lipopeptídeos/antagonistas & inibidores , Lipopeptídeos/farmacologia , Lipopolissacarídeos , Masculino , Pandemias , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia Viral/genética , Ratos , Transdução de Sinais/efeitos dos fármacos , Trombina/metabolismo , Receptores Toll-Like/metabolismo
20.
Nat Commun ; 11(1): 2361, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32398640

RESUMO

The development of thymic regulatory T cells (Treg) is mediated by Aire-regulated self-antigen presentation on medullary thymic epithelial cells (mTECs) and dendritic cells (DCs), but the cooperation between these cells is still poorly understood. Here we show that signaling through Toll-like receptors (TLR) expressed on mTECs regulates the production of specific chemokines and other genes associated with post-Aire mTEC development. Using single-cell RNA-sequencing, we identify a new thymic CD14+Sirpα+ population of monocyte-derived dendritic cells (CD14+moDC) that are enriched in the thymic medulla and effectively acquire mTEC-derived antigens in response to the above chemokines. Consistently, the cellularity of CD14+moDC is diminished in mice with MyD88-deficient TECs, in which the frequency and functionality of thymic CD25+Foxp3+ Tregs are decreased, leading to aggravated mouse experimental colitis. Thus, our findings describe a TLR-dependent function of mTECs for the recruitment of CD14+moDC, the generation of Tregs, and thereby the establishment of central tolerance.


Assuntos
Colite/imunologia , Células Dendríticas/imunologia , Células Epiteliais/imunologia , Linfócitos T Reguladores/imunologia , Timo/imunologia , Transferência Adotiva , Animais , Apresentação do Antígeno , Autoantígenos/imunologia , Separação Celular , Quimiocinas/imunologia , Quimiocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Citometria de Fluxo , Receptores de Lipopolissacarídeos/metabolismo , Camundongos , Receptores Imunológicos/metabolismo , Tolerância a Antígenos Próprios , Análise de Sequência de RNA , Transdução de Sinais/imunologia , Análise de Célula Única , Linfócitos T Reguladores/transplante , Timo/citologia , Receptores Toll-Like/metabolismo , Regulação para Cima
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