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1.
Int J Mol Sci ; 23(13)2022 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-35806134

RESUMO

Pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), induce the expression of intracellular adhesion molecule-1 (ICAM-1) by activating the nuclear factor κB (NF-κB) signaling pathway. In the present study, we found that cucurbitacin B decreased the expression of ICAM-1 in human lung adenocarcinoma A549 cells stimulated with TNF-α or interleukin-1α. We further investigated the mechanisms by which cucurbitacin B down-regulates TNF-α-induced ICAM-1 expression. Cucurbitacin B inhibited the nuclear translocation of the NF-κB subunit RelA and the phosphorylation of IκBα in A549 cells stimulated with TNF-α. Cucurbitacin B selectively down-regulated the expression of TNF receptor 1 (TNF-R1) without affecting three adaptor proteins (i.e., TRADD, RIPK1, and TRAF2). The TNF-α-converting enzyme inhibitor suppressed the down-regulation of TNF-R1 expression by cucurbitacin B. Glutathione, N-acetyl-L-cysteine, and, to a lesser extent, L-cysteine attenuated the inhibitory effects of cucurbitacin B on the TNF-α-induced expression of ICAM-1, suggesting that an α,ß-unsaturated carbonyl moiety is essential for anti-inflammatory activity. The present results revealed that cucurbitacin B down-regulated the expression of TNF-R1 at the initial step in the TNF-α-dependent NF-κB signaling pathway.


Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Células A549 , Adenocarcinoma de Pulmão/tratamento farmacológico , Humanos , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , NF-kappa B/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Transdução de Sinais , Triterpenos , Fator de Necrose Tumoral alfa/metabolismo
2.
Vaccines (Basel) ; 10(7)2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35891198

RESUMO

Uncovering the predictors of vaccine immunogenicity is essential for infection control. We have reported that the most prevalent polymorphism of the aldehyde dehydrogenase 2 gene (ALDH2), rs671, may be associated with an attenuated immune system. To test the inverse relationship between rs671 and antibody production after COVID-19 vaccination, the levels of anti-SARS-CoV-2 Spike protein S1 subunit (S1) IgG were repeatedly measured for four months before and after vaccination with BNT162b2 or mRNA-1273, in 88 Japanese workers and students (including 45 females, aged 21-56 years, with an rs671 variant allele frequency of 0.3). The mixed model including fixed effects of the vaccine type, weeks post vaccination (categorical variable), sex, age, height, smoking status, ethanol intake, exercise habit, perceived stress, steroid use, allergic diseases, and dyslipidemia, indicated an inverse association between log-transformed anti-S1 IgG levels and the number of rs671 variant alleles (partial regression coefficient = -0.15, p = 0.002). Our study indicated for the first time that the variant allele of ALDH2, rs671, is associated with the attenuated immunogenicity of COVID-19 mRNA vaccines. Our finding may provide a basis for personalized disease prevention based on a genetic polymorphism that is prevalent among East Asians.

3.
Int J Mol Sci ; 22(23)2021 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-34884902

RESUMO

The T-box transcription factor Eomesodermin (Eomes) promotes the expression of interferon-γ (IFN-γ). We recently reported that the small molecule inhibitors, TPCA-1 and IKK-16, which target nuclear factor κB (NF-κB) activation, moderately reduced Eomes-dependent IFN-γ expression in mouse lymphoma BW5147 cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In the present study, we investigated the direct effects of NF-κB on IFN-γ expression in mouse lymphoma EL4 cells and primary effector T cells. Eomes strongly promoted IFN-γ expression and the binding of RelA and NFATc2 to the IFN-γ promoter when EL4 cells were stimulated with PMA and IM. Neither TPCA-1 nor IKK-16 reduced IFN-γ expression; however, they markedly decreased interleukin (IL)-2 expression in Eomes-transfected EL4 cells. Moreover, TPCA-1 markedly inhibited the binding of RelA, but not that of Eomes or NFATc2 to the IFN-γ promoter. In effector CD4+ and CD8+ T cells activated with anti-CD3 and anti-CD28 antibodies, IFN-γ expression induced by PMA and A23187 was not markedly decreased by TPCA-1 or IKK-16 under conditions where IL-2 expression was markedly reduced. Therefore, the present results revealed that NF-κB is dispensable for IFN-γ expression induced by PMA and calcium ionophores in EL4 cells expressing Eomes and primary effector T cells.


Assuntos
Ionóforos de Cálcio/farmacologia , Interferon gama/genética , NF-kappa B/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Amidas/farmacologia , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Camundongos , Piperidinas/farmacologia , Cultura Primária de Células , Regiões Promotoras Genéticas/efeitos dos fármacos , Pirrolidinas/farmacologia , Proteínas com Domínio T/metabolismo , Tiofenos/farmacologia
5.
Eur J Pharmacol ; 890: 173651, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-33049301

RESUMO

α-Conidendrin is a lignan isolated from Taxus wallichiana and other species. In the present study, we demonstrated that α-conidendrin inhibited the cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) induced by tumor necrosis factor-α (TNF-α) at an IC50 value of 40-60 µM in human lung adenocarcinoma A549 cells. α-Conidendrin decreased ICAM-1 protein and mRNA expression levels at concentrations of 40-100 µM in TNF-α-stimulated A549 cells. The TNF-α-induced mRNA expression of vascular cell adhesion molecule-1, E-selectin, and cyclooxygenase-2 was also reduced by α-conidendrin. In the TNF-α-induced nuclear factor κB (NF-κB) signaling pathway, α-conidendrin did not influence the translocation of the NF-κB subunit RelA from the cytoplasm to the nucleus at concentrations up to 100 µM. A chromatin immunoprecipitation assay revealed that α-conidendrin at 100 µM reduced the binding of RelA to the ICAM-1 promoter in response to a stimulation with TNF-α. Collectively, these results indicated that α-conidendrin interfered with the DNA binding of RelA to the ICAM-1 promoter, thereby reducing ICAM-1 transcription.


Assuntos
Adenocarcinoma de Pulmão/metabolismo , Molécula 1 de Adesão Intercelular/biossíntese , Molécula 1 de Adesão Intercelular/genética , Lignanas/farmacologia , Neoplasias Pulmonares/metabolismo , Tetra-Hidronaftalenos/farmacologia , Células A549 , Adenocarcinoma de Pulmão/tratamento farmacológico , Sobrevivência Celular/efeitos dos fármacos , Cromanos/farmacologia , Ciclo-Oxigenase 2/metabolismo , Selectina E/efeitos dos fármacos , Selectina E/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Regiões Promotoras Genéticas/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/toxicidade
6.
Adv Exp Med Biol ; 1204: 31-56, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32152942

RESUMO

Mincle (macrophage inducible C-type lectin, Clec4e, Clecsf9) was originally identified as a member of the C-type lectin receptor family in 1999. Then, the function of Mincle to control antifungal immunity by binding to Candida albicans was reported in 2008. Around the same time, it was reported that Mincle recognized damaged cells and induced sterile inflammation by coupling with the ITAM-adaptor molecule FcRγ. In the following year, a breakthrough discovery reported that Mincle was an essential receptor for mycobacterial cord factor (trehalose-6,6'-dimycolate, TDM). Mincle gained increasing attention immediately after this critical finding. Although our understanding of the recognition of Mycobacteria has been advanced significantly, it was also revealed that Mincle interacts with pathogens other than Mycobacteria. In addition, endogenous ligands of Mincle were identified recently. Therefore, Mincle is now considered a danger receptor both for self and non-self ligands, so-called damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs). This chapter will give an overview of the accumulated knowledge of the multi-task danger receptor Mincle from its discovery to the latest findings.


Assuntos
Fatores Corda/imunologia , Lectinas Tipo C/imunologia , Mycobacterium/química , Mycobacterium/imunologia , Receptores Imunológicos/imunologia , Animais , Humanos
7.
J Biol Chem ; 294(45): 16776-16788, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31551352

RESUMO

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a family of cell-surface immune receptors that bind to sialic acid at terminal glycan residues. Siglecs also recognize nonsialic acid ligands, many of which remain to be characterized. Here, we found that Siglec5 and Siglec14 recognize lipid compounds produced by Trichophyton, a fungal genus containing several pathogenic species. Biochemical approaches revealed that the Siglec ligands are fungal alkanes and triacylglycerols, an unexpected finding that prompted us to search for endogenous lipid ligands of Siglecs. Siglec5 weakly recognized several endogenous lipids, but the mitochondrial lipid cardiolipin and the anti-inflammatory lipid 5-palmitic acid-hydroxystearic acid exhibited potent ligand activity on Siglec5. Further, the hydrophobic stretch in the Siglec5 N terminus region was found to be required for efficient recognition of these lipids. Notably, this hydrophobic stretch was dispensable for recognition of sialic acid. Siglec5 inhibited cell activation upon ligand binding, and accordingly, the lipophilic ligands suppressed interleukin-8 (IL-8) production in Siglec5-expressing human monocytic cells. Siglec14 and Siglec5 have high sequence identity in the extracellular region, and Siglec14 also recognized the endogenous lipids. However, unlike Siglec5, Siglec14 transduces activating signals upon ligand recognition. Indeed, the endogenous lipids induced IL-8 production in Siglec14-expressing human monocytic cells. These results indicated that Siglec5 and Siglec14 can recognize lipophilic ligands that thereby modulate innate immune responses. To our knowledge, this is the first study reporting the binding of Siglecs to lipid ligands, expanding our understanding of the biological function and importance of Siglecs in the innate immunity.


Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Proteínas Fúngicas/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Imunidade Inata , Lectinas/metabolismo , Receptores de Superfície Celular/metabolismo , Alcanos/química , Alcanos/metabolismo , Linhagem Celular , Humanos , Ligantes , Trichophyton/imunologia , Triglicerídeos/química , Triglicerídeos/metabolismo
8.
Sci Rep ; 9(1): 872, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696945

RESUMO

Increasing evidence indicates that pattern recognition receptors (PRRs) are involved in neuropathic pain after peripheral nerve injury (PNI). While a significant number of studies support an association between neuropathic pain and the innate immune response mediated through Toll-like receptors, a family of PRRs, the roles of other types of PRRs are largely unknown. In this study, we have focused on the macrophage-inducible C-type lectin (Mincle), a PRR allocated to the C-type lectin receptor family. Here, we show that Mincle is involved in neuropathic pain after PNI. Mincle-deficient mice showed impaired PNI-induced mechanical allodynia. After PNI, expression of Mincle mRNA was rapidly increased in the injured spinal nerve. Most Mincle-expressing cells were identified as infiltrating leucocytes, although the migration of leucocytes was also observed in Mincle-deficient mice. Furthermore, Mincle-deficiency affected the induction of genes, which are reported to contribute to neuropathic pain after PNI in the dorsal root ganglia and spinal dorsal horn. These results suggest that Mincle is involved in triggering sequential processes that lead to the pathogenesis of neuropathic pain.


Assuntos
Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Neuralgia/metabolismo , Traumatismos dos Nervos Periféricos/fisiopatologia , Animais , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Hiperalgesia/metabolismo , Imunidade Inata , Lectinas Tipo C/fisiologia , Macrófagos/metabolismo , Masculino , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neuralgia/fisiopatologia , Traumatismos dos Nervos Periféricos/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Corno Dorsal da Medula Espinal/metabolismo , Nervos Espinhais/patologia , Receptores Toll-Like/metabolismo
9.
Artigo em Inglês | MEDLINE | ID: mdl-30087858

RESUMO

Amoebiasis is caused by Entamoeba histolytica infection, a protozoan parasite belonging to the phylum Amoebozoa. This parasite undergoes a fundamental cell differentiation process from proliferative trophozoite to dormant cyst, termed "encystation." The cysts formed by encystation are solely responsible for the transmission of amoebiasis; therefore, Entamoeba encystation is an important subject from both biological and medical perspectives. Here, we have established a flow cytometry strategy for not only determining the percentage of formed cysts but also for monitoring changes in cell populations during encystation. This strategy together with fluorescence microscopy enables visualization of the cell differentiation process of Entamoeba encystation. We also standardized another flow cytometry protocol for counting live trophozoites. These two different flow cytometry techniques could be integrated into 96-well plate-based bioassays for monitoring the processes of cyst formation and trophozoite proliferation, which are crucial to maintain the Entamoeba life cycle. The combined two systems enabled us to screen a chemical library, the Pathogen Box of the Medicine for Malaria Venture, to obtain compounds that inhibit either the formation of cysts or the proliferation of trophozoites, or both. This is a prerequisite for the development of new drugs against amoebiasis, a global public health problem. Collectively, the two different 96-well plate-based Entamoeba bioassay and flow cytometry analysis systems (cyst formation and trophozoite proliferation) provide a methodology that can not only overcome the limitations of standard microscopic counting but also is effective in applied as well as basic Entamoeba biology.


Assuntos
Entamoeba/crescimento & desenvolvimento , Citometria de Fluxo/métodos , Parasitologia/métodos , Esporos de Protozoários/crescimento & desenvolvimento , Microscopia de Fluorescência/métodos
10.
PLoS One ; 13(3): e0195119, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29596528

RESUMO

Apaf1 is a critical component of the apoptosome and initiates apoptosis downstream mitochondrial damages. Although the importance of Apaf1 in embryonic development was shown, the role of Apaf1 in immune responses, especially T cell responses, has yet to be elucidated. We generated T cell-specific Apaf1-deficient mice (Lck-Cre-Apaf1f/f mice) and examined the antigen-specific delayed-type hypersensitivity (DTH). Lck-Cre-Apaf1f/f mice exhibited exacerbation of DTH responses as compared with Apaf1-sufficient control mice. In Lck-Cre-Apaf1f/f mice, antigen-specific T cells proliferated more, and produced more inflammatory cytokines than control T cells. Apaf1-deficient T cells from antigen-immunized mice showed higher percentages of activation phenotypes upon restimulation in vitro. Apaf1-deficient T cells from naive (non-immunized) mice also showed higher proliferation activity and cytokine production over control cells. The impact of Apaf1-deficiency in T cells, however, was not restored by a pan-caspase inhibitor, suggesting that the role of Apaf1 in T cell responses was caspase-independent/non-apoptotic. These data collectively demonstrated that Apaf1 is a negative regulator of T cell responses and implicated Apaf1 as a potential target for immunosuppressive drug discovery.


Assuntos
Fator Apoptótico 1 Ativador de Proteases/fisiologia , Hipersensibilidade Tardia/imunologia , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Animais , Apoptose , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
11.
Int Immunol ; 29(10): 479-485, 2017 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-29106539

RESUMO

Lineage-specific Cre Tg mice are widely used to delineate the functions of genes in a tissue-specific manner. Several T-cell-specific promoter cassettes have been developed; however, the activities of those promoters in non-T cells have not been investigated extensively. Here, we report that CD2-Cre-mediated deletion of Erk proteins by generating CD2-Cre × Erk1-/-Erk2flox/flox (Erk∆CD2-Cre) mice results in abnormal cartilage hyperplasia. Histological analysis revealed that this abnormality is caused by aberrant hyperplasia of chondrocytes. The presence of Erk-deficient T cells is not required for this chondroma formation, as it was similarly observed in the absence of T cells in a CD3ε-deficient background. In addition, adoptive transfer of bone marrow cells from Erk∆CD2-Cre mice to wild-type recipients did not cause chondroma formation, suggesting that Erk-deficient non-immune cells are responsible for this abnormality. By tracing Cre-expressed tissues using a ROSA26-STOP-RFP allele, we found that the chondroma emitted RFP fluorescence, indicating that functional Cre is expressed in hyperplastic chondrocytes in Erk∆CD2-Cre mice. Furthermore, RFP+ chondrocytes were also found in an Erk-sufficient background, albeit without aberrant growth. These results suggest that unexpected expression of CD2-driven Cre in chondrocytes generates Erk-deficient chondrocytes, resulting in hyperplastic cartilage formation. Recently, two independent reports showed that CD4-Cre-mediated Ras-Erk signaling ablation led to similar abnormal cartilage formation (Guittard, G., Gallardo, D. L., Li, W. et al. 2017. Unexpected cartilage phenotype in CD4-Cre-conditional SOS-deficient mice. Front. Immunol. 8:343; Wehenkel, M., Corr, M., Guy, C. S. et al. 2017. Extracellular signal-regulated kinase signaling in CD4-expressing cells inhibits osteochondromas. Front. Immunol. 8:482). Together with these reports, our study suggests that an unexpected link exists between T-like cell and chondrocyte lineages during ontogeny.


Assuntos
Antígenos CD2/imunologia , Condroma/metabolismo , Integrases/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Animais , Cartilagem/imunologia , Cartilagem/metabolismo , Cartilagem/patologia , Condrócitos/imunologia , Condrócitos/metabolismo , Condrócitos/patologia , Condroma/imunologia , Integrases/imunologia , Camundongos , Camundongos Knockout , Proteína Quinase 3 Ativada por Mitógeno/deficiência , Proteína Quinase 3 Ativada por Mitógeno/imunologia
12.
J Immunol ; 194(11): 5366-74, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25888641

RESUMO

C-type lectin receptors expressed in APCs are recently defined pattern recognition receptors that play a crucial role in immune responses against pathogen-associated molecular patterns. Among pathogen-associated molecular patterns, cord factor (trehalose-6,6'-dimycolate [TDM]) is the most potent immunostimulatory component of the mycobacterial cell wall. Two C-type lectin receptors, macrophage-inducible C-type lectin (Mincle) and macrophage C-type lectin (MCL), are required for immune responses against TDM. Previous studies indicate that MCL is required for TDM-induced Mincle expression. However, the mechanism by which MCL induces Mincle expression has not been fully understood. In this study, we demonstrate that MCL interacts with Mincle to promote its surface expression. After LPS or zymosan stimulation, MCL-deficient bone marrow-derived dendritic cells (BMDCs) had a lower level of Mincle protein expression, although mRNA expression was comparable with wild-type BMDCs. Meanwhile, BMDCs from MCL transgenic mice showed an enhanced level of Mincle expression on the cell surface. MCL was associated with Mincle through the stalk region and this region was necessary and sufficient for the enhancement of Mincle expression. This interaction appeared to be mediated by the hydrophobic repeat of MCL, as substitution of four hydrophobic residues within the stalk region with serine (MCL(4S)) abolished the function to enhance the surface expression of Mincle. MCL(4S) mutant failed to restore the defective TDM responses in MCL-deficient BMDCs. These results suggest that MCL positively regulates Mincle expression through protein-protein interaction via its stalk region, thereby magnifying Mincle-mediated signaling.


Assuntos
Lectinas Tipo C/metabolismo , Proteínas de Membrana/metabolismo , Mycobacterium tuberculosis/imunologia , Receptores Imunológicos/metabolismo , Tuberculose/imunologia , Sequência de Aminoácidos , Animais , Células da Medula Óssea/imunologia , Parede Celular/imunologia , Células Cultivadas , Fatores Corda/imunologia , Células Dendríticas/imunologia , Interações Hidrofóbicas e Hidrofílicas , Lectinas Tipo C/genética , Lipopolissacarídeos/imunologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas/imunologia , Estrutura Terciária de Proteína , RNA Mensageiro/biossíntese , Receptores Imunológicos/genética , Transdução de Sinais , Zimosan/imunologia
13.
Immunity ; 41(3): 402-413, 2014 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-25176311

RESUMO

Mycobacteria possess various immunomodulatory molecules on the cell wall. Mannose-capped lipoarabinomannan (Man-LAM), a major lipoglycan of Mycobacterium tuberculosis, has long been known to have both inhibitory and stimulatory effects on host immunity. However, the direct Man-LAM receptor that explains its pleiotropic activities has not been clearly identified. Here, we report that a C-type lectin receptor Dectin-2 (gene symbol Clec4n) is a direct receptor for Man-LAM. Man-LAM activated bone-marrow-derived dendritic cells (BMDCs) to produce pro- and anti-inflammatory cytokines, whereas it was completely abrogated in Clec4n(-/-) BMDCs. Man-LAM promoted antigen-specific T cell responses through Dectin-2 on DCs. Furthermore, Man-LAM induced experimental autoimmune encephalitis (EAE) as an adjuvant in mice, whereas Clec4n(-/-) mice were resistant. Upon mycobacterial infection, Clec4n(-/-) mice showed augmented lung pathology. These results demonstrate that Dectin-2 contributes to host immunity against mycobacterial infection through the recognition of Man-LAM.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Lectinas Tipo C/imunologia , Lipopolissacarídeos/imunologia , Infecções por Mycobacterium/imunologia , Animais , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Citocinas/biossíntese , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/genética , Inflamação/imunologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Lectinas Tipo C/genética , Lipopolissacarídeos/química , Manose/química , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Knockout , Infecções por Mycobacterium/genética , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Fator 88 de Diferenciação Mieloide/genética , Ligação Proteica/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores Imunológicos/genética , Linfócitos T/imunologia
14.
PLoS One ; 9(2): e88747, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24533147

RESUMO

Guinea pig is a widely used animal for research and development of tuberculosis vaccines, since its pathological disease process is similar to that present in humans. We have previously reported that two C-type lectin receptors, Mincle (macrophage inducible C-type lectin, also called Clec4e) and MCL (macrophage C-type lectin, also called Clec4d), recognize the mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM). Here, we characterized the function of the guinea pig homologue of Mincle (gpMincle) and MCL (gpMCL). gpMincle directly bound to TDM and transduced an activating signal through ITAM-bearing adaptor molecule, FcRγ. Whereas, gpMCL lacked C-terminus and failed to bind to TDM. mRNA expression of gpMincle was detected in the spleen, lymph nodes and peritoneal macrophages and it was strongly up-regulated upon stimulation of zymosan and TDM. The surface expression of gpMincle was detected on activated macrophages by a newly established monoclonal antibody that also possesses a blocking activity. This antibody potently suppressed TNF production in BCG-infected macrophages. Collectively, gpMincle is the TDM receptor in the guinea pig and TDM-Mincle axis is involved in host immune responses against mycobacteria.


Assuntos
Fatores Corda/metabolismo , Lectinas Tipo C/metabolismo , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Feminino , Regulação da Expressão Gênica , Cobaias , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Macrófagos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Fc/metabolismo
15.
Proc Natl Acad Sci U S A ; 110(43): 17438-43, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-24101491

RESUMO

Mincle [macrophage inducible Ca(2+)-dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6'-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor-TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle-citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca(2+)-dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.


Assuntos
Fatores Corda/química , Lectinas Tipo C/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptores Imunológicos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Cálcio/química , Cálcio/metabolismo , Ácido Cítrico/química , Ácido Cítrico/metabolismo , Fatores Corda/metabolismo , Cristalografia por Raios X , Humanos , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ligantes , Camundongos , Dados de Sequência Molecular , Mutação , Ligação Proteica , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície
16.
Immunity ; 38(5): 1050-62, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23602766

RESUMO

Cord factor, also called trehalose-6,6'-dimycolate (TDM), is a potent mycobacterial adjuvant. We herein report that the C-type lectin MCL (also called Clec4d) is a TDM receptor that is likely to arise from gene duplication of Mincle (also called Clec4e). Mincle is known to be an inducible receptor recognizing TDM, whereas MCL was constitutively expressed in myeloid cells. To examine the contribution of MCL in response to TDM adjuvant, we generated MCL-deficient mice. TDM promoted innate immune responses, such as granuloma formation, which was severely impaired in MCL-deficient mice. TDM-induced acquired immune responses, such as experimental autoimmune encephalomyelitis (EAE), was almost completely dependent on MCL, but not Mincle. Furthermore, by generating Clec4e(gfp) reporter mice, we found that MCL was also crucial for driving Mincle induction upon TDM stimulation. These results suggest that MCL is an FcRγ-coupled activating receptor that mediates the adjuvanticity of TDM.


Assuntos
Fatores Corda/imunologia , Encefalomielite Autoimune Experimental/imunologia , Lectinas Tipo C/imunologia , Proteínas de Membrana/metabolismo , Receptores de IgG/imunologia , Adjuvantes Imunológicos , Animais , Encefalomielite Autoimune Experimental/microbiologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Mycobacterium/imunologia , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia
17.
Adv Exp Med Biol ; 738: 144-52, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22399378

RESUMO

Multicellular organisms have developed ways to recognize potentially life-threatening events (danger signals). Classically, danger signals have been defined as exogenous, pathogen-associated molecular patterns (PAMPs) such as bacterial cell wall components (e.g., lipopolysaccharide and peptideglycan) or viral DNA/RNA. PAMPs interact with dedicated receptors on immune cells, so-called pattern recognition receptors (PRRs) and activate immune systems. A well-known family of PRRs is the toll-like receptors (TLRs) in which each member recognizes a specific set of PAMPs. However, not only exogenous pathogens but also several endogenous molecules released from necrotic cells (damaged self) also activate immune systems. These endogenous adjuvants are called damage-associated molecular patterns (DAMPs). It has been reported that high-mobility group box 1 protein (HMGB1), uric acid, heat shock proteins (HSPs) and nucleotides act as endogenous adjuvants. DAMPs are recognized by specific receptors (danger receptors) expressed mainly on antigen-presenting cells such as dendritic cells and macrophages and induce cell maturation and the production of inflammatory cytokines by activating the NF-kB pathway. In this chapter, we will review danger signals released from necrotic cells and its recognition receptors.


Assuntos
Células Dendríticas/imunologia , Imunidade Inata , Macrófagos/imunologia , Necrose/imunologia , Transdução de Sinais/imunologia , Animais , Bactérias/imunologia , Parede Celular/imunologia , Citocinas/imunologia , DNA Viral/imunologia , Células Dendríticas/patologia , Proteína HMGB1/imunologia , Humanos , Lipopolissacarídeos/imunologia , Macrófagos/patologia , Peptidoglicano/imunologia , RNA Viral/imunologia , Receptores Toll-Like/imunologia , Vírus/imunologia
18.
Immunity ; 34(1): 85-95, 2011 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-21194983

RESUMO

The generation of tumor-directed cytotoxic T lymphocytes is considered crucial for the induction of antitumor immunity. To activate these CD8(+) T cells, antigen-presenting cells (APCs) must initially acquire tumor cell-associated antigens. The major source of tumor antigens is dead tumor cells, but little is known about how APCs in draining lymph nodes acquire and crosspresent these antigens. Here we show that CD169(+) macrophages phagocytose dead tumor cells transported via lymphatic flow and subsequently crosspresent tumor antigens to CD8(+) T cells. Subcutaneous immunization with irradiated tumor cells protects mice from syngenic tumor. However, tumor antigen-specific CD8(+) T cell activation and subsequent antitumor immunity are severely impaired in mice depleted with CD169(+) macrophages. Neither migratory dendritic cells (DCs) nor lymph node-resident conventional DCs are essential for the crosspresentation of tumor antigens. Thus, we have identified CD169(+) macrophages as lymph node-resident APCs dominating early activation of tumor antigen-specific CD8(+) T cells.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Linfonodos/patologia , Linfoma de Células T/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/biossíntese , Receptores Imunológicos/biossíntese , Animais , Antígenos de Neoplasias/imunologia , Antígeno CD11c/biossíntese , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Movimento Celular/imunologia , Apresentação Cruzada , Imunização , Ativação Linfocitária , Linfoma de Células T/patologia , Macrófagos/imunologia , Macrófagos/patologia , Camundongos , Camundongos Transgênicos , Fagocitose/imunologia , Lectina 1 Semelhante a Ig de Ligação ao Ácido Siálico
19.
Proc Natl Acad Sci U S A ; 107(46): 19979-84, 2010 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-21030681

RESUMO

The pre-T-cell receptor (TCR) is crucial for the early T-cell development, but the ligand for pre-TCR remains unidentified. We recently proposed a model that pre-TCR complexes oligomerize spontaneously through interactions of the pre-TCRα chain. To investigate the mechanism underlying this ligand-independent signaling in vivo, we established knock-in mice that express a pre-TCRα mutant lacking charged amino acids (D(22)R(24)R(102)R(117) to A(22)A(24)A(102)A(117); 4A). CD4(+)CD8(+) thymocyte number was significantly reduced in invariant pre-TCRα (pTα(4A/4A)) mice, whereas CD4(-)CD8(-) thymocytes were unaffected. The percentages of double-negative 3 (DN3) cells and γδ T cells were increased in the pTα(4A/4A) thymus, indicating that ß-selection is impaired in pTα(4A/4A) mice. Pre-TCR-mediated tyrosine phosphorylation and clonal expansion into double-positive thymocytes were also defective in the knock-in mice. Pre-TCR was expressed at higher levels on pTα(4A/4A) cell surfaces than on those of the wild type, suggesting that the charged residues in pTα are critical for autonomous engagement and subsequent internalization of pre-TCR. Pre-TCR-mediated allelic exclusion of the TCRß gene was also inhibited in pTα(4A/4A) mice, and thereby, dual TCRßs were expressed on pTα(4A/4A) T cells. Furthermore, the TCRß chain variable region (Vß) repertoire of mature T cells was significantly altered in pTα(4A/4A) mice. These results suggest that charged residues of pTα are critical for ß-selection, allelic exclusion, and TCRß repertoire formation.


Assuntos
Eletricidade , Células Germinativas/imunologia , Glicoproteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Transdução de Sinais/imunologia , Alelos , Animais , Técnicas de Introdução de Genes , Células Germinativas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Timo/imunologia , Timo/patologia
20.
Proc Natl Acad Sci U S A ; 107(14): 6436-41, 2010 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-20308543

RESUMO

During the course of inflammation and its resolution, macrophages are exposed to various cytotoxic materials, including reactive oxygen species. Thus, macrophages require a protective machinery against oxidative stress to survive at the inflammatory site. Here, we showed that xCT, a component of transport system x(c)(-), was significantly up-regulated in activated infiltrating cells, including macrophages and neutrophils at the inflammatory site. System x(c)(-) mediates the uptake of extracellular L-cystine and is consequently responsible for maintenance of intracellular glutathione levels. We established a loss-of-function mouse mutant line of xCT by N-ethyl-N-nitrosourea mutagenesis. Macrophages from xCT(mu/mu) mice showed cell death in association with the excessive release of high mobility group box chromosomal protein 1 upon stimulation with LPS, suggesting that xCT deficiency causes unremitting inflammation because of the impaired survival of activated macrophages at the inflammatory site. Subcutaneous injection of 3-methylcholanthrene (3-MCA) induced the generation of fibrosarcoma in association with inflammation. When 3-MCA was injected s.c. into mice, xCT mRNA was up-regulated in situ. In xCT(mu/mu) mice, inflammatory cytokines (such as IL-1beta and TNFalpha) were overexpressed, and the generation of 3-MCA-induced fibrosarcoma was accelerated. These results clearly indicate that the defect of the protective system against oxidative stress impaired survival of activated macrophages and subsequently enhanced tumorigenecity.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Transformação Celular Neoplásica/imunologia , Transformação Celular Neoplásica/metabolismo , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Sistema y+ de Transporte de Aminoácidos/deficiência , Sistema y+ de Transporte de Aminoácidos/imunologia , Animais , Morte Celular , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Fibrossarcoma/induzido quimicamente , Fibrossarcoma/imunologia , Regulação Neoplásica da Expressão Gênica , Interleucina-1beta/imunologia , Metilcolantreno , Camundongos , Camundongos Knockout , Fator de Necrose Tumoral alfa/imunologia , Regulação para Cima
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