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1.
Front Immunol ; 12: 620494, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34122400

RESUMO

The innate and adaptive immune systems act in concert to protect us from infectious agents and other harmful substances. As a state of temporary or permanent immune dysfunction, immunosuppression can make an organism more susceptible to infection, organ injury, and cancer due to damage to the immune system. It takes a long time to develop new immunomodulatory agents to prevent and treat immunosuppressive diseases, with slow progress. Toll-like receptor 2 (TLR2) agonists have been reported as potential immunomodulatory candidates due to their effective activation of immune responses. It has been demonstrated that thymopentin (TP5) could modulate immunity by binding to the TLR2 receptor. However, the fairly short half-life of TP5 greatly reduces its pharmacological potential for immunosuppression therapy. Although peptide cathelicidin 2 (CATH2) has a long half-life, it shows poor immunomodulatory activity and severe cytotoxicity, which seriously hampers its clinical development. Peptide hybridization is an effective approach for the design and engineering of novel functional peptides because hybrid peptides combine the advantages and benefits of various native peptides. In this study, to overcome all these challenges faced by the parental peptides, six hybrid peptides (CaTP, CbTP, CcTP, TPCa, TPCb, and TPCc) were designed by combining the full-length TP5 with different active fragments of CATH2. CbTP, the most potent TLR2 agonist among the six hybrid peptides, was effectively screened through in silico analysis and in vitro experiments. The CbTP peptide exhibited lower cytotoxicity than either CATH2 or TP5. Furthermore, the immunomodulatory effects of CbTP were confirmed in a CTX-immunosuppressed mouse model, which showed that CbTP has increased immunopotentiating activity and physiological stability compared to the parental peptides. CbTP successfully inhibited immunosuppression and weight loss, increased immune organ indices, and improved CD4+/CD8+ T lymphocyte subsets. In addition, CbTP significantly increased the production of the cytokine TNF-α and IL-6, and the immunoglobulins IgA, IgM, and IgG. The immunoenhancing effects of CbTP were attributed to its TLR2-binding activity, promoting the formation of the TLR2 cluster, the activation of the TLR2 receptor, and thus activation of the downstream MyD88-NF-кB signaling pathway.


Assuntos
Peptídeos/metabolismo , Linfócitos T/imunologia , Timopentina/metabolismo , Receptor 2 Toll-Like/agonistas , Animais , Células Cultivadas , Ciclofosfamida , Citocinas , Feminino , Humanos , Imunidade , Imunidade Humoral , Hospedeiro Imunocomprometido , Imunomodulação , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Peptídeos/imunologia , Células RAW 264.7 , Timopentina/imunologia
2.
J Virol ; 95(17): e0081621, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34133900

RESUMO

The complete eradication of human immunodeficiency virus type 1 (HIV-1) is blocked by latent reservoirs in CD4+ T cells and myeloid lineage cells. Toll-like receptors (TLRs) can induce the reversal of HIV-1 latency and trigger the innate immune response. To the best of our knowledge, there is little evidence showing the "killing" effect of TLR1/2 agonists but only a small "shock" potential. To identify a new approach for eradicating the HIV latent reservoir, we evaluated the effectiveness of SMU-Z1, a novel small-molecule TLR1/2 agonist, in the "shock-and-kill" strategy. The results showed that SMU-Z1 could enhance latent HIV-1 transcription not only ex vivo in peripheral blood mononuclear cells from aviremic HIV-1-infected donors receiving combined antiretroviral therapy but also in vitro in cells of myeloid-monocytic origin targeting the NF-κB and mitogen-activated protein kinase pathways. Interestingly, the activation marker CD69 was significantly upregulated in natural killer (NK) cells, B cells, and monocytes 48 h after SMU-Z1 treatment. Furthermore, SMU-Z1 was able to activate T cells without global T cell activation, as well as increasing NK cell degranulation and gamma interferon (IFN-γ) production, which further block HIV-1-infected CD4+ lymphocytes. In summary, the present study found that SMU-Z1 can both enhance HIV-1 transcription and promote NK cell-mediated inhibition of HIV-1-infected autologous CD4+ T cells. These findings indicate that the novel TLR1/2 agonist SMU-Z1 is a promising latency-reversing agent (LRA) for eradication of HIV-1 reservoirs. IMPORTANCE Multiple in vivo studies showed that many LRAs used in the shock-and-kill approach could activate viral transcription but could not induce killing effectively. Therefore, a dual-function LRA is needed for elimination of HIV-1 reservoirs. We previously developed a small-molecule TLR1/2 agonist, SMU-Z1, and demonstrated that it could upregulate NK cells and CD8+ T cells with immune adjuvant and antitumor properties in vivo. In the present study, SMU-Z1 could activate innate immune cells without global T cell activation, induce production of proinflammatory and antiviral cytokines, and enhance the cytotoxic function of NK cells. We showed that SMU-Z1 displayed dual potential ex vivo in the shock of exposure of latently HIV-1-infected cells and in the kill of clearance of infected cells, which is critical for effective use in combination with therapeutic vaccines or broadly neutralizing antibody treatments aimed at curing AIDS.


Assuntos
Antirretrovirais/farmacologia , Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , Células Matadoras Naturais/imunologia , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Latência Viral , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/virologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Bibliotecas de Moléculas Pequenas/farmacologia , Carga Viral , Ativação Viral
3.
J Med Chem ; 64(11): 7371-7389, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34029463

RESUMO

The previous virtual screening of ten million compounds yielded two novel nonlipopeptide-like chemotypes as TLR2 agonists. Herein, we present the chemical optimization of our initial hit, 1-phenyl-3-(thiophen-2-yl)urea, which resulted in the identification of SMU-C80 (EC50 = 31.02 ± 1.01 nM) as a TLR2-specific agonist with a 370-fold improvement in bioactivity. Mechanistic studies revealed that SMU-C80, through TLR1/2, recruits the adaptor protein MyD88 and triggers the NF-κB pathway to release cytokines such as TNF-α and IL-1ß from human, but not murine, cells. To the best of our knowledge, it is the first species-specific TLR1/2 agonist reported until now. Moreover, SMU-C80 increased the percentage of T, B, and NK cells ex vivo and activated the immune cells, which suppressed cancer cell growth in vitro. In summary, we obtained a highly efficient and specific human TLR1/2 agonist that acts through the MyD88 and NF-κB pathway, facilitating cytokine release and the simultaneous activation of immune cells that in turn affects the apoptosis of cancer cells.


Assuntos
Desenho de Fármacos , Tioureia/análogos & derivados , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Animais , Apoptose/efeitos dos fármacos , Citocinas/metabolismo , Humanos , Imunoterapia , Células Matadoras Naturais/citologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Neoplasias/terapia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , Tioureia/metabolismo , Tioureia/uso terapêutico , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/metabolismo
4.
Sci Rep ; 11(1): 10770, 2021 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-34031466

RESUMO

In periodontitis, gingival fibroblasts (GFs) interact with and respond to oral pathogens, significantly contributing to perpetuation of chronic inflammation and tissue destruction. The aim of this study was to determine the usefulness of the recently released hTERT-immortalized GF (TIGF) cell line for studies of host-pathogen interactions. We show that TIGFs are unable to upregulate expression and production of interleukin (IL)-6, IL-8 and prostaglandin E2 upon infection with Porphyromonas gingivalis despite being susceptible to adhesion and invasion by this oral pathogen. In contrast, induction of inflammatory mediators in TNFα- or IL-1ß-stimulated TIGFs is comparable to that observed in primary GFs. The inability of TIGFs to respond directly to P. gingivalis is caused by a specific defect in Toll-like receptor-2 (TLR2) expression, which is likely driven by TLR2 promoter hypermethylation. Consistently, TIGFs fail to upregulate inflammatory genes in response to the TLR2 agonists Pam2CSK4 and Pam3CSK4. These results identify important limitations of using TIGFs to study GF interaction with oral pathogens, though these cells may be useful for studies of TLR2-independent processes. Our observations also emphasize the importance of direct comparisons between immortalized and primary cells prior to using cell lines as models in studies of any biological processes.


Assuntos
Infecções por Bacteroidaceae/imunologia , Gengiva/citologia , Interleucina-1beta/genética , Porphyromonas gingivalis/patogenicidade , Telomerase/genética , Fator de Necrose Tumoral alfa/genética , Aderência Bacteriana/efeitos dos fármacos , Infecções por Bacteroidaceae/genética , Células Cultivadas , Metilação de DNA , Dinoprostona/genética , Dinoprostona/metabolismo , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/imunologia , Fibroblastos/metabolismo , Gengiva/efeitos dos fármacos , Gengiva/imunologia , Gengiva/metabolismo , Humanos , Interleucina-1beta/metabolismo , Lipopeptídeos/farmacologia , Oligopeptídeos/farmacologia , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor Toll-Like 9/agonistas , Fator de Necrose Tumoral alfa/metabolismo
5.
Bioorg Chem ; 111: 104838, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33848722

RESUMO

A focused library of water soluble 1,2,3-triazole tethered glycopeptide conjugates derived from variety of azido-monosaccharides and aliphatic azido-alcohols were synthesized through manipulation at the C-terminus of Pam3CAG and screened for their potential as TLR2 agonistic adjuvants against HBsAg antigen. In vitro ligand induced TLR2 signal activation was observed with all the analogues upon treatment with HEK blue TLR2 cell lines. Conjugate derived from ribose (6e), which exhibited pronounced HBsAg specific antibody (IgG) titer also shown enhanced CD8+ population indicating superior cell mediated immunity compared to standard adjuvant Pam3CSK4. Further, docking studies revealed ligand induced heterodimerization between TLR1 and 2. Overall, the result indicates the usefulness of novel conjugates as potential vaccine adjuvant.


Assuntos
Adjuvantes Imunológicos/farmacologia , Receptor 2 Toll-Like/agonistas , Triazóis/farmacologia , Vacinas/farmacologia , Adjuvantes Imunológicos/química , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Imunidade Celular/efeitos dos fármacos , Estrutura Molecular , Relação Estrutura-Atividade , Triazóis/química , Vacinas/química
6.
Nat Commun ; 12(1): 1836, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758175

RESUMO

To prevent damage to the host or its commensal microbiota, epithelial tissues must match the intensity of the immune response to the severity of a biological threat. Toll-like receptors allow epithelial cells to identify microbe associated molecular patterns. However, the mechanisms that mitigate biological noise in single cells to ensure quantitatively appropriate responses remain unclear. Here we address this question using single cell and single molecule approaches in mammary epithelial cells and primary organoids. We find that epithelial tissues respond to bacterial microbe associated molecular patterns by activating a subset of cells in an all-or-nothing (i.e. digital) manner. The maximum fraction of responsive cells is regulated by a bimodal epigenetic switch that licenses the TLR2 promoter for transcription across multiple generations. This mechanism confers a flexible memory of inflammatory events as well as unique spatio-temporal control of epithelial tissue-level immune responses. We propose that epigenetic licensing in individual cells allows for long-term, quantitative fine-tuning of population-level responses.


Assuntos
Bactérias/imunologia , Células Epiteliais/imunologia , Imunidade Inata , Lipopeptídeos/imunologia , NF-kappa B/metabolismo , Receptor 2 Toll-Like/metabolismo , Animais , Bactérias/metabolismo , Linhagem Celular , Citocinas/metabolismo , Citocinas/farmacologia , Metilação de DNA/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Flagelina/farmacologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Processamento de Imagem Assistida por Computador , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/genética , Hibridização in Situ Fluorescente , Glândulas Mamárias Animais , Camundongos , Organoides/efeitos dos fármacos , Organoides/imunologia , Organoides/metabolismo , Regiões Promotoras Genéticas , RNA-Seq , Transdução de Sinais/imunologia , Análise de Célula Única , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/genética , Receptores Toll-Like/agonistas , Receptores Toll-Like/metabolismo
7.
Bioorg Med Chem Lett ; 40: 127861, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33636302

RESUMO

Toll-like receptors (TLRs) play key role in innate immune response to Damage Associated Molecular Patterns (DAMPs) and Pathogen Associated Molecular Patterns (PAMPs). DAMP/PAMP-mediated activation of TLRs triggers NFκB signaling resulting in pro-inflammatory cytokine release. Using TLR2-Pam2CSK4 agonist co-crystal structure information, we designed and synthesized a novel series of Toll-like Receptor 2 (TLR2) lipid antagonists and identified compounds 14, 15 and 17 with sub-micromolar potency. TLR2 antagonists that we identified are stable for > 1.0 h in both gastric juice and PBS buffer and could be used as research tools.


Assuntos
Lipídeos/química , Oligopeptídeos/química , Receptor 2 Toll-Like/agonistas , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor Toll-Like 9/agonistas , Cristalização , Citocinas/metabolismo , Descoberta de Drogas , Humanos , NF-kappa B/metabolismo , Ligação Proteica , Transdução de Sinais , Relação Estrutura-Atividade , Receptor 2 Toll-Like/química , Receptor Toll-Like 9/química
8.
J Cell Mol Med ; 25(8): 3785-3792, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33609010

RESUMO

Severe ionizing radiation causes the acute lethal damage of haematopoietic system and gastrointestinal tract. Here, we found CL429, the novel chimeric TLR2/NOD2 agonist, exhibited significant radioprotective effects in mice. CL429 increased mice survival, protected mice against the lethal damage of haematopoietic system and gastrointestinal tract. CL429 was more effective than equivalent amounts of monospecific (TLR2 or NOD2) and combination (TLR2 + NOD2) of molecules in preventing radiation-induced death. The radioprotection of CL429 was mainly mediated by activating TLR2 and partially activating NOD2. CL429-induced radioprotection was largely dependent on the activation of TLR2-MyD88-NF-κB signalling pathway. In conclusion, the data suggested that the co-activation of TLR2 and NOD2 could induce significant synergistic radioprotective effects and CL429 might be a potential high-efficiency selective agent.


Assuntos
Síndrome Aguda da Radiação/prevenção & controle , Sistema Hematopoético/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Proteína Adaptadora de Sinalização NOD2/agonistas , Protetores contra Radiação/farmacologia , Receptor 2 Toll-Like/agonistas , Irradiação Corporal Total/efeitos adversos , Síndrome Aguda da Radiação/etiologia , Síndrome Aguda da Radiação/patologia , Animais , Sistema Hematopoético/efeitos da radiação , Intestinos/lesões , Intestinos/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL
9.
Sci Rep ; 11(1): 2483, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33510265

RESUMO

Pneumonia caused by the intracellular bacterium Rhodococcus equi is an important cause of disease and death in immunocompromised hosts, especially foals. Antibiotics are the standard of care for treating R. equi pneumonia in foals, and adjunctive therapies are needed. We tested whether nebulization with TLR agonists (PUL-042) in foals would improve innate immunity and reduce the severity and duration of pneumonia following R. equi infection. Neonatal foals (n = 48) were nebulized with either PUL-042 or vehicle, and their lung cells infected ex vivo. PUL-042 increased inflammatory cytokines in BAL fluid and alveolar macrophages after ex vivo infection with R. equi. Then, the in vivo effects of PUL-042 on clinical signs of pneumonia were examined in 22 additional foals after intrabronchial challenge with R. equi. Foals infected and nebulized with PUL-042 or vehicle alone had a shorter duration of clinical signs of pneumonia and smaller pulmonary lesions when compared to non-nebulized foals. Our results demonstrate that host-directed therapy can enhance neonatal immune responses against respiratory pathogens and reduce the duration and severity of R. equi pneumonia.


Assuntos
Infecções por Actinomycetales , Doenças dos Cavalos , Cavalos , Imunidade Inata/efeitos dos fármacos , Lipopeptídeos/farmacologia , Oligodesoxirribonucleotídeos/farmacologia , Pneumonia Bacteriana , Rhodococcus equi/imunologia , Receptor 2 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Receptor Toll-Like 9/agonistas , Infecções por Actinomycetales/tratamento farmacológico , Infecções por Actinomycetales/imunologia , Infecções por Actinomycetales/patologia , Infecções por Actinomycetales/veterinária , Animais , Doenças dos Cavalos/tratamento farmacológico , Doenças dos Cavalos/imunologia , Doenças dos Cavalos/patologia , Cavalos/imunologia , Cavalos/microbiologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Pneumonia Bacteriana/tratamento farmacológico , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/patologia , Pneumonia Bacteriana/veterinária , Índice de Gravidade de Doença
10.
EBioMedicine ; 63: 103153, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33279857

RESUMO

BACKGROUND: The novel human coronavirus SARS-CoV-2 is a major ongoing global threat with huge economic burden. Like all respiratory viruses, SARS-CoV-2 initiates infection in the upper respiratory tract (URT). Infected individuals are often asymptomatic, yet highly infectious and readily transmit virus. A therapy that restricts initial replication in the URT has the potential to prevent progression of severe lower respiratory tract disease as well as limiting person-to-person transmission. METHODS: SARS-CoV-2 Victoria/01/2020 was passaged in Vero/hSLAM cells and virus titre determined by plaque assay. Challenge virus was delivered by intranasal instillation to female ferrets at 5.0 × 106 pfu/ml. Treatment groups received intranasal INNA-051, developed by Ena Respiratory. SARS-CoV-2 RNA was detected using the 2019-nCoV CDC RUO Kit and QuantStudio™ 7 Flex Real-Time PCR System. Histopathological analysis was performed using cut tissues stained with haematoxylin and eosin (H&E). FINDINGS: We show that prophylactic intra-nasal administration of the TLR2/6 agonist INNA-051 in a SARS-CoV-2 ferret infection model effectively reduces levels of viral RNA in the nose and throat. After 5 days post-exposure to SARS-CoV-2, INNA-051 significantly reduced virus in throat swabs (p=<0.0001) by up to a 24 fold (96% reduction) and in nasal wash (p=0.0107) up to a 15 fold (93% reduction) in comparison to untreated animals. INTERPRETATION: The results of our study support clinical development of a therapy based on prophylactic TLR2/6 innate immune activation in the URT, to reduce SARS-CoV-2 transmission and provide protection against COVID-19. FUNDING: This work was funded by Ena Respiratory, Melbourne, Australia.


Assuntos
Lipopeptídeos/administração & dosagem , Sistema Respiratório/virologia , SARS-CoV-2/patogenicidade , Receptor 2 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Eliminação de Partículas Virais , Administração Intranasal , Animais , COVID-19/tratamento farmacológico , COVID-19/patologia , Modelos Animais de Doenças , Feminino , Furões , Imunidade Inata , Lipopeptídeos/química , Lipopeptídeos/farmacologia , Cavidade Nasal/patologia , Cavidade Nasal/virologia , Faringe/patologia , Faringe/virologia , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sistema Respiratório/patologia , SARS-CoV-2/genética , SARS-CoV-2/isolamento & purificação , Carga Viral/efeitos dos fármacos
11.
J Med Chem ; 64(1): 233-278, 2021 01 14.
Artigo em Inglês | MEDLINE | ID: mdl-33346636

RESUMO

Toll-like receptors (TLRs) are the pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) in microbial species. Among the various TLRs, TLR2 has a special place due to its ability to sense the widest repertoire of PAMPs owing to its heterodimerization with either TLR1 or TLR6, broadening its ligand diversity against pathogens. Various scaffolds are reported to activate TLR2, which include naturally occurring lipoproteins, synthetic lipopeptides, and small heterocyclic molecules. We described a detailed SAR in TLR2 agonistic scaffolds and also covered the design and chemistry for the conjugation of TLR2 agonists to antigens, carbohydrates, polymers, and fluorophores. The approaches involved in delivery of TLR2 agonists such as lipidation of antigen, conjugation to polymers, phosphonic acids, and other linkers to achieve surface adsorption, liposomal formulation, and encapsulating nanoparticles are elaborated. The crystal structure analysis and computational modeling are also included with the structural features that facilitate TLR2 activation.


Assuntos
Bibliotecas de Moléculas Pequenas/farmacologia , Receptor 2 Toll-Like/agonistas , Animais , Cristalografia por Raios X , Humanos , Estrutura Molecular , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade
13.
Bioconjug Chem ; 31(11): 2499-2503, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33147965

RESUMO

Cyclic dinucleotides (CDNs), agonists of stimulator of interferon genes (STING), are promising agents for immunotherapy. However, the application of CDNs has been limited by their instability and low transmembrane efficiency. Here, we introduced a conjugated adjuvant of STING and TLR1/2, Pam3CSK4-CDGSF. Conjugating CDGSF with Pam3CSK4 increased the stability and intracellular delivery. In addition, by synergistically activating the STING and TLR pathways, Pam3CSK4-CDGSF was able to enhance immune activation. Both humoral and cellular immune responses were triggered by Pam3CSK4-CDGSF plus OVA (V4), and tumor growth was significantly inhibited after V4 administration. More importantly, V4 can also boost the antigen-specific CD8+ T cell response for cancer cell killing. Thus, the conjugated STING and TLR1/2 agonist Pam3CSK4-CDGSF can serve as a potent adjuvant for vaccine construction to augment antitumor immunotherapy.


Assuntos
Imunoterapia , Lipopeptídeos/farmacologia , Proteínas de Membrana/agonistas , Neoplasias/terapia , Receptor 2 Toll-Like/agonistas , Animais , Formação de Anticorpos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Humanos , Imunidade Celular , Camundongos , Neoplasias/imunologia , Receptor 1 Toll-Like/agonistas
14.
Front Immunol ; 11: 1899, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32983111

RESUMO

Background: Infection/inflammation is an important causal factor in spontaneous preterm birth (sPTB). Most mechanistic studies have concentrated on the role of bacteria, with limited focus on the role of viruses in sPTB. Murine studies support a potential multi-pathogen aetiology in which a double or sequential hit of both viral and bacterial pathogens leads to a higher risk preterm labour. This study aimed to determine the effect of viral priming on bacterial induced inflammation in human in vitro models of ascending and haematogenous infection. Methods: Vaginal epithelial cells, and primary amnion epithelial cells and myocytes were used to represent cell targets of ascending infection while interactions between peripheral blood mononuclear cells (PBMCs) and placental explants were used to model systemic infection. To model the effect of viral priming upon the subsequent response to bacterial stimuli, each cell type was stimulated first with a TLR3 viral agonist, and then with either a TLR2 or TLR2/6 agonist, and responses compared to those of each agonist alone. Immunoblotting was used to detect cellular NF-κB, AP-1, and IRF-3 activation. Cellular TLR3, TLR2, and TLR6 mRNA was quantified by RT-qPCR. Immunoassays were used to measure supernatant cytokine, chemokine and PGE2 concentrations. Results: TLR3 ("viral") priming prior to TLR2/6 agonist ("bacterial") exposure augmented the pro-inflammatory, pro-labour response in VECs, AECs, myocytes and PBMCs when compared to the effects of agonists alone. In contrast, enhanced anti-inflammatory cytokine production (IL-10) was observed in placental explants. Culturing placental explants in conditioned media derived from PBMCs primed with a TLR3 agonist enhanced TLR2/6 agonist stimulated production of IL-6 and IL-8, suggesting a differential response by the placenta to systemic inflammation compared to direct infection as a result of haematogenous spread. TLR3 agonism generally caused increased mRNA expression of TLR3 and TLR2 but not TLR6. Conclusion: This study provides human in vitro evidence that viral infection may increase the susceptibility of women to bacterial-induced sPTB. Improved understanding of interactions between viral and bacterial components of the maternal microbiome and host immune response may offer new therapeutic options, such as antivirals for the prevention of PTB.


Assuntos
Âmnio/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Miométrio/efeitos dos fármacos , Complicações Infecciosas na Gravidez/microbiologia , Complicações Infecciosas na Gravidez/virologia , Receptor 2 Toll-Like/agonistas , Receptor 3 Toll-Like/agonistas , Receptor 6 Toll-Like/agonistas , Vagina/efeitos dos fármacos , Âmnio/imunologia , Âmnio/metabolismo , Linhagem Celular , Citocinas/metabolismo , Dinoprostona/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Interações Hospedeiro-Patógeno , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , Miométrio/imunologia , Miométrio/metabolismo , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/metabolismo , Transdução de Sinais , Técnicas de Cultura de Tecidos , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/genética , Receptor 3 Toll-Like/metabolismo , Receptor 6 Toll-Like/genética , Receptor 6 Toll-Like/metabolismo , Vagina/imunologia , Vagina/metabolismo
15.
Am J Physiol Endocrinol Metab ; 319(5): E893-E903, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32954825

RESUMO

Saturated fatty acid (SFA) induces proinflammatory response through a Toll-like receptor (TLR)-mediated mechanism, which is associated with cardiometabolic diseases such as obesity, insulin resistance, and endothelial dysfunction. Consistent with this notion, TLR2 or TLR4 knockout mice are protected from obesity-induced proinflammatory response and endothelial dysfunction. Although SFA causes endothelial dysfunction through TLR-mediated signaling pathways, the mechanisms underlying SFA-stimulated inflammatory response are not completely understood. To understand the proinflammatory response in vascular endothelial cells in high-lipid conditions, we compared the proinflammatory responses stimulated by palmitic acid (PA) and other canonical TLR agonists [lipopolysaccharide (LPS), Pam3-Cys-Ser-Lys4 (Pam3CSK4), or macrophage-activating lipopeptide-2)] in human aortic endothelial cells. The expression profiles of E-selectin and the signal transduction pathways stimulated by PA were distinct from those stimulated by canonical TLR agonists. Inhibition of long-chain acyl-CoA synthetases (ACSL) by a pharmacological inhibitor or knockdown of ACSL1 blunted the PA-stimulated, but not the LPS- or Pam3CSK4-stimulated proinflammatory responses. Furthermore, triacsin C restored the insulin-stimulated vasodilation, which was impaired by PA. From the results, we concluded that PA stimulates the proinflammatory response in the vascular endothelium through an ACSL1-mediated mechanism, which is distinct from LPS- or Pam3CSK4-stimulated responses. The results suggest that endothelial dysfunction caused by PA may require to undergo intracellular metabolism. This expands the understanding of the mechanisms by which TLRs mediate inflammatory responses in endothelial dysfunction and cardiovascular disease.


Assuntos
Aorta/metabolismo , Coenzima A Ligases/metabolismo , Células Endoteliais/metabolismo , Endotélio Vascular/metabolismo , Inflamação/metabolismo , Ácido Palmítico/farmacologia , Aorta/efeitos dos fármacos , Selectina E/metabolismo , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Humanos , Insulina/farmacologia , Lipopolissacarídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistas , Triazenos/farmacologia
16.
Anticancer Res ; 40(10): 5361-5369, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988855

RESUMO

BACKGROUND/AIM: The aim of this study was to evaluate the role of toll-like receptor 2 (TLR2) in the proliferation of human lung cancer cells and identify the signaling pathway that mediates this effect. MATERIALS AND METHODS: Adenocarcinoma (A549 and H1650) and adenosquamous (H125) cells were treated with increasing doses of Pam3CSK4, a TLR2 agonist. Cell proliferation and NF-ĸB activation were evaluated. NF-ĸB was inhibited prior to treatment with Pam3CSK4 and proliferation was assessed. RESULTS: TLR2 expression was significantly higher in A549 and H1650 cells compared to H125 cells (p<0.001). TLR2 stimulation induced proliferation in adenocarcinoma cells only and led to a corresponding increase in NF-ĸB activity (p<0.05). Inhibition of NF-ĸB prior to treatment with Pam3CSK4 attenuated this proliferative response. CONCLUSION: TLR2 activation induced proliferation of lung adenocarcinoma cells through activation of NF-ĸB. Thus, the TLR2 signaling pathway may be a potential therapeutic target in lung adenocarcinoma.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Lipopeptídeos/farmacologia , Receptor 2 Toll-Like/genética , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/patologia , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , NF-kappa B/genética , Receptor 2 Toll-Like/agonistas
17.
Front Immunol ; 11: 1693, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32793243

RESUMO

Chronic exposure to periodontopathogenic bacteria such as Porphyromonas gingivalis and the products of these bacteria that interact with the cells of the tooth surrounding tissues can ultimately result in periodontitis. This is a disease that is characterized by inflammation-related alveolar bone degradation by the bone-resorbing cells, the osteoclasts. Interactions of bacterial products with Toll-like receptors (TLRs), in particular TLR2 and TLR4, play a significant role in this chronic inflammatory reaction, which possibly affects osteoclastic activity and osteogenic capacity. Little is known about how chronic exposure to specific TLR activators affects these two antagonistic activities. Here, we studied the effect of TLR activation on gingival fibroblasts (GF), cells that are anatomically close to infiltrating bacterial products in the mouth. These were co-cultured with naive osteoclast precursor cells (i.e., monocytes), as part of the peripheral blood mononuclear cells (PBMCs). Activation of GF co-cultures (GF + PBMCs) with TLR2 or TLR4 agonists resulted in a weak reduction of the osteoclastogenic potential of these cultures, predominantly due to TLR2. Interestingly, chronic exposure, especially to TLR2 agonist, resulted in increased release of TNF-α at early time points. This effect, was reversed at later time points, thus suggesting an adaptation to chronic exposure. Monocyte cultures primed with M-CSF + RANKL, led to the formation of bone-resorbing osteoclasts, irrespective of being activated with TLR agonists. Late activation of these co-cultures with TLR2 and with TLR4 agonists led to a slight decrease in bone resorption. Activation of GF with TLR2 and TLR4 agonists did not affect the osteogenic capacity of the GF cells. In conclusion, chronic exposure leads to diverse reactions; inhibitory with naive osteoclast precursors, not effecting already formed (pre-)osteoclasts. We suggest that early encounter of naive monocytes with TLR agonists may result in differentiation toward the macrophage lineage, desirable for clearing bacterial products. Once (pre-)osteoclasts are formed, these cells may be relatively insensitive for direct TLR stimulation. Possibly, TLR activation of periodontal cells indirectly stimulates osteoclasts, by secreting osteoclastogenesis stimulating inflammatory cytokines.


Assuntos
Fibroblastos/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Leucócitos Mononucleares/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Oligopeptídeos/farmacologia , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Receptor 2 Toll-Like/agonistas , Receptor 4 Toll-Like/agonistas , Receptor Toll-Like 9/agonistas , Adulto , Células Cultivadas , Técnicas de Cocultura , Fibroblastos/metabolismo , Gengiva/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Transdução de Sinais , Fatores de Tempo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Adulto Jovem
18.
Int Immunopharmacol ; 85: 106619, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32485352

RESUMO

Toll-like receptor 2 (TLR2) is a primary sensor for pathogens, including those derived from gram-positive bacteria. It can also mediate the effects of endogenous inflammatory signals such as ß-amyloid peptide (Aß), thus promoting the microglial activation and subsequent neuronal dysfunction, characteristic of chronic neuroinflammatory conditions. More recently, a role for TLR2 has been proposed in the pathogenesis of disorders associated with acute inflammation, including anxiety and depression. The current study aims to characterise the acute effects of the TLR2 agonist lipoteichoic acid (LTA) on microglial activation and neuronal integrity, and to evaluate the influence of LTA exposure on sensitivity to the inflammation and neuronal dysfunction associated with Aß. Using BV2 and N2a cells as an in vitro model, we highlight that acute exposure to LTA robustly promotes inflammatory cytokine and nitric oxide (NO) production in microglia but also in neurons, similar to that reported under longer-term and chronic inflammatory conditions. Moreover, we find that exposure to LTA can enhance sensitivity to subthreshold Aß, promoting an 'M1'-like phenotype in microglia and provoking dysregulation of neuronal activity in acute hippocampal slices. Anti-inflammatory agents, including mimetics of brain-derived neurotrophic factor (BDNF), have proven effective at alleviating chronic neuroinflammatory complications. We further examined the effects of 7,8,3-trihydroxyflavone (7,8,3-THF), a small-molecule TrkB agonist, on LTA-induced microglial activation. We report that 7,8,3-THF can significantly ameliorate interleukin (IL)-6 and NO production in LTA-stimulated BV2 cells. Taken together, our findings offer support for exploration of TLR2 as a potential target for therapeutic intervention into acute neuroinflammatory conditions. Moreover we propose that exposure to gram-positive bacterial pathogens may promote sensitivity to the inflammatory changes characteristic of the aged brain.


Assuntos
Inflamação/metabolismo , Inflamação/fisiopatologia , Lipopolissacarídeos/toxicidade , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/fisiopatologia , Ácidos Teicoicos/toxicidade , Receptor 2 Toll-Like/agonistas , Doença Aguda , Peptídeos beta-Amiloides/toxicidade , Animais , Linhagem Celular , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Flavonas/farmacologia , Inflamação/induzido quimicamente , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Modelos Teóricos , Doenças do Sistema Nervoso/induzido quimicamente , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Nitritos/metabolismo , Receptor trkB/agonistas , Fator de Necrose Tumoral alfa/metabolismo
19.
Bioconjug Chem ; 31(6): 1685-1692, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32510940

RESUMO

Toll-like receptors (TLRs) are key pathogen sensors of the immune system. Their activation results in the production of cytokines, chemokines, and costimulatory molecules that are crucial for innate and adaptive immune responses. In recent years, specific (sub)-cellular location and timing of TLR activation have emerged as parameters for defining the signaling outcome and magnitude. To study the subtlety of this signaling, we here report a new molecular tool to control the activation of TLR2 via "click-to-release"-chemistry. We conjugated a bioorthogonal trans-cyclooctene (TCO) protecting group via solid support to a critical position within a synthetic TLR2/6 ligand to render the compound unable to initiate signaling. The TCO-group could then be conditionally removed upon addition of a tetrazine, resulting in restored agonist activity and TLR2 activation. This approach was validated on RAW264.7 macrophages and various murine primary immune cells as well as human cell line systems, demonstrating that TCO-caging constitutes a versatile approach for generating chemically controllable TLR2 agonists.


Assuntos
Ciclo-Octanos/química , Receptor 2 Toll-Like/metabolismo , Animais , Desenho de Fármacos , Humanos , Ligantes , Camundongos , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Estereoisomerismo , Receptor 2 Toll-Like/agonistas
20.
Rheumatology (Oxford) ; 59(11): 3533-3539, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32594150

RESUMO

OBJECTIVE: RA is an autoimmune inflammatory joint disease. Both RF and ACPA are associated with more progressive disease and higher levels of systemic inflammation. Monocyte activation of toll-like receptors (TLRs) by endogenous ligands is a potential source of increased production of systemic cytokines. RA monocytes have elevated TLRs, some of which are associated with the disease activity score using 28 joints (DAS28). The aim of this study was to measure TLR-induced cytokine production from monocytes, stratified by autoantibody status, to assess if their capacity to induce cytokines is related to autoantibody status or DAS28. METHODS: Peripheral blood monocytes isolated from RA patients and healthy controls were stimulated with TLR1/2, TLR2/6, TLR4, TLR5, TLR7, TLR8 and TLR9 ligands for 18 h before measuring IL-6, TNFα and IL-10. Serum was used to confirm the autoantibody status. Cytokine levels were compared with RF, ACPA and DAS28. RESULTS: RA monocytes demonstrated significantly increased IL-6 and TNFα upon TLR1/2 stimulation and IL-6 and IL-10 upon TLR5 activation. TLR7 and TLR9 activation did not induce cytokines and no significant differences were observed between RA and healthy control monocytes upon TLR2/6, TLR4 or TLR8 activation. When stratified by ACPA or RF status there were no correlations between autoantibody status and elevated cytokine levels. However, TLR1/2-induced IL-6 did correlate with DAS28. CONCLUSIONS: Elevated TLR-induced cytokines in RA monocytes were not related to ACPA or RF status. However, TLR1/2-induced IL-6 was associated with disease activity.


Assuntos
Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Citocinas/imunologia , Monócitos/imunologia , Fator Reumatoide/imunologia , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 5 Toll-Like/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antirreumáticos/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/fisiopatologia , Estudos de Casos e Controles , Feminino , Humanos , Interleucina-10/imunologia , Interleucina-6/imunologia , Ligantes , Masculino , Pessoa de Meia-Idade , Receptor 1 Toll-Like/agonistas , Receptor 2 Toll-Like/agonistas , Receptor 5 Toll-Like/agonistas , Receptores Toll-Like/agonistas , Receptores Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia
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