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1.
Front Immunol ; 12: 727161, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34603298

RESUMO

Plasmacytoid dendritic cells (pDCs) are known to respond to viral infections. However, the activation of pDCs by bacterial components such as lipopolysaccharides (LPS) has not been well studied. Here, we found that pDCs, conventional dendritic cells (cDCs), and B cells express high levels of toll-like receptor 4 (TLR4), a receptor for LPS. Moreover, LPS could effectively bind to not only cDCs but also pDCs and B cells. Intraperitoneal administration of LPS promoted activation of splenic pDCs and cDCs. LPS treatment led to upregulation of interferon regulatory factor 7 (IRF7) and induced production of interferon-alpha (IFN-α) in splenic pDCs. Furthermore, LPS-dependent upregulation of co-stimulatory molecules in pDCs did not require the assistance of other immune cells, such as cDCs. However, the production levels of IFN-α were decreased in cDC-depleted splenocytes, indicating that cDCs may contribute to the enhancement of IFN-α production in pDCs. Finally, we showed that activation of pDCs by LPS requires the TLR4 and myeloid differentiation factor 2 (MD2) signaling pathways. Thus, these results demonstrate that the gram-negative component LPS can directly stimulate pDCs via TLR4/MD2 stimulation in mice.


Assuntos
Células Dendríticas/imunologia , Lipopolissacarídeos , Antígeno 96 de Linfócito/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Feminino , Fator Regulador 7 de Interferon/imunologia , Interferon-alfa/imunologia , Antígeno 96 de Linfócito/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Baço/citologia , Baço/imunologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética
2.
Mol Immunol ; 139: 202-210, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34583098

RESUMO

A balance between the positive and negative regulation of toll-like receptor (TLR) signaling pathways is required to avoid detrimental and inappropriate inflammatory responses. Although some protein post-translational modifications (PTMs) such as phosphorylation and ubiquitination have been demonstrated to potently modulate innate immune responses, the role of methylation, an important PTM, control of TLR4 signaling pathway remains unclear. In this study, we found that protein arginine methyltransferase 1, 2 and 3 (PRMT1, 2 and 3) were recruited to methylate TLR4-CD (cytoplasmic domain) after lipopolysaccharide (LPS) stimulation respectively, but the effect of PRMT2 on arginine methylation of TLR4-CD is the most significant among above three PRMTs, which prompted us to focus on PRMT2. Reduction of PRMT2 expression down-regulated arginine (R) methylation level of TLR4 with or without LPS treatment. Methionine 115 (M115) mediated PRMT2 catalyzed-arginine methylation of TLR4 on R731 and R812. Furthermore, PRMT1, 2 and 3 was recruited to methylate interferon regulatory factor 3 (IRF3) after LPS stimulation respectively, but the effect of PRMT2 on arginine methylation of IRF3 is the most significant among the above three PRMTs. Arginine methylation of TLR4 on R812 or arginine methylation of IRF3 on R285 mediated the interaction between TLR4 and IRF3 respectively. Arginine methylation of IRF3 on R285 induced by LPS led to its dimerization and promoted its translocation from the cytoplasm to the nucleus. In addition, the enhancement of arginine methylation of TLR4 induced by PRMT1 or 2 increased IRF3 transcription activity with or without LPS treatment, while PRMT2 with histidine 112 glutamine (H112Q) or methionine 115 isoleucine (M115I) mutation and TLR4 with arginine 812 lysine (R812K) mutation decreased it. Arginine methylation of TLR4 on R812 or PRMT2 enhanced interferon-ß (IFN-ß) production. Our study reveals a critical role for PRMT2 and protein arginine methylation in the enhancement of IFN-ß production via TLR4/IRF3 signaling pathway and may provide a therapeutic strategy to control endotoxemia.


Assuntos
Arginina/metabolismo , Regulação da Expressão Gênica/imunologia , Processamento de Proteína Pós-Traducional/fisiologia , Proteína-Arginina N-Metiltransferases/metabolismo , Transdução de Sinais/fisiologia , Animais , Endotoxemia/imunologia , Endotoxemia/metabolismo , Células HEK293 , Humanos , Fator Regulador 3 de Interferon/imunologia , Fator Regulador 3 de Interferon/metabolismo , Interferon beta/imunologia , Interferon beta/metabolismo , Metilação , Camundongos , Proteína-Arginina N-Metiltransferases/imunologia , Células RAW 264.7 , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo
3.
Sci Rep ; 11(1): 16569, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34400677

RESUMO

Maternal immune adaptation to accommodate pregnancy depends on sufficient availability of regulatory T (Treg) cells to enable embryo implantation. Toll-like receptor 4 is implicated as a key upstream driver of a controlled inflammatory response, elicited by signals in male partner seminal fluid, to initiate expansion of the maternal Treg cell pool after mating. Here, we report that mice with null mutation in Tlr4 (Tlr4-/-) exhibit impaired reproductive outcomes after allogeneic mating, with reduced pregnancy rate, elevated mid-gestation fetal loss, and fetal growth restriction, compared to Tlr4+/+ wild-type controls. To investigate the effects of TLR4 deficiency on early events of maternal immune adaptation, TLR4-regulated cytokines and immune regulatory microRNAs were measured in the uterus at 8 h post-mating by qPCR, and Treg cells in uterus-draining lymph nodes were evaluated by flow cytometry on day 3.5 post-coitum. Ptgs2 encoding prostaglandin-endoperoxide synthase 2, cytokines Csf2, Il6, Lif, and Tnf, chemokines Ccl2, Cxcl1, Cxcl2, and Cxcl10, and microRNAs miR-155, miR-146a, and miR-223 were induced by mating in wild-type mice, but not, or to a lesser extent, in Tlr4-/- mice. CD4+ T cells were expanded after mating in Tlr4+/+ but not Tlr4-/- mice, with failure to expand peripheral CD25+FOXP3+ NRP1- or thymic CD25+FOXP3+ NRP1+ Treg cell populations, and fewer Treg cells expressed Ki67 proliferation marker and suppressive function marker CTLA4. We conclude that TLR4 is an essential mediator of the inflammation-like response in the pre-implantation uterus that induces generation of Treg cells to support robust pregnancy tolerance and ensure optimal fetal growth and survival.


Assuntos
Retardo do Crescimento Fetal/imunologia , Reabsorção do Feto/imunologia , Prenhez/imunologia , Receptor 4 Toll-Like/deficiência , Animais , Quimiotaxia de Leucócito , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Citocinas/biossíntese , Citocinas/genética , Feminino , Retardo do Crescimento Fetal/genética , Reabsorção do Feto/genética , Idade Gestacional , Mutação com Perda de Função , Linfonodos/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/biossíntese , MicroRNAs/genética , Tamanho do Órgão , Placenta/anatomia & histologia , Gravidez , Resultado da Gravidez , Taxa de Gravidez , Sêmen/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Útero/metabolismo
4.
Cells ; 10(8)2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34440913

RESUMO

The C1q/TNF-related protein 3 (CTRP3) represents a pleiotropic adipokine reciprocally associated with obesity and type 2 diabetes mellitus and exhibits anti-inflammatory properties in relation to lipopolysaccharides (LPS)-mediated effects in adipocytes, as well as monocytes/macrophages. Here, we focused on the influence of CTRP3 on LPS-mediated effects in endothelial cells in order to expand the understanding of a possible anti-inflammatory function of CTRP3 in a setting of endotoxemia. An organ- and tissue-specific expression analysis by real-time PCR revealed a considerable Ctrp3 expression in various adipose tissue compartments; however, higher levels were detected in the aorta and in abundantly perfused tissues (bone marrow and the thyroid gland). We observed a robust Ctrp3 expression in primary endothelial cells and a transient upregulation in murine endothelial (MyEND) cells by LPS (50 ng/mL). In MyEND cells, CTRP3 inhibited the LPS-induced expression of interleukin (Il)-6 and the tumor necrosis factor (Tnf)-α, and suppressed the LPS-dependent expression of the major endothelial adhesion molecules Vcam-1 and Icam-1. The LPS-induced adhesion of monocytic cells to an endothelial monolayer was antagonized by CTRP3. In C57BL/6J mice with an LPS-induced systemic inflammation, exogenous CTRP3 did not affect circulating levels of TNF-α, ICAM-1, and VCAM-1. In conclusion, we characterized CTRP3 beyond its function as an adipokine in a setting of vascular inflammation. CTRP3 inhibited LPS-induced endothelial expression of adhesion molecules and monocyte cell adhesion, indicating an important vascular anti-inflammatory role for CTRP3 in endotoxemia.


Assuntos
Adipocinas/imunologia , Tecido Adiposo/imunologia , Células Endoteliais/imunologia , Perfilação da Expressão Gênica , Inflamação/imunologia , Adipocinas/genética , Adipocinas/metabolismo , Tecido Adiposo/metabolismo , Animais , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Adesão Celular/imunologia , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Humanos , Inflamação/genética , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34445142

RESUMO

It is difficult to treat allergic diseases including asthma completely because its pathogenesis remains unclear. House dust mite (HDM) is a critical allergen and Toll-like receptor (TLR) 4 is a member of the toll-like receptor family, which plays an important role in allergic diseases. The purpose of this study was to characterize a novel allergen, Der f 38 binding to TLR4, and unveil its role as an inducer of allergy. Der f 38 expression was detected in the body and feces of Dermatophagoides farinae (DF). Electron microscopy revealed that it was located in the granule layer, the epithelium layer, and microvilli of the posterior midgut. The skin prick test showed that 60% of allergic subjects were Der f 38-positive. Der f 38 enhanced surface 203c expression in basophils of Der f 38-positive allergic subjects. By analysis of the model structure of Der p 38, the expected epitope sites are exposed on the exterior side. In animal experiments, Der f 38 triggered an infiltration of inflammatory cells. Intranasal (IN) administration of Der f 38 increased neutrophils in the lung. Intraperitoneal (IP) and IN injections of Der f 38 induced both eosinophils and neutrophils. Increased total IgE level and histopathological features were found in BALB/c mice treated with Der f 38 by IP and IN injections. TLR4 knockout (KO) BALB/c mice exhibited less inflammation and IgE level in the sera compared to wild type (WT) mice. Der f 38 directly binds to TLR4 using biolayer interferometry. Der f 38 suppressed the apoptosis of neutrophils and eosinophils by downregulating proteins in the proapoptotic pathway including caspase 9, caspase 3, and BAX and upregulating proteins in the anti-apoptotic pathway including BCL-2 and MCL-1. These findings might shed light on the pathogenic mechanisms of allergy to HDM.


Assuntos
Alérgenos/imunologia , Antígenos de Dermatophagoides/imunologia , Proteínas de Artrópodes/imunologia , Dermatophagoides farinae/imunologia , Hipersensibilidade/imunologia , Ligação Proteica/imunologia , Receptor 4 Toll-Like/imunologia , Sequência de Aminoácidos , Animais , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina E/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pyroglyphidae/metabolismo , Testes Cutâneos/métodos
6.
Biomolecules ; 11(8)2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34439812

RESUMO

Intra-amniotic infections (IAI) are one of the reasons for preterm birth. High mobility group box 1 (HMGB1) is a nuclear protein with various physiological functions, including tissue healing. Its excessive extracellular release potentiates inflammatory reaction and can revert its action from beneficial to detrimental. We infected the amniotic fluid of a pig on the 80th day of gestation with 1 × 104 colony forming units (CFUs) of E. coli O55 for 10 h, and evaluated the appearance of HMGB1, receptor for glycation endproducts (RAGE), and Toll-like receptor (TLR) 4 in the amniotic membrane and fluid. Sham-infected amniotic fluid served as a control. The expression and release of HMGB1 were evaluated by Real-Time PCR, immunofluorescence, immunohistochemistry, and ELISA. The infection downregulated HMGB1 mRNA expression in the amniotic membrane, changed the distribution of HMGB1 protein in the amniotic membrane, and increased its level in amniotic fluid. All RAGE mRNA, protein expression in the amniotic membrane, and soluble RAGE level in the amniotic fluid were downregulated. TLR4 mRNA and protein expression and soluble TLR4 were all upregulated. HMGB1 is a potential target for therapy to suppress the exaggerated inflammatory response. This controlled expression and release can, in some cases, prevent the preterm birth of vulnerable infants. Studies on suitable animal models can contribute to the development of appropriate therapy.


Assuntos
Infecções por Escherichia coli/veterinária , Escherichia coli/patogenicidade , Proteína HMGB1/genética , Complicações Infecciosas na Gravidez/veterinária , RNA Mensageiro/genética , Receptor para Produtos Finais de Glicação Avançada/genética , Receptor 4 Toll-Like/genética , Âmnio/imunologia , Âmnio/microbiologia , Âmnio/patologia , Líquido Amniótico/imunologia , Líquido Amniótico/microbiologia , Animais , Modelos Animais de Doenças , Escherichia coli/crescimento & desenvolvimento , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/imunologia , Infecções por Escherichia coli/microbiologia , Feminino , Regulação da Expressão Gênica , Proteína HMGB1/imunologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Gravidez , Complicações Infecciosas na Gravidez/genética , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/microbiologia , Nascimento Prematuro/prevenção & controle , RNA Mensageiro/imunologia , Receptor para Produtos Finais de Glicação Avançada/imunologia , Transdução de Sinais , Suínos , Receptor 4 Toll-Like/imunologia
7.
FASEB J ; 35(9): e21866, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34416031

RESUMO

Macrophage migration inhibitory factor (MIF), an immunoregulatory cytokine plays an important role in inflammation and the immune response, and has been described as having a potential role in immune evasion by parasites. Thelazia callipaeda, a vector-borne zoonotic eye worm with a broad host range, has been documented as an agent of ocular infection of thelaziosis. The ability of T. callipaeda to persist in an immunologically competent host has led to the suggestion that it has evolved specific measures to counter immune defenses. To date, whether the immune evasion of T. callipaeda is related to MIF and the possible related signaling pathway and molecular mechanism have remained unclear. In the present study, we examined the effect of T. callipaeda MIF (T. cp-MIF) on macrophages. We analyzed the antigenic epitopes of the candidate T. cp-MIF and found that it exhibited an ideal antigenic index. Morphology, Flow cytometry, and cytokine analysis showed that T. cp-MIF induced the dynamic polarization of THP-1 macrophages from the M1-like phenotype to the M2-like phenotype. The chemotaxis assay revealed an inhibitory effect of T. cp-MIF on THP-1 macrophages. Western blotting suggested that, compared to the control, THP-1 macrophages exposed to T. cp-MIF had higher TLR4 protein expression and the phosphatidylinositol 3'-kinase (PI3K) -Akt pathway activation. In conclusion, T. cp-MIF induces M2-like macrophage polarization through TLR4-mediated activation of the PI3K-Akt pathway, which might provide a basis for future research on how it affects the immune system of the host.


Assuntos
Fatores Inibidores da Migração de Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia , Epitopos , Humanos , Células THP-1
8.
Carbohydr Polym ; 269: 118345, 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34294352

RESUMO

This work reports novel chitosan functionalized graphene oxide (GO) nanocomposites combined fluorescence imaging and therapeutic functions in one agent, which can serve as a promising alternative to alleviate related diseases caused hyperinflammation. Briefly, GO was designed to be conjugated with chitosan, fluorescein-labeled peptide, toll-like receptor 4 antibody and hydroxycamptothecin/aloe emodin. We have demonstrated that such nanocomposites could effectively achieve active targeted delivery of pro-apoptotic and anti-inflammatory drugs into inflammatory cells and cause cells apoptosis by acid-responsive drug release. Moreover, confocal fluorescence imaging confirms that the drug-induced inflammatory cells apoptosis could be visualized the light-up fluorescence of fluorescein activated by caspase-3. Meanwhile, inflammatory-related biomarkers have down-regulated after the nanocomposites' treatment in both vitro and vivo experiments consistent with the results in histological sections. In summary, the bifunctional nanocomposites that possess anti-inflammation and fluorescence imaging could serve as a promising therapeutic agent for reducing hyperinflammation caused by numerous diseases.


Assuntos
Anti-Inflamatórios/uso terapêutico , Apoptose/fisiologia , Portadores de Fármacos/química , Inflamação/tratamento farmacológico , Nanocompostos/química , Animais , Anti-Inflamatórios/química , Anticorpos/imunologia , Camptotecina/análogos & derivados , Camptotecina/química , Camptotecina/uso terapêutico , Bovinos , Linhagem Celular , Quitosana/química , Liberação Controlada de Fármacos , Emodina/química , Emodina/uso terapêutico , Corantes Fluorescentes/química , Grafite/química , Humanos , Lipopolissacarídeos , Glândulas Mamárias Humanas/efeitos dos fármacos , Glândulas Mamárias Humanas/patologia , Mastite/induzido quimicamente , Mastite/tratamento farmacológico , Mastite/patologia , Camundongos , Receptor 4 Toll-Like/imunologia
9.
Front Immunol ; 12: 705080, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34282358

RESUMO

Respiratory viral infections have been a long-standing global burden ranging from seasonal recurrences to the unexpected pandemics. The yearly hospitalizations from seasonal viruses such as influenza can fluctuate greatly depending on the circulating strain(s) and the congruency with the predicted strains used for the yearly vaccine formulation, which often are not predicted accurately. While antiviral agents are available against influenza, efficacy is limited due to a temporal disconnect between the time of infection and symptom development and viral resistance. Uncontrolled, influenza infections can lead to a severe inflammatory response initiated by pathogen-associated molecular patterns (PAMPs) or host-derived danger-associated molecular patterns (DAMPs) that ultimately signal through pattern recognition receptors (PRRs). Overall, these pathogen-host interactions result in a local cytokine storm leading to acute lung injury (ALI) or the more severe acute respiratory distress syndrome (ARDS) with concomitant systemic involvement and more severe, life threatening consequences. In addition to traditional antiviral treatments, blocking the host's innate immune response may provide a more viable approach to combat these infectious pathogens. The SARS-CoV-2 pandemic illustrates a critical need for novel treatments to counteract the ALI and ARDS that has caused the deaths of millions worldwide. This review will examine how antagonizing TLR4 signaling has been effective experimentally in ameliorating ALI and lethal infection in challenge models triggered not only by influenza, but also by other ALI-inducing viruses.


Assuntos
Lesão Pulmonar Aguda/imunologia , COVID-19/imunologia , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Lesão Pulmonar Aguda/prevenção & controle , Lesão Pulmonar Aguda/virologia , Antivirais/uso terapêutico , COVID-19/epidemiologia , COVID-19/virologia , Síndrome da Liberação de Citocina/imunologia , Síndrome da Liberação de Citocina/metabolismo , Síndrome da Liberação de Citocina/prevenção & controle , Humanos , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/virologia , Pandemias , SARS-CoV-2/fisiologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo
10.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34206009

RESUMO

Toll-like receptor (TLR) signaling plays a critical role in the induction and progression of autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematous, experimental autoimmune encephalitis, type 1 diabetes mellitus and neurodegenerative diseases. Deciphering antigen recognition by antibodies provides insights and defines the mechanism of action into the progression of immune responses. Multiple strategies, including phage display and hybridoma technologies, have been used to enhance the affinity of antibodies for their respective epitopes. Here, we investigate the TLR4 antibody-binding epitope by computational-driven approach. We demonstrate that three important residues, i.e., Y328, N329, and K349 of TLR4 antibody binding epitope identified upon in silico mutagenesis, affect not only the interaction and binding affinity of antibody but also influence the structural integrity of TLR4. Furthermore, we predict a novel epitope at the TLR4-MD2 interface which can be targeted and explored for therapeutic antibodies and small molecules. This technique provides an in-depth insight into antibody-antigen interactions at the resolution and will be beneficial for the development of new monoclonal antibodies. Computational techniques, if coupled with experimental methods, will shorten the duration of rational design and development of antibody therapeutics.


Assuntos
Anticorpos Monoclonais/imunologia , Artrite Reumatoide/imunologia , Encefalite/imunologia , Epitopos/genética , Doença de Hashimoto/imunologia , Doenças Neurodegenerativas/imunologia , Receptor 4 Toll-Like/genética , Sequência de Aminoácidos/genética , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/uso terapêutico , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/genética , Técnicas de Visualização da Superfície Celular , Encefalite/genética , Encefalite/patologia , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Doença de Hashimoto/genética , Doença de Hashimoto/patologia , Humanos , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Ligação Proteica/genética , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/imunologia
11.
Front Immunol ; 12: 660065, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34234775

RESUMO

Toll-like receptors (TLRs) play a crucial role in the recognition of pathogen-derived components as a first line of defense against infections. It has been suggested that depending on the nature of the pathogens, TLRs activation induce a distinct cytokine profile that may contribute to the polarization of the acquired immune response. Here, we investigated the early MAPK signaling activation via TLR4 and TLR2 receptors and its impact in differential cytokine profile by macrophages. We found that TLR2 ligands activated MAPKs p38 and ERK earlier compared to the TLR4 ligand LPS in macrophages. Higher IL-10/IL-12 and IL-10/TNF-α ratios were also observed at later time points in response to TLR2 ligands compared to LPS. The results also indicate an earlier activation of the phosphatase MKP-1 and that MKP-1 KO macrophages show a prolongation in p38 phosphorylation in response to TLR2 stimulation. Furthermore, p38 is critical for IL-10 expression in response to TLR2 ligands, which triggers the macrophage change to a M2 and regulatory phenotype in contrast to the M1 phenotype induced by TLR4 activation. Therefore, the early TLR2-mediated p38 induction contributes for the high IL-10 production, likely as a virulence strategy to suppress host Th1 response against certain types of pathogens.


Assuntos
Fosfatase 1 de Especificidade Dupla/imunologia , Interleucina-10/imunologia , Macrófagos/imunologia , Receptor 2 Toll-Like/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Células Cultivadas , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Diglicerídeos/farmacologia , Fosfatase 1 de Especificidade Dupla/genética , Fosfatase 1 de Especificidade Dupla/metabolismo , Ativação Enzimática/imunologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Interleucina-10/genética , Interleucina-10/metabolismo , Lipopeptídeos/farmacologia , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oligopeptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Células RAW 264.7 , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
PLoS Pathog ; 17(7): e1009781, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34280250

RESUMO

Cytokines made by macrophages play a critical role in determining the course of Legionella pneumophila infection. Prior murine-based modeling indicated that this cytokine response is initiated upon recognition of L. pneumophila by a subset of Toll-like receptors, namely TLR2, TLR5, and TLR9. Through the use of shRNA/siRNA knockdowns and subsequently CRISPR/Cas9 knockouts (KO), we determined that TRIF, an adaptor downstream of endosomal TLR3 and TLR4, is required for full cytokine secretion by human primary and cell-line macrophages. By characterizing a further set of TLR KO's in human U937 cells, we discerned that, contrary to the viewpoint garnered from murine-based studies, TLR3 and TLR4 (along with TLR2 and TLR5) are in fact vital to the macrophage response in the early stages of L. pneumophila infection. This conclusion was bolstered by showing that i) chemical inhibitors of TLR3 and TLR4 dampen the cytokine output of primary human macrophages and ii) transfection of TLR3 and TLR4 into HEK cells conferred an ability to sense L. pneumophila. TLR3- and TLR4-dependent cytokines promoted migration of human HL-60 neutrophils across an epithelial layer, pointing to the biological importance for the newfound signaling pathway. The response of U937 cells to L. pneumophila LPS was dependent upon TLR4, a further contradiction to murine-based studies, which had concluded that TLR2 is the receptor for Legionella LPS. Given the role of TLR3 in sensing nucleic acid (i.e., dsRNA), we utilized newly-made KO U937 cells to document that DNA-sensing by cGAS-STING and DNA-PK are also needed for the response of human macrophages to L. pneumophila. Given the lack of attention given them in the bacterial field, C-type lectin receptors were similarly examined; but, they were not required. Overall, this study arguably represents the most extensive, single-characterization of Legionella-recognition receptors within human macrophages.


Assuntos
Doença dos Legionários/imunologia , Macrófagos/imunologia , Padrões Moleculares Associados a Patógenos/imunologia , Receptor 3 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Proteínas de Bactérias/imunologia , Humanos , Legionella pneumophila/imunologia , Lipopolissacarídeos/imunologia , Macrófagos/metabolismo , Camundongos , Padrões Moleculares Associados a Patógenos/metabolismo , Receptor 3 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
13.
Front Immunol ; 12: 691039, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34322122

RESUMO

Background: Clostridioides difficile is the leading cause of nosocomial infectious diarrhea. Toll-like receptors (TLRs) are the major components of innate immunity that sense pathogens. The relationship between TLRs and C. difficile infection (CDI) was analyzed in clinical patients and a mouse model. Materials and Methods: A prospective investigation was conducted in medical wards of Tainan Hospital, Ministry of Health and Welfare, Tainan, Taiwan, from January 2011 to January 2013. Adult patients were followed up for the development of CDI. Single nucleotide polymorphisms (SNPs) of TLR2 and TLR4 were analyzed to assess the relationship between genetic polymorphisms and the development of CDI. A mouse model of CDI was used to investigate the pathogenic role of TLRs in CDI, TLR2 and TLR4 knockout (Tlr2-/- and Tlr4-/-) mice. Results: In the prospective study, 556 patients were enrolled, and 6.5% (36) of patients, accounting for 3.59 episodes per 1000 patient-days, developed CDI. Of 539 patients with available blood samples, the TLR2 rs3804099 polymorphism was more often noted in those with CDI than in those without CDI (64.5% vs. 46.1%; P = 0.046) but was not significant in multivariate analysis. Because the TLR2 rs3804099 polymorphism was moderately associated with CDI, the role of TLR2 and TLR4 was further evaluated in a mouse model. Both Tlr2-/- and Tlr4-/- mice showed more severe CDI disease than wild-type mice in terms of body weight change and fecal content five days after oral challenge with C. difficile. Furthermore, Tlr2-/- mice suffered from more severe disease than Tlr4-/- mice, as evidenced by stool consistency, cecum weight, and survival rate. Conclusion: The TLR2 rs3804099 polymorphism is marginally associated with the development of CDI, and the pathogenic role of TLR2 is further supported by a mouse model.


Assuntos
Infecções por Clostridium/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Grupo com Ancestrais do Continente Asiático/genética , Clostridioides difficile , Infecções por Clostridium/genética , Infecções por Clostridium/patologia , Colo/imunologia , Colo/patologia , Modelos Animais de Doenças , Feminino , Genótipo , Humanos , Masculino , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia
14.
Infect Immun ; 89(10): e0009121, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34152806

RESUMO

Of the 486,000 burn injuries that required medical treatment in the United States in 2016, 40,000 people were hospitalized, with >3,000 fatalities. After burn injury, humans are at increased risk of sepsis and mortality from infections caused by Pseudomonas aeruginosa, an opportunistic pathogen. We hypothesize that systemic events were initiated from the burn that increased the host's susceptibility to P. aeruginosa. A nonlethal 10% total body surface area (TBSA), full-thickness flame burn was performed in CD-1 mice without and with subsequent P. aeruginosa (strain M2) infection. The 50% lethal dose for subcutaneous infection with P. aeruginosa M2 at the burn site immediately after the burn decreased by 6 log, with mortality occurring between 18 and 26 h, compared with P. aeruginosa-infected mice without burn injury. Bacteria in distal organs were detected by 18 h, concurrent with the onset of clinical symptoms. Serum proinflammatory cytokines (interleukin-6 [IL-6], IL-1ß, gamma interferon, and tumor necrosis factor alpha) and the anti-inflammatory cytokine IL-10 were first detected at 12 h postburn with infection and continued to increase until death. Directly after burn alone, serum levels of HMGB1, a danger-associated molecular pattern and TLR4 agonist, transiently increased to 50 ng/ml before returning to 20 ng/ml. Burn with P. aeruginosa infection increased serum HMGB1 concentrations >10-fold (250 ng/ml) at the time of death. This HMGB1-rich serum stimulated TLR4-mediated NF-κB activation in a TLR4 reporter cell line. Treatment of infected burned mice with P5779, a peptide inhibitor of HMGB1, increased the mean survival from 23 to 42 h (P < 0.0001). We conclude that the high level of serum HMGB1, which preceded the increase in proinflammatory cytokines, is associated with postburn mortality.


Assuntos
Queimaduras/imunologia , Queimaduras/microbiologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Modelos Animais de Doenças , Feminino , Proteína HMGB1/imunologia , Inflamação/imunologia , Inflamação/microbiologia , Interferon gama/imunologia , Interleucina-10/imunologia , Interleucina-6/imunologia , Camundongos , NF-kappa B/imunologia , Sepse/imunologia , Sepse/microbiologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Fator de Necrose Tumoral alfa/imunologia
15.
Chem Commun (Camb) ; 57(50): 6209-6212, 2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34059855

RESUMO

Toll-like receptor 4 (TLR4) recognizes various protein ligands; however, the protein-TLR4 binding model is unclear. Here we demonstrate a Crenomytilus grayanus lectin (CGL)-TLR4/MD2 model to show that CGL interacts with a TLR4/myeloid differentiation factor 2 (MD2) complex independently of sugar-binding properties. CGL could suppress lipopolysaccharide-induced immune responses significantly, suggesting that TLR4 itself has potential as a therapeutic target.


Assuntos
Carboidratos/química , Lectinas/química , Antígeno 96 de Linfócito/química , Receptor 4 Toll-Like/química , Animais , Sítios de Ligação , Bivalves , Carboidratos/imunologia , Humanos , Lectinas/imunologia , Antígeno 96 de Linfócito/imunologia , Receptor 4 Toll-Like/imunologia
16.
Nat Commun ; 12(1): 3519, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112781

RESUMO

TLR4 signaling plays key roles in the innate immune response to microbial infection. Innate immune cells encounter different mechanical cues in both health and disease to adapt their behaviors. However, the impact of mechanical sensing signals on TLR4 signal-mediated innate immune response remains unclear. Here we show that TLR4 signalling augments macrophage bactericidal activity through the mechanical sensor Piezo1. Bacterial infection or LPS stimulation triggers assembly of the complex of Piezo1 and TLR4 to remodel F-actin organization and augment phagocytosis, mitochondrion-phagosomal ROS production and bacterial clearance and genetic deficiency of Piezo1 results in abrogation of these responses. Mechanistically, LPS stimulates TLR4 to induce Piezo1-mediated calcium influx and consequently activates CaMKII-Mst1/2-Rac axis for pathogen ingestion and killing. Inhibition of CaMKII or knockout of either Mst1/2 or Rac1 results in reduced macrophage bactericidal activity, phenocopying the Piezo1 deficiency. Thus, we conclude that TLR4 drives the innate immune response via Piezo1 providing critical insight for understanding macrophage mechanophysiology and the host response.


Assuntos
Infecções Bacterianas/imunologia , Imunidade Inata , Canais Iônicos/metabolismo , Macrófagos/imunologia , Fagossomos/metabolismo , Receptor 4 Toll-Like/metabolismo , Actinas/metabolismo , Animais , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Citoesqueleto/genética , Citoesqueleto/metabolismo , Infecções por Escherichia coli/imunologia , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Canais Iônicos/genética , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Fagocitose/imunologia , Fagossomos/imunologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
17.
Emerg Microbes Infect ; 10(1): 1358-1368, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34132167

RESUMO

Respiratory infections caused by Bordetella pertussis are reemerging despite high pertussis vaccination coverage. Since the introduction of the acellular pertussis vaccine in the late twentieth century, circulating B. pertussis strains increasingly lack expression of the vaccine component pertactin (Prn). In some countries, up to 90% of the circulating B. pertussis strains are deficient in Prn. To better understand the resurgence of pertussis, we investigated the response of human monocyte-derived dendritic cells (moDCs) to naturally circulating Prn-expressing (Prn-Pos) and Prn-deficient (Prn-Neg) B. pertussis strains from 2016 in the Netherlands. Transcriptome analysis of moDC showed enriched IFNα response-associated gene expression after exposure to Prn-Pos B. pertussis strains, whereas the Prn-Neg strains induced enriched expression of interleukin- and TNF-signaling genes, as well as other genes involved in immune activation. Multiplex immune assays confirmed enhanced proinflammatory cytokine secretion by Prn-Neg stimulated moDC. Comparison of the proteomes from the Prn-Pos and Prn-Neg strains revealed, next to the difference in Prn, differential expression of a number of other proteins including several proteins involved in metabolic processes. Our findings indicate that Prn-deficient B. pertussis strains induce a distinct and stronger immune activation of moDCs than the Prn-Pos strains. These findings highlight the role of pathogen adaptation in the resurgence of pertussis as well as the effects that vaccine pressure can have on a bacterial population.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Bordetella pertussis/imunologia , Células Dendríticas/imunologia , Transcriptoma , Fatores de Virulência de Bordetella/genética , Adaptação Biológica , Proteínas da Membrana Bacteriana Externa/metabolismo , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Bordetella pertussis/patogenicidade , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/metabolismo , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Inflamação , Vacina contra Coqueluche/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 4 Toll-Like/imunologia , Fatores de Virulência de Bordetella/metabolismo , Coqueluche/microbiologia
18.
Mol Med Rep ; 24(2)2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34184086

RESUMO

A humanized anti­Toll­like receptor 4 (TLR4) monoclonal antibody (mAb) was previously produced using phage antibody library technology, and it was found that the mAb could effectively ameliorate lipopolysaccharide (LPS)­induced damage in macrophages. The present study investigated the protective effects exerted by the humanized anti­TLR4 mAb against LPS­induced acute kidney injury (AKI), as well as the underlying mechanisms. Female C57BL/6 mice were randomly divided into four groups (n=8 per group): i) Control; ii) LPS; iii) LPS + humanized anti­TLR4 mAb (1 µg/g); and iv) LPS + humanized anti­TLR4 mAb (10 µg/g). Serum creatinine, blood urea nitrogen, IL­6, TNFα and IL­1ß levels were then examined, followed by renal pathology assessment, immunohistochemical staining, reverse transcription­quantitative PCR and western blotting to assess apoptosis/survival/inflammation­related molecules and kidney injury molecule (KIM)­1. The humanized anti­TLR4 mAb successfully ameliorated LPS­induced AKI and renal pathological damage. The humanized anti­TLR4 mAb also dose­dependently suppressed LPS­induced elevations in serum IL­6, TNFα and IL­1ß, and decreased the renal expression levels of myeloid differentiation primary response 88 (MyD88), IKKα/ß, IκB, p65 and KIM­1. Compared with the LPS group, renal Bax and KIM­1 expression levels were significantly downregulated, and Bcl­2 expression was notably upregulated by the humanized anti­TLR4 mAb. Moreover, the humanized anti­TLR4 mAb also significantly decreased the protein expression levels of MyD88, phosphorylated (p)­IKKα/ß, p­IκB and p­p65 in the renal tissues compared with the LPS group. Therefore, the present study indicated that the anti­inflammatory effects of the humanized anti­TLR4 mAb against LPS­related AKI in mice were mediated via inhibition of the TLR4/NF­κB signaling pathway.


Assuntos
Injúria Renal Aguda/tratamento farmacológico , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais Humanizados/uso terapêutico , NF-kappa B/antagonistas & inibidores , Substâncias Protetoras/farmacologia , Substâncias Protetoras/uso terapêutico , Receptor 4 Toll-Like/imunologia , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/patologia , Animais , Citocinas/sangue , Modelos Animais de Doenças , Feminino , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Quinase I-kappa B/genética , Quinase I-kappa B/metabolismo , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Lipopolissacarídeos/toxicidade , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Proteína X Associada a bcl-2/metabolismo
19.
Biochim Biophys Acta Mol Basis Dis ; 1867(8): 166155, 2021 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-33932524

RESUMO

Glioblastoma (GB) is the most common and aggressive form of primary brain tumor, in which the presence of an inflammatory environment, composed mainly by tumor-associated macrophages (TAMs), is related to its progression and development of chemoresistance. Toll-Like Receptors (TLRs) are key components of the innate immune system and their expression in both tumor and immune-associated cells may impact the cell communication in the tumor microenvironment (TME), further modeling cancer growth and response to therapy. Here, we investigated the participation of TLR4-mediated signaling as a mechanism of induced-immune escape in GB. Initially, bioinformatics analysis of public datasets revealed that TLR4 expression is lower in GB tumors when compared to astrocytomas (AST), and in a subset of TAMs. Further, we confirmed that TLR4 expression is downregulated in chemoresistant GB, as well as in macrophages co-cultured with GB cells. Additionally, TLR4 function is impaired in those cells even following stimulation with LPS, an agonist of TLR4. Finally, experiments performed in a cohort of clinical primary and metastatic brain tumors indicated that the immunostaining of TLR4 and CD45 are inversely proportional, and confirmed the low TLR4 expression in GBs. Interestingly, the cytoplasmic/nuclear pattern of TLR4 staining in cancer tissues suggests additional roles of this receptor in carcinogenesis. Overall, our data suggest the downregulation of TLR4 expression and activity as a strategy for GB-associated immune escape. Additional studies are necessary to better understand TLR4 signaling in TME in order to improve the benefits of immunotherapy based on TLR signaling.


Assuntos
Neoplasias Encefálicas/imunologia , Regulação para Baixo/imunologia , Glioblastoma/imunologia , Glioblastoma/metabolismo , Evasão da Resposta Imune/imunologia , Receptor 4 Toll-Like/imunologia , Macrófagos Associados a Tumor/imunologia , Idoso , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/metabolismo , Microambiente Tumoral/imunologia , Macrófagos Associados a Tumor/metabolismo
20.
Nat Commun ; 12(1): 2776, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986291

RESUMO

Radiation therapy (RT) is used in the management of several cancers; however, tumor radioresistance remains a challenge. Polymorphonuclear neutrophils (PMNs) are recruited to the tumor immune microenvironment (TIME) post-RT and can facilitate tumor progression by forming neutrophil extracellular traps (NETs). Here, we demonstrate a role for NETs as players in tumor radioresistance. Using a syngeneic bladder cancer model, increased NET deposition is observed in the TIME of mice treated with RT and inhibition of NETs improves overall radiation response. In vitro, the protein HMGB1 promotes NET formation through a TLR4-dependent manner and in vivo, inhibition of both HMGB1 and NETs significantly delays tumor growth. Finally, NETs are observed in bladder tumors of patients who did not respond to RT and had persistent disease post-RT, wherein a high tumoral PMN-to-CD8 ratio is associated with worse overall survival. Together, these findings identify NETs as a potential therapeutic target to increase radiation efficacy.


Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/imunologia , Tolerância a Radiação/imunologia , Neoplasias da Bexiga Urinária/radioterapia , Idoso , Idoso de 80 Anos ou mais , Animais , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Proteína HMGB1/antagonistas & inibidores , Proteína HMGB1/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Microambiente Tumoral/imunologia , Neoplasias da Bexiga Urinária/patologia
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